Mechanisms of Vascular Smooth Muscle Contraction and the Basis for Pharmacologic Treatment of Smooth Muscle Disorders

1The smooth muscle cells of blood vessels display considerable plasticity in their phenotype. In healthy, young blood vessels, the phenotype is largely contractile and blood pressure is well autoregulated. However, during the life span of an individual, vascular cells can switch to a synthetic, largely noncontractile phenotype in response to proinflammatory stimuli, diet or other factors that result in the development of atherosclerosis or vessel remodeling. We will not focus on these processes here but refer the reader to several recent reviews on this topic.

2Here we will focus on the contractile phenotype, which also can display plasticity of function through a range of more subtle adaptations to aging, biomechanical stress, and vasoactive physiologic and pathophysiologic molecules. The current review will focus on these responses and especially focus, as a prototype disease of contractile vascular smooth muscle, on the complex role of this cell type in hypertension and where many opportunities exist for the exploration of untapped potential therapeutic targets.

3In humans, the diagnosis of hypertension is widespread, but typically asymptomatic; 20–50% of the world’s population has hypertension and in the United States ∼30% of the population is hypertensive. Furthermore, hypertension is a major risk factor for cardiovascular disease, stroke, and end-stage renal disease, and thus, there is significant morbidity and mortality associated with this disease. Because blood pressure (BP) is related to the cardiac output (CO) and systemic vascular resistance (SVR) by the equation BP = CO × SVR, increases in either CO or SVR should produce hypertension. Thus, although the molecular mechanism(s) that produce hypertension would be expected to be relatively straightforward, over 50 years of investigation have not defined the molecular mechanism(s) that underlies this medical condition.

4The control of blood pressure is an integrated response that includes regulation by neural receptors, hormones, and renal fluid balance. However, the handling of sodium within the kidney is well accepted to be the major factor that regulates blood pressure (Fig. 1), and hence, in the pathogenesis of hypertension renal Na+ excretion, which regulates intravascular volume, is the primary determinant of cardiac output (CO) and therefore blood pressure. The role of control of intravascular volume by the kidney for the pathogenesis of hypertension is supported by the results of an elegant series of studies by Lifton’s group (reviewed in ). These investigators demonstrated that in humans, rare genetic causes of hypertension all arise from a defect in the handling of Na+ in the kidney; mutations that increase Na+ reabsorption (volume expansion) result in severe hypertension, whereas mutations that decrease Na+ resorption (volume contraction) produce hypotension. We will not discuss the well-accepted role of renal fluid balance in regulation blood pressure, because this topic has been the subject of a number of reviews.

5More than 90% of patients are diagnosed with essential hypertension, or hypertension of unknown etiology. Fortunately, despite the lack of a clear mechanism, there are a number of classes of antihypertensive agents that effectively lower blood pressure. Intuitively, one would expect that changes in vascular tone would result in changes in systemic vascular resistance (SVR) and result in either hyper- and/or hypotension. And, although a number of the classes of antihypertensive agents target the vascular smooth muscle [α-blockers, angiotensin converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), calcium channel blockers (CCBs)], until recently, there was little experimental evidence consistent with the regulation of vascular tone being an important factor for the molecular mechanism that produces hypertension (Fig. 1). However, a number of studies have demonstrated the importance of changes in vascular reactivity or the regulation of vascular smooth muscle contraction and/or vascular tone for the control of blood pressure. For these experiments, investigators have genetically modified a mouse to produce abnormalities in the regulation of vascular tone and/or vascular dysfunction; these mouse models include the BKCa2+ channel β1 subunit knockout (KO), estrogen receptor β KO, vascular smooth muscle cell Sur2 K(ATP) channel KO, endothelial nitric oxide synthase (eNOS) KO, RGS2 KO, PKGI KO, PKGIα leucine zipper mutant, and the MYPT1 KO. All of these mice have both vascular dysfunction and hypertension, and these data suggest that vascular dysfunction produces hypertension. However, in these transgenic models, vascular dysfunction within the kidney could alter fluid balance and a resulting increase in intravascular volume and the resulting increase in CO could be responsible for producing hypertension. The most compelling argument that isolated vascular dysfunction results in hypertension are the results of. These investigators demonstrated that mice with a KO of the angiotensin type 1 (AT1) receptor were hypotensive. Furthermore, these investigators produced mice with the KO of the AT1 receptors in the kidney with normal AT1 expression in the peripheral vasculature, as well as the KO of AT1 receptors in the peripheral vascular smooth muscle with normal AT1 expression in the kidney. The blood pressure in these two strains was equal and intermediate between the AT1 KO and wild-type (WT) mice. These results demonstrate that in isolation, an abnormality in the regulation of vascular smooth muscle contraction produces a change in blood pressure, and therefore, an isolated increase in vascular smooth muscle tone will produce hypertension. Thus the regulation of vascular smooth muscle contraction is important in both health and disease.

6Further complicating investigation of the mechanism underlying the pathogenesis of hypertension are racial differences in the effectiveness of the various classes of antihypertensives, including the response to β-blockers, ACE inhibitors, and ARBs. White compared with black patients with hypertension are more likely to respond to β-blockers, ACE inhibitors, and ARBs, whereas for black patients, treatment with a diuretic or calcium channel blocker (CCB) is more likely to be effective. Additionally, there also appears to be regional differences in the response to antihypertensive agents; there is a 10 state region in the Southeastern U.S., referred to as the Stroke Belt, in which the mortality from cerebral vascular accidents is 10% greater than the rest of the country. In this region, compared with the rest of the U.S., treatment of hypertension with diuretics, β-blockers, ACE inhibitors, and clonidine is less effective, whereas there is no difference in the effectiveness of CCBs and prazosin. After controlling for race, the differences in the therapeutic success of diuretics and clonidine is still present. Furthermore, for black patients with hypertension in this region, similar to the rest of the U.S., CCBs are more likely to control blood pressure and the effectiveness of β-blockers and prazosin therapy is poor.

7These racial differences in response to therapy are also present for the treatment of heart failure. Analysis of the results of the V-HeFT (Vasodilator-Heart Failure) trial demonstrated that treatment of black patients with heart failure with the combination of hydralazine and isosorbide dinitrate reduced mortality, whereas this regimen did not change mortality compared with placebo for white patients. In contrast to these results, treatment of heart failure with enalapril reduced mortality in white, but not black, patients, and in white patients, enalapril produced a larger reduction in blood pressure and regression of cardiac size than hydralazine and isosorbide dinitrate.

8These racial and regional differences in the response to antihypertensive regimens could be due to polymorphisms. A number of genome wide-association studies (GWAS) have investigated this question (reviewed in; ), and these studies as well as their implications will be discussed later in this review. However, changes in the vascular smooth muscle phenotype could be responsible for diversity in the effectiveness of the different classes of antihypertensive agents. Defining the role of the vascular smooth muscle phenotype in the pathogenesis of hypertension could identify novel therapeutic targets, which could be exploited in rational drug design. Furthermore, comparing the vascular smooth muscle phenotype between races and regions could potentially define the mechanism that governs the heterogeneity in the response to antihypertensive therapy and form the basis for an individualized approach for selecting an effective antihypertensive regimen.

9Vascular smooth muscle cells (VSMC), like all other muscle cells, depend on Ca2+ influx to initiate contraction. However, the VSMC intracellular Ca2+ concentration does not only determine the contractile state, but also affects the activity of several Ca2+ dependent transcription factors and thereby determines VSMC phenotype. To govern the various Ca2+-dependent functions and in reaction to different stimuli, VSMCs use a variety of plasmalemmal and sarcoplasmic reticulum (SR) Ca2+ channels to produce a large repertoire of Ca2+ signals, which differ in their spatial and temporal distribution (reviewed by ). These signals range from cell-wide changes in [Ca2+] to highly localized Ca2+ entry or release events. Ca2+ can enter the cell from the extracellular space or be released from the largest intracellular Ca2+ store, the sarcoplasmic reticulum (SR). Extracellular Ca2+ influx is mainly mediated by the opening of voltage dependent L-type Ca2+ channels (LTCC), but there are a number of other channels that modulate intracellular Ca2+, including transient receptor potential (TRP) cation channels. Because of their high single-channel conductance and expression in VSMCs, LTCCs have the largest influence on global [Ca2+]i, and their activity largely determines the VSMC’s contractile state and ultimately vessel diameter. In response to agonist activation of SR-bound inositol trisphosphate (IP3) or ryanodine receptors (RyR), Ca2+ is released into the cytoplasm from the SR. Local Ca2+ signals from the plasmalemma or the junctional SR can augment or oppose increases in global Ca2+ through the activation of Ca2+-dependent ion channels and their regulatory signaling molecules that ultimately affect plasma membrane potential and therefore LTCC activity.

10The concept of Ca2+ compartmentalization was introduced when it was demonstrated that local increases in Ca2+ could activate the contractile apparatus without influencing other Ca2+-dependent signaling pathways. Ca2+ is slow to diffuse across the cytoplasm and a large flux of Ca2+ is required to achieve the high Ca2+ concentration necessary for activation of Ca2+-dependent processes. Therefore, to compartmentalize and regulate Ca2+ signals, VSMCs arrange their organelles in a fashion that limits the space for diffusion and thereby increases the effect of local changes in [Ca2+] (Fig. 2). The effects of Ca2+ entry hence depend on the way that organelles, Ca2+ pumps, channels, and Ca2+-dependent signaling molecules are organized in signaling microdomains around the source of the Ca2+ signal, as well as its duration and amplitude. More on the organization of such microdomains in VSMCs and how they affect VSMC contractility and phenotype can be found in the review on regulation of cellular communication by signaling microdomains by.

11Local increases in cytoplasmic Ca2+ resulting from influx through single or small clusters of LTCCs are called Ca2+ sparklets (reviewed by ). Because of the steep voltage sensitivity of LTCCs, the sparklet frequency and persistence are closely linked to membrane potential. Thus local changes in membrane potential will result in alterations of local sparklet activity, whereas cell-wide depolarization leads to extensive opening of LTCCs and global influx of Ca2+. Increases in [Ca2+]i and Ca2+ sensitivity of the contractile apparatus in VSMCs are considered hallmarks of essential hypertension, and it has been widely assumed that the increase in intracellular Ca2+ is mediated by increased influx through LTCCs. Consistent with this are results in the rat where banding was used to produce a sudden high intravascular pressure in the right renal artery. After only 2 days, VSMCs from the right renal artery showed increased expression of α1C subunits of the LTCC and increased Ca2+ currents compared with VSMCs from the left renal artery. However, the ratio of right renal artery/left renal artery α1C subunit expression decreased over time, which may indicate a dynamic adjustment to this sudden pressure overload occurring within the VSMCs.

12Surprisingly, overall LTCC expression and cell-wide Ca2+ influx was recently found to be decreased in a mouse model of essential hypertension. However, although there was a decrease in the number of LTCCs present on the plasma membrane, the LTCCs showed increased local sparklet activity. These investigators demonstrated that fewer, but highly active, LTCCs were able to increase [Ca2+]i locally as well as cell-wide. The activity of Ca2+ sparklets has been shown to depend on whether the LTCC is part of a pentad complex bound to the plasma membrane by the scaffolding protein AKAP150. LTCCs that are not coupled in such complexes have a higher probability of producing stochastic sparklets with low flux and short duration, whereas AKAP-associated channels can produce high-activity persistent sparklets.

13The dynamics of these persistent sparklets are regulated by kinases and phosphatases that are targeted to a subpopulation of LTCCs by the plasmalemmal anchor AKAP150. Under physiologic conditions in these signaling microdomains, the formation of persistent sparklets mainly relies on protein kinase C (PKC) activity and is counteracted by the serine phosphatase calcineurin. In pathologic conditions such as diabetes, however, protein kinase A (PKA) becomes a mediator of enhanced sparklet activity. In a study by it was shown that the inhibition of cytoplasmic calcineurin with cyclosporine A in AKAP−/− mice had no effect on LTCC sparklet activity, whereas the inhibition of AKAP150-anchored calcineurin in wild-type mice yielded an increase in persistent sparklets. This confirmed the hypothesis that there was a negative relationship between calcineurin and LTCC sparklet activity but highlighted the importance of calcineurin being targeted to the plasmalemma by AKAP150. The relevance of PKC interaction with LTCCs in the development of ATII-induced hypertension has been demonstrated in a number of experiments, in which not only the KO of PKC but also the ablation of AKAP150 lead to an inability of ATII infusion to produce hypertension. In this model, the level of cellular PKC was unchanged in AKAP150−/− VSMCs. These data suggest that recruitment of PKC to the LTCC by AKAP150 is crucial for the development of this form of hypertension. AKAP150 is also thought to play a role in the functional coupling of LTCCs to each other, which amplifies Ca2+ influx and is, similar to persistent sparklet activity, increased in hypertension. Although the mechanism of coupled gating is still under investigation, a model has been proposed by which coupled gating is mediated by calmodulin (CaM)-dependent interactions between the carboxy-terminals of AKAP150-coupled LTCCs and is increased with PKC activation and calcineurin inhibition.

14It should be noted, that in some studies, there was significant PKC activation in agonist-mediated vasoconstriction, but not in pressure-mediated vasoconstriction, suggesting that PKC has a negligible role in myogenic tone. However, other groups reported PKC involvement in the modulation of the arteriolar myogenic response to increased intravascular pressure. This study demonstrated that inhibition of PKC led to inhibition of the myogenic response, whereas a stimulator of PKC activity increased myogenic responsiveness.

15In a recent study, investigators demonstrated that AKAP150-recruited PKC also regulates the activity of Ca2+-permeable, nonselective TRPV4 channels. These channels can produce Ca2+ sparklets with 100-fold higher Ca2+ flux compared with LTCCs, yet they have been linked to VSMC relaxation. This association results from the high Ca2+ flux that enables TRPV4 to stimulate SR-membrane bound RyRs in relative proximity to the plasmalemma as a form of Ca2+-induced Ca2+ release (CICR) found in VSMCs.

16In contrast to TRPV4 sparklets, LTCC sparklet flux is much lower and therefore not sufficient to trigger Ca2+ release from the SR, but overall LTCC sparklet activity is higher and hence LTCCs have a much greater effect on global Ca2+. Through the effect on global Ca2+, LTCC sparklet activity determines the rate at which the SR can refill its Ca2+ stores. However, neither the SR Ca2+ content nor the number and amplitude of SR Ca2+ release events appear to be directly linked to LTCC sparklet activity.

17Other important members of the TRP channel family include TRPC1, TRPC3, TRPC6, and TRPM4. They have been found to have a role in regulating myogenic tone as well as the myogenic response and are known to be involved in the mechanism of action of vasoconstrictors (refer to reviews by, ) and will be discussed in the section II.B.4. However, for details on the role of TRP channels in vascular function and how the dysregulation of vascular as well as endothelial TRP channels is related to vascular-related pathologies, please see the recent review by.

18Highly restricted and large Ca2+ release events through SR RyRs are called Ca2+ sparks, and Ca2+ sparks have an important regulatory role in VSMCs. Similar to sparklets, their spatial reach is small, so they have no direct effect on contractility; however, the proximity of RyRs to the plasma membrane allows them to affect global [Ca2+]i indirectly (reviewed by ). The nature of the VSMC’s response to Ca2+ sparks depends on the Ca2+-activated plasmalemmal ion channels that are spatially coupled to the RyRs. In many VSMC, tissues sparks are targeted to large conductance Ca2+-dependent K+ channels (BK) that oppose vasoconstriction by allowing hyperpolarizing outward K+ currents. On the other hand, Ca2+ gated Cl− channels (CaCCs) depolarize the plasmalemma and thereby enhance Ca2+ influx through LTCCs.

19A single Ca2+ spark increases the open probability of about 30 BK channels in its proximity by 100-fold. Sparks can occur spontaneously or be triggered by TRPV4 sparklets in the form of a CICR mechanism. Structurally, plasmalemmal BK channels in VSMCs are formed by four pore forming alpha subunits encoded by the slo gene and regulatory β1 subunits that are not necessary for the formation of a functional channel. However, the β1 subunits play a significant role in modulating the Ca2+ sensitivity and hence functional coupling to RyRs. It has been demonstrated by several groups that ablation of the β1 subunit in mice leads to desensitization to Ca2+ and functional uncoupling of BK channels from Ca2+ sparks, causing membrane depolarization, increases in arterial tone, and eventually hypertension. Furthermore for ATII-induced hypertension, it has been reported that the β1, but not the pore-forming alpha subunit, is downregulated, which mediates a decrease in the sensitivity of BK channels and thereby contributes to vascular dysfunction. Consistent with these results, associations between gain of function mutations of the β1 subunit and a lower prevalence of diastolic hypertension have been described. Additionally, it has been demonstrated that β1 subunit downregulation in ATII-induced hypertension is mediated by enhanced activity of the transcription factor NFATc3. However in hypertensive animals, there have also been studies that have found higher expression of the α subunit in VSMCs, suggesting that the BK channel is primarily involved in a compensatory response to increased VSMC tone from enhanced LTCC or decreased Kv activity (reviewed in ). Hence BK channels appear to be involved in the pathogenesis in some as well as compensation and protection in other forms of hypertension.

20Using different strategies to modulate plasmalemmal K+ channel activity to inhibit β1 downregulation in developing hypertension or to increase β1 expression in VSMCs would appear to be a promising approach for the treatment of hypertension. In addition, a number of BK channel openers are currently in development; however, the use of BK channel openers for the treatment of hypertension is limited by concerns for off-site effects in other smooth muscle tissues. As the β1 subunit of the BK channel seems to be expressed predominantly in VSMCs, targeting β1 expression through gene therapy or modulation of the NFATc3 pathway represents a possible alternative (reviewed by ).

21In some VSMCs, Ca2+ sparks are coupled to CaCCs, and their activation is followed by “spontaneous transient inward currents” or STICs. The two families of CaCCs that have only recently been identified are called bestrophins and TMEM16A. They are also expressed in renal tubular epithelium as well as the heart and are hence thought to have a multidimensional role in blood pressure regulation (reviewed by ). Because the activation of these channels results in plasma membrane depolarizing currents, they are thought to have an amplifying effect on vascular contractile stimuli by indirectly causing the opening of LTCCs. Indeed, downregulation or inhibition of TMEM16A led to decreased arterial constriction in a variety of studies, and a smooth muscle KO of TMEM16A in mice lead to a decrease in the ability of ATII infusion to produce hypertension. As CaCCs are also permeable to other anions such as HCO3−, it is also possible that some effect may be due to changes in intracellular pH that would affect pH-sensitive enzymes including Rho kinase. Although there are a number of substances that can inhibit CaCC activity in vitro, the unknown molecular identity of TMEM16A as well as its expression in various tissues would suggest that there is poor pharmacological specificity in vivo and there would be many off target effects.

22Activation of CaCCs is also often mediated by Ca2+ waves, a Ca2+ signal in which subsequent openings of IP3Rs and in some tissues RyRs on the SR cause a wave of Ca2+ release events across the entire length of the VSMC, usually close to the plasma membrane. Westcott and colleagues described the contrasting roles of RyRs and IP3Rs for the effects of Ca2+ waves in arterioles and upstream feed arteries. Although arteriolar Ca2+ waves are solely IP3R mediated and therefore not inhibited by ryanodine, RyR inhibitors decreased Ca2+ waves in feed arteries. In both tissues, Ca2+ waves were inhibited with phospholipase C (PLC) inhibitors and IP3R blockers, which led to a decrease in [Ca2+i] and vasodilation. Therefore, IP3Rs contribute to Ca2+ waves in both tissues as part of a positive feedback loop for myogenic tone. In contrast, despite the inhibition of Ca2+ sparks and waves in feed arteries, inhibition of RyRs caused an increase in [Ca2+i] and led to vasoconstriction. This was abolished in the presence of BK-channel blocker paxilline, which supports the hypothesis that RyRs, which are involved in Ca2+ waves, are also coupled to BK channels and part of a negative feedback regulation of myogenic tone.

