Oxidative Stress: A Major Player in Cerebrovascular Alterations Associated to Neurodegenerative Events

1The brain integrates and regulates several central and peripheral signals to maintain body homeostasis. So, it is not surprising that the brain is an organ with a high energy demand, although it represents only 2% of the body weight. In fact, it is widely known that proper neuronal activity entails high amounts of energy. However, the capability of the brain to store energy is very reduced, requiring a constant supply of energy substrates, namely glucose, through blood flow to fulfill its energy needs. For that reason the brain receives about 15% of cardiac output and accounts for 20% of total body oxygen consumption. At this point, it is worth mentioning the neurovascular coupling, where neurons, glial cells and blood vessels communicate to each other to regulate cerebral blood flow (CBF) and vessels permeability depending on location and neuronal activity in order to efficiently maintain energy substrates supply to satisfy the metabolic needs. Cerebral blood vessels comprise unique properties forming the blood–brain barrier (BBB), a physical barrier that permits the passage of water, some gases and lipophilic molecules by passive diffusion and the selective transport of certain molecules (e.g., glucose) and protects against external toxins and pathogens. Thus, even slight alterations in BBB properties can be responsible for the onset/progression of neurological diseases. From all the BBB constituents (endothelial, mural and glia cells, astrocytes and macrophages), endothelial cells that form blood vessels, play a major role in BBB proper functioning. Blood vessels control the influx and efflux transport allowing, for example, the entry of glucose and amino acids from the blood into the central nervous system (CNS) and the removal of specific waste products from the CNS into the blood. More recently,, performed a mapping of brain microvessels obtained from two healthy human adult brain samples and found that in certain brain areas microvessels density is higher than others, which can reflect the different susceptibilities to vascular damage. Even slight alterations in brain vasculature can underlie different neurodegenerative events. Indeed, it is widely described that conditions interfering with brain vasculature, such as stiffening of cerebral arteries or increased vessel tortuosity, caused by diabetes and hypertension among other conditions, can induce BBB breakdown underlying the development of neurodegenerative conditions such as Alzheimer’s disease (AD). It is also known that in many cases BBB integrity is deeply affected by oxidative stress. In fact, increased reactive oxygen species (ROS) production contribute to endothelium dysfunction and increased permeability of BBB. These alterations are mainly attributed to the redistribution and/or altered expression of critical tight junction proteins such as claudin-5 and occludin.

2Although it is widely accepted that vascular changes play a crucial role in neurodegenerative diseases, none of the available therapies are effective when translated to clinical trials revealing some gaps in the mechanisms behind the vascular and tissue brain changes under pathological conditions. Thus, studies aimed to develop new non-invasive techniques to better understand why and when changes occur bring a new hope for the treatment of conditions characterized by cerebrovascular alterations. In the next section we will discuss the role of oxidative stress in brain vascular alterations putting the focus on mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox).

3It is widely known that cells homeostasis depends on the regulated levels of ROS. Low/moderate levels of ROS can act as signaling molecules, which are crucial to maintain normal cells function, while uncontrolled generation of ROS causes oxidative damage contributing to cells dysfunction and damage.

4Physiological ROS levels can play important roles in cerebral vasculature. Studies performed in animal models show that ROS can contribute to the regulation of brain perfusion through their action in vascular tone control. Indeed, physiological levels of ROS play a major role as cerebral vasodilators (Figure 1). For example, it has been shown that the addition of NADPH, the substrate for Nox, in cerebral vessels in vitro and in vivo cause hydrogen peroxide (H2O2)-dependent vasodilatation.

5The neurovascular unit (NVU), containing neurons, astrocytes, pericytes, microglia and endothelial cells, is equipped with a powerful antioxidant defense systems that include glutathione (GSH), glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase. GSH in particular has been shown to play an important role in the maintenance of BBB integrity (Figure 1). Also, the nuclear factor erythroid 2-related factor 2 (NRF2) seems to play a major defense role by modulating microglial dynamics, by protecting astrocytes and neurons from toxic insults and by regulating the expression of antioxidant enzymes. Additionally, NRF2 induces secondary defense proteins via interaction with the antioxidant response element (ARE) in the promoter region of target genes (Figure 1). Interestingly, astrocytes have a higher capability of efficiently increase GSH and ARE-linked gene expression, which allows astrocytes to be more protected than neurons against moderate levels of oxidative stress.

