Dysfunctional Vascular Endothelium as a Driver of Atherosclerosis: Emerging Insights Into Pathogenesis and Treatment

1Atherosclerosis is a chronic inflammatory process in which the accumulation of cholesterol-laden plaque restricts blood flow within the arterial vasculature. The occlusion of arteries by luminal encroachment of expanding plaque or emboli from plaque rupture underlies a spectrum of cardiovascular diseases (CVDs) including myocardial infarction, ischemic cardiomyopathy, stroke, and peripheral vascular disease. Although CVD has remained a leading cause of morbidity and mortality in high-income countries, an epidemiological shift has occurred in recent decades, where improvements in vaccination and treatment of infectious diseases have led to a notable increase in CVD prevalence within low- and middle-income nations. The prominent and evolving burden of atherosclerotic CVD has stimulated continued interest in the identification of cellular mechanisms that govern its pathogenesis, which may aid in the discovery of novel biomarkers and therapeutic targets for CVD prevention, detection, and treatment.

2Endothelial cells (ECs) comprise the vascular endothelium, the inner lining of all blood vessels, which forms the interface between systemic circulation and underlying tissues. The quiescent or non-proliferating endothelium, once considered to be dormant outside the settings of vascular development or disease, is now understood to play an active role in maintaining vascular homeostasis by receiving and generating diverse biochemical (i.e., autocrine, paracrine, and endocrine) and mechanical signals. The systemic functions of the endothelium are numerous, and include the provision of oxygen and nutrients to tissues, regulation of vascular tone and permeability, maintenance of hemostasis and coagulation, induction of angiogenesis, and coordination of the inflammatory response. These essential processes are modulated by rich crosstalk between ECs and other vascular cell populations, including smooth muscle cells (SMCs), monocytes, and macrophages, which contribute to normal vascular function in physiological settings. Likewise, dysregulated communication between ECs and other vascular cell types is associated with vascular dysfunction and pathological remodeling in CVDs such as hypertension, atherosclerosis, and aneurysm.

3The pathophysiology of atherosclerosis begins with the perturbed endothelium and is mediated by a cascade of intra- and intercellular signaling events that shape the behaviour of cells within the vasculature. Vascular ECs facilitate the active transport of low-density lipoprotein (LDL) to the subendothelial space through transcytosis pathways, and LDL accumulation initiates a vascular inflammatory response. Early in atherosclerosis, the endothelium transitions from a quiescent to an activated state in response to proatherogenic stimuli, including oxidized LDL (oxLDL), proinflammatory cytokines, and disturbed flow. In turn, the activated endothelium plays a critical role in the recruitment of inflammatory cells including T lymphocytes, neutrophils, and monocytes to the arterial intima, the first of which induces the adaptive immune response, and the latter of which gives rise to intimal macrophages. Subsequent lipid engulfment by macrophages produces foam cells, which undergo necrosis and apoptosis to form the lipid core of the progressing atherosclerotic lesion. Vascular SMCs that comprise the medial layer of arteries migrate to the intima, form fibrous tissue through the production of collagen and elastin, and can also differentiate into macrophage-like foam cells in the developing plaque. In this proatherogenic environment, communication between the endothelium and other vascular cell populations stimulates the release of proinflammatory signals, which augment local inflammation and contribute to sustained plaque progression. Intimal thickening occurs during plaque development and creates a hypoxic intraplaque environment, which stimulates angiogenesis of the vasa vasorum – adventitial blood vessels that supply larger arteries – and promotes neovascularization into the vascular wall. Progressive thinning of the fibrous cap results from an inflammation-associated decrease in collagen synthesis and increase in degradation, which in combination with erosion of the endothelium, contributes to plaque rupture, thrombosis, and obstruction of the affected vessel. Although beyond the scope of this review, the role of endothelial dysfunction in plaque rupture, as well as the contribution to pathogenesis and the therapeutic potential of the vasa vasorum in treating atherosclerosis, has been previously discussed in detail. The pathophysiological relevance of the arterial endothelium has been similarly outlined, however, the specific and modifiable intra- and intercellular mechanisms that mediate endothelial dysfunction and atherogenesis remain to be elucidated.

4In this review, we present emerging studies that implicate the vascular endothelium as a driver of atherosclerotic CVD by directing site-specificity of plaque formation and governing plaque progression through intracellular processes. Furthermore, we highlight recent studies that describe intercellular communication between ECs and key vascular cell types involved in atherogenesis (Figure 1), and discuss how ECs participate in resolution biology during plaque development. Finally, we outline outstanding questions and future directions for atherosclerosis treatments, including epigenetic interventions and the targeted delivery of therapeutics to the activated endothelium to resolve vascular inflammation and limit atherosclerotic plaque progression.

