Mechanisms and clinical implications of endothelium-dependent vasomotor dysfunction in coronary microvasculature

Abstract

Coronary microvascular disease (CMD), which affects the arterioles and capillary endothelium that regulate myocardial perfusion, is an increasingly recognized source of morbidity and mortality, particularly in the setting of metabolic syndrome. The coronary endothelium plays a pivotal role in maintaining homeostasis, though factors such as diabetes, hypertension, hyperlipidemia, and obesity can contribute to endothelial injury and consequently arteriolar vasomotor dysfunction. These disturbances in the coronary microvasculature clinically manifest as diminished coronary flow reserve, which is a known independent risk factor for cardiac death, even in the absence of macrovascular atherosclerotic disease. Therefore, a growing body of literature has examined the molecular mechanisms by which coronary microvascular injury occurs at the level of the endothelium and the consequences on arteriolar vasomotor responses. This review will begin with an overview of normal coronary microvascular physiology, modalities of measuring coronary microvascular function, and clinical implications of CMD. These introductory topics will be followed by a discussion of recent advances in the understanding of the mechanisms by which inflammation, oxidative stress, insulin resistance, hyperlipidemia, hypertension, shear stress, endothelial cell senescence, and tissue ischemia dysregulate coronary endothelial homeostasis and arteriolar vasomotor function.

INTRODUCTION

The coronary microvasculature comprises the smallest caliber vessels (generally, <200 µm diameter) of the coronary vasculature that extend from a single arteriole to a network of capillaries to a draining venule. These microvessels are critically important for gas and nutrient exchange between blood and myocardial tissue, as well as in regulating coronary blood flow. Within the microvasculature, the key structural components involved in vasomotor regulation are arterioles, which are resistance microvessels lined by vascular smooth muscle, and endothelial cells, which line the innermost layer of arterioles and the majority of capillaries (Fig. 1). The interplay between endothelial cells and arteriolar smooth muscle cells (SMCs) is fundamental to microvascular vasomotor regulation. Disruption of normal endothelial function leads to vasomotor dysfunction that impairs coronary blood flow and thereby contributes to cardiovascular morbidity and mortality. Therefore, understanding the mechanisms of coronary endothelium-dependent vasomotor dysfunction is critically important. The following review will begin with an overview of normal coronary microvascular physiology and modalities of measuring microvascular function, followed by a discussion of the clinical implications of coronary microvascular disease. The subsequent sections will discuss recent literature that highlight mechanisms by which inflammation, oxidative stress, insulin resistance, hyperlipidemia, hypertension, shear stress, endothelial cell senescence, and tissue ischemia dysregulate coronary endothelial homeostasis and arteriolar vasomotor function.

Figure 1.

Components of coronary microcirculation and mechanisms of endothelium-dependent vasomotor dysfunction. Created with BioRender.com and published with permission.

Overview of Coronary Arteriolar and Endothelial Physiology

Vasomotor physiology in the coronary microvasculature is determined by arteriolar smooth muscle and endothelial function. Coronary arteriolar SMCs undergo constriction and relaxation responses that alter myocardial blood flow, with contraction triggered by increases in intracellular calcium and relaxation promoted by cAMP and cGMP activities (1). The coronary endothelium plays a pivotal role in maintaining homeostasis and directing normal arteriolar vasomotor function. Several vasoactive compounds that act on the coronary arterioles to regulate vasomotor tone are derived from the endothelium (Table 1). One of the most important endothelium-derived vasoactive compounds is nitric oxide (NO), a potent vasodilator that is synthesized in the transformation of l-arginine to citrulline as catalyzed by endothelial nitric oxide synthase (eNOS). This enzymatic reaction requires NADPH and oxygen as additional substrates, as well as tetrahydrobiopterin (BH₄) as a cofactor. eNOS activity is stimulated by several factors, including increased intracellular calcium concentrations and calmodulin. After synthesis, NO exits endothelial cells and locally diffuses into vascular SMCs, where it activates guanylyl cyclase to convert GTP to cGMP, thereby promoting vasodilation. Other endothelium-derived vasodilating compounds include prostacyclin, which increases intracellular cAMP, and endothelium-derived hyperpolarizing factor (EDHF), which makes the vascular SMC membrane potential more negative, inducing relaxation. Of note, endothelium-dependent vascular SMC hyperpolarization may also occur in a contact-dependent manner, via rises in intracellular calcium concentrations that activate small- (SK) and intermediate- (IK) conductance calcium-activated potassium channels. Activation of these channels results in endothelium-dependent hyperpolarization (EDH) that is directly transmitted to vascular SMCs via gap junctions at endothelial fenestrae in the internal elastic lamina, with a final effect of smooth muscle relaxation (2, 3). Endothelium-derived vasoconstricting compounds include endothelins and thromboxane A₂. Endothelins act on endothelin receptors on vascular SMCs, which increase intracellular calcium concentrations to promote contraction. Thromboxane A₂, which is produced and released by endothelial cells from arachidonic acid via the cyclooxygenase pathway, acts on thromboxane-A₂ receptors that promote contraction by both increasing intracellular calcium concentrations, as well as increasing the levels of superoxide anion radicals that interact with NO to reduce its vasodilating effects.