23In arterioles, Ca2+ waves are initiated by IP3-dependent opening of an IP3R creating a Ca2+ “blip,” a single IP3R opening, or a “puff,” short Ca2+ release from a small cluster of IP3Rs that is biophysically different from a RyR-mediated spark. Subsequently, clusters of IP3Rs open in response to the Ca2+ released by adjacent IP3Rs (CICR) and are inactivated as the [Ca2+] rises further. The IP3R’s Ca2+-dependent activation/inactivation properties are reflected in the wave-like pattern of IP3R-mediated Ca2+ release. Ca2+ wave initiation depends on IP3 synthesis by PLC, which occurs after activation of G-protein-coupled receptors by their respective agonists, including norepinephrine and endothelin-1. However, Ca2+ waves are also seen in the absence of agonists and depend on the spontaneous basal production of IP3 by PLC, which varies in different vascular beds, and thus will affect the frequency of Ca2+ release through RyRs via CICR. In arterioles, the wave leads to VSMC contraction by directly increasing [Ca2+]I, and this effect is amplified by the Ca2+-dependent opening of CaCCs in the plasma membrane that leads to membrane depolarization and increased Ca2+ influx through LTCCs.

24When SR Ca2+ stores are depleted after release through IP3Rs, the SR Ca2+ sensor STIM (stromal interaction molecule) relocates to the SR-plasmalemmal junction and physically interacts with and activates the selective Ca2+ channel Orai [CRAC (calcium release activated calcium channel); reviewed by ]. For VSMCs in the normal physiologic contractile phenotype, the expression of STIM/Orai is relatively low, but its expression is upregulated when the VSMC changes its phenotype to the proliferative state. In a rodent STIM/Orai knockdown model, nuclear factor activated T-cells (NFAT) translocation to the nucleus was decreased and VSMC proliferation in response to vascular injury was impaired. In spontaneously hypertensive rats, STIM/Orai is upregulated and depletion of SR stores lead to greater SOCE, which may represent an independent mechanism leading to increased VSMC [Ca2+]i in hypertension. Furthermore, these investigators found evidence suggesting that increased STIM/Orai activity may underlie sex differences in the development of hypertension. They determined that inactivation of the STIM/Orai mechanism with CRAC inhibitors as well as antibodies to STIM or Orai during and after store depletion abolished sex differences in spontaneous contractions of VSMCs. Thus CRACs represent a novel target in the treatment of hypertension.

25However, there are a number of studies also suggesting a role of TRPC channels in SOCE (reviewed by ). Both TRPCs and Orai channels can be activated by STIM after store depletion; however, their individual contribution to SOCE is variable. Studies have demonstrated a partial suppression of SOCE by Orai and TRPC siRNA, respectively. The nature of TRPC-Orai interaction, or if there is in fact one, is currently unresolved (refer to ), but both channels can also be activated independently from store depletion or Ca2+ release and their downstream effects on activation differ. TRPs exhibit multiplicity of gating and hence have been suggested to integrate various cellular signals including store depletion. TRPC1 mediates Ca2+ influx after store depletion with thapsigargin and is thought to be involved in contractile modulation and regulation of cell proliferation; however, more data are needed to determine its exact function. TRPC6 is a channel that mediates cation movement in a variety of experimental settings. In some tissues, inhibition of TRPC6 leads to a decrease in SOCE, but it also appears to be involved in SR-independent signaling. Studies demonstrated that TRPC6 is store and receptor operated, as well as stretch and osmotically activated in VSMCs. It can associate and form heteromultimers with TRPC3, which leads to tonic channel activation. TRPC3 and TRPC6 are upregulated in idiopathic pulmonary hypertension and an siRNA-induced decrease of TRPC6 expression decreases proliferation of cultured pulmonary artery VSMC isolated from patients with pulmonary hypertension. Furthermore, chronic hypoxia increases TRPC6 expression, whereas the ET-1 antagonist bosentan, a common treatment of PAH, lowers TRPC6 expression in pulmonary VSMCs. These are merely examples of the various roles TRPC channels occupy in VSMC signaling and a complete discussion of TRPC channels in health and disease has been recently presented in a number of reviews.

26An important mode in which Ca2+ can regulate VSMC contractility is by regulating the composition of the contractile apparatus, ion channels, and cellular signaling molecules by influencing VSMC gene transcription (reviewed by ). In certain cytoplasmic locations, high [Ca2+] activates specific kinases or phosphatases that in turn lead to activation and translocation of transcription factors to the nucleus. In the nucleus, Ca2+ can bind helix-loop-helix-loop structural domain or motif (EF hand) containing transcription factors directly or modulate transcription factor binding via Ca2+/CaM-S100 complexes. Although in many disease states VSMCs can completely lose their contractile function due to a phenotype switch toward a proliferative ECM-producing phenotype, more subtle changes within the contractile phenotype are also thought to play a role in the increased VSMC contractility observed in hypertension.

27NFAT is a major target of calcineurin, and it translocates to the nucleus upon calcineurin-mediated dephosphorylation. Calcineurin activation is enhanced by the activity of the AKAP150-bound LTCC signaling pentad. Hence, NFAT activity is regulated by the level of persistent sparklet activity, but is also dependent on simultaneous inhibition of its nuclear export. Interestingly, although membrane-depolarizing signals such as IP3R-mediated Ca2+ waves are thought to cause an increase in NFATc3 activation via enhanced LTCC activity, RyR-mediated Ca2+ release from the SR decreases NFAT activity by an LTCC independent mechanism. SOCE has also been implicated in NFAT activation, and its disruption led to reduced hypoxia-induced NFAT nuclear translocation in pulmonary VSMCs. Although a variety of Ca2+ signals lead to NFAT activation, persistently raised levels of intracellular Ca2+ fail to induce NFAT. Therefore it is thought that oscillating Ca2+ signals and concomitant inhibition of nuclear export leads to nuclear NFAT accumulation. It is well documented that inhibition of the calcineurin/NFAT pathway reduces VSMC proliferation, neointima formation, and vascular remodeling in response to injury. However there are also studies indicating a role for NFAT within the contractile phenotype, altering the expression of plasmalemmal ion channels including BK and Kv channels and thereby increasing VSMC contractility and ultimately arterial tone.

28In contrast to NFAT, CREB is regulated by the Ca2+ dependent kinases CaMKII and CaMKIV. Ca2+ influx through LTCCs is important for activated phospho-CREB to accumulate in the nucleus. Signals that increase LTCC activity including IP3R-mediated Ca2+ waves and SOCE lead to increased CREB-induced transcription, whereas Ca2+ sparks counteract CREB activity by hyperpolarizing the plasmalemma and reducing LTCC flux. Because CREB activates genes involved in the contractile, as well as the proliferative phenotype, the ultimate effect of CREB activation on VSMC phenotype has not yet been determined. However in contrast to NFAT, CREB is induced by any signal that causes a sustained increase in Ca2+ entry through LTCCs.

29In addition to controlling transcription indirectly through CREB, LTCCs have also been found to directly influence gene expression in VSMCs. In a study by it was determined that when the C-terminal end of the LTCC (CCt) is cleaved, it either reassociates with LTCCs and reduces LTCC sparklet activity or it relocates to the nucleus and inhibits the transcription of LTCCs. The CCt thus acts as a bimodal vasodilator by decreasing LTCC transcription and reducing voltage-dependent LTCC opening. However, the enzyme responsible for CCt cleavage and the mechanism(s) for regulation have yet to be determined. Potentiating the effects of CCt through increased cleavage or possibly stimulation of CCt promoter sequences may offer another novel approach to controlling vascular contractility.

30There are a variety of Ca2+-mediated mechanisms that increase VSMC contractility and are possible targets in antihypertensive therapy, some of which are well understood and can be specifically inhibited in vitro. However the development of novel treatments is often limited by the expression of the targets in nonvascular smooth muscle tissues and thus the many off site effects. A possible solution to this issue could be targeting specific therapies to VSMCs using viral vectors. There are a number of successful proof of concept studies using this technique that were recently reviewed by. Another issue that limits progress in the effort to find novel pharmacologic therapies in Ca2+ signaling is that the composition of Ca2+ signaling microdomains differs in various vascular beds, and hence results cannot always be generalized for VSMCs. This problem again highlights the importance of genetic KO and knockdown studies as a tool to explore targets for gene therapy for the treatment of hypertension.

31Many potential therapeutic strategies are designed to activate or inhibit specific signaling pathways in the vascular smooth muscle cell. It is clear that multiple vascular signaling pathways coexist as spatially separate signaling compartments in individual differentiated vascular smooth muscle (dVSM) cells and coordinated by a multitude of scaffolding proteins. However, these pathways are often overlapping, multilayered, and tissue specific. The tissue-specific nature of these pathways, even between different vessels or sizes of vessels, has led to much controversy on the relative importance of one pathway versus another. Ultimately, however, the possibility of multiple pathways that could be activated or inhibited in various disease states or as functional compensation to physiologic stress gives the system considerable functional plasticity.

32At the simplest level, it is well established that vascular tone can be increased either by increasing activation of myosin (Pathways #1 & #2, Fig. 3) or, in a manner analogous to that in striated muscle, by removal of inhibition of actin (Pathway #3, Fig. 3). Either mechanism will lead to an increase in actomyosin activation and crossbridge cycling. Recently, several laboratories have reported more controversial mechanisms by which agonists or biomechanical forces can regulate both vascular and airway smooth muscle contractility by remodeling cytoskeletal attachments (Pathway #4, Fig. 3). These four pathways are discussed in more detail below.

33This has been a very active area of investigation by vascular smooth muscle biologists and, as discussed below, has already identified many potential pharmacologic target molecules and in some cases led to possible drug candidates.

34Smooth muscle myosin differs from skeletal and cardiac myosins in that it lacks intrinsic myosin ATPase activity in the pure state. Smooth muscle myosin requires a posttranslational modification, phosphorylation of Ser 19 of the 20-kDa regulatory light chain to display enzymatic activity. This phosphorylation is caused by a dedicated Ser/Thr kinase, myosin light chain kinase (MLCK).

35MLCK is a Ca/CaM-dependent kinase and is most simply activated by increases in cytoplasmic ionized Ca ([Ca2+i]) levels (Pathway #1, Fig. 3) such as occurs with a large number of G-protein coupled receptor-mediated agonists, such as alpha agonists or by depolarization of the cell membrane by channel activity or experimentally by equimolar replacement of NaCl with KCl in physiologic saline solution. It has also been reported that increases in the free CaM level or Ca-independent changes in the kinase activity of MLCK can also occur by phosphorylation-mediated events.

36Dephosphorylation of myosin by myosin phosphatase (MP) decreases its activity, and conversely, inhibition of MP will increase its activity. A large number of pathways, such as those activated by PGF2a and lysophosphatidic acid (Pathway #2, Fig. 3), have been reported to inhibit MP through either Rho-associated protein kinase (ROCK)-dependent mechanisms or those involving Zipper-interacting protein kinase. These pathways are discussed in detail in section IV.

37CaMKinase II is another Ca/CaM-dependent kinase with the interesting property, when activated, of autophosphorylating itself on T287, which leads to a sustained activity after Ca is removed, giving it a chemical “memory” of having been activated. Conversely, when S26 in the catalytic domain is autophosphorylated, it can terminate sustained kinase activity, making it “forget” prior activation.

38There are four main isoforms of CaMKII, the alpha, beta, gamma, and delta isoforms. The gamma (especially the G-2 variant) and delta (especially the d2 variant) isoforms have been shown to play important roles in smooth muscle, with the gamma isoforms primarily regulating contractility and the delta isoforms regulating proliferation. To a large degree the gamma/delta ratio represents the degree of a phenotype switch between the contractile/proliferative phenotypes displayed by smooth muscle in different settings. Vascular injury reduces gamma isoform expression and upregulates delta expression. Conversely, siRNA-mediated knock down of the delta isoform attenuates VSM proliferation and neointimal formation. The conditional smooth muscle knockout of CaMKIIdelta significantly delays the progression of airway smooth muscle hyperresponsiveness to an ovalbumin challenge and this isoform is upregulated in the wild-type mouse in response to the same challenge. Thus the delta isoform may play a role in smooth muscle inflammatory responses.

39With respect to the gamma isoform and its regulation of smooth muscle contractility, six smooth variants of the gamma isoform of CaMKII have been described with varying kinetics of Ca/CaM-dependent activation/deactivation. One variant, the G-2 variant, which has unique sequence in the association domain of the kinase, has been shown to have scaffolding properties with respect to ERK (extracellular regulated kinase). Antisense specific for the G-2 variant or generic against the gamma isoform or small molecule inhibitor studies have all demonstrated roles for gamma CaMKII in the regulation of contractility. The CaMKII gamma G-2 variant is reported to be associated with vimentin intermediate filaments and dense bodies in unstimulated vascular smooth muscle cells and on activation by depolarization-mediated increases in cytosolic Ca2+ levels the G-2 variant translocates to the cortical focal adhesions. This variant also has been shown to be specifically dephosphorylated by an SCP3 phosphatase. SCP3 is a PP2C typed protein phosphatase, primarily expressed in vascular tissues and specifically binds to the unique association domain sequence in CaMKII gamma G-2. G-2 is bound to this phosphatase in unstimulated vascular smooth muscle tissue but is released upon depolarization-mediated Ca2+ influx. This phosphatase does not appear to regulate kinase activity but rather is thought to result in the exposure of a SH3 domain targeting of the kinase, which leads to targeting to focal adhesions.

40Although CaMKIIgamma is known to be activated by stimuli that increase the free Ca2+ level in dVSM, and antisense or inhibitors to CaMKII decrease the amplitude of the contraction to KCl PSS, the exact pathways that regulate contractility are still being confirmed. It has been shown that knock down of the gamma isoform or the G-2 variant specifically, as well as small molecule inhibitors of the kinase, lead to an inhibition of ERK activation and an inhibition of MLCK. ERK has been shown to be capable of activating MLCK in other systems, but whether this is the link in smooth muscle has not been definitively shown. Additionally in cultured vascular cells, CaMKII is rapidly activated after upon adherence of the cells upon plating onto ECM or poly-lysine. Adherence led to CaMKII-dependent tyrosine phosphorylation of paxillin and ERK activation. The CaMKII delta 2 variant has also been shown to regulate vascular smooth muscle cell motility in culture through a Src-family tyrosine kinase, Fyn. Because focal adhesions are known to serve as ERK scaffolds in contractile smooth muscle, this is an appealing possible link. Thus, at the present time, although CaMKII is clearly an important regulator of Ca2+-dependent vascular contractility, the complete molecular details of the CaMKII pathway used by contractile vascular smooth muscle to regulate contractility are not yet resolved. It is clear, however, that these details represent considerable untapped potential as future therapeutic targets for the modulation of Ca2+-dependent vascular contractility and hence blood pressure. Additionally, the wealth of information on isoform specific effects of CaMKII, especially the gamma G-2 variant, offers the potential of considerable tissue and smooth muscle phenotype specificity of such therapeutics.

41In contrast to the pathways described above, ex vivo studies have demonstrated that phorbol esters, or alpha agonists, by activating PKC can trigger increases in contractile force that in some tissues are either Ca2+ independent or cause leftward shifts in the [Ca2+i]-force relationship. The Ca2+ dependence of phorbol ester contractions varies in different smooth muscle tissues, dependent on the isoforms of PKC present in those tissues. The alpha, beta, and gamma isoforms are calcium dependent, but delta and epsilon are calcium independent. Thus, phorbol ester and alpha agonist-induced contractions have been described as being Ca2+ independent in experiments using the aorta of the ferret, which contains significant amounts of the epsilon isoform of PKC, but tissues containing more PKC alpha, such as the portal vein of the ferret, show an increased Ca2+ sensitivity but are still Ca2+ sensitive and lead to the activation of Pathway #3 in Fig. 3.

42Pathway 3 can be activated by diacylglycerol (DAG) release, generated by the activation of GPCRs in vascular smooth muscle, or by myometrial stretch, RU486, and labor in the rat and human myometrium. Interestingly, in the presence of extracellular Ca2+ (Cae), phenylephrine (PE) will activate pathways 1, 3, and 4 but in the absence of Cae and in the absence of changes in 20kda light chain phosphorylation, a contraction persists in aorta of the ferret. Phorbol esters give a maximal tonic contraction in the absence of changes in 20kda light chain phosphorylation, even in the presence of Cae in this system.

43When activated, Pathway #3 leads, indirectly, to the PKC-dependent activation of MEK, a dual activity kinase that phosphorylates ERK on a tyrosine and threonine, resulting in activation of ERK. ERK activation can have multiple downstream effects, largely controlled by output-specific scaffolding proteins (see below). In contractile smooth muscle, these downstream effects include phosphorylation of the actin binding protein caldesmon. Caldesmon has been described as being functionally analogous to the troponin complex in striated muscle in that it blocks the access of myosin to actin and hence impairs crossbridge cycling. The C-terminal end of caldesmon is responsible for the direct inhibition of myosin ATPase activity. Investigators have demonstrated that the interaction of the actin-binding domain of caldesmon with actin is responsible for the inhibition the actomyosin ATPase (AMATPase) by decreasing the rate of Pi release by 80%.

44Caldesmon has an NH2-terminal myosin-binding domain, in addition to the COOH-terminal actin-binding domain, and thus, in theory, could crosslink actin and myosin. However, also observed a tethering effect of the N-terminal region of caldesmon to myosin that has the proposed agonist-dependent functional effect of positioning caldesmon so that its C-terminal end no longer inhibits myosin activity. The binding of caldesmon to myosin is regulated by Ca2+-calmodulin, whereas the interaction with actin is regulated by ERK phosphorylation at Ser789 on caldesmon.

45In general, in most systems it appears that phosphorylation of caldesmon on Ser789 by ERK, PAK, or other serine kinases can reverse caldesmon-mediated inhibition of myosin ATPase activity (Pathway #3, Fig. 3). However, results from mechanical experiments examining caldesmon function are variable. In smooth muscle from caldesmon KO mice, compared with WT controls, both the rate of force activation and the steady-state force in response to depolarization, phorbol esters, and carbachol were similar, but the rate of force relaxation was reduced. In contrast to these results, an siRNA-induced decrease in caldesmon expression lowered both isometric force and muscle shortening velocity.

46In cultured smooth muscle cells, p42/44 MAPK has been clearly demonstrated to phosphorylate caldesmon at Ser789, but for agonist activation of intact smooth muscle, the kinase responsible for caldesmon phosphorylation remains a matter of controversy or may involve different kinases in different settings. In skinned smooth muscle strips, ERK-induced phosphorylation of caldesmon did not alter the force-Ca2+ relationship. Porcine carotid artery preparations did not display detectable phosphorylation of caldesmon at the ERK sites during phorbol ester stimulation,, but PAK phosphorylation at Thr627, Ser631, Ser635, and Ser642 was demonstrated to reduce caldesmon’s inhibition of the AMATPase. On the other hand, ERK-mediated phosphorylation of caldesmon at 789 has been clearly shown in ferret aorta preparations as well as mouse aorta and rat myometrium. Furthermore, although an increase in caldesmon phosphorylation was observed by in porcine carotid artery during both depolarization and histamine stimulation, experiments using inhibitors suggested that a second kinase in addition to ERK also phosphorylates caldesmon.

47In contrast to the myosin regulatory pathways, this is a relatively untapped area of investigation for the discovery of new target molecules with therapeutic potential. The relative importance of these pathways are definitely tissue and species specific. Interestingly, the strongest evidence of the importance of these pathways appears to have come from myometrial smooth muscle in the setting of preterm labor. Thus, the potential is there for novel and possibly quite specific therapeutic targets within these pathways.

48The vast majority of known protein phosphorylation events in the contractile, differentiated smooth muscle cell are serine/threonine events. Where phosphotyrosine screening with immunoblots of contractile vascular as well as myometrial smooth muscle tissue has been performed, the reactive bands have been almost exclusively focal adhesion-associated proteins. These tyrosine phosphorylations are largely sensitive to Src inhibitors, pointing to the presence of focal adhesion remodeling in nonproliferating, nonmigrating smooth muscle. These mechanisms have been especially studied in vascular and airway smooth muscles, resulting in pathways extending from Pathway #4 (Fig. 3). These mechanisms will be discussed in further detail in section V below.

49When our group first (Bradley and Morgan, 1982, ) measured intracellular Ca levels ([Ca2+]i) in dVSM with the photoprotein aequorin, we noticed that agonists often cause tonic contractions with only transient increases in [Ca2+]i or differing magnitudes of [Ca2+]i, reflecting apparent changes in “Ca2+ sensitivity” of the contractile apparatus. This dissociation between [Ca2+]i and force has been confirmed with many agonists and many different Ca2+ indicators in contractile smooth muscle tissues and with permeabilized smooth muscle preparations where leftward shifts in the Ca2+-force relationship in response to agonists and various agents are seen. Mechanistically, we now have molecular explanations for this phenomenology. Changes in the apparent Ca2+ sensitivity of the contractile apparatus have been partially explained by the ability of agonists to regulate the activity of myosin phosphatase (MP) (Pathway #2, Fig. 3), partially by the ability of ERK to regulate the action of caldesmon (CaD) to inhibit acto-myosin interactions (Pathway #3, Fig. 3) and clearly also by yet to be defined pathways.