5The vascular endothelium is subjected to both tangential (i.e., shear stress) and circumferential forces (i.e., pulsatile stretch) that result from circulating blood flow. ECs at this interface convert mechanical stimuli to biochemical signals through mechanotransduction, which modulates key cellular processes in response to fluctuations in the vascular environment, including proliferation and turnover. The development of early atherosclerotic lesions is characteristically localized to regions of the vasculature where laminar blood flow is disturbed (e.g., arterial branch points and lesser curvatures of vessels), which have previously been associated with decreased expression of endothelial nitric oxide synthase (eNOS) and increased nuclear factor kappa B (NF-κB) activation.

6In the setting of undisturbed laminar flow, nitric oxide (NO) is produced by elevated levels of eNOS in ECs and diffuses across cell membranes to modulate the activity of vascular cells, including SMCs and leukocytes. NO-mediated activation of soluble guanylate cyclase and S-nitrosylation has been observed to inhibit SMC proliferation, suppress EC inflammation, and regulate vascular tone, blood flow, and oxygen delivery to tissues. Hydrogen sulfide, a gaseous signaling molecule and upstream regulator of NO, is similarly produced under laminar flow conditions and has a critical role in reducing vascular inflammation, promoting antioxidative activity, and limiting the formation of foam cells. Laminar flow conditions have also been found to promote EC alignment, and foster a reducing environment that limits oxidative stress, tumor necrosis factor α- and signal transducer and activator of transcription 3-induced inflammation, and EC turnover via apoptosis. The transcription factor Kruppel-like factor 2 (KLF2) has emerged as a critical transcriptional mediator of vascular homeostasis that is induced under laminar shear stress and increases eNOS expression and represses adhesion proteins such as vascular cell adhesion molecule 1 (VCAM-1) and E-selectin, which are induced by NF-κB in regions of disturbed flow. In contrast to regions of laminar flow, altered hemodynamics in atheroprone regions has been associated with increased oxidative stress, reorganization of cytoskeletal and cell-cell junction proteins, and enhanced senescence and turnover in ECs. Curiously, hydrogen sulfide has been observed to impair dilation of coronary arteries by reducing NO production under disturbed flow, although hydrogen sulfide administration has also been shown to inhibit leukocyte adhesion to the endothelium in regions of shear stress by upregulating Akt/eNOS signaling.

7Recent studies continue to elucidate a plethora of EC regulatory pathways that induce a proinflammatory and proatherogenic phenotype in response to disturbed flow (Figure 1). The mechanosensitive cation channel Piezo1, though essential for coordinating vascular morphogenesis under physiological shear stress in embryogenesis and adulthood, has been previously linked to altered flow-induced inflammation and leukocyte recruitment to the endothelium. Indeed, pathological activation of Piezo1 has been associated with induction of downstream integrin α5 and NF-κB pathways, which contributes to endothelial inflammation and the progression of murine atherosclerosis, and elevation of intracellular calcium, which results in actin disruption and increased monocyte adhesion in cultured ECs. Likewise, integrin α5β1-associated phosphorylation of yes-associated protein (YAP) by c-Abl kinase, the coupling of integrin α5 to annexin A2, and the guidance receptor plexin D1, upstream of EC integrins, have been identified as novel mechanisms for endothelial activation under oscillatory shear stress. In contrast, several EC processes confer in vivo protection against atherosclerosis under disturbed flow, including inhibition of integrin α5 by cartilage oligomeric matrix protein, increased glycolysis by protein kinase AMP-activated catalytic subunit alpha 1, decreased lipid uptake by the transcription factor inhibitor of DNA binding 1, and increased EC alignment by Rho GTPase activating protein 18. Many of these preclinical findings hold promise for the development of atherosclerosis treatments through pharmacological modulation of the endothelium (e.g., mitigating atherogenic responses or promoting atheroprotective functions in ECs). However, further investigation is required in vitro to determine the generalizability of endothelial responses between vascular cell models (e.g., shared and distinct behaviors of different EC lines under varying flow conditions), as well as in vivo to identify translatability in preclinical models and long-term safety and efficacy in individuals with CVD.