6A failure in the ability of NVU cells to maintain the proper balance between ROS production and their neutralization causes the disruption of NUV and brain homeostasis predisposing to neurodegenerative conditions. It has been shown that following an injury microglia and astrocytes produce high levels of ROS via Nox, which seem to have harmful effects in the expression of important molecules involved in BBB integrity (e.g., ZO-1, claudin-5 and occluding). Pericytes, which are in close proximity to endothelial cells, play a vital role in the integrity of BBB. Under pathological conditions, pericytes are highly susceptible to oxidative stress resulting from overproduction of mitochondrial ROS. Moreover, it has been demonstrated that ROS production by activated microglia causes pericytes apoptosis. However, there is a lack of information regarding the role of ROS in pericytes under physiological conditions. Endothelial cells signaling seems to be crucial in the regulation of NVU proper functioning with increased ROS formation playing a major role in the alteration of NVU function and coupling.

7It was also shown that ROS-induced changes in microcirculation can have profound implications in brain vascular pathophysiology due to alterations in blood flow resistance and, consequently, in regulation of blood pressure (Figure 1). Both superoxide (O2•-) and H2O2 are able to cause both relaxation as well as contraction of cerebral blood vessels depending on the concentration and presence of other species. In fact, oxidative stress can impair cerebral vascular function via the disruption of endothelium-dependent nitric oxide (•NO) signaling (Figure 1). It is widely described that the reaction of O2•- with •NO leads to a decrease in •NO bioavailability and, consequently, a decrease in its vasodilator, anti-proliferative, and anti-inflammatory properties. Furthermore, increased levels of H2O2, a common vasodilator in cerebral circulation, can act as a pro-apoptotic agent in cerebral vascular cells (Figure 1). Increased levels of ROS can also lead to an increase in Rho kinase signaling interfering with endothelial nitric oxide synthase (eNOS) expression and activity, affecting •NO production (Figure 1).

8Considering vascular cells, there are several ROS sources such as mitochondrial electron-transport chain, cyclooxygenases (COXs), lipoxygenases, cytochrome P450 reductases, xanthine oxidase, nitric oxide synthase (NOS) and Nox. However, in this review, we only discuss mitochondria and Nox, both of them widely described as main producers of ROS either in physiological and pathological conditions in endothelial cells.

9Reporting to the history of biology evolution, mitochondria are described as organelles derived from aerobic bacteria that in ancient times invaded proto-eukaryotic cells as parasites. From there, a symbiotic relationship evolved with mutual benefits and mitochondria became intracellular organelles. Although mitochondria are widely described as energy producers through the highly conserved oxidative phosphorylation (OXPHOS) process, nowadays the role of mitochondria reached substantially higher importance in cell homeostasis due to their involvement in several vital processes such as cell growth and differentiation, cell cycle control and death, intermediary metabolism, calcium (Ca2+) homeostasis and signaling, and apoptosis.

10However, mitochondrial energy production can be a double-edge sword. Indeed, OXPHOS is not 100% efficient and during this process an electron leak occurs between mitochondrial complexes leading to the production of ROS such as O2•-, •NO, hydroxyl radical (HO•), peroxynitrite (ONOO-), and H2O2. As previously mentioned, low/moderate ROS levels exert a beneficial role by activating protective mechanisms. However, when ROS levels reach critical values they lead to oxidative stress and activate anomalous signaling mechanisms that can lead to cells degeneration and death.

11Brain endothelial cells seem to possess a number of mitochondria higher than that observed in peripheral endothelial cells. However, in comparison with other cell types with higher energy requirements, mitochondria content in endothelial cells is modest. In rodent models, mitochondria compose 2–6% of the cell volume as opposed to 28% in hepatocytes and 32% in cardiac myocytes. These observations support the idea that mitochondria is not a major source of energy in brain endothelial cells. In fact, several studies support the idea that brain endothelial cells obtain a large proportion of their energy from anaerobic glycolytic metabolism of glucose. Actually, mitochondria are more likely to serve primarily as essential signaling organelles in the vascular endothelium.