50The complexity of signaling pathways in the smooth muscle cell raises the issue of how kinases connect with their complex specific upstream activators and downstream substrates in an agonist-specific manner within the three-dimensional space of the interior of a cell. Scaffold proteins are now recognized to play important roles in coordinating mammalian signal transduction. Protein scaffolds are defined as docking platforms that colocalize components of kinase cascades and facilitate activation of the kinases. The scaffolds themselves generally lack enzymatic activity but promote specific outcomes of the pathway. Protein scaffolds can be thought of as “traffic cops” in what would otherwise be the chaos of multiple competing intracellular signaling pathways. Because scaffold proteins add specificity to the cellular pathways, they also present very attractive targets for drug discovery programs. Two major types of scaffolds relevant for the smooth muscle cell are ERK scaffolds and scaffolds for regulators of myosin phosphatase.

51ERK is known to often be targeted to the intranuclear space in proliferative cells and to regulate nuclear signaling, especially to transcription factors. In the smooth muscle cell these pathways can lead to a proliferative phenotype for the smooth muscle cell. These pathways will not be discussed here, but rather we will focus on those most relevant for the fully differentiated contractile cell. Even so, much of this work has been performed using cell culture models and no doubt needs further work in specific contractile smooth muscle tissue systems.

52Calponin is a bit of an enigma and its function in smooth muscle is still debated. It has been reported to serve both cytoskeletal and signaling functions. Both PKC and CAM kinase II phosphorylate calponin at Ser175, and after phosphorylation, calponin loses its ability both to bind actin and inhibit the AMATPase. Calponin has been reported directly to regulate contractility but others have reported negative results. Calponin phosphorylation increases during carbachol stimulation of smooth muscle. Consistent with a physiologic role for calponin in the regulation of contractility are results in skinned smooth muscle; the addition of exogenous calponin reduces both Ca2+ activated force and maximal shortening velocity. In the smooth muscle isolated from calponin KO mice, compared with WT controls, muscle-shortening velocity is significantly higher, but there is no difference in the force produced by Ca2+, carbachol, or phorbol esters. However, the addition of exogenous calponin reduces force in skinned single smooth muscle cells, and the Ser175Ala calponin mutant has no effect on force. In intact smooth muscle during agonist-induced activation, calponin redistributes from the contractile filaments to the cell surface, which is attenuated with the inhibition of PKC. Thus, these results are consistent with a role for calponin in the regulation of smooth muscle contraction; agonist stimulation leads to the activation of PKC, which phosphorylates calponin at Ser-175 to decrease calponin’s interaction with actin to relieve calponin’s inhibition of the AMATPase.

53Three isoforms exist for calponin. h1CaP/CNN1/basic calponin is one of the most specific and rigorous markers for the differentiated smooth muscle phenotype. h2CaP/CNN2/neutral calponin and h3/acidic CaP/CNN3/aCaP are more widely distributed but appear to also be expressed in some smooth muscles. Work in our group has led us to propose that calponin is an adaptor protein for ERK. Antisense knock down of calponin led to decreased ERK activity and contractile force after alpha agonist activation but not after a depolarizing stimulus. Also, protein chemistry studies and cellular immunoprecipitation studies demonstrated that CaP directly binds both PKC and ERK and in intact vascular smooth muscle cells and is bound to the thin filaments but translocates to the cortex of the cell in response to alpha agonists. A detailed model has been suggested where agonists activate PKC, which phosphorylates CaP, releasing it from the thin filaments. Colocalization of ERK and CaP is seen in unstimulated vascular smooth muscle cells and agonist-activation leads to the translocation of a PKC/CaP/ERK complex to the cell cortex, likely meeting up with SmaV (see below), Raf, and MEK, which leads to the activation of ERK, at which point it is seen to return to the contractile filaments and CaD phosphorylation of the ERK sites is observed.

54SmAV is the smooth muscle isoform of a major scaffolding protein supervillin. SmAV was initially cloned and identified SmAV as a CaP binding partner in a two-hybrid assay with CaP as bait, and it was found that SmAV acts as an ERK scaffold, leading to the regulation of CaD phosphorylation. Data have been published indicating that CaP, SmAV and CaV function as scaffolds coordinating Pathway #3.

55Paxillin, better known as a focal adhesion protein, is also known to bind the classic ERK “signaling module” of Raf, MEK, and ERK. In quiescent cultured cells, paxillin is constitutively associated with MEK, but showed that when cells are stimulated with HGF, Src-mediated phosphorylation of paxillin at Y118 leads to the recruitment of ERK, followed by Raf, which leads to ERK phosphorylation and activation. Shortly thereafter focal adhesion kinase (FAK) is recruited to the complex, leading to FA remodeling in both cultured cell systems and airway smooth muscle. Thus, paxillin provides a signaling hub in the vicinity of focal adhesions that can have specific cytoskeletal outcomes.

56Caveolin is an extensively studied protein but there are still many mysteries regarding its function. A caveolin-associated protein has also discovered and named cavin. The exact way in which caveolin and cavin interact and the role of cavin specifically in smooth muscle is not yet clear; however, a cavin knockout mouse has been produced. In this knockout animal not only were arterial expression of cavin-1, cavin-2, and cavin-3 reduced but also all isoforms of caveolin were reduced. As a result, caveolae were absent from both smooth muscle and endothelial cells. An enhanced contractile response to an alpha 1 adrenergic agent was seen, but was likely to be due to the increased thickness of the vascular wall. In contrast, myogenic tone was essentially absent. Surprisingly, blood pressure of the knockout mouse was well maintained, presumably due to opposing influences from smooth muscle and endothelial effects.

57Inhibition of caveolin function by a caveolin decoy peptide or by methyl-beta-cyclodextrin has been shown to disrupt ERK activation in vascular smooth muscle. Work using cultured vascular smooth muscle cell models has suggested that caveolin-mediated scaffolding of ERK leads to different functional outputs than actin/calponin-mediated scaffolding. This concept has not yet been pursued in contractile smooth muscle but illustrates the idea of scaffold proteins regulating the output of kinase cascades toward separate purposes and serving as traffic cops for complex cellular signaling pathways.

58IQGAP (IQ motif containing GTPase activating protein) is an ERK-binding and actin-binding protein that has been extensively studied in nonmuscle systems but little studied in smooth muscle systems. In cultured vascular smooth muscle cells, knockdown of IQGAP prevents the phosphorylation and activation of an actin-associated pool of ERK in response to PKC activation. Proximity ligation assays demonstrated direct tethering of ERK1/2 to actin by IQGAP. Interestingly caveolin is also required for activation of this pathway unless ERK is already associated with actin. Caveolin appears to be required specifically for upstream C-raf activation.

59Myosin regulation is discussed in detail in section IV below, but multiple pathways have been suggested to coordinate signaling associated with myosin phosphatase (MP), and hence, myosin activity (Pathway #2, Fig. 1), and it seems likely that scaffolds play a role to regulate/facilitate these pathways. One MP putative scaffold, M-RIP, also called p116RIP, is thought to link active Rho/ROCK to the inhibition of MP. reported that another scaffold/adaptor protein, Par-4, also regulates myosin phosphatase activity in contractile smooth muscle. We have described a “padlock” model to explain the actions of Par-4, whereby binding of Par-4 to MYPT1 activates MP. This is postulated to occur by the physical blockade by Par-4 of the MYPT1 inhibitory phosphorylation sites. Conversely, this model indicates that inhibitory phosphorylation of MYPT1 by Zipper-interacting protein kinase requires “unlocking” of the blockade by phosphorylation and displacement of Par-4. Whether M-RIP and Par-4 facilitate or antagonize each other’s actions is not known.

60The complexity of this system is impressive, but it is expected that the multiple scaffolding proteins and signaling molecules involved in regulating myosin phosphorylation will lead to the development of rational and selective therapeutic approaches to cardiovascular disease.

61We describe here a number of pathways by which vascular smooth muscle contraction and stiffness are directly regulated and hence will affect blood pressure. It should be mentioned that many other indirect pathways are also involved, with a major mechanism being the development of inflammation and subsequent reduction-oxidation reaction (REDOX) signaling pathways. These pathways are triggered by angiotensin-induced signaling, and as a result, inhibitors of the effects of and production of angiotensin are major ways of regulating blood pressure, including blood vessel contraction. For further details, we refer you to for a review of this topic.

62Y27632, the first ROCK inhibitor described, decreases blood pressure in 11-Deoxycorticosterone acetate (DOCA)-salt rat model of hypertension. A similar effect was obtained with the newer ROCK inhibitors fasudil, SAR07899, in other animal models of hypertension, including the spontaneously hypertensive rat (SHR), angiotensin II-induced hypertension in several animals, and L-NG-Nitroarginine Methyl Ester (L-NAME)-induced hypertension. Of note, this class of inhibitors also has a major part of their effect on hypertension through inhibition of inflammatory pathways and cardiovascular remodeling. For more details we refer you to a recent review by.

63The endothelin pathway, linked to PLC and ERK signaling, has been identified as an effective antihypertensive target.

64Of interest is the fact that beta receptor mediated relaxation of vascular smooth muscle has been reported to decline with age in both the human and animal models. In aortas from Fischer 344 rats, an increase in the level of G-protein receptor kinase-2, which desensitizes the beta adrenergic receptor by phosphorylation of the receptor has been reported to increase with age, and thus inhibitors of G-protein receptor kinase-2 may promote beneficial restoration of beta receptor mediated vasodilation.

65The crossbridge cycle describes the development of force through a series of complexes between actin (A), myosin (M), ATP, and its hydrolysis products, ADP and Pi (Fig. 4 and the termination of Pathway #1; Fig. 3). Beginning in the rigor state (AM), ATP binding to AM results in rapid dissociation of AM, forming an A+M-ATP state, and then ATP is hydrolyzed by myosin. After hydrolysis, the crossbridge enters a weakly attached, pre-powerstroke AM-ADP-Pi state, and then transitions to a strongly bound, force producing AM-ADP-Pi state. After Pi release from the AM-ADP-Pi state, the crossbridge enters a AM-ADP state, which then isomerizes to a high force generating state (AM-ADP) followed by ADP release and returning to the rigor state (AM). MgATP subsequently binds to the AM state, causing rapid crossbridge detachment, and then another crossbridge cycle commences. The duty cycle is defined as the proportion of time crossbridges spend in strongly attached states divided by the time for the total crossbridge cycle; high duty cycle motors are capable of processive movement (i.e., dynein, myosin V), whereas skeletal muscle myosin has a low duty cycle that prevents the development of an internal load from strongly bound crossbridges, which would decrease shortening velocity. Although the crossbridge cycle for all types of myosin is frequently described in this generic manner, differences exist between the kinetics of skeletal, cardiac, and smooth muscle and even within different smooth muscle tissues, requiring changes in the crossbridge cycle to explain the differences in AMATPase rates.

66The smooth muscle AMATPase is similar to that of striated muscle, albeit the kinetics are slower. The kinetics and individual rate constants of the steps in the actomyosin ATPase have been defined in a number of studies, and similar to other myosin IIs, the ATPase is limited by phosphate release or the transition from weak to strong binding states. Both cardiac and skeletal muscle myosin is functionally on, i.e., myosin will hydrolyze ATP in the presence of actin. Smooth muscle (SM) myosin will hydrolyze ATP in the presence of actin, albeit very slowly; however, after phosphorylation of the 20-kDa regulatory myosin light chain (RLC), the rate of hydrolysis is increased due to an ∼1000-fold increase in the rate of product release. Thus, changes in RLC phosphorylation regulate smooth muscle activation and relaxation.

67In smooth muscle, in addition to RLC phosphorylation regulating the AMATPase, it also controls the structure of SM myosin and filament formation. In the absence of RLC phosphorylation, myosin is in the 10S conformation [high sedimentation velocity and low ATPase, with the tail of myosin bending back over the head neck junction interacting with the regulatory light chain]. After RLC phosphorylation, the interaction of the myosin tail with the RLC is perturbed, and myosin exists in an extended conformation [6S, low sedimentation velocity, high ATPase] and also forms filaments. Other investigators have suggested that in the absence of RLC phosphorylation, the interaction of the NH2-terminal region of caldesmon (see Fig. 3 and section III.A.1) with the myosin crossbridge disrupts the interaction of the myosin head with its neck-tail region to promote a transition from the 10S to the active, 6S conformation. Nonetheless, in human smooth muscle, there is significant pool of 10S myosin that can be converted by changes in cellular conditions to 6S myosin that can then assemble into side polar thick filaments. These data could suggest that in addition to the regulation of the AMATPase, changes in RLC phosphorylation could regulate the formation of myosin filaments within the smooth muscle during activation and relaxation; however in vivo, the ability of RLC phosphorylation to regulate filament formation is controversial.

68The level of RLC phosphorylation is defined by the relative activities of myosin light chain kinase (MLCK) and MLC phosphatase, i.e., RLC phosphorylation is related to MLCK/(MLCK+MLC phosphatase). Thus, changes in the activity of either MLCK or MLC phosphatase will change RLC phosphorylation and force or vascular tone. MLCK is regulated by Ca2+-calmodulin, whereas MLC phosphatase activity is regulated by a number of signaling pathways. At a constant [Ca2+], a decrease in MLC phosphatase activity increases SM RLC phosphorylation and force to produce Ca2+ sensitization, whereas an increase in MLC phosphatase activity decreases SM RLC phosphorylation and force to produce Ca2+ desensitization.

69MLC phosphatase is a holoenzyme consisting of a catalytic subunit, a 20-kDa subunit of unknown function, and a myosin targeting subunit (MYPT1). Alternative splicing of a 123-bp central exon results in MYPT1 isoforms that differ by a 41-aa central insert (CI), referred to as M130 and M133. Additionally, alternative splicing of the 3′ exon is responsible for generating MYPT1 isoforms that differ by the presence or absence of a carboxy-terminal leucine zipper (LZ). Thus in humans and other species, alternative splicing generates four MYPT1 isoforms that differ by the presence or absence of a CI and LZ: CI+LZ+, CI-LZ+, CI+LZ−, CI-LZ−.

70The Rho GTPases are within the RAS superfamily of small G proteins, which exist as either active GTP-bound and inactive GDP-bound forms. The conversion between active and inactive forms is controlled by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and guanine dissociation inhibitors. The role of RhoA for the regulation of smooth muscle tone has been well described (Fig. 5; Ca2+ sensitization and Pathway #2, Fig. 3). RhoA/Rho kinase signaling is activated by G protein-coupled receptors, and the role of this pathway for the inhibition of MLC phosphatase and Ca2+ sensitization has been the subject of a number of reviews. The activation of Rho kinase has been demonstrated to phosphorylate CPI-17 at Thr38, PHI-1 at Thr57, and MYPT1 at both T696 and T850. MYPT1 phosphorylation at T696 has been demonstrated to decrease MLC phosphatase activity to increase force at a constant Ca2+, whereas the phosphorylation of MYPT1 at T850 dissociates the holoenzyme, which results in a decrease in MLC phosphatase activity, which produces Ca2+ sensitization. Similarly, agonist activation of G-protein-coupled receptors has been demonstrated to lead to the activation of PKC, which phosphorylates both CPI-17 at Thr38 PHI-1 at Thr57. When phosphorylated, these proteins will bind to the catalytic core of the catalytic subunit of MLC phosphatase to decrease phosphatase activity. In addition to Rho kinase, Zip kinase will phosphorylate MYPT1 as well as CPI-17, and integrin-linked kinase will phosphorylate MYPT1 as well as CPI-17 and PHI-1.

71There are a number of studies demonstrating Rho kinase signaling mediates both MYPT1 phosphorylation and Ca2+ sensitization. However, in smooth muscle of the guinea pig ilium, although inhibition of Rho kinase decreased the carbachol-induced increase in Ca2+ sensitivity, it had no effect on MYPT1 phosphorylation. In this study, although staurosporin prevented MYPT1 phosphorylation, specific PKC inhibitors had no effect on the phosphorylation of MYPT1, which could suggest in this tissue Zip kinase is involved in a physiologically important signaling pathway for Ca2+ sensitization. In bladder smooth muscle, others have demonstrated that there is constitutive phosphorylation of MYPT1 at T696, which was unaffected by inhibition of either Rho kinase or PKC, whereas MYPT1 phosphorylation at T850 was primarily mediated by Rho kinase. These investigators generated T696A and T850A MYPT1 mutant mice to demonstrate that the MYPT1 phosphorylation at T696, but not T850, is important for increasing RLC phosphorylation and Ca2+ sensitization during the sustained phase of force maintenance. These results demonstrate that during activation of smooth muscle, the physiologically important signaling pathways mediating both MYPT1 phosphorylation as well as Ca2+ sensitization are agonist as well as tissue specific.

72Although the role of RhoA in the regulation of Ca2+ sensitization of smooth muscle is established, the role of other Rho GTPases for force regulation in smooth muscle is not well defined. Pak1, a downstream target of Rac1, has been demonstrated to inhibit MLCK and relax permeabilized intestinal smooth muscle. However in airway smooth muscle, both the knockout or inhibition of Pak reduces tone, and Pak3 has been shown to induce a Ca2+-independent contraction of permeabilized smooth muscle. Furthermore, Pak also has been demonstrated to phosphorylate CPI-17. Recently, the role of Rac1 in force regulation has been further delineated. Using a smooth muscle-specific, conditional Rac1 KO, these investigators demonstrated that the decrease in Rac1 reduced force in both bladder and saphenous arterial smooth muscle. Furthermore, the inhibition of Rac1 with EHT1864, which affects nucleotide binding, decreased the Ca2+ transient and force produced in response to depolarization, agonist activation, and activation of PKC. However, inhibition of Rac1 with NSC23766, which blocks the interaction of Rac1 with GEFs, decreased force for phenylephrine (α-agonist) activation but increased the force produced by stimulation with both prostaglandin F2α and thromboxane (U46619). These results suggest that Rac1 signaling is agonist dependent and can result in either an enhancement of the Ca2+ transient and force or an inhibition of Ca2+ sensitization.

73The well documented role of RhoA/Rho kinase signaling for the inhibition of MLC phosphatase and Ca2+ sensitization could suggest that Ca2+ sensitization of vascular smooth muscle cells contributes to the molecular mechanism for the increase in vascular tone and/or systemic vascular resistance that produces hypertension. Consistent with this hypothesis are the results demonstrating that in SHR compared with control rats, both the sensitivity to G-protein-coupled agonists and the magnitude and sensitivity of Ca2+ sensitization are increased as well as studies defining the role of G12-G13-induced activation of Rho kinase-mediated Ca2+ sensitization for the development of DOCA salt-sensitive hypertension. Furthermore, the infusion of the Rho kinase inhibitor Y-27632 reduced blood pressure in normal Wistar rats, as well as several animal models of hypertension including the SHR and DOCA salt-sensitive rat models of hypertension, as well as L-NG-Nitroarginine Methyl Ester (L-NAME)-induced hypertension. Additionally, the expression and activity of Rho kinase are higher in vascular smooth muscle isolated from the SHR compared with controls, and the specific Rho kinase inhibitor fasudil reduced blood pressure in the SHR model of essential hypertension.

74Rho/Rho kinase signaling has also been implicated as an important contributor for the regulation of vascular tone in other mammals; the Rho kinase inhibitor HA1077 has been shown to dilate canine coronary arteries. Furthermore, Rho kinase is upregulated in a porcine model of coronary vasospasm, and in this model, inhibition of Rho kinase with Y-27632 decreases coronary vasospasm. In humans, fasudil has been demonstrated to be effective in treating the cerebral vasospasm associated with subarachnoid hemorrhage, and fasudil is approved for treating patients with pulmonary hypertension. In humans, demonstrated that brachial artery infusion of fasudil did not decrease systemic blood pressure, but did produce a dose-dependent increase in forearm blood flow in hypertensive patients, but not controls without hypertension. Sodium nitroprusside, on the other hand, resulted in comparable increases in forearm blood flow in both hypertensive and normal humans. These results suggest that an increase in RhoA/Aho kinase signaling may be involved in the molecular mechanism that produces essential hypertension, and development of drugs that target this pathway could represent an effective, novel class of therapeutic agents.