8Emerging evidence has also implicated disturbed blood flow in the dysregulation of EC proliferation and turnover during early atherosclerosis. Under physiological conditions, ECs that become senescent or apoptotic are replaced by the replication of neighboring cells, and more significant disruptions to the endothelium (e.g., during injury) are mitigated by circulating endothelial progenitor cells. Atherosclerosis is characterized by cellular processes that promote EC turnover and induce proliferation of the activated endothelium. However, whether this proliferation is protective or detrimental has not been fully resolved. The mechanosensitive transcription factor zinc finger and BTB domain containing 46, for instance, inhibits proliferation in quiescent ECs and is downregulated by disturbed flow in vitro. Hemodynamic signaling also impacts epigenetic pathways, and we have reviewed the impact of epigenetics on atherosclerosis elsewhere. For example, altered flow has been shown to induce DNA methyltransferase 1-mediated hypermethylation of the endothelium and its subsequent inhibition limits atherosclerosis via the cell cycle regulator cyclin A. Furthermore, laminar flow-induced autophagy and expression of the deacetylase sirtuin 1 (SIRT1) together inhibit Hippo/YAP signaling to attenuate atherosclerotic plaque formation, and hypermethylation of eNOS promoter elements under chronic disturbed flow has been shown to contribute to repressed eNOS expression in wildtype mice.

9Flow-sensitive microRNAs have also been identified as key modulators of endothelial proliferation and turnover in atherosclerosis, including miR-126-5p, which has been shown to contribute to proliferative reserve in ECs and prevents plaque development through upregulation of Notch signaling. Curiously, increased expression of miR-126 has been found in atheroprone regions of the endothelium. Furthermore, physiological flow conditions have also been observed to promote antiproliferative microRNA activity within the endothelium, whereby KLF2-induced miR-23b represses cyclin H to reduce activity of the cyclin-dependent kinase–activating kinase complex and limit cell cycle progression. The regulatory interplay of antagonistic, flow-sensitive microRNAs may serve as a mechanism for fine-tuning EC proliferation and warrants further characterization in both quiescent and activated endothelial states.

10A robust characterization of the transcriptional and epigenetic regulatory elements that contribute to endothelial dysfunction has remained an important challenge for atherosclerosis research and has been recently addressed with next-generation approaches for global EC profiling. Of note, the integration of chromatin immunoprecipitation, chromatin accessibility, and RNA sequencing has enabled identification of diverse DNA regulatory elements associated with disturbed flow and proinflammatory activation (e.g., NF-κB and hypoxia inducible factor 1α, as well as ETS, zinc finger, and activator protein 1 transcription factor families). Moreover, combined single-cell RNA sequencing and genome-wide chromatin accessibility assays have been used to profile the genome- and epigenome-wide changes associated with proatherogenic ECs under oscillatory shear stress. Likewise, these technologies have allowed for characterization of EC heterogeneity in human atherosclerotic plaque and identification of coronary artery disease-associated genetic variants in the open chromatin regions of activated ECs. The future integration of global EC profiling with functional assays will aid in the validation of putative modulators of atherogenesis, which may serve as prospective therapeutic targets for CVD treatment. Notably, real-time monitoring of the vascular endothelium has been enabled with organ-on-a-chip technologies, allowing for specific and quantifiable testing to elucidate the impact of environmental stimuli on endothelial function. Nevertheless, the complex cellular milieu of the atherosclerotic plaque remains difficult to fully model.

11Metabolic pathways prominently contribute to EC phenotypes in health and disease. Despite direct exposure to oxygen in the blood, ECs do not utilize oxidative phosphorylation as a primary means of energy production, perhaps because this might enhance oxidative stress and would hamper angiogenesis in hypoxic environments. Instead, glycolysis serves as the primary method of energy delivery for ECs, in which 75–85% of ATP is generated via hexokinase 2-mediated phosphorylation of glucose to glucose-6-phosphate and conversion to lactate. Fatty acid oxidation, used as a secondary source of energy by ECs, is modulated by carnitine palmitoyltransferase 1A-mediated shuttling of fatty acids to the mitochondria and ATP production via adenosine monophosphate activated protein kinase signaling. Alternatively, fatty acids can be generated within ECs via fatty acid synthase, which has important functions for EC migration, permeability, and eNOS-mediated activity. Moreover, the proliferative and vasodilatory capacities of the endothelium can also be regulated by the metabolism of amino acids, in which inhibition of glutaminase-mediated conversion of glutamine to α-ketoglutarate represses angiogenesis, and the eNOS-induced conversion of arginine to NO controls vascular tone.