12The activation of mitochondria by physiological stimuli or pharmacological agents leads to the liberation of vasoactive factors by the endothelium, which exert a major role in the modulation and maintenance of BBB integrity and brain homeostasis. Moreover, it is far known that aging leads to a decrease in mitochondria number in cerebral endothelial cells associated with the loss of BBB integrity. The BBB high demand of mitochondrial activation comes with a price, the prospect for an increased ROS production that in physiological conditions is regulated by antioxidant enzymes such as glutathione reductase, manganese superoxide dismutase, catalase, among others. Additionally, it has been demonstrated that vascular endothelial growth factor (VEGF) exerts its functions in endothelial cells migration through mitochondrial ROS, a process involved in several physiological processes such as wound healing and vascular repair. Moreover, studies in human coronary resistance arteries showed that mitochondrial ROS are involved in endothelium regulation of vascular homeostasis. In the same study, the authors reported that mitochondrial ROS have an influence in vascular regulation and health. Indeed, the authors tested mitochondrial complexes I and III and Nox inhibitors and observed that only the mitochondrial inhibitors were able to exert effects in the regulation of flow-induced dilation. Besides the above evidence more studies are needed to explore the role of mitochondrial ROS in cerebral vasculature during disease progression, in contrast to the extensive literature concerning systemic vessels.

13The Nox family is also broadly studied due to its main catalytic function of ROS production by transference of an electron to molecular oxygen. The Nox family is composed by different isoforms: Nox1 to 5, Duox1 and Duox2 with a broad expression in different organs and tissues and with different cellular locations at vascular walls. Except for Nox-5 and DUOX-1 and -2, Nox are phagocytic oxidases, whose main task is to generate ROS to kill foreign pathogens at homeostasis. Of note, the major source of endothelial cells ROS comes from NOX-1, -2, -4, and -5. Different Nox isoforms seem to produce different forms of ROS; Nox1 and 2 mainly produce O2•-; Nox4 mainly produces H2O2 and Nox5 seems to produce both O2•- and H2O2. Under physiological conditions, Nox-derived ROS seem to exert an important role in the regulation of vasodilatation. Although it is considered that under physiological conditions Nox activity is constitutively low, when its function increases with the consequent increase in ROS production, it can trigger ROS production by other sources. Indeed, it has been described that Nox-derived ROS play a major role in coordinating some physiological processes such as innate immunity, modulation of redox-dependent signaling cascades, and can act as cofactors in the production of hormones. Furthermore, it is described that H2O2 produced by Nox family can exert an endothelium-derived hyperpolarizing role causing vasodilatation and reducing blood pressure in mice. The Nox activity depends on the stimuli, such as cytokines, growth factors, hyperlipidemia, and high glucose. Additionally, Nox family is known for its role as oxygen sensors, modulating the different responses to hypoxia through hypoxia-inducible factor 1 alpha (HIF-1α) mRNA induction and HIF-1α stabilization, a process that is responsible for alterations in gene expression due to the oxidation of target proteins.

14However, the overproduction of ROS by Nox family plays a major role in disruption of vascular homeostasis, which can underlie the development of neurodegenerative diseases. Of notice, so far only Nox2 and Nox4 were associated with endothelium dysfunction probably due to their major role as vascular ROS producers, in comparison to the other Nox isoforms.

15Compelling evidence shows that Nox-derived oxidative stress causes many of the deleterious effects of angiotensin II on the cerebral vasculature. It was observed that angiotensin II acutely and chronically, increases O2•- production by Nox in rodent cerebral vessels. Also, functional alterations on rodents’ cerebral arterioles following angiotensin II treatment are prevented by co-treatment with the ROS scavenger MnTBAP and the Nox2 peptide inhibitor gp91ds-tat. It was also reported that angiotensin II can also activate and increase O2•- production in cerebral vessels by activating Nox1.

16Interestingly, although not extensively studied, the Nox family activation and expression seems to be seasonal, a fact that could be correlated with seasonal alterations in oxidative stress and endothelium dysfunction observed in humans and rats unveiling a new strategic pathway to future studies of clinical relevance.