Table 1.

Endothelium-derived and endothelium-dependent vasoactive compounds

Endothelium Derived		Endothelium Dependent
Vasoactive compound	Effect*	Vasoactive compound	Effect*
Nitric oxide	−	Acetylcholine	+/−**
Prostacyclin	−	Substance P	−
EDHF	−	Bradykinin	−
Endothelin	+	ADP	−
Thromboxane A2	+	Serotonin	+/−**

ADP, adenosine diphosphate; EDHF, endothelial-derived hyperpolarizing factor.

*(−), vasodilation; (+), vasoconstriction; **vasodilates with normal endothelium, vasoconstricts with impaired endothelium.

Other vasoactive compounds are important in regulating vasomotor tone in the coronary microvasculature that are not produced in the endothelium but are still dependent on healthy endothelium to function normally. Acetylcholine (ACh), for example, acts on endothelial cells to stimulate eNOS, as well as to release EDHF resulting in vasodilation in healthy endothelium, though notably in the setting of endothelial dysfunction, ACh triggers paradoxical vasoconstriction (4). Substance P, also dependent on endothelial cells, is a potent vasodilator in the coronary circulation via stimulation of eNOS to produce NO (5, 6). Bradykinin is another potent vasodilator in the coronary circulation that acts on B₂ receptors on the endothelium to stimulate eNOS and NO release and to promote prostacyclin and EDHF release (7). Other endothelium-dependent vasoactive substances include ADP, which binds to P2Y1 receptors on the endothelium to promote arteriolar vasodilation via gap junction-mediated conducted responses and release of NO (8), and serotonin, which has both vasoconstricting and NO-mediated vasodilating effects in the setting of intact endothelium (9).

Modalities of Measuring Coronary Microcirculatory Vasomotor Function

With technological advancements, there are increasing modalities by which coronary microcirculatory vasomotor function can be assessed, both noninvasively and invasively. Noninvasive techniques include Doppler or contrast echocardiography (echo), positron emission tomography (PET) scans, and cardiac magnetic resonance (CMR) imaging. Importantly, all these modalities must exclude patients with obstructive coronary artery disease (CAD) to gather isolated microvascular functional data. Doppler echo measures diastolic flow in the epicardial arteries [typically only the left anterior descending (LAD)] at rest and with drug-induced vasodilators such as adenosine to provide the coronary flow velocity ratio (CFVR), a surrogate for coronary flow reserve (CFR) (10). However, measurements that rely on CFVR or CFR are poor indicators of microvascular vasomotor function in the presence of obstructive CAD, hemodynamic changes, and elevated metabolic rates (11). Furthermore, an assumption is made that the microvascular function in the LAD territory represents the entire myocardium (10, 12). Contrast echo is an important noninvasive technique that provides resolution of capillary blood flow using microbubble contrast agents, which are smaller than erythrocytes and can traverse the microvasculature (13). This technique allows for measurement of myocardial blood flow (MBF) and myocardial perfusion reserve (MPR) with pharmacological stress, important indicators of microvascular function in the absence of obstructive CAD (10, 13). However, limitations of both Doppler and contrast echo include variations in body habitus and ability to obtain a clear window, necessary exclusion of obstructive CAD to draw conclusions about microvascular function, and requirement of technical expertise to obtain reproducible data. Similar to contrast echo, PET imaging, currently the gold standard noninvasive modality to measure microvascular function, provides information on MBF and MPR, but is more reproducible and clinically available (10). CMR performed with and without pharmacological stressors similarly provides quantitative data on MBF and MPR, as well as the semiquantitative measurement of MPR index (14). CMR has high spatial resolution with no radiation exposure, but is limited by availability, high costs, and longer times for image acquisition.