12Altered EC metabolism has been identified as both a consequence of, and a contributor to, endothelial dysfunction in atherosclerosis. In the quiescent endothelium, laminar shear stress reduces glucose uptake and glycolytic and mitochondrial activity in ECs via KLF2. Conversely, in the diabetic and proatherogenic environment, elevated levels of circulating glucose induce the production of reactive oxygen species, DNA damage, and the accumulation of advanced glycation end products, which contribute to endothelial dysfunction via NF-κB signaling and increased vascular permeability. Disturbed flow similarly induces NF-κB and hypoxia inducible factor 1α expression, EC proliferation, and inflammation via upregulation of glycolytic enzymes, and can also promote EC activation and atherosclerosis through YAP/tafazzin (TAZ) signaling. Importantly, YAP/TAZ signaling has been shown to induce EC glycolysis, and glycolytic activity can in turn upregulate the YAP/TAZ pathway, which has been hypothesized to result in a cyclical and sustained pro-inflammatory response in the perturbed endothelium. Emerging preclinical studies propose altered EC metabolism as a therapeutic target for mitigating the development of atherosclerosis, including coenzyme Q10-mediated activation of the AMP-activated protein kinase-YAP-optic atrophy protein 1 pathway to promote mitochondrial function and energy metabolism, and inhibition of the glycolytic regulator 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 to improve plaque stability in atheroprone mice. Although repressing glycolysis in the activated endothelium remains a promising strategy for treating patients with atherosclerotic CVD, consideration must be given to the complex interactions between genetics and environment (e.g., age, diet, diabetes, and cardiovascular fitness) that shape individual metabolic profiles to allow for patient-tailored therapies.

13In its quiescent state, the vascular endothelium forms a semipermeable barrier between luminal and abluminal environments that allows for selective bidirectional movement of molecules via EC cell-cell junctions, vesicle-mediated transport within ECs, and diffusion between ECs or across endothelial gaps. In pioneering studies, LDL and other serum macromolecules were shown to enter the vessel wall using a paracellular route in permeable regions of the vasculature. However, more recently, ECs have been implicated in the active transcytosis of LDL via caveolae, scavenger receptor B1, activin receptor-like kinase 1, LDL receptor, and high mobility group box protein 1, which contribute to the proinflammatory accumulation of LDL within the subendothelial space and promote atherogenesis. Loss of barrier function also potentiates atherosclerosis by facilitating leukocyte extravasation into the vessel wall through paracellular diapedesis from the vascular lumen, and may promote inflammatory infiltration through the vasa vasorum, contributing to subsequent plaque instability. Vascular insults including atherosclerosis, ischemia, and trauma are characterized by the accumulation of pathological proinflammatory mediators, which can induce acute (e.g., due to vascular injury) or chronic disruptions in endothelial permeability (e.g., due to plaque progression). Numerous mechanisms that underlie endothelial barrier disruption have been characterized, including protein kinase C-induced phosphorylation of cell junction proteins, which promotes actin reorganization and increased paracellular flux, and stimulation of myosin light chain kinase by inflammatory factors, which promotes EC retraction via actin-myosin network dynamics. Mediators including histamine, thrombin, and the proinflammatory cytokines interleukin (IL) 1 beta (IL-1β) and tumor necrosis factor α also increase permeability via modulation of tight junctions (e.g., zonula occludens 1 and occludins) and adhesion complexes. Furthermore, prolonged inflammation and oxidative stress can act to disrupt adherens junctions [e.g., vascular endothelial (VE)-cadherin] and gap junctions (e.g., connexins) via decreased NO.

14Emerging studies continue to uncover novel causal roles for established atherosclerosis mediators in the disruption of endothelial permeability. The mechanosensitive channel Piezo1, previously discussed as a coordinator of vascular structure and activator of integrin α5/NF-κB pathways, also promotes VE-cadherin degradation and increased vascular permeability under altered hemodynamics. Furthermore, the NOD like receptor family pyrin domain containing 3 (NLRP3) inflammasome, discussed in greater detail below as a therapeutic target for inflammation resolution in atherosclerosis, induces a loss of barrier in the diabetic vascular environment. Recent findings have also implicated the chemokine C–C motif ligand 8 in promoting atherosclerosis through enhanced permeability via NADPH oxidase 2 and reactive oxygen species, while the inhibition of insulin-like growth factor-1 signaling has been associated with disrupted endothelial barrier function in vitro and elevated atherosclerotic burden in mice. The multifaceted roles of these atherogenic factors offer potential advantages for prospective therapies, which may simultaneously target multiple aspects of the atherosclerotic disease processes to limit plaque development (e.g., combined anti-inflammatory and pro-barrier effects of pharmacological agents that inhibit Piezo1 or NLRP3).