17The aging process causes several alterations in brain blood vessels such as decreased elasticity, increased cerebrovascular remodeling and calcification, gradual cerebrovascular wall stiffness, low-grade and widespread inflammation and oxidative stress, factors that increase the risk for cerebrovascular diseases. So far, there is no agreement of how aging process occurs although several theories can be found in the literature. One of the most accepted theory for aging was first proposed by and is known as “free radical theory of aging,” which postulates that ROS levels increase with age, being responsible for deoxyribonucleic acid (DNA), proteins and lipids oxidative damage.

18Recent findings demonstrated the existence of BBB permeability alterations during normal aging in human hippocampus using advanced dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). It is also known that aging increases BBB susceptibility to different challenges and its permeability is less controlled allowing the entrance of neurotoxic substances, which affect brain homeostasis. In fact, BBB dysfunction is more pronounced under pathological conditions, including in mild cognitive impairment (MCI) individuals, supporting the idea that vascular alterations are early events in cognitive deficits (Figure 2). Using a multiphoton microscope, observed increased levels of O2•- in the cerebral vessels of aged PS1/APP transgenic mice, a model of AD. Also, electron and confocal microscopy studies revealed that in F344 rats aortas, the expression of the mitochondrial biogenesis factors, such as mitochondrial transcription factor A and peroxisome proliferator-activated receptor-γ coactivator-1-alpha (PGC-1α), decreases with aging. Moreover, an increase in mitochondrial H2O2 production was observed in aged arterial rat vessels leading to an activation of nuclear factor-κB (NF-κB) and induction of inflammatory phenotypic changes in aged vasculature. Furthermore, an age-dependent decline in mitochondrial complexes I, III, and IV subunits expression was observed in rat aortas, although the authors did not specify if all the subunits of the three complexes were evaluated or not. Also, were able to show an age-dependent increase in mitochondrial ROS production and mitochondrial DNA lesions causing aorta vascular dysfunction in two different mice models, the ALDH-2–/– and MnSOD–/– models, which are deficient in aldehyde dehydrogenase-2 (ALDH-2) and manganese superoxide dismutase (MnSOD), respectively. Furthermore, the in vitro use of mitochondrial complex I inhibitor rotenone, after angiotensin II-induced O2•- production, showed significant improvements in endothelial dysfunction and tolerance in human aortic endothelial cells. Furthermore, the use of preconditioning strategies as a therapeutic intervention is emerging and under extensive scrutiny. Indeed, studies from our laboratory were able to show that treating rat brain endothelial cells with non-lethal cyanide concentrations, which lead to increased mitochondrial ROS production, was able to confer protection against a posterior high glucose-mediated damage, preventing apoptotic cell death. Importantly, were able to prove that in the absence of mitochondrial DNA, using mitochondrial-depleted DNA human teratocarcinoma NT2 cells, this effect was abrogated, emphasizing mitochondrial role in endothelium protection by preconditioning. Moreover, the use of mitochondrial-targeted antioxidants such as MitoTempo, showed promising results in counteracting age-related and microgravity-induced and hyperglycemia-induced endothelial dysfunction. Indeed, Mitotempo was able to protect endothelial primary cultures of diabetic mouse model (db/db) and rat cerebral arteries against amyloid β (Aβ) toxicity and lead to improvements in spatial working memory and motor skill learning in young 5xFAD mouse models of AD. Moreover, it was also able to recover mitochondrial dysfunction observed in rat cerebral arteries of animals exposed to microgravity conditions through reduction of mitochondrial ROS levels, increased mitochondrial potential and improvement in mitochondrial respiratory chain function.

19Mitochondrial-derived oxidative stress can also activate vascular inflammation in aged carotid arteries and vessels leading to atherosclerosis through activation of endothelial NF-κB, which is responsible for the upregulation of adhesion molecules and increased monocyte adhesiveness in aged aortic arteries. Moreover, changes in Nox activity and/or expression is widely described in aged brain vessels. Nevertheless, the mechanistic role of Nox in the aging process remains obscure.