75Four isoforms of the smooth muscle myosin heavy chain (SM MHC) are produced by alternative splicing of a single gene. Alternative splicing of a 5′-site produces two isoforms that express a unique aa sequence of either 43 (SM1, 204 kDa) or 9 (SM2, 200 kDa) residues at the carboxy terminus of the SM MHC tail, whereas alternative splicing of a 21-bp insert produces a difference of 7 aa near the ATP binding site of the SM MHC. Although SM1 or SM2 homodimers are more common than herterodimers in a single myosin rod, SM1 and SM2 homodimers will copolymerize and assemble into side polar thick filaments. Estrogen has been demonstrated to increase the ratio of SM1/SM2 expression, and this shift in the expression of SM1 MHC isoform has been suggested to contribute to changes in the sensitivity to the agonist norepinephrine and KCl depolarization. However, others have demonstrated in the motility assay, there is no difference in the ability of SM1 and SM2 to translate actin. These investigators also demonstrated that although there is no difference in the length of SM1 and SM2 filaments, the differences in SM1 and SM2 at the carboxy terminus of the myosin tail influenced filament packing and stability; SM1 filaments have greater stability. Consistent with these results, others have demonstrated that the smooth muscle thick filaments of the SM2 KO are similar in length to that of WT mice, but the SM1 thick filaments isolated from SM2 KO mice are smaller in diameter and there are fewer thick filaments per high-powered field. However, despite the decrease in the number of smooth muscle myosin thick filaments and a concomitant decrease in the expression of SM1, the force produced by both KCl depolarization and carbachol activation was higher in the SM2 KO.

76The other SM MHC isoform is due to a 7-aa insert at the amino terminus, near the ATP binding site of the SM MHC. The 7-aa insert is expressed in both SM1 and SM2 SM MHCs, and the SM MHC with the 7-aa insert has been referred to as SMB, whereas SM MHC lacking the insert is SMA. SMB, when compared with SMA, has been demonstrated to have a twofold higher AMATPase activity and a 2.5-fold faster velocity of actin translation in the in vitro motility assay. Whether the presence of the insert confers a functional difference to the mechanical properties of smooth muscle was assessed using bladder smooth muscle isolated from a transgenic mouse line; the maximum velocity of muscle shortening of bladder smooth muscle strips from WT (SMB+/+) is higher than that of either of heterozygous (SMB+/−) animals or the SMB KO mice. Further analysis of the mechanical responses of Ca2+-activated skinned bladder strips to elevated Pi and ADP suggested that the lower shortening velocity of the SMA isoform is due to a slower rate of ADP dissociation or an additional force producing isomerization of the AM-ADP state in the crossbridge cycle. These results demonstrate that the insert near the SM MHC ATP binding site will alter the mechanical properties of smooth muscle, and the duty cycle of SMA should be higher than SMB, which would be predicted to increase vascular tone and/or vascular resistance and thus blood pressure. Consistent with this prediction are the results demonstrating that compared with WT mice, the isometric force for mesenteric vessels of SMB KO mice was increased. However, the blood pressure of SMB KO animals compared with WT controls has not been reported, and whether SMA/SMB isoform expression is altered during hypertension has not been investigated.

77For the 17-kDa essential myosin light chain (ELC17), alternative splicing of exon six, which encodes 44 bp, also produces two isoforms, which differ in the expression of 9 aa at the carboxy terminus of the ELC17. Exon 6 is excluded in the nonmuscle, more basic isoform (ELC17a), whereas exon 6 is included in the smooth muscle, more acidic isoform (ELC17b). Changes in the ELC17 could affect the stiffness of the SM MHC lever arm, and thus myosin step size and or unitary force. However for ELC17 isoforms, the motility assay does not show any difference in the velocity of actin translocation. Nonetheless in fast smooth muscle, the expression of ELC17a and SMA is higher than in slow smooth muscle.

78Alternative mRNA splicing produces four splice variant MYPT1 isoforms, formed by the presence or absence of a 43 aa central insert (CI+/−) and carboxy-terminal leucine zipper (LZ+/−). The expression of the CI (residues 512–552) is both developmentally regulated and tissue specific, and phosphorylation of MYPT1 at Thr696 has been demonstrated to inhibit phosphatase activity. Although there is evidence that the CI+ MYPT1 isoform may be preferentially phosphorylated during Ca2+ sensitization, a role for the CI for the regulation of smooth muscle contractile properties has not been established. The MYPT1 LZ+ isoform is generated by the exclusion of a 3′- to 31-bp exon, whereas exon inclusion generates a LZ− MYPT1 isoform. The aa sequence of the MYPT1 LZ domain is identical from avians to mammals and 75% identical in mammals and worms, which could suggest that this domain is important for the regulation of MLC phosphatase activity. were the first to demonstrate an important functional role for the MYPT1 LZ domain. These investigators demonstrated that the LZ MYPT1 domain was important for the interaction of MYPT1 and PKG. Subsequently it has been demonstrated that PKG interacts with the LZ domain as well as a MYPT1 domain between aa 888 and 928, but the LZ domain is necessary for the PKG-mediated activation of the MLC phosphatase during Ca2+ desensitization (Fig. 6,; Ca2+ desensitization) and/or nitric oxide (NO)-mediated vasodilatation. PKG phosphorylates MYPT1 at Ser695 and Ser849, which excludes the Rho kinase-mediated MYPT1 phosphorylation at Thr696 and Thr850, to prevent a Rho kinase-mediated decrease in MLC phosphatase activity, but MYPT1 phosphorylation at these sites by PKG does not increase MLC phosphatase activity. The mechanism for the increase in MLC phosphatase activity during NO/cGMP-mediated Ca2+ desensitization was recently defined; PKG phosphorylates only LZ+ MYPT1 isoforms at Ser668, and the Ser668 MYPT1 phosphorylation increases the activity of MLC phosphatase. MYPT1 LZ+/− isoform expression is developmentally regulated, tissue specific, and modulated in animal models of heart failure, pre-eclampsia, portal hypertension, pulmonary hypertension, nitrate tolerance, and sepsis. Furthermore, a decrease in LZ+ expression decreases the sensitivity to NO-mediated vasodilatation. Thus, the molecular mechanism that underlies the differential response of the vasculature to NO and NO-based vasodilators is in part due to differential expression of LZ+/− MYPT1 isoforms, and furthermore, changes in the relative LZ+/− expression could tune the vasculature between a low-resistance vascular bed (relatively vasodilated), which is NO responsive and Rho kinase/PKC resistant, and a high-resistance vascular bed (relatively vasoconstricted) that is resistant to NO but responsive to Rho kinase/PKC.

79The importance of the LZ domain of MYPT1 for the regulation of vascular tone has been established using two different transgenic mice. produced a transgenic mouse with mutations in the LZ domain of PKG, which disrupts the interaction of PKG with MYPT1. Compared with control littermates, the vascular smooth muscle isolated from these mice were less sensitive to NO-mediated vasodilatation, and thus these mice were hypertensive. In addition and also consistent with an important role for LZ+ MYPT1 isoform expression for the regulation of vascular tone are the results demonstrating that transgenic mice expressing only the LZ+ MYPT1 isoform are more sensitive to NO-mediated relaxation and hypotensive compared with WT littermates.

80Because NO-mediated vasodilation is a fundamental property of the vasculature, preservation of the normal response to NO by maintaining the normal LZ+ MYPT1 isoform expression could improve the outcome for the treatment of diseases of the vasculature. For the treatment of heart failure, both ACE inhibitors and ARBs have been demonstrated to preserve both LZ+ MYPT1 expression and the sensitivity to NO-mediated vasodilatation, which could underlie the beneficial effects of inhibition of angiotensin II signaling in the treatment of heart failure compared with other vasodilators. Additionally, differential expression of LZ+/− MYPT1 isoforms in patients with heart failure could underlie the mortality benefit of ACE inhibitors in white compared with black patients. ACE inhibitors reduced mortality in white patients, whereas in black patients, although ACE inhibitors did not show a benefit, treatment with the combination of hydralazine and isosorbide dinitrate reduced mortality.

81The regulation for LZ+/− MYPT1 expression is unknown, but LZ+/− MYPT1 expression is both developmental regulated and tissue specific, as well as modulated during disease states. During heart failure (HF), p42/44 MAP kinase is activated and the expression of LZ+ MYPT1 decreases, and further losartan therapy prevents the activation of p42/44 MAP kinase and preserves LZ+ MYPT1 expression. Additionally, the expression of LZ+ MYPT1 isoforms as well as Tra-2β, an atypical member of RNA binding proteins, are higher in fast (phasic) compared with slow (tonic) smooth muscle, and in an animal model of portal hypertension, Tra-2β is downregulated coincident with the decrease in LZ+ MYPT1 expression (E23 exon exclusion) and the shift to the expression of LZ− MYPT1 isoforms. These investigators demonstrated both that Tra-2β binds to E23 and transactivates E23 splicing (exon inclusion) to generate a LZ− MYPT1 isoform and deletion or mutation of the Tra2β binding site abolished E23 splicing (exon exclusion) to produce a LZ+ MYPT1. These investigators also showed that an siRNA-induced decrease of Tra-2β decreased E23 splicing to produce an increase in LZ+ MYPT1 expression, which is consistent with Tra-2β expression regulating E23 splicing and LZ+/− MYPT1 expression. Further investigation of the signaling pathway for the regulation of LZ+/− MYPT1 isoform expression could reveal novel targets in this pathway. Small molecules could be designed to modulate either total MYPT1 or LZ+ MYPT1 expression, which would both improve both the vascular response to endogenous NO and pharmacological response during the treatment of hypertension as well as a number of other diseases of the vasculature.

82In most systems, flow and pressure are linearly related; as pressure increases, so does flow. However, small arteries vasoconstrict in response to an increase in pressure and vasodilate in response to a decrease in pressure, and the change in vessel diameter in response to changes in intravascular pressure is referred to as the myogenic response. The myogenic response is an intrinsic property of the small resistance vessels and does not require flow. Furthermore, because of the intrinsic myogenic response as well as the modulatory actions of vasoactive substances, the resistance vessels maintain a constant blood flow over a wide range of perfusion pressures, and this property is termed the autoregulation of blood flow.

83Hypertension is associated with abnormalities of the myogenic response, and thus, the mechanism underlying the myogenic response is important in both health and disease. There are significant regional as well as vessel dimension differences in the magnitude and mechanism governing the autoregulation of blood flow. Further complicating investigation of the mechanism(s) responsible for the myogenic response are the small size (<200 μm diameter) of the resistance vessels; until recently, investigation of the signaling pathways regulating the myogenic response of the small resistance arteries was not possible. But, recent improvements in the sensitivity of protein phosphorylation levels have allowed for direct investigation of signaling pathways that regulate the myogenic response.

84Although the signaling pathway for the myogenic response is still being investigated, the first element is the mechanosensor that responds to the changes in intraluminal pressure. Martinez-Lemus et al. demonstrated that blocking integrin function with either anti-integrin antibodies or integrin-specific peptides results in a significant inhibition of the myogenic response. Furthermore, both the activation of integrins and the myogenic response are associated with tyrosine phosphorylation and the activation of focal adhesion kinase (FAK) and Src family tyrosine kinases. Additionally, agonist stimulation of smooth muscle lead to a tyrosine phosphorylation of the protein paxillin as well as activation of p42/44 MAPK and the L-type Ca2+ channel. These data suggest that because of their ability to link the extracellular matrix to the cytoskeleton of the smooth muscle cell, integrins participate in the sensing and transmission of changes in intravascular pressure.

85There are a number of studies that have demonstrated an important role for TRP channels, specifically TRPC6 and TRPM4 in the myogenic response. demonstrated that selective inhibition of TRPM4 decreased the transient inward cation current induced by membrane stretch. These investigators demonstrated that the generation of IP3 by PLCγ1 and the subsequent Ca2+ release from internal stores is required for both TRPM4 activity and myogenic tone. Furthermore, both TRPC6 inhibitors and antibodies that bind to an extracellular epitope of TRPC6, which block TRPC6 currents, attenuated the stretch-induced activation of TRPM4 current. These investigators also demonstrated that the inhibition of Src nonreceptor tyrosine kinases, which signal through PLCγ, decreased myogenic tone. These results suggest that Src tyrosine kinase activity is important in the stretch-induced increases PLCγ, which generates IP3. Subsequently, IP3 binds to the IP3 receptor, which stimulates Ca2+ release from the SR. Thus, in response to stretch, a local increase in Ca2+ generated by both TRPC6 and IP3 are important for the activation of TRPM4, which changes membrane potential and increases the conductance of voltage-dependent Ca2+ channels, which results in smooth muscle cell contraction to generate the myogenic response.

86Additionally, both Ca2+ release and Ca2+ sensitization of the contractile filaments contribute to the myogenic response. The pressure-induced increase in wall tension leads to depolarization of the smooth muscle cells, which results in opening of voltage-gated Ca2+ channels and an increase in intracellular Ca2+. However, in addition to the pressure-induced increase in Ca2+, other mechanisms contribute to the myogenic response. Using a highly sensitive biochemical technique, demonstrated that only in the presence of myogenic tone, serotonin stimulation produced a Rho kinase-mediated phosphorylation of MYPT1, which induced a Ca2+ sensitization of the contractile filaments. Furthermore, this group has also demonstrated that both a Rho kinase-mediated phosphorylation of MYPT1, as well as a Rho kinase- and PKC-mediated increase in actin polymerization are important determinants of the myogenic response. The activation of the Ca2+-dependent tyrosine kinase, Pyk2, has also been demonstrated to occur with KCl depolarization, and although the inhibition of Pyk2 did not change the rapid increase in force and RLC phosphorylation in response to KCl depolarization, Pyk2 inhibition did depress RLC phosphorylation, Thr-696, and Thr-850 MYPT1 phosphorylation and force during the sustained phase of the contraction. These results suggest that a Ca2+-induced activation of Pyk2 leads to an activation of RhoA/Rho kinase and Ca2+ sensitization, which is important for force maintenance, or the tonic phase of smooth muscle contraction.

87Because hypertension is associated with abnormalities of the myogenic response, antihypertensive agents aimed at the signaling pathway for the myogenic response should be effective for the control of blood pressure. Drugs that decrease Ca2+ influx as well as agents that decrease and/or block the activation of Rho kinase signaling such as ACE inhibitors, ARBs, Rho kinase inhibitors should be and are effective antihypertensives. However, novel agents designed to decrease the sensitivity of the mechanosensor such as blocking the activation of integrins or decreasing tyrosine kinase activation could reduce blood pressure. However, tyrosine kinase inhibitors used for the treatment of carcinomas are known to be cardiotoxic, and hypertension is the most common cardiovascular side effect. The mechanism by which tyrosine kinase inhibition produces hypertension is not well understood, but hypothesized to be due to fluid retention, endothelial dysfunction, and an inhibition of NO. These data demonstrate the complex interplay between the myogenic response, circulating vasoactive substances, and the kidney in the regulation of blood pressure.

88There are three classes of nonmuscle (NM) myosin, NMIIA, NMIIB, and NMIIC, and both NMIIA and NMIIB are expressed in smooth muscle, whereas NMIIC is only expressed in neuronal tissue. Both NMIIA and NMIIB are able to form bipolar thick filaments, and similar to SM, myosin RLC phosphorylation promotes filament formation. In smooth muscle, NM myosin expression represents ∼10–15% of total myosin. Furthermore, NMIIA has been demonstrated to form mixed bipolar filaments with myosin 18A, and SM1 and SM2 will copolymerize to form filaments. However, it is unclear if NM myosin and SM myosin copolymerize to form mixed filaments or whether two distinct pools of SM and NM myosin thick filaments exist within the smooth muscle cells.

89The kinetics of the individual steps of the AMATPase of NMIIB and NMIIA are slower than other types of class II myosin. NMIIA and NMIIB have a high ADP affinity, and for NMIIB, the rate of ADP release is similar to that of the steady-state ATPase. Additionally for NMIIB, actin augments ADP binding rather than accelerating ADP release, which results in NMIIB spending the majority of its kinetic cycle in states that are strongly bound to actin. On the other hand, the rate of ADP release for NMIIA is one order of magnitude faster than the ATPase, which results in NMIIA spending much less of its ATPase cycle in strongly actin bound states. Although the slow kinetics of NMIIA suggest that NMIIA could contribute to force maintenance, the high duty ratio make NMIIB an ideal candidate for the so called “latch crossbridge”, and NM myosin may be an important component for the sustained phase of smooth muscle contraction.

90recently extended his kinetic studies and demonstrated that ADP release from NMIIB is slow and strain dependent; positive strain increases the rate of ADP release by a factor of fourfold, whereas negative strain decreases ADP release by 12-fold. Load dependence of ADP release prevents NMIIB from slowing either shortening or the rate of force generation by smooth muscle myosin. However for NMIIB, negative strain increases the duty ratio, which would contribute to force maintenance; i.e., for NMIIB, rapid ADP binding and load-dependent ADP release prolongs the attachment of NMIIB to actin at 10–100 μM ADP (at normal MgATP), which would decrease the rate of ATP usage to <0.01 ATP per head per second during force maintenance. Furthermore, during force maintenance both heads of the NMIIB would be attached to actin, which is ideal for a crossbridge that maintains tone.

91Similar to NM myosin, the kinetics of SM myosin have been demonstrated to depend on load. Using optical tweezers, these investigators demonstrated that the displacement of the SM crossbridge occurs in successive steps of 4 and 2 nm. The duration of the first phase (4-nm displacement) is strain dependent, increasing by twofold with a negative strain and decreasing by twofold with positive strain. These results could suggest that the increase in attachment time of SM myosin due to the negative strain on the SM crossbridge during force maintenance could contribute to the latch state. Furthermore, recent data from optical trap experiments demonstrated that the attachment time of smooth muscle myosin to actin varies with SM RLC phosphorylation; when only one of the two heads of smooth muscle myosin is phosphorylated, the dwell time was fit with a double exponential with rates of 24 second−1 and 1 second−1, compared with a single rate of 29 second−1 when both heads were phosphorylated. These investigators suggested that the long attachment time of the singly phosphorylated myosin could explain the latch state, or force maintenance, in smooth muscle. However, experiments in the optical trap are performed at low ionic strength to both promote actin-myosin interaction and increase interaction times; at physiologic conditions, rates are much faster. also demonstrated that the ATP turnover rate of singly phosphorylated myosin was 30% of that compared with doubly phosphorylated myosin, which contrasts with the results of who demonstrated that the ATP turnover rate of myosin with a single phosphorylated head was over 50% of that when both heads were phosphorylated. The reason for the discrepancy between the results for the ATP turnover is unclear. However, it is likely that the single molecule mechanics of NM myosin with the RLC of one or both heads phosphorylated would be similar to smooth muscle myosin, although much slower, making NM myosin a more attractive candidate for a latch crossbridge.

92The regulation of NM myosin has been studied in nonmuscle cells, and similar to smooth muscle myosin, NM myosin is regulated by phosphorylation of its regulatory light chain (NM RLC); NM RLC phosphorylation promotes NM myosin filament assembly and also results in a 10-fold increase in the Vmax of the NM myosin AMATPase. In nonmuscle cells, Rho kinase phosphorylates the NM RLC at Ser19, and in epithelial cells, results are consistent with both MLCK and Rho kinase as important for the phosphorylation of NM RLC. In epithelial carcinoma cell lines, Rho kinase increases the phosphorylation of the NM RLC at Ser19 phosphorylation of both NMIIA and NMIIB. During KCl depolarization of smooth muscle, both SM RLC and NM RLC phosphorylation increase, and the increase in RLC phosphorylation is not dependent on either Rho kinase or PKC. However for angiotensin II (Ang II) activation, inhibition of either Rho kinase or PKC blunted SM RLC phosphorylation, whereas only a Rho kinase-dependent pathway regulated NM RLC phosphorylation. These results demonstrate that similar to SM myosin, NM myosin is regulated in smooth muscle, and both MLCK and Rho kinase regulate the activation of NM myosin (Fig. 7).