15The endothelium is an essential regulator of vascular homeostasis, defined as the balance of vascular injury and repair, and dynamic changes in endothelial phenotype can both potentiate and limit atherogenesis and associated complications. In the setting of chronic inflammatory diseases, including atherosclerosis, ECs undergo complete or partial endothelial-to-mesenchymal transition (EndMT), during which they lose endothelial properties and gain mesenchymal cell characteristics (e.g., extracellular matrix production and contractile function). EndMT is driven by proatherogenic stimuli including inflammation and disturbed flow and early activation of transforming growth factor beta signaling, and has garnered interest as an important pathophysiological mechanism for atherosclerotic CVD. Indeed, previous work has linked EndMT to neointimal hyperplasia, increased leukocyte migration, lipid uptake by lesional macrophages, and oxidative stress in the developing plaque (Figure 1). Moreover, statin therapy, histone deacetylase inhibitors, and microRNA inhibition have recently been investigated as potential strategies for limiting EndMT, the latter of which was observed to reduce plaque formation in atherosclerotic mice.

16Future study is warranted to address several outstanding research areas in EndMT and atherosclerosis, including the characterization of EndMT as a discrete versus continuous process, as well as the potential beneficial versus detrimental role of EndMT in plaque vulnerability. First, the proatherogenic endothelium has recently been shown to exist in a “metastable or partial” state of EndMT, where perturbed ECs perform both endothelial and mesenchymal functions. The metastable nature of EndMT may be further elucidated with next-generation technologies that profile the atherosclerotic plaque and neighboring regions at the single-cell level, as recently used in studies of disturbed flow and endothelial reprogramming and diabetic atherogenesis, as well as through the incorporation of computational models that predict endothelial activation and EndMT in various genetic and environmental conditions. Second, although augmentation of EndMT has previously been associated with increased plaque progression, emerging studies have highlighted a potential protective role of EndMT by maintaining plaque stability in the absence of SMC-derived myofibroblast-like cells. Evidently, a more nuanced understanding of EndMT in the context of plaque development and rupture is required before therapeutic inhibition of EndMT is considered for the treatment of atherosclerotic CVD.

17Vascular cell populations including ECs, monocytes, macrophages, and SMCs have well-established roles in driving atherosclerosis. ECs orchestrate the cellular interactions between these cell populations through the local expression of adhesion proteins and secretion of signaling molecules. The intercellular processes that mediate this pathophysiology have been a focus of previous studies, which have characterized EC-EC and EC-leukocyte communication via adherens junctions (e.g., VE-cadherin), tight junctions (e.g., junctional adhesion molecules), and other adhesion proteins including occludin, claudins, and platelet endothelial cell adhesion molecule. Furthermore, a diverse group of EC-derived cytokines, chemokines, and other molecules (e.g., NO and endothelin-1) regulate vascular function by controlling proatherogenic processes such as leukocyte activation and SMC proliferation. Inflammatory cell recruitment to the activated endothelium is coordinated by the expression of EC adhesion proteins such as E-selectin, VCAM-1, and intercellular adhesion molecule-1 (ICAM-1), which facilitate leukocyte rolling and extravasation into the intimal layer, as well as the chemotactic factor monocyte chemoattractant protein-1, which attracts circulating monocytes, and the mitogen macrophage colony-stimulating factor, which stimulates monocyte proliferation and differentiation to intimal macrophages. Additionally, the proinflammatory polarization of macrophages has been associated with EndMT and sprouting angiogenesis, the latter of which supports plaque progression through increased supply of oxygen and nutrients. The developing atherosclerotic lesion is further characterized by intimal hyperplasia and neointimal formation, which are mediated by decreased production of EC-derived NO and a resultant increase in SMC proliferation, in conjunction with EndMT.

18In recent years, vascular cell-cell communication mediated by the secretion of extracellular vesicles (i.e., EVs; nano-sized packages of proteins, mRNAs, noncoding RNAs, and lipids) has emerged as an important research area for the pathogenesis and treatment of atherosclerosis. Three broad categories of EVs – exosomes (30–150 nm in diameter), microparticles (100 nm–1 μm in diameter), and apoptotic bodies (1–5 μm in diameter) – have been shown to modulate vascular function in atherogenesis via intercellular signaling among ECs, as well as between ECs and other immune and non-immune cell types (Figure 1). Within the vascular endothelium, EC-derived EVs with distinct microRNA content have been observed to both induce and inhibit angiogenesis (;, 2) via paracrine regulation of nearby ECs, whereby EVs fuse to recipient cells, and the release of EV-derived microRNAs allows for silencing of complementary mRNA targets. Moreover, the uptake of EC-secreted microparticles by neighboring ECs was shown to promote ICAM-1 expression and monocyte adhesion via miR-222 in diabetic conditions, and EV-derived apoptotic bodies containing miR-126 were found to reduce macrophage content in plaque and limit atherosclerotic burden. The specific role of the proatherogenic environment in governing EV loading and function remains of interest for future study, and characterization of this environment has begun with exposure of donor ECs to proatherogenic stimuli (i.e., oxLDL and IL-6), which were observed to decrease thrombospondin 1 and increase angiogenesis in recipient ECs via EV-derived miR-92a-3p.