20Data from the literature also show that in cultured aged cerebromicrovascular endothelial cells (CMVECs) the mRNA expression levels of Nox2 and 4 subunits were upregulated compared with young CMVECs, a phenomenon similar to that observed in the myocardium, leading to increased levels of oxidative stress. Although further studies must be performed in order to corroborate this hypothesis, it seems that, similarly to the myocardium, in CMVECs the increased expression of Nox 2 and 4 causes oxidative stress, which activates the renin–angiotensin–aldosterone system (RAAS). This activation can occur through a ROS-induced increase in angiotensinogen (AGT), a 60-kDa α2-globulin glycoprotein that constitutes the precursor of RAAS; stimulation of renin, the enzyme responsible for the initiation of the RAAS pathway; and release or regulation of angiotensin converting enzyme (ACE) activity, crucial for the formation of angiotensin II, the major effector of RAAS. Furthermore, since Nox 4 is usually localized in mitochondria, its increased expression can also contribute to increased levels of mitochondrial ROS potentiating the aging process as well as age-related diseases. The use of Nox inhibitors showed promising results in reducing oxidative stress levels and in improving endothelial function in several disease models. Also the use of resveratrol seems to be effective in restoring cerebromicrovascular endothelial function through a downregulation of Nox-derived ROS production in aged mice.

21Despite the extensive literature stating that age-associated oxidative stress can lead to cerebrovascular dysfunction and cognitive decline, there is a significant gap in our knowledge regarding the exact mechanisms underlying these defects.

22Although the previous alterations seem to be common features in physiological aging of vessels, somewhere over the way those defects can become more pronounced triggering neurodegenerative events. Indeed, recent reports suggest a major role for the NVU in the initiation of neurodegeneration. In fact, NVU dysfunction leads to an increased BBB permeability with the subsequent entry of neurotoxic molecules into the brain disturbing its homeostasis. A decrease in the removal of neurotoxic substances from the brain, and a deficient nutrient delivery system eventually culminate in neuronal loss and synaptic dysfunction. Thus, a decline in cerebrovascular function in age-associated diseases, such as AD, is easily understandable. Indeed, it is estimated that about 60–90% of AD patients also present cerebrovascular alterations including cerebral amyloid angiopathy (CAA), microinfarcts and ischemic lesions and microvascular degeneration.

23It has been recently reported the existence of BBB and vascular alterations in several diseases such as Parkinson’s disease (PD), Huntington’s disease and cerebral small vessel disease although there is a gap concerning the mechanisms underlying those alterations and the role of vascular oxidative stress in these pathologies.

24Concerning AD, the most common form of dementia in the elderly, more information exists about the vascular alterations that occur in this disease. Several clinical and basic evidence points to the existence of a major contribution of both large artery and small vessel disease in the pathogenesis of AD. Having this into account, the two-hit vascular hypothesis of AD emerged, postulating that cerebrovascular damage is an initial insult that is self-sufficient to initiate neuronal injury and neurodegeneration, but can also promote accumulation of Aβ peptide, a neurotoxic peptide, in the brain. Thus, events that compromise vascular health can initiate a cascade of deleterious events that culminate in AD development. In fact, previous studies from our laboratory demonstrated that some risk factors for AD, such as hyperglycemia, increase the susceptibility of brain endothelial cells to Aβ peptide, a phenomenon related to ROS overproduction. Moreover, several genetic [apolipoprotein E4 (APOE4), phosphatidylinositol-binding clathrin assembly protein (PICALM), clusterin, presenilin 1, amyloid precursor protein (APP), mesenchyme homeobox gene 2 (MEOX2)] and non-genetic (hypertension, metabolic syndrome, hypercholesterolemia, atherosclerosis, alcohol and substance abuse, among others) risk factors for AD seem to be linked with alterations in vascular function supporting the idea that vascular alterations play a major role in AD pathology.