93Recent evidence from mechanical studies also suggests that NM myosin contributes to the force maintenance phase of smooth muscle tissue contraction. showed that bladder smooth muscle from transgenic mice lacking smooth muscle myosin still contract, albeit with a very slow tonic response, as opposed to the rapid phasic contraction (with transient peaks in force and Vmax) characteristic of wild-type bladder smooth muscle. The force produced by NM myosin in KO tissues is low, which would suggest that NM myosin will not participate in the rapid phase of force activation, but rather the kinetics of NM myosin are tuned for force maintenance. Consistent with this hypothesis are results demonstrating that the inhibition of the NM AMATPase with blebbistatin reduced force maintenance; for phasic smooth muscle, blebbistatin did not affect the rapid rise in force, but decreased maintained force, and for tonic smooth muscle, blebbistatin decreased force maintenance. However, although blebbistatin is thought to be specific for the inhibition of the NM myosin AMATPase, the specificity of blebbistatin for the NM versus SM AMATPase has been questioned. Nonetheless, consistent with a role of NM myosin for force maintenance are the results with heterozygous NMIIB KO mice; when compared with WT control mice, force maintenance is depressed by 25% in smooth muscle of heterozygous NMIIB KO. Interestingly, demonstrated for carbachol activation of guinea pig ileum that inhibition of Rho kinase did not affect peak force but decreased force maintenance, and Rho/Rho kinase signaling regulates NM RLC phosphorylation. These results are consistent with NM myosin participating in force maintenance, and the inhibition of Rho kinase would lead to a decrease in the activation of NM myosin, which results in a reduction in both the force maintenance phase of smooth muscle contraction and vascular tone (SVR). It is also interesting to speculate on the contributions of other pathways, which have been demonstrated to be important contributors for changes in vascular tone for the regulation of NM myosin including tyrosine kinase.

94Smooth muscle contractile properties have been classified as phasic or tonic; after activation, for phasic smooth muscle, force rises rapidly to a peak before falling to a lower steady-state level, whereas for tonic smooth muscle, force slowly increases to a sustained steady state. Smooth muscle has also been termed “fast” and “slow” because of the differences in Vmax. The molecular mechanism that governs tonic and/or phasic contractile properties has yet to be elucidated, although emerging evidence suggests that there are tonic and phasic contractile phenotypes (see ). In general, the fast isoforms of the SM MHC (SMB) and ECL17 (ECL17a) are expressed in phasic smooth muscle while slow isoforms (SMA and ECL17b) expression predominates in tonic smooth muscle. Similarly, the expression of NM myosin is significantly higher in tonic smooth muscle. In addition, there are differences in the expression of the regulatory proteins in phasic and tonic smooth muscle. The expression of MLCK, MYPT1, and the LZ− MYPT1 isoform is higher in phasic compared with tonic smooth muscle. Furthermore, in phasic versus tonic smooth muscle, the expression of CPI-17 is lower, whereas telokin expression is higher. These results could suggest that a gene program exists that governs the differential expression of contractile proteins in fast (phasic) versus slow (tonic) smooth muscle, and the contractile phenotype regulates systemic vascular resistance.

95Resistance is inversely related to the vessel radius to the forth power (1/r4), and thus, SVR is predominantly regulated at the level of the small resistance arteries with a diameter of 50–300 μm. Because of their small size, the molecular contractile phenotype of the resistance vessels have not been fully characterized, but the small resistance vessels express a mixture of fast and slow contractile proteins and exhibit a mixture of tonic and phasic contractile activity, which is referred to as vasomotion. The molecular basis of essential hypertension is unknown, but the molecular contractile phenotype is known to be modulated during disease in both the large conduit vessels, as well smaller resistance vessels. In the small resistance vessels, an increase in the relative expression of protein isoforms associated with the tonic contractile phenotype (i.e., an increase in NM myosin expression) or decrease in LZ+ MYPT1 isoform expression would produce an increase in vascular tone and/or SVR, which would produce hypertension.

96Flow is governed by the simple equation, flow = pressure/resistance, and the ability of NO, or flow, to mediate changes in vascular tone is considered a fundamental property of the vasculature. The autoregulation of blood flow to a vascular bed maintains a constant flow over a wide range of pressures to ensure that perfusion is maintained despite either hypo- or hypertension. The mechanism governing the autoregulation of blood flow has yet to be fully elucidated but is known to be dependent on the intrinsic myogenic response as well as the modulatory actions of vasoactive substances, which importantly includes the vascular response to NO. NO is produced by the vascular endothelium in response to shear stress. The NO produced by the endothelium diffuses into the smooth muscle cells where it activates the soluble pool of guanylate cyclase and results in an increase in cGMP. cGMP activates protein kinase G (PKGI), which has a number of targets that produce smooth muscle relaxation, including myosin light chain (MLC) phosphatase. As perfusion pressure increases, the subsequent increase in blood flow will increase shear stress on the endothelial cells, which will stimulate NO production and a subsequent vasodilatation to decrease flow. Conversely, a decrease in perfusion pressure will decrease endothelial shear stress and NO production, and the resulting vasoconstriction will increase flow. Therefore, the NO-induced changes in vascular resistance can be viewed as part of a negative feedback loop that blunts the myogenic response; the myogenic response generates a vasoconstriction with an increase in pressure and vasodilatation with a decrease in pressure. Thus, NO production regulates vascular resistance and is essential for the normal regulation of blood flow.

97The sensitivity and response of the vasculature to NO and NO-based vasodilators are well known to be heterogenous, and the molecular basis for this variable response to NO is controversial. Nonetheless, during NO signaling, activation of the MLC phosphatase requires the expression of a LZ+ MYPT1 isoform and in both health and disease. The sensitivity of the vasculature to NO-mediated vasodilation is regulated by the relative expression of LZ+/LZ− MYPT1 isoforms; i.e., an increase and/or decrease in LZ+ MYPT1 expression will produce an increase and/or decrease in the sensitivity to NO, respectively. Nitrates and nitrate-based vasodilators are a well-known class of antihypertensive agents that will decrease blood pressure in the acute setting. However, tolerance to nitrates is a well-known phenomenon, which may limit the efficacy of nitrates for the treatment of hypertension, and a decrease in LZ+ MYPT1 expression has been demonstrated to contribute to the molecular mechanism of nitrate tolerance. In the treatment of heart failure, both ACE inhibitors and ARBs have been demonstrated to maintain the normal expression of LZ+ MYPT1 expression and sensitivity to NO-mediated vasodilatation. In essential hypertension, both whether changes in LZ+ MYPT1 expression contribute to the molecular mechanism producing this disease and strategies to improve LZ+ MYPT1 expression and the sensitivity to NO-based vasodilators for the treatment of hypertension have not been investigated.

98Several strains of genetically modified mice have been produced to evaluate the contribution of abnormalities in the regulation of vascular tone and/or vascular dysfunction to hypertension, and experimental results and their implications will be discussed in this section.

99Ca2+ signaling is well known to be important for the regulation of vascular tone; the activation of voltage-gated Ca2+ channels increases cytoplasmic Ca2+ and results in vasoconstriction. However, the increase in Ca2+ also activates Ca2+-activated potassium channels (BK channels), and Ca2+ activation of these channels results in a membrane hyperpolarizing current that opposes vasoconstriction and K+ channel openers, such as nicorandil, hyperpolarize smooth muscle to produce vasodilatation. Brenner et al. (2003) deleted the β1 subunit of the BK channel in mice and demonstrated that, compared with controls, the open probability of BK channels was 100-fold lower and the BK current in response to Ca2+ sparks was impaired in vascular smooth muscle from the β1 KO animals. Furthermore, myography demonstrated that cerebral arteries from the β1 KO animals mice were more constricted at all pressures, which resulted in hypertension in these animals. Additionally, BK channels have been demonstrated to increase their conductance in response to NO/cGMP signaling.

100In smooth muscle, contractile agonists have been demonstrated to activate the G-proteins Gq and G11 and stimulate phospholipase C, which leads to an increase intracellular Ca2+ and activation of MLCK to increase RLC phosphorylation and force. However, many vasoconstrictors also couple with G12 and G13 to activate Rho kinase-mediated signaling, which inhibits MLC phosphatase to produce a Ca2+-independent increase in force. produced mice with smooth muscle-specific ablation of Gq-G11 or G12-G13 to investigate the relative contributions of Gq-G11 versus G12-G13 on vascular tone and the development of hypertension. In aortic smooth muscle isolated from the Gq-G11 KO mice, the contractile response to both phenylephrine and Ang II was completely blocked, whereas the response to serotonin, endothelin, vasopressin, and the thromboxane analog U46619 was inhibited. Ablation of G12-G13 had no effect on the dose-response relationship for phenylephrine or serotonin, but it reduced the steady-state force produced by Ang II, endothelin, vasopressin, and U46619. Furthermore, compared with WT mice, blood pressure was no different in the mice with ablation of G12-G13, but significantly lower in the Gq-G11 KO mice, indicating that the normal regulation of blood pressure requires Gq-G11 signaling. Additionally, ablation of either Gq-G11 or G12-G13 attenuated the increase in blood pressure produced by DOCA-salt treatment, which suggests that although G12-G13 signaling and the subsequent activation of a Rho kinase pathway leading to Ca2+ sensitization is not required for maintenance of normotension, it contributes to the development of DOCA-salt-dependent hypertension. These investigators also demonstrated that the RhoGEF protein LARG is important for the G12-G13-mediated activation of Rho kinase and DOCA-induced hypertension.

101The importance of the NO/cGMP signaling pathways for the maintenance of a normal blood pressure has been established with several different models. In mice, the disruption eNOS has been demonstrated to eliminate ACh-mediated relaxation of aortic smooth muscle rings, and as would be predicted, the relaxation produced by sodium nitroprusside was no different in eNOS KO and WT animals. This defect in vascular reactivity resulted in hypertension in the eNOS KO mice. Similarly, PKGI KO mice were hypertensive compared with WT littermates. These investigators demonstrated compared with WT, that although the response to contractile agonist was not different in aortic rings from the PKGI KO, ACh- and 8Br-cGMP-mediated relaxation was significantly attenuated. Further preincubation of aortic cells with 8Br-cGMP attenuated the Ca2+ transient in response to NE in WT aortic smooth muscle cells, but had no effect on the Ca2+ transient in the PKGI KO aortic smooth muscle cells.

102Most contractile agonists activate Gq-coupled receptors, resulting in the activation of phospholipase C, the generation of IP3, Ca2+ release, and the activation of MLCK as well as Rho/Rho kinase and a resulting inhibition of the MLC phosphatase. Mendelsohn’s group demonstrated that the dose-dependent increase in IP3 production by thrombin was inhibited by both S-nitrosocysteine and 8Br-cGMP. Because NO and cGMP inhibit thromboxane signaling via a PKG-mediated phosphorylation of the cytoplasmic tail of the thromboxane receptor and RGS-2 terminates G-protein receptor signaling by accelerating the GTP hydrolysis rate by Gα subunits, these investigators examined the function of RGS-2 in NO/cGMP signaling cascade. They demonstrated that phosphorylation of RGS-2 by PKG resulted in a translocation of RGS-2 from the cytosolic to particulate fractions and decreased Gαq GTPase activity. These data show that NO/cGMP-mediated activation of PKG results in a phosphorylation of RGS-2, which increases the activation of Gq-coupled receptors. Consistent with these data are the results that show agonist-induced force production was increased and the vasodilatory response to NO was reduced in aortic smooth muscle from RGS-2 KO mice, and the resulting increase in vascular tone (relative vasoconstriction) was responsible for the increase in blood pressure in RGS-2 KO mice compared with WT littermates. The importance of the NO/cGMP signaling cascade for the regulation of vascular tone and blood pressure has also been demonstrated in three recent studies. produced a knock-in mouse in which the initial four leucine/isoluecine residues of PKGIα were replaced by alanine. This PKGIα LZ mutant does not interact with MYPT1, and as would be predicted, when compared with WT, aortic rings isolated from the PKGIα LZ mutant were less sensitive to ACh- and 8Br-cGMP-mediated relaxation, which produced hypertension. produced a conditional MYPT1 KO, which were hypertensive compared with WT controls. The lack of MYPT1, which would disrupt the ability of the MLC phosphatase to target to its substrate, the RLC, resulted in an increase in the phosphorylation of the RLC as well as force in response to both KCl depolarization and contractile agonists. However, surprisingly, the sensitivity of relaxation produced by NO/cGMP signaling was similar in the control and MYPT1 KO mice. The lack of a decrease in the sensitivity to NO of secondary branches of the mesenteric arteries from MYPT1 KO animals could be explained if the conditional KO of MYPT1 increased RGS-2 expression or decreased NO/cGMP-mediated PKG phosphorylation of RGS-2 or the functional replacement of MYPT1 by MBS85, which is a member of the MYPT family that is also expressed in smooth muscle. generated a conditional deletion of MYPT1 E24 to produce a LZ+ MYPT1 KI mouse. Vascular tissue isolated for the LZ+ MYPT1 mice was more sensitive to NO/cGMP-mediated relaxation, and as would be expected, the mice were hypotensive compared with controls. Of course, it must be kept in mind that NO, cGMP, and PKG also can dilate vascular smooth muscle by more than one mechanism, thus pathways not involving MYPT1 are also possible but we are not aware of appropriate animal models yet available to test those possibilities.

103Mendelsohn’s group has also demonstrated that NO signaling is regulated by estrogens; compared the vascular responses in WT, iNOS KO, and estrogen receptor β KO mice. Estrogen was found to decrease PE-induced contraction of aortic smooth muscle in WT, but not iNOS KO mice. Furthermore, iNOS is upregulated by estrogen, as well as transfection with estrogen receptor β. In vascular rings from estrogen receptor β KO mice, compared with WT mice, the contractile response to PE was reduced, which could be the result of a decrease in iNOS in the estrogen receptor β KO tissues. The sensitivity of smooth muscle relaxation to sodium nitroprusside was similar in estrogen receptor β KO and WT animals. Nonetheless, in the estrogen receptor β KO animals, the abnormalities in vascular tone produced hypertension.

104The data presented in this section demonstrate the importance of the regulation of vascular tone for the regulation of blood pressure and consistently demonstrate that disruption in the NO/cGMP signaling pathway reduces the sensitivity to NO-mediated vasodilatation, and this decrease in the vascular response to NO and/or NO-based vasodilators produces hypertension.

105Diuretics reduce blood pressure by decreasing intravascular volume, which mechanistically fits with a Guytonian view for the regulation of blood pressure, and diuretics, as a class, are useful for treating essential hypertension. Given the importance of Ca2+ signaling for the regulation of vascular tone, the benefit of CCB for treating humans with essential hypertension is not surprising. Similarly, ACE inhibitors and ARBs will decrease the activation of the Ang II G-protein-coupled receptor and inhibit Ang II-stimulated increase in intracellular Ca2+ and activation of Rho kinase signaling to decrease vascular tone. demonstrated that the KO of the AT1 receptor in only the peripheral vasculature decreases SVR and blood pressure, and thus, these agents would be expected to and have been demonstrated to be effective therapies of essential hypertension. In fact, drugs that block any G-protein-coupled receptor, i.e., α-receptor blockers, would be expected to decrease vascular tone and lower blood pressure. β-Blockers decrease the activation of the cardiac β-adrenergic receptor, and the subsequent decrease in cAMP and PKA signaling produce negative chronotropic and inotropic response, which decreases blood pressure. However, in the vascular smooth muscle, an increase PKA activity produces a vasodilatation (, and β-blocker therapy could potentially result in an increase in vascular tone, but because of the inhibition of central sympathetic output, SVR decreases with β-blocker therapy. Drugs designed to increase K+ channel conductance produce smooth muscle hyperpolarization and a resulting vasodilatation of vascular smooth muscle, and these agents have been demonstrated to both decrease blood pressure and be effective treating angina. Although NO and NO-based vasodilators will result in a decrease in vascular tone and are effective for treating angina, they have not been demonstrated to be useful in treating essential hypertension, possibly because of the phenomenon of nitrate tolerance.

106Although there are a number of effective classes of antihypertensives, when treating patients with essential hypertension, the selection of an antihypertensive agent does not consider the possible changes in the smooth muscle contractile phenotype that may contribute to the molecular mechanism for essential hypertension. This could explain the variable racial and individual response to drug classes. Thus, a critical need for a personalized approach for the treatment of essential hypertension exists.

107Despite the effectiveness of current antihypertensive, many patients with essential hypertension either do not respond to one or more therapeutic agents or have a resistant hypertension that requires treatment with multiple classes of antihypertensives for adequate control of blood pressure. Other than the well-known racial and regional differences in response to various classes of antihypertensives, there is no known method to determine for any individual drug class whether a patient with essential hypertension will have a therapeutic response or the magnitude of the response to therapy. Because the molecular mechanism that produces essential hypertension is unknown, the diversity in the response to treatment is not unexpected and demonstrates the importance of identifying the mechanism producing hypertension in each individual. As outlined in the preceding sections, there are a number of changes in the contractile phenotype that could contribute to hypertension, and the contribution of the contractile phenotype could be variable among patients.

108NO-mediated vasodilatation is a fundamental property of the vasculature, and studies of transgenic mice have demonstrated the importance of NO/cGMP signaling for maintenance of normal SVR and blood pressure. However, nitrate-based vasodilators have not been demonstrated to be effective for the treatment of essential hypertension, possibly because of nitrate tolerance. A number of mechanisms contribute to this phenomenon, but recently a decrease in LZ+ MYPT1 isoform has been suggested to play a role in the molecular mechanism for nitrate tolerance. Because PKG-induced phosphorylation of LZ+ MYPT1 isoforms and subsequent activation of the MLC phosphatase is a key component in the NO/cGMP signaling pathway leading to vasodilatation, increasing MYPT1 LZ+ expression should be effective for both for reversing nitrate tolerance and the treatment of essential hypertension. The regulation for LZ+/LZ− MYPT1 expression is unknown, but investigators have demonstrated that Tra-2β appears to be an important regulator for LZ+/LZ− MYPT1 expression. Furthermore, both ACE-inhibitors and ARBs were demonstrated to preserve both the normal LZ+ MYPT1 expression and sensitivity to NO-mediated vasodilation in heart failure, which may explain the benefit of these agents in treating heart failure compared with other vasodilators, as well as the mortality differences for treatment of heart failure with hydralazine and isosorbide dinitrate versus enalapril in white and black patients. Investigation of the signaling pathway for the regulation of total MYPT1 as well as LZ+/− MYPT1 isoform expression could reveal novel targets in this pathway that would increase LZ+ MYPT1 expression. Small molecules could be designed to modulate either total MYPT1 or LZ+ MYPT1 expression in vascular smooth muscle, and therapies designed to increase LZ+ MYPT1 expression in vascular smooth muscle would improve both the vascular response to endogenous NO and pharmacological response during the treatment of hypertension. Other targets in the NO signaling pathway could also be exploited for the treatment of essential hypertension; i.e., novel activators of RGS-2 would be expected to decrease the activation of Gq-coupled receptors and ultimately decrease vascular tone and blood pressure.

109NM myosin could represent another novel target for drug development for the treatment of hypertension. The inhibition, as well as the KO of NM myosin has been demonstrated to decrease force during the tonic phase of smooth muscle contraction, and thus inhibition of NM myosin would be expected to decrease both vascular tone and blood pressure. As outlined above, there are significant differences in the expression of contractile proteins between tonic and phasic smooth muscle, and resistance vessels express a mixture of fast and slow contractile proteins, as well as tonic and phasic contractile properties. The contractile phenotype is known to be modulated during disease in both the large conduit vessels and smaller resistance vessels, and thus essential hypertension could be due to changes in the gene program governing the contractile phenotype and a resulting change in the contractile properties of the resistance vessels from a phasic to more tonic phenotype. This could suggest that targeting the gene program to enhance the expression of phasic contractile proteins would decrease blood pressure and represent a novel therapy for essential hypertension.

110Regulation of smooth muscle function by myosin isoforms, particularly nonmuscle myosin isoforms is discussed above in section IV. Here we will discuss the cytoskeletal proteins that until recently were assumed to serve a primarily structural role in smooth muscle. There are three types of cytoskeletal proteins in this category: intermediate filaments, microtubules, and actin filaments.