19Beyond the endothelium, EC-EVs regulate both immune and non-immune vascular cell populations involved in the development of atherosclerosis and circulating EVs may have systemic effects at distant sites. Exosomal microRNAs mediate crosstalk between ECs and macrophages by suppressing (e.g., miR-10a) or promoting monocyte activation (e.g., miR-155), reducing macrophage infiltration, and delaying plaque progression (e.g., miR-126, miR-210, and miR-216). In turn, monocyte- and macrophage-derived EVs have been shown to induce EC apoptosis and promote murine atherosclerosis through elevated blood lipids, oxidative stress, and inflammation, as well as by reducing proliferation and angiogenesis in human coronary artery ECs via miR-503-5p. Reciprocal communication between cells of the vasculature has been similarly observed in ECs and SMCs, where EC-secreted EVs induce both atheroprone and atheroprotective SMC phenotypes, while SMC-derived EVs govern endothelial migration under stimulation by platelet-derived growth factor and can increase endothelial permeability and potentiate atherosclerosis via miR-155.

20EC-EVs that are secreted into systemic circulation may serve as biomarkers for CVD and have the potential to govern cell behaviour at distant areas of the vasculature, although definitive evidence for this regulation is lacking. Preliminary animal studies have demonstrated that microRNAs secreted by circulating blood cells, and presumably contained within EVs, can modulate SMC activity in atherogenesis and have proapoptotic and antiproliferative effects on ECs. Further investigation is warranted to determine whether circulating EVs are present in sufficient quantities to elicit systemic effects in patients and if their regulatory functions extend to other CVDs (e.g., cardiac fibrosis and ischemic heart disease), as well as to identify the precise mechanisms by which circulating EVs promote vascular dysfunction in recipient cells. Notably, future study is also required to determine whether EVs undergo transcytosis or pass through intercellular or intracellular gaps to the subendothelial space and elicit direct effects on medial cells, or are endocytosed by ECs, which then serve as indirect mediators of altered vascular function. The continued integration of EV profiling in patients with atherosclerosis (i.e., local EVs in plaque; circulating EVs in plasma) with experimental models of EV activity will shed light on the causal role of EVs and EV-contents in the setting of atherosclerotic CVD. Such models will require the transition from in vitro co-culture towards in vivo tracking approaches to allow for a robust, physiological characterization of EV activity, which will be made possible with advances in the visualization of EV release and uptake. With respect to the diagnostic potential of vascular EVs, the application of machine learning strategies to predict CVD from circulating EV biomarkers has garnered recent interest and will likely play an important role in the development of precision cardiovascular medicine over the coming decades.

21Present therapeutic strategies to limit atherosclerosis and plaque rupture are broadly categorized as those which reduce atherosclerotic risk (e.g., lipid-lowering and antihypertensive agents) and those which prevent associated complications (e.g., antithrombotic agents). Lipid-lowering therapies, including 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (i.e., statins) and proprotein convertase subtilisin/kexin type 9 inhibitors, reduce circulating levels of LDL-cholesterol and thus oxLDL in the vessel wall, and decrease the incidence of severe events in CVD, although these agents are not sufficient to prevent plaque formation. Moreover, a spectrum of anti-atherogenic effects of statins on the endothelium has been previously discussed in detail, and includes reduced inflammation through NF-κB blockade, inhibition of EC apoptosis via Janus kinase 2/signal transducer and activator of transcription 3 signaling, protection against EndMT via Kruppel-like factor 4/miR-483, epigenetic modulation of ECs through histone modification, and increased NO production via hydrogen sulfide and eNOS. Notably, the statin-mediated mechanisms that contribute to elevated eNOS activity include increased eNOS transcription via KLF2, improved eNOS mRNA stability via polyadenylation, and increased eNOS phosphorylation via phosphatidylinositol 3-kinase/Akt signaling. Other anti-atherogenic treatments have also been observed to mitigate endothelial dysfunction by increasing NO production, such as antihypertensive agents (e.g., angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers), antihyperglycemic drugs (e.g., insulin), and antioxidants (e.g., streptozotocin). In experimental settings, treatments for atherosclerosis have largely focused on antagonizing broad inflammatory pathways (e.g., IL-1β and 5-lipoxygenase). Therapeutic agents that specifically target the perturbed endothelium to resolve inflammation and limit atherosclerosis, however, have not yet been fully realized in clinical studies.