25Recent studies in wild type mice showed that, even under normal conditions, there is a different regional susceptibility of the cerebrovasculature to oxidative stress. Vessels from cortex and hippocampus, the two main areas compromised in AD, contain significantly higher levels of intracellular O2•- and increased protein levels of both Nox 2 and 4. Other studies revealed significant changes in blood vessels morphology, decreased vascular density and increased vessels tortuosity in AD brains. Moreover, the use of recent technologies such as arterial spin labeling magnetic resonance imaging (MRI), functional blood-oxygen-level-dependent (BOLD)-MRI, fluorodeoxyglucose-positron emission tomography (FDG-PET), and single-photon emission computerized tomography (SPECT), showed decreased levels of CBF in early phases of human AD progression. Recently, showed that reduced CBF changes found in triple transgenic for AD (3xTg-AD) mice preceded memory dysfunction thus suggesting that cerebrovascular dysfunction could be the primary cause of neurovascular uncoupling in AD. Those observations are supported by MRI, transcranial doppler and SPECT studies revealing different patterns of decreased CBF coincident with the brain regions where neuropathological alterations are more relevant in AD. Those alterations seem to be closely related with the levels of oxidative stress in the cerebrovasculature. Studies from our laboratory showed that 3xTg-AD mice present an increased BBB permeability, in cortex and hippocampus, these alterations being correlated with decreased aconitase activity, an enzyme whose activity is inhibited by O2•-. The increased levels of ROS appeared to be the result of impaired activity of mitochondrial enzymatic complexes I–III (Figure 2). Moreover, and similarly to our observations in mitochondria isolated from whole brain of 3xTg-AD mice, Golgi silver impregnation studies using multiple samples from the hippocampus and cortices of AD patients showed that brain endothelial cells present enlarged mitochondria, disruption of the mitochondrial cristae and reduced abundance.

26An interesting fact is that even though mitochondrial morphology is very similar in male and female rodent cerebral arteries, major differences can be observed concerning mitochondrial protein mass, respiration, and function, which is in accordance with sex/gender differences observed in several brain-related studies including the susceptibility to develop AD.

27Mitochondrial ROS overproduction also increases Aβ deposition in vessels walls, which is a common feature in many cases of AD (Figure 2). Indeed, 34–35% of human AD cases present CAA, predominantly in the occipital lobe. Aβ deposition in vessels walls seems to initiate a vicious cycle, Aβ increases ROS levels, which in turn potentiate Aβ deposition (Figure 2). There is also evidence showing that Aβ can induce Nox activation and ROS production, which are responsible for the activation of multiple mechanisms involved in vascular dysfunction and decreased levels of tight junction proteins mRNA. However, Nox driven ROS production does not seem to affect Aβ production in Tg2576 mice, a mouse model of AD, lacking the key Nox 2 subunit.

28Endothelial ROS overproduction upregulates the endothelial production and release of endothelin-1, a well know vasoconstrictor that appears to be increased in AD. Likewise, ROS overproduction seems to be responsible for reduced •NO bioavailability mainly by its fast reaction with O2•- generating ONOO- and for a decreased activity of potassium channels, two major factors required for endothelial-mediated dilations in brain vasculature. Moreover, Aβ-induced nitrosative stress in endothelial cells is also responsible for DNA damage, resulting in poly (ADP-ribose) polymerase (PARP) activation, ultimately leading to a large increase in intracellular Ca2+ through transient receptor potential melastatin-2 channels activation (Figure 2).

29Reactive oxygen species overproduction also interferes with HIF-1α reducing its expression and activity which will restrain the stimulus to promote angiogenesis and new vessels formation leading to a vicious cycle of impaired capillary perfusion, hypoxia and oxidative stress.

30Recognizing vascular ROS production as one of the main causes of BBB dysfunction in aging and dementia, particularly in AD, raises the prospect that pharmacotherapy targeting the major ROS producers, namely mitochondria and/or Nox, might open a new venue to stop or, at least, slow dementia progression. Indeed, relying on the overwhelming evidence that mitochondrial dysfunction could be in the genesis of neurovascular dysfunction, several molecules with potential therapeutic effects were designed to target ROS, boost mitochondrial function and decrease free radical production and oxidative damage in an attempt to improve neurovascular health. Some of those molecules ameliorated the cognitive performance of mouse models of cerebrovascular disease. It was observed that the steroid hormone 17β-estradiol (estrogen) modulates the expression of several transcriptional regulators causing a decrease in PGC-1α, and an increase in PGC-1β, PGC-1-related coactivator (PRC), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), which protect cerebral blood vessels (Table 1). These protective effects were due to increased mitochondrial biogenesis and antioxidant enzymes. However, the use of antioxidant therapy in cerebrovascular disease still needs validation. Indeed, several promising approaches failed in clinical trials, opening an intense debate about the reasons for the failure of those molecules in humans. Merely as an example, the use of tempol showed promising results in improving vasodilator responses of cerebral arterioles in aged APP transgenic mice with CAA but so far there is no evidence of its efficacy in clinical trials. Furthermore, two major concerns arise from the use of antioxidants as therapeutic approaches: (1) physiological ROS concentrations are important in normal cell functioning and the use of antioxidants may interfere with ROS signaling and (2) the concentrations of antioxidants revealed to be suboptimal doses in certain situations. Indeed, it is known now that supra-physiological concentrations are required to compete with the constant reaction that usually occurs between O2•- and •NO. Large part of the studies failed to prove that antioxidants reach the vasculature at appropriate/therapeutic concentrations.