111Intermediate filaments have been studied relatively little in contractile smooth muscle but of note is literature that suggests that intermediate filaments may be the glue that sticks together different functional domains of the smooth muscle cellular cytoskeleton. This is based on anatomic studies of several types of smooth muscle showing that a population of actin filaments that are not directly contacting myosin surround the actomyosin filament bundles and insert into cytoplasmic “dense bodies.” These cytoplasmic dense bodies in turn are connected to “dense bodies” at the surface of the cell (focal adhesions) by intermediate filaments. Studies on airway smooth muscle have also indicated that intermediate filaments form cable-like structures that connect dense bodies and that these structures have a functional plasticity due to mechanisms, yet to be defined, by which the cable length can be adjusted in a regulatory manner. Intermediate filaments in contractile vascular smooth muscle have also been suggested to serve functional roles that are not simply structural. For example, these filaments bind CaMKII and are phosphorylated by this kinase, but whether the intermediate filaments are serving a signaling scaffolding role or whether CaMKII is somehow changing the function of the intermediate filaments is not known. Similarly, in airway smooth muscle, PAK has been shown to phosphorylate intermediate filaments. Interestingly, antisense knockdown of vimentin in smooth muscle inhibits agonist-induced force development, but, again, the exact molecular mechanism involved is not clear.

112Of interest is the fact that smooth muscle contains large quantities of dystrophin and utrophin. These proteins have been little studied in smooth muscle, but in lung and vascular smooth muscle they and the dystroglycan complexes they form connect with caveolin and cavin in caveoli as well as the actin cytoskeleton. Knockout of dystrophin in a mouse model decreases contractility of tracheal rings.

113With respect to microtubules, as expected, the microtubular network is sparse in nonproliferative, nondividing smooth muscle. They could serve a transport purpose, but we are unaware of such functions yet being demonstrated in contractile smooth muscle.

114A far more extensive literature has developed with respect to actin in smooth muscle. The actin in contractile smooth muscle can be divided into that associated with smooth muscle myosin, generally called the thin filaments, and the nonmuscle actin cytoskeleton. The mechanisms of regulation of actomyosin have been discussed above. Here we will focus on the emerging role of the nonmuscle cytoskeleton in the regulation of vascular function.

115Several groups have demonstrated that vasoconstrictors and myogenic contractions regulate the structure of the actin cytoskeleton and its connection to focal adhesions (FAs) (Pathway #4, Fig. 3). These smooth muscle FAs appear to be essentially identical FAs in cultured cells except for their less dynamic nature. Increasing evidence indicates that tension modulates the function of signaling pathways in the smooth muscle tissue/cell and that these biomechanical functions are mediated by remodeling of the smooth muscle focal adhesions and its nonmuscle actin cytoskeletal connections. It is important to emphasize that smooth muscles generally lack tendons for the transmission and summation of contractile forces generated by the muscles. In contrast, in smooth muscles the extracellular matrix (ECM) forms a sort of “intramuscular tendon” to which the integrins attach. It is one function of the focal adhesions, then, to channel, somehow, all contractile force generated by the contractile apparatus through the integrins and to the ECM. The exact mechanisms involved are now an active area of investigation in smooth muscle cells.

116have reported that a decline in G-actin content occurs in response to pressurization or agonist activation of cerebral resistance arteries, resulting in an increase in contractility in the absence of detectable myosin or actin phosphorylation that could be blocked by Rho kinase and PKC inhibitors. These results pointed to cytoskeletal remodeling contributing to the contractile responses observed. Smooth muscle actin exists as four different isoforms. Alpha smooth muscle actin and gamma smooth muscle actin are the dominant actins associated with myosin in the contractile filaments, what has been called “mini sarcomers,” in vascular and gastrointestinal smooth muscle, respectively. Beta nonmuscle and gamma nonmuscle actins are thought to be present in all smooth muscles. These four isoforms are separate gene products and clearly have different functions and different expression patterns, but interestingly they have highly similar protein sequences with no isoform sharing less than 93% identity with any other isoform. mapped the individual actin isoforms in vascular smooth muscle during contraction in response to an alpha agonist and found that the overall F/G ratio increases by about twofold in response to the alpha agonist phenylephrine, but only gamma nonmuscle actin displays a statistically significant increase in polymerization. Interesting, in response to a phorbol ester, 12-deoxyphorbol 13-isobutyrate 20-acetate, only beta nonmuscle actin showed significant evidence of remodeling.

117The actin isoforms also appear to define distinct subcellular domains within the geography of the cell. It has been found that alpha smooth muscle actin is associated with contractile filaments, beta nonmuscle actin is associated with cytoplasmic dense bodies and focal adhesions and gamma nonmuscle actin describes in interesting submembranous cortical domain in vascular smooth muscle. Truly specific gamma nonmuscle antibodies have only recently become available, but earlier studies described qualitatively similar organizations in gut smooth muscle.

118The smooth muscle FA, by connecting with the nonmuscle actin and intermediate filament cytoskeleton, is thought to serve the purpose of transmitting force generated in the contractile filaments through the integrins to the extracellular matrix and the tissue as a whole. FAs in contractile smooth muscle, like those in cultured, migrating cells, are thought to undergo considerable plasticity and remodeling in response to biomechanical forces, agonists, and hormones. When this has been directly investigated in contractile VSMCs, some, but not all, proteins have been shown to dissociate from the bulk of the FA during vasoconstrictor stimulation. Zyxin and vasodilator-stimulated phosphoprotein (VASP), but notably not FAK, undergo Src-dependent endosomal processing and regulate, through processes not yet fully understood, vascular contractility and stiffness. In contrast, in airway smooth muscle, FAK has been shown to be quite mobile, shuttling between FAs at the plasmalemma and the cell interior. This highlights differences not only in organ function but also in cellular mechanisms between different types of smooth muscle.

119Zyxin and vasodilator-stimulated phosphoprotein (VASP) are FA proteins associated with regulation of actin polymerization. This is consistent with a model whereby in VSMCs, only proteins at the edge of the focal adhesion, furthest from the plasmalemma, are capable of breaking off and undergoing remodeling in smooth muscle. This model is consistent with recent nanoscale super-resolution imaging of cultured fibroblast focal adhesion function described by. In the Kanchanawong model, their data describe three sublayers within individual FAs: 1) the “integrin signaling layer,” containing integrin and its direct connections such as FAK closest to the plasmalemma; 2) the “force transduction layer,” including vinculin and talin, which are more interior; and 3) the “actin regulatory layer,” containing alpha actinin and zyxin, which is deepest into the cytoplasm.

120An interesting scenario occurs immediately after birth when there is a drastic demand for a decrease in pulmonary vascular resistance. This triggers a transition from the intrauterine pulmonary vascular requirement for only 8–10% of cardiac output, whereas the placenta performs the function of the primary site of gas exchange to the independent support of the newborn’s lung function. As a result, in the first days to weeks of the newborn’s life, dramatic vascular remodeling is required. If this fails it is associated with the disorder of persistent pulmonary hypertension of the newborn. Interestingly, the remodeling is not just of the cells of the vascular tissue but also a subcellular remodeling of the actin isoforms within the vascular cells with a persistence of excessive gamma actin being a marker of persistent pulmonary hypertension of the newborn. Cortical gamma actin polymerization is reported to prevent nuclear translocation of the transcription factor YY1, which downregulates SM22, important for smooth muscle specific differentiation.

121A similar link between actin polymerization at a subcellular level within the vascular smooth muscle cell and blood pressure has been suggested by the effect of vasoconstrictor agonists to cause both actin polymerization and inward remodeling of resistance vessels associated with many forms of hypertension. Furthermore, inhibition of actin polymerization prevented the agonist-induced inward remodeling of the resistance vessels. It is well known that increased intraluminal pressure elicits a myogenic vasoconstriction from resistance vessels and a further increase in blood pressure. This myogenic response has been associated with increased Ca2+ entry to the cell, activation of myosin light chain kinase, as well as increased activation of ROCK and protein kinase C pathways that regulate actin polymerization.

122Further investigation of the signaling pathway for the myogenic response has implicated integrins as the link between the extracellular matrix and the cytoskeleton that senses changes in pressure. Because the activation of integrins and the myogenic response is associated with tyrosine phosphorylation and the activation of focal adhesion kinase (FAK) and Src family tyrosine kinases, blocking the activation of integrins or decreasing the activation of tyrosine kinase could be novel targets for treating essential hypertension. Although nonreceptor tyrosine kinases can phosphorylate paxillin and induce actin polymerization and a PKC-mediated pathway will lead to the phosphorylation, or activation, of PYK2 and Src tyrosine kinases, there is also evidence that receptor tyrosine kinases participate in the regulation of vascular tone. ephrin (EPH) kinases are the largest family of receptor tyrosine kinases, and the ligand for EPH tyrosine kinases are ephrins. Ephrins are also cell surface molecules that transduce signals into cells, and multiple EPHs will interact with multiple ephrins. Recently, the smooth muscle-specific deletion of the tyrosine kinase EPHB4 was demonstrated to result in decreased contractile responses to the agonist phenylephrine and hypotension in male, but not female, mice. Contrasting with these results are those demonstrating that blood pressure was unaffected by deletion of the tyrosine kinase EPHB6 in male and female mice, but blood pressure was elevated and the contractile response to phenylephrine was enhanced in castrated male EPHB6 KO mice. These results demonstrate that there is interplay between the various members of the non-receptor and receptor tyrosine kinase families as well as other factors that ultimately contribute to the regulation of blood pressure that may provide untapped targets for the development of novel antihypertensive therapeutics.

123Increased arterial stiffness is a prominent concept in studies of cardiovascular disease (CVD), especially in the context of aging, as an independent biomarker and predictor of hypertension and atherosclerosis, which are associated with increased mortality rates and severe damage to organ systems via stroke, renal failure, and heart disease. Given the overwhelming predominance and persistence of CVD as the leading cause of human death worldwide, there is great interest and urgency to develop a thorough understanding of arterial stiffness as a possible cause of, and thus a potential therapeutic target for preventing or treating, CVD in humans.

124Assessment of arterial stiffness exists on multiple scale levels. Macroscale techniques that characterize the hemodynamics of pulsatile blood flow are available clinically in humans and more easily interpretable, whereas microscale techniques better elucidate the underlying mechanisms causative of changes in the physical properties of blood vessels. In studying arterial stiffness, the predominant challenge moving forward is to establish an integrated model of increased arterial stiffness across macro- and microscopic scales that enable the development of treatments to alleviate or prevent later-onset CVD. Recent studies suggest that vascular smooth muscle represents an attractive therapeutic target for such designs.

125In vivo arterial stiffness can be measured representatively and noninvasively as pulse wave velocity (PWV). This measurement of the pulse wave generated by cardiac systole is the ratio of the distance it travels along the vascular wall to the time delay between its arrivals at different points along the circulatory pathway. Most commonly, PWV is measured via ultrasound between the carotid and femoral arteries as representative of aortic stiffness. As the current “gold standard” of arterial stiffness measurements, increased PWV is strongly correlated to both aging and CVD.

126The validity of PWV as an in vivo approximation of aortic stiffness is described by the Moens-Korteweg equation:where E is the incremental Young’s modulus of the vessel, h and a are the thickness and radius of the aortic wall, respectively, and ρ is blood density. The value of E represents a material stiffness of the wall that is more precisely descriptive of its biomechanical properties. The Moens-Korteweg equation is itself dependent upon key assumptions: that the aortic wall is isotropic and homogeneous and that the aorta undergoes small isovolumetric changes in response to pulse wave propagation. Unfortunately, these assumptions do not hold, as demonstrated by previous studies; therefore, whereas PWV is an undoubtedly useful prognosticative tool, its accuracy as a biomechanical indicator is unclear.

127It is appropriate here to distinguish between “functional stiffness”—a relation between pulse pressure applied to a vessel and its resultant deformation, or strain—and “material stiffness,” the normalization of functional stiffness to vessel geometry, most notably vessel diameter and wall thickness. Material stiffness corresponds most directly to the value of E in the Moens-Korteweg relation and is the most independent representation of an intrinsic property to resist deformation in response to an applied force.

128Therefore, PWV can more accurately be described as a standard for functional but not material stiffness. Functional stiffness is derived from material stiffness, but the opposite is not true. In understanding this subtle difference, the diverse terminology used in reference to aortic biomechanical properties may present significant difficulties, compounded by the prevalence of terms reciprocal to stiffness such as “compliance” and “distensibility,” which are sometimes used interchangeably among studies in the context of independence from vessel geometry. The most concise discussion of this complicated canvas is provided by, who cite conflicting results attributable to imprecise terminology and summarize their own study, showing that changes to PWV in brachial arteries, in response to smooth muscle relaxation, are not necessarily predictive of any corresponding change in material stiffness.

129Analysis of arterial mechanical properties is integral to understanding more broadly the epidemiologic link between arterial stiffness and negative cardiovascular outcomes. Although arterial PWV is an easily demonstrable and a clinically valuable measure of functional stiffness, there is also a strong impetus to evaluate material stiffness of the aortic wall directly to explain how different components of the aortic wall, a highly complex and layered network of interconnected cellular and extracellular elements, contribute to altered or defective mechanisms in disease models. This is most conveniently and commonly done ex vivo by subjecting arterial tissue to mechanical stretch.

130Although numerous studies and quantitative models have highlighted the extracellular matrix as the primary effector of dramatic changes in arterial stiffness due to aging or other cardiovascular defects, vascular smooth muscle cells (VSMCs) are implicated to be major regulatory factors of arterial stiffness overall.

131All major biologic components of the arterial wall play integral roles in maintaining normal cardiovascular function. Abnormalities in cellular mechanotransduction, which is regulated by cytoskeletal structures, transmembrane receptors, matrix proteins, and cell-cell and cell-matrix adhesions, have been linked to changes in cell mechanics or sensory machinery, as well as structural alterations in extracellular matrix, which are commonly featured in a wide range of diseases. Indeed, there is a critical codependence between medial elastin and VSMCs in maintaining structure and function in the arterial wall, with studies showing both that elastin regulates VSMC proliferation and that VSMC knockout phenotypes result in severe degradation of elastin. VSMCs have been implicated in recruitment of both elastin and collagen during their contractile response.

132Physiologic factors such as aging and hypertension induce observable physical changes in the wall due to VSMC remodeling. With aging in particular, vascular smooth muscle cells (VSMCs) exhibit increased stiffness and are capable of switching their phenotype from contractile to synthetic, resulting in fundamental changes to cellular function as well as wall structure, because remodeled VSMCs migrate and proliferate in concert with alterations to other wall components, most notably elastin and collagen.

133The endothelium has also become an increasingly visible nexus of aging-induced changes, with widespread effects on cellular and extracellular processes throughout the arterial wall and especially those linked to VSM function. Nitric oxide (NO) produced by endothelial cells induces vasodilation and relaxation in VSM. NO bioavailability is reduced with aging, which attenuates this benefit, increasing VSM stiffness as a result. This downstream effect may characterize a powerful positive feedback loop where the increased stiffness leads to further reduction of NO.

134Recently, attempts to quantify the constituency of total arterial stiffness attributable to VSMC activity have become more prevalent. Ex vivo studies have shown that VSMCs contribute significantly to total stiffness, potentially in conjunction with recruitment of collagen. Under optimally physiologic conditions for smooth muscle viability, up to 50% of maximal ex vivo aortic stiffness is observable only after activating VSMCs by alpha agonist and eliminating NO-mediated vasodilation, quantifiable as shown in Fig. 8.

135Therefore, it is of great interest to probe the cellular mechanisms that produce this effect to identify underlying origins of dysfunction and degeneration. A series of studies highlighting stimulated VSMCs have found that focal adhesions connecting the cortical cytoskeleton to the matrix play a critical role in the regulation of signaling cascades triggered by smooth muscle activation, independently of actomyosin crossbridges. In particular, disruption of the FAK-Src signaling complex reduces contractility and stiffness, as well as biochemical markers of focal adhesion signaling, induced by smooth muscle activation. Interestingly, these mechanisms are defective with aging, and may not be limited to the focal adhesions, but are also processes inherent in the cortical actin cytoskeleton.The normal plasticity of the cytoskeleton may be an important sort of “shock absorber” for the aortic wall that is lost with aging. A model for the focal adhesion and actin cytoskeleton in the VSMC as core machinery regulating arterial stiffness, which is compromised with aging, has emerged from these findings, as shown in Fig. 9.

136It has become increasingly clear that VSM may play a major role in the regulation of arterial stiffness.

137Epigenetic changes refer to the heritable alterations in gene expression that occur without changes in genome sequence. The conventional epigenetic mechanisms include DNA methylation and histone modification, which alter the accessibility of transcription factors at DNA regulatory regions, such as promoters or enhancers. Over the last decade, RNA-based modifications, which alter the translation of genetic information, have emerged as important regulators of development and disease. Collectively, these mechanisms likely play a role in the remarkable plasticity exhibited by vascular smooth muscle cells (VSMCs). This section will discuss the role of epigenetics in vascular smooth muscle (VSM) biology and describe ways by which these changes may influence the risk of cardiovascular diseases, such as hypertension.

138DNA methylation involves the covalent addition of a methyl group to the 5′-position of the pyrimidine ring of cytosine residues to form 5-methylcytosine. This process, catalyzed by DNA methyltransferases, leads to transcriptional repression by blocking the access of transcription factors to response elements in the promoter region of genes. DNA methylation had long been considered irreversible until the recent discovery of the members of the ten-eleven translocation (TET 1-3) family of proteins. TET proteins oxidize 5-methylcytosine to 5-hydroxymethyl cytosine, which ultimately revert to cytosine via the demethylation pathway.

139Variable methylation patterns are observed in the genes of VSMCs originating from different tissues. Abnormalities in DNA methylation patterns have been observed to associate with vascular diseases and have been studied extensively in atherosclerosis. SMCs of atherosclerotic lesions in humans and animal models exhibit reduced levels of 5-methylcytosine. The resulting DNA hypomethylation enhanced switching of VSMCs from a contractile to synthetic phenotype.

140The modifications at protruding N-terminal tails of histones act as an integral epigenetic tag for chromatin remodeling. The histone tails can be modified by acetylation, methylation, ubiquitylation, and phosphorylation. The unique pattern of histone modifications in SMCs can alter chromatin packaging, which leads to the differential expression of crucial VSMC genes. For example, the chromatin structure of the CArG-box [CC(A/T)6GG] motif, essential for tissue-specific expression of SMC marker genes, exists in an easily accessible euchromatin-like form in SMCs but not in non-SMCs. Various histone modifications, such as dimethylation of H3K4 and H3K79 and acetylation of H4 and H3K9, have been observed in the CArG elements of SM α-actin and SM MHC in VSMC gene loci. In contrast, these histone modifications are associated with transcriptional silencing in non-SMCs, such as endothelial cells (ECs), embryonic stem cells, and fibroblasts. ECs exhibit high levels of H4 acetylation and H3K4 dimethylation at the EC-selective vascular endothelial cadherin gene locus. Increased levels of H4K20 dimethylation of this locus in VSMCs, however, are associated with transcriptional silencing. These studies implicate histone modifications as important epigenetic tags in the regulation of SMC determination and differentiation.

141Histone acetylation is characterized by the posttranslational modification of histones, which lead to chromatin remodeling by loosening histone-DNA contacts. These modifications affect many SMC processes, such as differentiation, phenotypic switching, proliferation/apoptosis, and migration. Acetylation and deacetylation of histones are catalyzed by enzymes known as histone acetylases (HATs) and histone deacetylases (HDACs), respectively. HATs are classified into three families, including GNAT, MYST, and CBP/p300 [cAMP response element-binding protein (CREB)]. HDACs are categorized into four classes in mammals: class I (HDAC1-3, HDAC8), class II (HDAC4-7, HDAC9-10), class III sirtuins (SIRT1-7), and class IV (HDAC11). The class I, II, and IV HDACs are Zn2+-dependent deacetylases, whereas the class III HDACs, sirtuins, possess nicotinamide adenine dinucleotide (NAD)-dependent deacetylase activity.

142HATs and HDACs work in a dynamic manner to regulate cellular gene expression. Histone acetylation by p300 HAT allows binding of serum response factor to previously inaccessible CArG-containing regions of SMC-specific genes enabling the recruitment of coactivators, such as myocardin, to stimulate expression of α-actin, γ-actin, calponin, SM22α, h-caldesmon, MLC, and SM MHCs (SM1 and SM2). Retinoic acid treatment of A404 cells, an SMC embryonic cell line, enhances acetylation of histones H3 and H4 at SM α-actin and SM MHC CArG-containing promoter regions. Unlike myocardin, KLF4 (Kruppel-like factor 4) is a transcription factor associated with myogenic repression that regulates SMC phenotypic switching both in vitro and in vivo after vascular injury. KLF4 acts through epigenetic mechanisms to decrease histone H4 deacetylase by recruiting HDAC2 and HDAC5, which block the binding of serum response factor to methylated histones and CArG box chromatin during repression of SMC gene expression.