22The NLRP3 inflammasome is now recognized as a key player in the coordination of vascular inflammation and onset of atherogenesis. NLRP3-mediated induction of atherosclerosis has been associated with a host of causal factors including hypoxia, cholesterol crystals and oxLDL, and disturbed flow. Activation of NLRP3 subsequently leads to the maturation of proinflammatory cytokines (e.g., IL-1β and IL-18), increases the migration and lipid loading of macrophages, and promotes pyroptosis, a proinflammatory form of programmed cell death that contributes to the release of additional inflammatory mediators. Therapeutic agents that directly inhibit the NLRP3 inflammasome, including the natural compound arglabin, colchicine, and the small molecule inhibitor MCC950, have been shown to ameliorate endothelial inflammation and atherosclerosis in preclinical studies. Moreover, adjunct colchicine therapy has entered clinical trials for repressing vascular inflammation in acute coronary syndrome and coronary artery disease. Previous trials have employed several outcomes related to improved cardiovascular health, including CVD endpoints, serum inflammatory markers, and proxy measures for endothelial dysfunction including flow-mediated vasodilation and exercise-induced coronary blood flow. Although the latter two of these measures remain the gold standard for clinical assessment of the perturbed endothelium, they rely on endothelial responsiveness to vasodilatory or vasoconstrictive manipulation and do not adequately capture changes in EC phenotype during atherogenesis (e.g., permeability, metabolism, EndMT, or intercellular communication). Emerging studies have begun to address this limitation by applying machine learning techniques to predict CVD events from clinical data, CT imaging, and circulating biomarkers, including matrix metalloproteinase 9 and polymeric immunoglobulin receptor, very low-density lipoprotein and leucine, and serum EVs. Alongside functional assessment of the endothelium, robust models for CVD prediction have the potential to identify subclinical disease, aid in risk stratification, and inform treatment and prevention strategies.

23Recent studies have also highlighted the potential of endogenous specialized proresolving mediators (SPMs) as anti-inflammatory therapies for limiting endothelial inflammation in CVD. Resolvin D1 (RvD1) belongs to a family of SPMs formed from metabolic processing of polyunsaturated docosahexaenoic acid and eicosapentaenoic acid, and is synthesized by ECs under proinflammatory conditions including stimulation with oxLDL. Treatment of ECs with RvD1 has been observed to repress lipopolysaccharide- and cholesterol crystal-stimulated EC-monocyte interactions in vitro by downregulating NFκB-ICAM-1/VCAM-1 signaling, and attenuates leukocyte trafficking to the endothelium in mice fed a cholesterol rich diet. Beyond the resolvin family, the SPMs maresin 1 and lipoxin A4 have likewise been observed to disrupt leukocyte adhesion to activated ECs, suggesting that endogenous production or exogenous administration of diverse SPM metabolites may have therapeutic benefits for limiting endothelial activation in the setting of atherosclerosis.

24Notably, the potential therapeutic impact of promoting inflammation resolution through efferocytosis (i.e., the clearing of apoptotic cells) is being established with preclinical studies, wherein atherosclerotic plaque progression has been attenuated by proefferocytic cluster of differentiation 47 (CD47)-blocking therapies or activation of the macrophage cell surface receptor MER proto-oncogene, tyrosine kinase. While these effects appear to be mediated by lesional macrophages, ECs could facilitate the transport of inflammation resolving agents to cells within the developing plaque. Indeed, a proefferocytic and anti-atherosclerotic nanoparticle therapy has been previously hypothesized to function by uptake within circulating monocytes or delivery to macrophages within the intima, the latter of which would require the monocyte-independent transport of nanoparticles across the endothelium. In this role, ECs may contribute to therapeutic delivery by modulating vascular permeability and trafficking nanoparticles to macrophages via EC-secreted EVs. Clinically, the promise of inflammation resolving strategies has been highlighted in a subset of patients enrolled in a phase 1b-2 trial of the proefferocytic anti-CD47 antibody magrolimab for treating B-cell non-Hodgkin’s lymphoma, where vascular inflammation was significantly reduced in the carotid arteries of patients with CVD after 9 weeks of treatment.