31To overcome the failure of the traditional antioxidants, researchers are developing new specific-targeted antioxidants. However, evidence about the specific-target antioxidants effects in endothelial cells is scarce. Moreover, mitochondria-targeted antioxidants decrease the dose required and limit toxic side effects rendering them a promising therapeutic approach. It was observed that MitoTempo, a mitochondria-targeted antioxidant, was able to counteract Aβ-induced damage under hyperglycemic conditions in rat and mouse primary cultures of endothelial cells. Additionally, it was shown that MitoTempo was able to improve mitochondrial function and coronary collateral growth after ischemia/reperfusion in Zucker fatty rats (Table 1). Recent studies also showed that MitoTempo was able to decrease vascular O2•- and increase vascular •NO production improving endothelial-dependent relaxation in angiotensin II-induced hypertensive C57Bl/6 mice. It was also observed that MitoTempo improves endothelial function and reduces mitochondrial O2•- levels in subcutaneous arterioles isolated from type 2 diabetic patients (Table 1). Moreover, MitoQ, another mitochondria-targeted antioxidant was able to reduce oxidative stress, without causing adverse effects, on wild-type mice and animal models of neurodegenerative diseases such as AD and PD. MitoQ was able to improve age-related arterial endothelial dysfunction in C57BL/6 mice (Table 1). Furthermore, MitoQ has been shown to prevent cocaine-induced cardiac dysfunction in Wistar rats. However, the use of mitochondrial-directed drugs should take in account two major limitations: first, the lack of organ-specificity, leading to a greater accumulation in mitochondria-rich tissues; and second the typically used chemicals tend to accumulate in the matrix and the matrix-facing surface of the inner mitochondrial membrane, over other important mitochondrial compartments. The majority of mitochondria-targeted compounds were already able to identify and get ahead of these limiting factors in human clinical trials, defining the amount of the compound that can be administered safely. For example, MitoQ, was already developed as a pharmaceutical by Antipodean Pharmaceuticals Inc. and clinical trials are running, namely in type 2 diabetic patients, where MitoQ treatment was able to decrease ROS levels and significantly reduced the adhesion of leukocytes to endothelial cells in type 2 diabetic individuals.

32Likewise, approaches designed to target Nox-derived ROS overproduction are also under intense scrutiny. Indeed, the genetic deletion or inhibition of Nox2 seems effective in improving cerebral vessels functioning in aged APP mice. Apocynin and diphenyleniodonium are widely used to ameliorate Nox-related cerebrovascular dysfunction, however, both compounds present non-specific effects and cannot be used in the clinic (Table 1). In this line, several attempts are being made to synthesize new Nox inhibitors including fulvene-5, triphenylmethane derivatives, grindelic acid, and ML171 (Table 1). However, results are scarce and further studies are needed to demonstrate that those compounds are potential therapeutics for the treatment of neurodegenerative conditions characterized by oxidative stress-associated vascular alterations.

33In sum, despite the existing information about the role of cerebrovascular oxidative stress in neurodegenerative conditions, a long way is still ahead to clarify the mechanisms of age-associated vascular damage and subsequent neurodegenerative conditions. New information is crucial for the design of more effective therapeutic strategies.

34CC performed literature search and wrote the paper. PM provided a critical revision of the paper.

35The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a shared affiliation, though no other collaboration, with the authors.