143Although the studies identifying the role of HATs and HDACs in the regulation of SMC functions warrant further in vivo studies, they highlight the relevance of inhibitors as epigenetic therapeutics in the treatment of vascular diseases. One such study demonstrated that inhibition of p300 HAT activity by Lys-CoA-TAT decreases the effects of retinoic acid on the differentiation of A404 cells toward an SMC lineage. In a separate study in A404 cells, overexpression of HDACs increased CREB-CArG-dependent SM22 promoter activity, whereas the HDAC inhibitor trichostatin A had the opposite effect. In contrast, HDAC inhibition after tributyrin treatment inhibited migration of cultured VSMCs after hyperacetylation of histone H3 and reduced expression of HDAC7. Pharmacological inhibition of HDAC activity or knockdown of HDAC expression with small interfering RNAs (siRNA) prevents platelet-derived growth factor-induced VSMC proliferation.

144Recently, HATs and HDACs have been found to act on the lysine (K) residues of a wide range of nonhistone proteins and are therefore also denoted as KATs and KDACs, respectively. Nonhistone targets for KATs and KDACs include large cellular macromolecular complexes involved in the actin nucleation complex and other proteins participating in microfilament formation and dynamics. A majority of proteins involved in smooth and striated muscle contraction have been shown as substrates undergoing acetylation. KDAC8 regulates the differentiation and contraction of SMCs by interacting with α-actin, tropomyosin, and cortactin proteins. KDAC6 is involved in the modulation of the cytoskeleton, cell migration, and cell-cell interactions by interacting with proteins such as SIRT2. KDAC inhibition by trichostatin A and hydroxamic acid was associated with increased protein acetylation and reduction of agonist-stimulated contractions in VSM tissues. In a separate study, during contraction, the cytoskeletal proteins were dynamically regulated through HDAC inhibition and activation, resulting in the regulation of cytoskeletal structure.

145Epigenetic mechanisms influence the changes in vascular structure that occur during hypertension. Angiotensin II (Ang II) at least partially influences vascular remodeling via HDACs in the pathogenesis of hypertension. Specifically, HDAC5 and HDAC4, after phosphorylation by Ang II, export from the nucleus where they activate myocyte enhancer factor-2, leading to SMC hypertrophy. Treatment of spontaneously hypertensive rats with HDAC inhibitors, such as trichostatin A or valproic acid, reduced SMC hypertrophy, blood pressure, and vascular inflammation. All these studies highlight an important therapeutic potential for HDAC inhibitors in the treatment of vascular diseases.

146Sirtuins are a class of proteins that have multiple functions including NAD+-dependent deacetylation. Currently, there are seven known mammalian sirtuins (SIRT1-7), located in the nucleus (SIRT1, 6, and 7), cytoplasm sirtuin (SIRT2), or mitochondria sirtuins (SIRT3, 4, and 5). Because SIRTs are multifunctional, they have a plethora of protein targets, including p53, nuclear factor κB, Ku70, FOXO, tubulin, eNOS, BAD, CytoC, Ndufa9, GDH, ACS2, and ISDH2. Extensive reviews are available on the role of sirtuins in aging, calorie restriction, mitochondrial biogenesis, and neurodegenerative diseases,

147Sirtuins have largely been shown to have protective effects on the vasculature. For instance, SIRT1 modulates vascular biology during hypoxia-induced redox stress by deacetylating the transcription factor, HIF (Hypoxia-inducible factor)-2α, which modulates vascular tone and enhances cell survival through induction of antioxidant enzymes to promote angiogenesis. The deacetylase activity of SIRT1 has also been shown to regulate the proliferation and migration of VSMCs via the suppression of cellular senescence mediator, p21 protein, and enhancement of senescence-resistant cell replication. Overexpression of SIRT1 increases the activity of metalloproteinase-3 inhibitor, which inhibits cell migration. In addition, SIRT1 also deacetylates and activates eNOS, which enhances NO-induced vasodilation.

148Interestingly, recent studies implicated SIRT1 in blood pressure regulation through targeting VSMCs. Overexpression of SIRT1 in VSMCs has been found to modulate blood pressure by downregulation of the Ang II type 1a receptor gene. Moreover, resveratrol treatment activated SIRT1, resulting in the repression of Ang II type 1a receptor gene transcription to counteract Ang II-induced hypertension in mice. SIRT1 expression in human VSMCs was demonstrated to correlate inversely with donor age. This was associated with functional defects, such as reduced migratory capacity and increased senescence. Interestingly, Ang II infusion significantly decreased the expression of SIRT1 in mouse aorta, which was associated with increased blood pressure and elevated vascular remodeling. Importantly, a VSMC-specific SIRT1 transgene attenuated both Ang II-induced increases in blood pressure and vascular remodeling. These recent studies point to the potential of therapeutically improving SIRT1 function in VSMCs to treat hypertension.

149Noncoding RNAs comprise many functional RNA transcripts that are not transcribed into proteins, but regulate the transcription, stability, or translation of protein-encoding genes. Noncoding RNAs were once assumed only to regulate generic functions of cells, such as transcription, translation, and splicing. It is now recognized, however, that a wide variety of noncoding RNA transcripts are transcribed and have diverse biologic activity. The complex nature of this RNA-based regulatory network may partially explain the vast diversity of the characteristics of mammals, despite possessing relatively similar proteomes. In addition, epigenetic mechanisms, such as DNA methylation, can regulate the expression of noncoding RNAs, adding a further level of complexity. This section discusses microRNAs and long noncoding RNAs, which function in the broadly termed RNA silencing machinery.

150MicroRNAs (miRs) are a family of short (21–25 nucleotide) RNAs, which typically negatively regulate protein translation by direct binding to the 3′ untranslated (3′ UTR) region of mRNA targets. The first miR, lin-4, was discovered to have a role in Caenorhabditis elegans larval development more than 20 years ago. To date, over 30,000 miRs have been discovered in 206 species, including 2578 in humans. It is now certain that miRs play an important role in many cellular and developmental processes. Crucially, miRs are dysregulated in many pathophysiological conditions, including cardiovascular disease.

151MicroRNAs are transcribed in the nucleus by RNA polymerase II to long primary transcripts (Fig. 10). They are subsequently processed by the RNAse III enzyme Drosha in the nucleus to a 70 nucleotide hairpin structure termed preliminary miRNAs (pre-miRNA). After export into the cytoplasm, pre-miRNAs are further processed by a separate RNAse III enzyme, Dicer, into a 21–25 nucleotide duplex. The mature miR strand is then incorporated onto Argonaute protein to form the RNA-induced silencing complex (RISC), leading to unraveling and degradation of the complimentary strand. The miR then guides the RISC to complimentary sequences in the 3′ UTR of target mRNA. In plants, the near-perfect complementarity between the miR and target sequence promotes mRNA cleavage of the target, akin to the mechanism for siRNA-induced silencing. In mammals, however, this level of complementarity is rare. In contrast, association of a 6–8 nucleotide ("seed") sequence with the 3′ UTR of target mRNA leads to repression of mRNA translation through inhibition of translation initiation and/or promotion of mRNA decay (reviewed by).

152There are several excellent reviews that discuss the role of miRs in vascular structure, function, and disease. The current review will mainly focus on miRs that have a role in regulating vascular smooth muscle cell (VSMC) contractility, thus influencing the risk of hypertension. The importance of miRs in vascular smooth muscle was demonstrated in experiments using smooth muscle-specific Dicer KO mice. Knockout of Dicer in vascular smooth muscle (VSM) induced embryonic lethality, which was associated with widespread hemorrhaging, loss of contractile function, and reduced VSMC proliferation. A further study using a tamoxifen-inducible SMC-specific knockout of Dicer, suggested miRs are necessary for blood pressure regulation and contractile function. Blood pressure was reduced after Dicer knockdown, with no change in cardiac dimensions. This was associated with impairment of both receptor- and calcium-mediated contraction of small mesenteric arteries and reduction of expression of contractile proteins. Furthermore, the stretch-dependent increase in contractile gene expression in the portal veins of mice was dependent on Dicer expression. Loss of the miR-143/-145 cluster is thought to be at least partially responsible for this reduced vascular contractility. Pathways that promote the vascular smooth muscle contractile phenotype, such as Notch and bone morphogenic protein signaling, function partially through promoting the expression of miR-143/-145. In addition to miR-143/-145, the expression of miR-21 and miR-24 is similarly induced by bone morphologic protein (BMP) signaling, promoting the contractile phenotype by targeting Programmed Cell Death 4 and Tribbles-like protein-3, respectively.

153The Dicer KO model has also demonstrated an important role for miRs in the regulation of small arterial myogenic tone, therefore modulating peripheral arterial resistance, which is raised in hypertension. Myogenic tone of mesenteric arteries was abolished from Dicer KO mice, which was associated with a loss of calcium influx through the L-type calcium channel. It is therefore possible that the aberrant expression of miRs may increase the development of myogenic constriction of small arteries, thereby increasing peripheral resistance and systemic blood pressure.

154Several miRs have been found to posttranscriptionally modulate the expression of VSMC contractile proteins. Overexpression of miR-143/-145 in VSMCs increases the expression of contractile proteins, such as SM-MHC, calponin, and SM22α, through the targeted downregulation of KLF4 and 5. VSMCs from Type 2 diabetic rats and humans display an increased expression of miR-145, which contributes to the enhanced expression of vascular contractile proteins in Type 2 diabetes. Myocardin increases the expression of miR-1 in human aortic SMCs, which blocks the expression of the contractile proteins α-SMA and SM22, possibly acting as a negative feedback mechanism to counter myocardin-induced increase in contractility. Additionally, although miR-21 may promote the contractile phenotype, it may also negatively regulate VSMC contraction through the targeted downregulation of myosin phosphatase, Rho-interacting protein, and cofilin-2.

155MicroRNAs have also been demonstrated to posttranscriptionally regulate the expression of VSMC ion channels, thereby influencing vascular contractility and thus blood pressure regulation. For example, miR-145 indirectly increases VSM contractility by targeted downregulation of CamKIIδ, which results in increased expression of the α1C subunit of the L-type calcium channel. Similarly, miR-328 has been demonstrated to directly target the α1C subunit of the L-type calcium channel. Overexpression of the miR-424/322 resulted in decrease in cyclin D1 and calcium-regulating proteins calumenin and stromal-interacting molecule 1, which reduces store-operated calcium entry in human and rat VSMCs. Further studies have elucidated a role for miR-9a-3p and miR-190 in the downregulation of the VSMC KATP and potassium channel Kv7.5, respectively, causing an increase in contractility.

156MicroRNAs have also been implicated in the downregulation of proteins involved in the ERK 1/2 signaling pathway in proliferating, but not contractile, VSMCs. The miRs-21, -31, -132, and -155 all affect ERK activation. It remains to be determined if those miRs similarly affect ERK activation in fully differentiated VSMCs.

157Long noncoding RNAs (LncRNAs), arbitrarily classified as longer than 200 nucleotides, have a role in regulating the expression of neighboring genes. LncRNAs have a much more widespread mode of action than miRs, and their function cannot currently be inferred from sequence or structure. LncRNAs can recruit chromatin-remodeling complexes to epigenetically regulate specific genomic loci. They can also influence transcriptional regulation of genes by recruiting RNA binding proteins to gene promoters, acting as cofactors to modulate transcription factor and RNA polymerase II activity. Finally, LncRNAs can also regulate posttranscriptional processing events, by recognizing complimentary sequences of RNA. Like miRs, they have the ability to bind to mRNA targets, affecting their translation and degradation, but they can also influence other posttranscriptional processes such as splicing, editing, and transport. For a deeper description of LncRNA function the reader may refer to the following references.

158Although the field is newer than that for miRs, LncRNAs are emerging as potentially important players in cardiovascular disease (recently reviewed by and ). Through RNA sequencing approaches in human coronary arteries, VSMCs have been demonstrated to express several LncRNAs, including smooth muscle and endothelial cell-enriched migration/differentiation-associated long noncoding RNA. Depletion of endothelial cell-enriched migration/differentiation-associated long noncoding RNA in VSMCs was associated with decreased expression of myocardin and other contractile genes and increased expression of the promigratory proteins MDK and PTN, suggesting a role in maintenance of the contractile phenotype. The Lnc-Ang362 was increased in Ang II-treated rats, which was associated with an increased VSMC proliferation. Interestingly, Lnc-Ang362 was cotranscribed with miR-221 and -222 and was required for their expression. This study provided evidence for the regulation of LncRNAs by Ang II, which may have important implications in the pathogenesis of Ang II-associated cardiovascular diseases, such as hypertension.

159Recent evidence suggested the expression of LncRNAs may affect VSMC proliferation and apoptosis, therefore influencing the risk of aortic aneurysm and atherosclerosis. For example, LncRNA-p21 was demonstrated to inhibit cell proliferation and neointimal hyperplasia by releasing the mouse double minute 2 repression of p53. Importantly, this LncRNA was downregulated in atherosclerotic plaques from ApoE−/− mice. Furthermore, the expression of the LncRNA ANRIL was influenced by several atherosclerosis-associated single nucleotide polymorphisms (SNPs) in the 9p21 locus. These data alert one to the possibility that previous genome-wide association studies (GWAS) may have misinterpreted SNPs in regions that encode nonprotein coding RNA as without effect on disease risk.

160MicroRNAs continue to provide great therapeutic potential, and the strategies to alter miR function will be discussed below. Because the functions of LncRNAs in VSM biology are still being determined, approaches to modify their function have not yet been extensively examined in this setting.

161One of the benefits of miRs is that they are strongly conserved in species (albeit potentially targeting differing mRNA targets), thus aiding the design of preclinical studies to determine their efficacy and safety. In addition, miRs have the potential to target many members of the same molecular pathway and as a result having a greater combined effect than siRNAs, which typically target only a single gene. For example, the miR-200 family has been implicated in regulating many targets that control actin filament organization and dynamics. Furthermore, miR therapy will not completely knock down their target protein’s expression. Rather, they will result in a "fine-tuning" effect on target protein expression, making them an attractive proposition for therapeutic intervention.

162There are two main techniques to modulate miR activity in vivo (reviewed in; ) (Fig. 10): 1) restoring the expression of a downregulated miR by use of a miR-mimic and 2) inhibiting the activity of an abnormally expressed miR by use of an anti-miR. A miR-mimic is a synthetically designed RNA duplex, containing a passenger strand, chemically modified to enhance uptake and disengage once inside the cell, and a guide strand, which is identical to a miR of interest. Conversely, an anti-miR is designed to be complementary to the endogenous miR and therefore binds and inhibits it from acting on its mRNA target. Several modifications, such as locked nucleic acid and phosphodiester additions, have improved the in vivo stability of anti-miRs.

163The great strength of miRs, their ability to target multiple mRNAs, is also their greatest weakness because they could potentially result in many off-target effects. In addition, miR mimics often increase the miR concentration to supraphysiologic levels, again increasing the risk of off-target actions. To lower such risk, viral constructs have been developed to re-express the miRs to endogenous levels. Recently, lipid nanoparticle delivery has proved promising to deliver miR regulators to target cells and reduced delivery to nontarget cells (Fig. 10). For instance, anti-miR-145 therapy in rats with Sugen-5416/hypoxia-induced pulmonary arterial hypertension (PAH) using the Star:Star-mPEG delivery system, reduced the severity of pulmonary hypertension without significant off-target effects.

164Extracellular vesicles, such as exosomes, microvesicles, and apoptotic bodies, allow long-distance delivery of cellular information, including noncoding RNA. They recently emerged as important regulators of cardiovascular diseases, such as atherosclerosis (recently reviewed by ) and may serve as important diagnostic and prognostic biomarkers for these conditions.

165Several miRs have been found to contribute to clinical and experimental pulmonary hypertension, which is characterized by enhanced proliferation and constriction of pulmonary artery smooth muscle cells (PASMCs). For example, miR-130a promotes PASMC proliferation through the negative regulation of its targets CDKN1A and growth arrest-specific homeobox. Interestingly, the miR-130/-301 family also regulates the expression of peroxisome proliferation receptor gamma, which influences the expression of many vasoactive factors, such as endothelin-1. The expression of miR-190 was augmented in PASMCs of the hypoxic rat, which acts as a model for pulmonary hypertension. Vasoconstriction to both potassium chloride and phenylephrine was enhanced after transfection of pulmonary arterial rings with a miR-190 mimic. The same study demonstrated that miR-190 downregulates the protein expression of the potassium channel Kv7.5, which results in raised intracellular calcium in PASMCs. The miR-328 has been demonstrated to directly target the α1C subunit of the L-type calcium channel and was reduced in the pulmonary artery from patients with pulmonary hypertension, thus enabling an increase in vasoconstriction.

166There have been fewer animal studies focusing on the role of miRs in the development of essential hypertension. Clinical studies, however, have highlighted the possibility of therapeutic intervention to prevent or treat systemic hypertension. A study from Greece found that the expression of miR-145, -143, and -133 was upregulated, and miR-21 and -1 were downregulated in the peripheral blood of patients presenting with essential hypertension. These miRs are all important regulators of VSMC phenotype plasticity. A further study in a Chinese population identified the SNP rs12731181 (A to G) in the prostaglandin F2α receptor that was more prevalent in hypertensive individuals. This polymorphism reduces the likelihood of miR-590-3p binding, thus increasing prostaglandin F2α receptor expression and enhancing prostaglandin-mediated contractility of VSMCs.

167Several studies have demonstrated the feasibility of miR therapies to slow the development of abdominal aortic aneurysms. In particular, the miR-29 family has been heavily implicated in aneurysm development in mouse models. Murine in vivo delivery of miR-29 was found to decrease elastin mRNA and increase MMP activity and aneurysm development. Importantly, blockade of miR-29 reduced aneurysm development in Ang II-infused mice, a mouse model for Marfan syndrome, and ApoE−/− mice, signifying its therapeutic potential. In addition, miR-21, -712/-205, and -195 have all been demonstrated to promote aneurysm development, whereas miR-24 slows aneurysm progression in mouse models.

168Interestingly, miR-29 was found to increase with aging in the aorta from mice, which correlated with aneurysm development. The targets affected by miR-29 are strongly implicated in aortic stiffness, which is also known to increase with age. Few studies have focused on the role of miRs in vascular stiffness. A clinical study found that two SNPs, rs978906 (A allele) and rs9808232 (C allele), were associated with high pulse wave velocity in 856 individuals in a Chinese population. These polymorphisms rendered the expression of the aortic stiffness-associated ROCK2 less responsive to changes in miR-1183 expression, thus enhancing ROCK2 expression. In addition, miR-145 was strongly reduced in ApoE−/− mice, which was associated with an increase in collagen and stiffer arteries.

169These studies demonstrate the capacity of miR therapeutics to improve treatments for vascular complications such as hypertension, abdominal aortic aneurysm, and arterial stiffness. Indeed, anti-miR-33 (atherosclerosis, Regulus Therapeutics, (Carlsbad, CA)), anti-miR-92 (peripheral artery disease, miRagen Therapeutics Boulder, CO), and anti-miR-145 (vascular occlusion, miRagen Therapeutics) have already reached preclinical trials. This has clearly been a rapidly expanding field and we expect to see many further developments, including approaches to target LncRNAs, in the coming years.

170Excess vasoconstriction and the resulting increase in vascular tone and SVR are an important contributing factor to the pathogenesis of essential hypertension. CCBs, ACE inhibitors, ARBs, and direct vasodilators (i.e., hydralazine) all target the smooth muscle cell to decrease vascular tone and blood pressure. CCBs decrease the activation of SM myosin by decreasing intracellular Ca2+, whereas ACE inhibitors and ARBs will decrease the activation of the RhoA/Rho kinase signaling pathway to reduce Ca2+ sensitization. K+ channel openers produce vasodilatation; nicorandil acts on BK channels and hyperpolarizes smooth muscle cells, whereas minoxidil opens ATP-sensitive K+ channels and hyperpolarizes the smooth muscle cells. The mechanism of action for hydralazine, on the other hand, is unclear, but evidence suggests that hydralazine both activates K+ channels and also stimulates NO production by the vascular endothelium.

171In addition to essential hypertension, there are a number of other diseases of the vasculature and diseases with associated abnormal vascular reactivity. These defects in vascular reactivity contribute to morbidity and mortality. In this section, we will outline these diseases, their mechanism, current therapies, and suggest novel targets for both the treatment of essential hypertension as well as these other diseases of the vasculature (Table 1).