25Epigenetic interventions allow for broad targeting of the diverse mechanisms underlying endothelial dysfunction and have shown recent promise as anti-atherosclerotic therapies. The methyltransferase enhancer of zeste homologue 2 (EZH2) and histone deacetylase SIRT1 respectively induce and limit endothelial dysfunction in murine models of CVD and represent novel avenues for epigenetic treatment. EZH2 antagonism and SIRT1 agonism similarly increase NO production and decrease EC activation and EndMT in experimental models of CVD (Figure 2). More recently, natural antioxidants including epigallocatechin and naringenin have been shown to reduce oxidative damage in ECs via SIRT1, and an intermittent fasting regimen was found to ameliorate vascular dysfunction in a murine model of diabetes by activating the SIRT1 pathway. Small molecule agents that target EZH2 and SIRT1 are currently available or under clinical study in several disease areas (e.g., oncology, dermatology, and nephrology), and may be investigated as potential anti-atherosclerotic agents by incorporating CVD endpoints into ongoing clinical trials. Beyond their desired therapeutic potential, the unintended effects of genome-wide modification via epigenetic agents also warrant consideration. Future studies may improve upon the specificity of these agents by making use of targeted CRISPR/Cas9 epigenome-editing techniques for epigenetic modifications associated with CVD risk loci. Alternatively, targeted EC therapies, as discussed below, may be considered for the resolution of vascular inflammation at the site of the activated endothelium in atherosclerosis. Prospective agents for targeted resolution include ICAM-1- or VCAM-1-targeted nanocarriers, which have previously been employed to limit neurovascular and pulmonary inflammation in experimental models of disease.

26Advances in the development of nanoparticles (on a scale of <0.1 μm) have likewise afforded new opportunities for anti-atherosclerotic therapies that resolve endothelial dysfunction. Vascular nanotherapies have been broadly classified as those which resolve inflammation and dysfunction in efferocytosis, limit plaque neovascularization and neointimal growth, modulate lipid metabolism, and decrease thrombosis. Several of these therapies have been shown to target the perturbed endothelium in experimental models of CVD, including the encapsulation of five cell adhesion molecule small interfering RNA, which reduced leukocyte migration to plaque and suppressed post-myocardial infarction inflammation in murine models. Integrin-targeted nanoparticles containing the antiangiogenic compound fumagillin have likewise been shown to limit neovascularization in mice and demonstrate prolonged activity when combined with statin treatment (Figure 2). More recently, engineered endothelial adrenoreceptor-, VCAM-1-, and P-selectin-targeted nanoparticles, which respectively carried the genes eNOS and IL-10 and receptor for advanced glycation end products-silencing RNA, have been found to localize to ECs and resolve inflammation within the murine atherosclerotic plaque. Nano-selenium particles have also been observed to improve endothelial dysfunction in murine atherosclerosis via Na+/H+ exchanger 1 inhibition. Despite the therapeutic advantages offered by nanoparticles (e.g., reduced off-target effects and increased efficacy per dose), several methodological and biological challenges remain for clinical translation. Importantly, robust protocols are required to determine the composition and purity of nanoparticles derived using various formulations (e.g., lipid or polymeric). Furthermore, the long-term stability of nanoparticles, as well as their safety and selectivity for the activated endothelium in patients with atherosclerosis, must be assessed before these agents are clinically adopted. Nevertheless, vascular nanotherapies remain a promising avenue for the targeted resolution of endothelial dysfunction and reduction of plaque progression in atherosclerotic CVD, with encouraging evidence being generated for the systemic administration of nanoparticle-enveloped small interfering RNA in nonhuman primates to inhibit EC gene expression in multiple organs.

27An emerging body of literature has implicated the vascular endothelium as an essential driver of atherosclerosis, a disease process that contributes to substantial mortality and healthcare burden among aging populations. These discoveries shed light on diverse cellular mechanisms that underlie endothelial activation, dysfunctional cell-cell communication, and perturbed vascular homeostasis in atherogenesis. Moreover, they highlight novel therapeutic targets and delivery methods with significant potential to prevent or limit plaque development in patients with atherosclerotic CVD. Although a broad spectrum of intra- and intercellular processes has been implicated in preclinical models of endothelial dysfunction and atherosclerosis, several methodological challenges remain to effectively translate this research to clinical practice. First, the generalizability of findings between diverse in vitro and in vivo models of endothelial activation and atherosclerosis remains an important consideration to identify strategies that hold significant therapeutic potential for distinct plaque locations and diverse patient groups. Additionally, the safety, efficacy, timing, and sustained response of candidate treatments must be considered, as the atherosclerotic disease process develops over decades and necessitates long-term intervention. A robust integration of experimental techniques (e.g., high-throughput EC profiling with functional validation), data sources (e.g., human samples with experimental models), and cutting-edge computational methods (e.g., machine learning) will be required for the creation of next-generation biomarkers and therapies that effectively mitigate atherosclerotic CVD in vulnerable patients.