172Considerable evidence from both human subjects and animal models have shown that HF is associated with an impaired vasodilatory response to acetylcholine, via an endothelium-dependent mechanism, as well as to nitroglycerin, via an endothelial-independent mechanism. Both MYPT1 LZ+ expression and the vasodilatory response to NO/cGMP has been demonstrated to decrease in HF, which demonstrates that the fall in LZ+ MYPT1 expression contributes to the decrease in sensitivity to NO-mediated vasodilatation associated with HF. Additionally, treatment of HF with either ACE inhibitors or ARBs has been demonstrated to preserve the normal expression of the LZ+ MYPT1 isoform and sensitivity to NO/cGMP-mediated vasodilatation. These results are similar to those of; this group demonstrated that both endothelium-dependent and -independent responses in renal blood flow were impaired in HF rats compared with control, and Ang II receptor blockade normalized these vasodilatory responses.

173The mechanism for the regulation of LZ+/LZ− MYPT1 expression by Ang II-induced activation of the AT1 receptor is unknown, but evidence suggests that both the activation of p42/44 MAPK and Tra2β are important. Ang II produces vasoconstriction through its effect on both the endothelium and vascular smooth muscle. Additionally, Ang II activates nuclear factor κB to produce a proinflammatory state by inducing expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α and also activates membrane NADH/NADPH oxidases, generating reactive superoxide anions, which increase NO catabolism and therefore decrease NO bioavailability. In heart failure, inhibition of Ang II signaling counter these deleterious effects and could contribute to the reduction in cardiovascular morbidity and mortality observed with ACE inhibitors and ARBs. Furthermore, similar beneficial effects on survival have not been observed with other vasodilators such as prazosin, although prazosin has also been shown to improve cardiac index and fractional shortening for heart failure patients, which suggests that ACE inhibitor have other beneficial effects beyond reducing afterload and improving left ventricular remodeling.

174In the setting of advanced heart failure, marked activation of the renin-angiotensin system and worsening functional status have been associated with a progressive elevation of the proinflammatory marker TNF-α, which has been demonstrated to contribute to endothelial dysfunction and also an increase in vascular tone that is produced by a RhoA/Rho kinase-mediated inhibition of MLC phosphatase. Additionally, the inflammatory marker IL-1β, which is also elevated in heart failure, increases Ser850 MYPT1 phosphorylation to inhibit MLC phosphatase. It has been demonstrated that impaired endothelial and vascular smooth muscle dysfunction are associated with a poor prognosis in heart failure. Potentially IL-1β and TNF-α could be involved in the regulation of MYPT1 isoform expression, and defining the role of inflammatory cytokines for the regulation of MYPT1 isoform expression could reveal a novel therapy to reverse the vascular dysfunction and improve prognosis in heart failure.

175In addition to inhibiting the RhoA/Rho kinase pathway, ACE inhibitors and ARBs also preserve the LZ+ MYPT1 isoform expression. The variability in LZ+ MYTP1 expression among different vascular smooth muscle cells and the changes that occur during heart failure could provide an explanation as to why racial differences play an important role in predicting responses to ACE inhibitor therapy compared with the combination of vasodilators and nitrates. There are racial variations in plasma norepinephrine and renin, and this could influence both MYPT1 isoform expression as well as MLC phosphatase activity. These factors could explain the more significant blood pressure and mortality reduction seen in white compared with black patients. MYPT1 polymorphisms may also exist, which could contribute to the diversity of symptoms in patients with similar reductions in LVEF. Hence, the ability of ACE inhibitors and ARBs to alter the vascular smooth muscle cell phenotype could contribute to the improvement in survival in patients with heart failure, and thus enhancing LZ+ MYPT1 expression and normalizing the vascular response to NO may represent another novel target for the treatment of heart failure.

176Pulmonary arterial hypertension (PAH) is a rare, incurable disease with a poor prognosis. In patients with PAH, the pulmonary vasculature is characterized by a resting vasoconstriction and an abnormal response to NO, and in the majority of patients, NO-mediated vasodilatation is severely blunted. Clinically, the initial assessment of patients with PAH includes the evaluation of the pulmonary response to NO prognosis is improved if NO decreases pulmonary arterial pressure and PVR by 20%. However, less than 10% of patients with PAH demonstrate a significant vasodilatory response to NO, and although there are a number of proposed mechanisms for the progressive increase in PVR, the molecular mechanism that results in the lack of vasodilatation to NO in PAH is unknown.

177There are two components that produce PAH: the extent of the structural changes (smooth muscle cell proliferation, smooth muscle cell hypertrophy, and the deposition of matrix proteins within the media of pulmonary arterial vessels and excess vasoconstriction. Structural changes have been documented in animal models of PAH, which have demonstrated that chronic hypoxia leads to pulmonary vascular remodeling that increases PVR and decreases the vasodilatory response to NO (reviewed in;; ). However, despite the fact that for PAH the target and aim of all current therapeutic agents [prostaglandins (epoprostenol), phosphodiesterase inhibitors (sildenafil), guanylate cyclase stimulators (riocigaut), endothelin antagonists (bosentan), NO inhalation and Rho kinase inhibitors (fasudil)] is the smooth muscle cell to reduce pulmonary vascular resistance, the fundamental changes in the pulmonary smooth muscle contractile phenotype that contribute to the excess vasoconstriction, or the increase in PVR, as well as the decrease in vasodilatory response to NO, are poorly characterized. Evidence supports a contribution of changes in the pulmonary smooth muscle contractile phenotype in PAH; an increase in the expression and activity of Rho kinase has been demonstrated to contribute to the pathogenesis of PAH, and Rho kinase inhibition reduces pulmonary pressure in some animal models of PAH. The importance of the NO/cGMP signaling pathway for the pathogenesis of PAH is highlighted by data demonstrating that PKG KO mice develop pulmonary hypertension, and mice with mutations in the PKG LZ domain, which disrupts PKG interaction with MYPT1, develop progressive increases in pulmonary pressures and right ventricular hypertrophy. Furthermore, activation of guanylate cyclase has been demonstrated to improve exercise capacity and reduce pulmonary pressures in patients with PAH. All these studies are consistent with a defect in NO/cGMP signaling contributing to the pathogenesis of pulmonary hypertension, but do not isolate the step in the NO signaling pathway that produces PAH.

178In smooth muscle, investigators have demonstrated that NM myosin (Fig. 7) contributes to the sustained force response and a decrease in NM myosin expression will result in a decrease in vascular tone. Additionally, changes in the sensitivity to NO-mediated vasodilatation are due to changes in the expression of LZ+/LZ− MYPT1. Thus, an increase in NM myosin and decrease in LZ+ MYPT1 expression will produce abnormalities of pulmonary vascular reactivity and may participate in the molecular mechanism that produces PAH. demonstrated that with the development of severe PAH, there is a 3.5-fold increase in NM myosin expression, which could contribute to the prolongation in the rates of both force activation and relaxation, as well as the increase in force maintenance. Similarly, an increase in NM myosin expression has also been reported for hypoxia induced PAH, and therefore drugs that target NM myosin expression or activation could represent a new class of therapeutic agents for the treatment of PAH.

179Konik’s data also demonstrated that a decrease in LZ+ MYPT1 expression is associated with severe PAH. The sensitivity of smooth muscle to NO is regulated, in part, by relative LZ+ MYPT1 expression. In severe PAH, these data would suggest that the decrease in LZ+ MYPT1 expression would result in a decrease in the sensitivity of the pulmonary vasculature to NO. Similarly, a decrease in LZ+ MYPT1 expression has been reported for cultured pulmonary SMC exposed to hypoxia. Additionally, the transition from a phasic to tonic SM contractile phenotype has been suggested to be associated with a decrease in the ratio of LZ+ MYPT1/MYPT1 expression. Tonic contractile properties would produce an increase in vascular tone, similar to that observed in patients with PAH. These results are consistent with a decrease in LZ+ MYPT1 expression contributing to the pathogenesis of PAH; a decrease in LZ+ MYPT1 expression would reduce the sensitivity to cGMP, and riociguat treatment would increase cGMP to counteract and overcome the decrease in sensitivity to NO to reduce PVR and pulmonary pressures. If a decrease in LZ+ MYPT1 expression contributes to the molecular mechanism for PAH, these data could provide a unifying mechanism to explain the variable response to drugs that act on the NO/cGMP signaling pathway (NO, PDE5 inhibitors, and riociguat), i.e., patients with a mild decrease in LZ+ MYPT1 expression would represent “responders,” whereas patients in which LZ+ MYPT1 expression is severely depressed would not respond to this class of therapeutics. Developing therapeutics that increase LZ+ MYPT1 expression should normalize the pulmonary vascular response to NO and decrease PVR, which would represent a novel target for treating patients with PAH.

180Vascular reactivity is known to be altered in portal hypertension, with increased sensitivity to dilatation and decrease sensitivity to constriction. The mechanism that contributes to this altered reactivity is unclear, but evidence suggests that splice variant expression of the contractile proteins is modulated by flow and/or pressure. examined the expression of SM MHC, ELC17, and MYPT1 in the portal vein stenosis model of portal hypertension. The ligature of the portal vein produces dynamic changes in flow and pressure; there is an initial increase in both splanchnic pressure and flow, which fall with the development of porto-systemic shunting. Similar to these dynamic changes in flow, these investigators demonstrated that in both the portal veins and mesenteric arteries within 1 day of the increase in portal pressure, although total MYPT1 expression decreased, the expression of the LZ+ MYPT1 isoform increased. These changes in MYPT1 expression were maintained for ∼7 days before returning to preportal hypertension levels by 14 days after the ligature of the portal vein. Similar to MYPT1 isoform expression, ELC17 and SM MHC expression were modulated with an increase in the expression of the slow isoforms (ELC17b and SMA) 3 days after ligature, but SMA expression returned to baseline at day 14.

181Others have altered flow in the mesenteric circulation by ligating alternative pairs of second order mesenteric arteries to produce either low or high flow in the upstream first order mesenteric arteries. These investigators demonstrated that there was an initial increase in LZ+ MYPT1 expression in both low and high flow vessels, but by 1 month LZ+ MYPT1 expression returned toward baseline in the low flow vessels, whereas in the high flow vessels, LZ+ MYPT1 expression continued to increase. As would be predicted, the sensitivity to NO/cGMP-mediated relaxation paralleled LZ+ MYPT1 expression. Furthermore, total MYPT1 expression rapidly fell in both the low and high flow mesenteric vessels, and pretreatment with an inhibitor of the proteosome attenuated the decrease in MYPT1. These data suggest that the rapid decrease in total MYPT1 is due to degradation by the proteosome, which is similar to the results in the mesenteric arterioles in heart failure. These data demonstrate that flow and/or pressure is also important in regulating the smooth muscle contractile phenotype, and thus vascular reactivity.

182Raynaud’s phenomenon is described as a transient cessation of blood flow in the fingers and toes and can be triggered by either cold or emotional stress. The phenomenon is the result of a transient vasospasm of the digital arteries in the hands and/or feet that leads to a cessation of blood flow, which produces pallor, followed by cyanosis and pain due ischemia of the sensory nerves. As the vasospastic attack subsides, blood flow to the digits is restored, and the resulting hyperemia produces a red phase. The estimates of the prevalence of this disease vary, but it is more common in women, and the onset and severity of the disease peaks between menarche and menopause. Treatment of Raynaud’s involves keeping the digits warm and pharmacologic therapy with CCBs can be helpful; however the usefulness of CCBs is often limited by hypotension.

183There are a number of mechanisms proposed for the transient vasoconstriction of the digital vessels; these have been reviewed and there is evidence supporting roles for hyperactivity of adrenergic nervous system, α2 adrenoceptors, central stress responses, 5-HT, endothelin, oxidative stress, cyclo-oxygenase, and NO. However, it is interesting that the vasodilation of the digital vessels produced by both sodium nitroprusside and ACh are reduced in patients with Raynaud’s, which suggest that the defect lies within the cGMP/PKG signaling cascade. Consistent with this hypothesis are the results that show l-arginine does not restore the normal vasodilatory response to ACh, which indicates that the vasodilatory response to NO is depressed in Raynaud’s disease, and the mechanism lies beyond the bioavailability of NO within the cGMP/PKG signaling cascade. These data could suggest that a decrease in LZ+ MYPT1 expression in the digital vessels could contribute to the abnormal vascular reactivity.

184During a normal pregnancy, vascular resistance falls, which is thought to be the results of increased synthesis and activity of NO and prostacyclin. However, approximately 5–15% of pregnancies are complicated by hypertension, which increases perinatal morbidity and mortality of both the mother and fetus. Antihypertensive therapy is limited due to the effects on the fetus, i.e., ACE inhibitors and ARBs are fetotoxic. A number of mechanisms have been proposed for the increase in PVR and blood pressure, including both a decrease in NO produced by endothelial dysfunction and an increase in circulating vasoconstrictors including Ang II, thromboxane, and endothelin. NOS inhibition is commonly used to produce a model of pregnancy-induced hypertension, and studies have suggested that the increase in blood pressure is due to altered vascular reactivity produced by RhoA, PKC, and Ca2+ signaling.

185recently examined MYPT1 isoform expression in pregnancy-induced hypertension. These investigators demonstrated that during a normal pregnancy total MYPT1 expression increased, but there was no change in MYPT1 isoform expression. However, during pregnancy-induced hypertension, although there were no changes in MYPT1 expression in mesenteric arteries or the aorta, in uterine arteries, pregnancy-induced hypertension was associated with an increase in LZ+ MYPT1 expression but an ∼50% decrease in total MYPT1 expression. As would be predicted, the increase in LZ+ MYPT1 expression was associated with an increase in the sensitivity of uterine artery relaxation to both sodium nitroprusside and cGMP. The increase in LZ+ MYPT1 expression did not occur when the blood pressure of the pregnant hypertensive rats was normalized with hydralazine. It is interesting to speculate that during pregnancy-induced hypertension the changes in MYPT1 expression are compensatory, and the resulting reduction in uterine vascular resistance maintains blood flow despite the inward remodeling of the arteries.

186A number of studies demonstrate that blood pressure is a genetically determined trait with heritability estimates of 31–68%. There are several, rare monogenic syndromes, which are due to abnormalities in renal fluid balance that result in hypertension. However, because the regulation of blood pressure is due to complex interactions of fluid balance, cardiac contractility, and vascular tone, the genetic basis of essential hypertension has yet to be elucidated. Recently, a number of genome wide-association studies (GWAS) have identified SNPs in a number of genes that could contribute to the development of essential hypertension. These studies have focused on populations of European decent, but GWAS have examined East Asia as well as African American cohorts. A complete discussion of GWAS methods as well as the results of these studies are beyond the scope of the present review, and we would refer readers to recent reviews on this subject. In addition to SNPs, gene expression is also regulated by miR and DNA methylation. The importance of miRs in the regulation of blood pressure is highlighted by a recent study demonstrating that mice lacking miR-142 and miR-145 have reduced vascular tone and blood pressure. Additionally, hcmv-miR-UL122 is highly expressed in hypertensive patients, a number of miRs are increased in hypertensive nephrosclerosis, and furthermore, renin expression appears to be regulated by mi-RNA-181a and miR-663 renin. These factors illustrate that SNPs, miRs, and posttranslational modifications all play a role in the development of hypertension.

187In addition to revealing candidate genes important in the pathogenesis of essential hypertension, pharmacogenomic GWAS have the potential to reveal the likelihood of a patient to respond to therapy or even to develop a rare adverse drug reaction. There are several reports of an association between polymorphisms in the β1-adrenergic receptor gene and the lowering of blood pressure (reviewed in ). An association of Ser49Gly and Arg389Gly polymorphisms has been demonstrated to be associated with a significant reduction in blood pressure with β-blocker therapy, and furthermore, one or both of these SNPs were carried by 54% of Chinese and 44% of whites, perhaps suggesting an etiology of the ethnic differences in response to β-blocker therapy. The PEAR study evaluated the association of 39 SNPs known to be associated with hypertension from GWAS studies with a response to monotherapy with atenolol or HCTZ in 768 hypertensive patients (60% white and 40% black). The response to atenolol therapy was greater in the white patients; six SNPs were associated with a response to atenolol therapy, with greater responses with in those with all six BP lowering alleles. HCTZ lowered blood pressure less in white compared with black patients. In the white patients, three alleles were associated with BP lowering for HCTZ monotherapy, and similarly, the association was strongest for those with all three alleles, but none of these were associated with BP lowering in blacks. Interestingly for HCTZ therapy, one SNP was associated with a BP reduction in white hypertensive patients, whereas it was associated with an increase in BP in the black patients. A G825C polymorphism in the gene encoding the G-protein β3-subunit has been associated with a response to HCTZ therapy, and this genotype was a stronger predictor of response to diuretics than ethnicity. There have been two large trials examining polymorphisms in the genes encoding ACE and the AT1 receptor and the response to ACE inhibition. No association was found for the insertion/deletion polymorphism in the ACE gene and the response to lisinopril therapy, and similarly for all ethnic groups examined, no genetic association was present for the 1166A-C genotype of the AT1 receptor and response to trandolapril. These data suggest that pharmacogenomic GWAS have the potential to identify genotypes that not only contribute to the development of hypertension but will also predict a positive response to antihypertensive therapy. An individual’s genotype could be then used for a personalized approach for selecting an effective antihypertensive with minimal chance of the patient developing an adverse reaction.

188There is no question that essential hypertension is due, in part, to changes at the level of vascular smooth muscle that lead to excess vasoconstriction and a concomitant increase in SVR and blood pressure. In this review, we suggested have several novel targets for the treatment of essential hypertension. Details and the strategy for the each target can be found in the individual sections. These targets include those that affect Ca2+, including 1) downregulation of AKAP150, 2) increasing the expression of the β1 subunit of the BK channel, 3) novel BK channel openers, 4) inhibiting or decreasing the expression of STIM/Orai (Ca2+ release-activated Ca2+ channels), and 5) increasing cleavage of the C terminal of LTCC or increasing CCt expression. One could also consider inhibition of MLCK as a novel target; the soybean isoform of calmodulin (SCaM-4) has been demonstrated to bind Ca2+ but not activate MLCK and thus inhibit the activation of smooth muscle. Thus, targeted expression of SCaM-4 or a CaM fragment in vascular smooth muscle should reduce SVR and blood pressure. There are also a number of scaffolds including those for ERK (calponin, SmAV, and paxillin) and MLC phosphatase (M-RIP and Par-4) that could be examined as potential therapeutic targets, which could result in a reduction of vascular tone. Inhibition of the signaling pathways for Ca2+ sensitization including Rho kinase and GEF signaling could represent another area for rational drug design, and similarly, activation of pathways leading to Ca2+ desensitization including guanylate cyclase and increasing the expression of LZ+ MYPT1 would enhance NO/flow mediated vasodilatation, which should decrease blood pressure. Other attractive targets would be pathways leading to remodeling of the actin cytoskeleton including integrins, focal adhesion proteins, as well as the activation of tyrosine kinases. Targeted delivery of miRs to the vasculature also may prove to be an effective strategy for the treatment of hypertension; miRs could be used as a strategy to change vascular smooth muscle gene program from high force (tonic) and NO unresponsive to a lower force (phasic) and NO responsive and hence produce a reduction in vascular tone and blood pressure.

189These novel targets could form the basis for rationale drug design, and could be exploited for development of therapeutic agents that are effective for the treatment of hypertension. For some targets, small molecule inhibitors/activators would appear to be a reasonable choice, whereas for other targets, i.e., to reverse the decreases in MYPT1 expression, adenoviral delivery of MYPT1 to the vasculature could also be used as a strategy to increase the sensitivity to NO or flow-mediated vasodilation, which would decrease in vascular tone and blood pressure.

190The contribution of alteration in the vascular phenotype from Ca2+ signaling, regulation of the cytoskeleton and contractility, and biomechanics to the pathogenesis of essential hypertension may vary among ethnic groups and/or individuals. Currently, antihypertensive therapy is generally approached by a method that could be best summarized as trial and error: an agent is selected, usually without consideration of the patient’s ethnic background, and if control is not adequate, either the dose is increased or another agent is added.

191In this review, we discussed the importance of the vascular phenotype in the mechanism that leads to essential hypertension. Therefore, mechanisms to identify the etiology of the increase in blood pressure in each patient are important for ultimately selecting an individualized and effective therapeutic agent. These future studies will hopefully lead to a more personalized approach to antihypertensive therapy.