Invasive techniques for measuring coronary microvascular function include coronary angiography, intracoronary temperature-pressure wires, and intracoronary Doppler flow-pressure wires. Coronary angiography can quantify not only obstructive CAD, but also microvascular function via different techniques such as quantifying dye blush at the distal coronaries or the speed at which contrast reaches the distal coronary artery. Intracoronary infusion of ACh during angiography can provide an indication of microvascular spasm if ischemic electrocardiogram (EKG) changes or symptoms occur in the absence of epicardial spasm (15). Intracoronary temperature-pressure or Doppler flow-pressure wires provide more detailed assessments of coronary microvascular function. Though CFR can be measured using both wires, as previously mentioned CFR is of limited value in the setting of epicardial obstructive CAD. Newer measurements are available including thermodilution-based index of microvascular resistances (IMR), calculated as the product of distal coronary pressure and hyperemic mean transit time of a saline bolus. IMR provides a more accurate assessment of coronary microvascular vasomotor function that is independent of the epicardial artery and hemodynamic variation (11). Hyperemic microvascular resistance (HMR) is a similar parameter using Doppler flow velocity rather than thermodilution-based mean transit time to estimate blood flow (10). Minimal microvascular resistance (mMR), which is calculated in the same way as HMR but only during the wave-free period of diastole rather than the entire cardiac cycle, has been proposed as a more isolated measure of microcirculatory dysfunction (16), though IMR remains the standard modality. In summary, there are several noninvasive and invasive modalities for measuring coronary microvascular function, though PET imaging remains the gold standard noninvasive modality whereas IMR is the gold standard invasive measurement of coronary microvascular function.

Clinical Implications of Coronary Endothelium-Dependent Vasomotor Dysfunction

Coronary microvascular dysfunction (CMD) is a growing area of investigation given its role in clinical heart disease. Endothelial injury typically results in impaired production of endothelium-derived vasoactive compounds and/or impaired arteriolar responses to endothelium-dependent vasoactive compounds. These alterations in microvascular signaling and vasomotor activity have adverse consequences to the myocardium thereby contributing to multiple cardiac disease processes as discussed below.

For instance, CMD can contribute to ischemic heart disease even in the absence of obstructive CAD. Indeed, patients with diabetes and signs of coronary microvascular dysfunction have similar rates of cardiac death as those with frank CAD (17). The contribution of CMD to ischemic heart disease may be secondary to impaired augmentation of coronary blood flow in the setting of increased myocardial demand and/or coronary microvascular spasm (15). Although the absolute prevalence of CMD is unknown, ∼30%–50% of patients with nonobstructive CAD are found to have CMD based on invasive coronary testing (18). The diagnosis of “microvascular angina” (MVA) has recently been designated to describe patients with symptoms and objective documentation of myocardial ischemia in the absence of obstructive CAD (<50% coronary diameter reduction), with evidence of impaired coronary microvascular function such as reduced CFR, abnormal HMR or IMR, and/or inducible microvascular spasm (18). This broad definition may be somewhat problematic given limitations of CFR as a measurement of microvascular function as previously discussed, particularly with inclusion of patients with some degree of coronary stenosis (up to 50%). However, this definition of MVA is an important starting point to identify patients with ischemic heart disease with at least some contribution from microvascular dysfunction. Two clinical entities in which MVA may be implicated but which carry even broader definitions are “ischemia and no obstructive coronary artery disease” (INOCA) and “myocardial infarction and nonobstructive coronary arteries” (MINOCA), defined as <50% coronary diameter stenosis in the setting of signs and symptoms of myocardial ischemia and myocardial infarction, respectively. CMD is a proposed mechanism in the pathophysiology of INOCA and MINOCA, though other etiologies such as epicardial coronary vasospasm, plaque disruption, coronary embolism/thrombus with partial or complete lysis, and coronary dissection may also contribute (19). Nonetheless, these clinical entities highlight the burden of disease that CMD may contribute to patients without significant obstructive CAD. For instance, INOCA affects an estimated three to four million people in the United States. Unfortunately, a lack of obstructive coronary disease may be interpreted as reassuring despite INOCA being associated with major adverse cardiac events including late onset myocardial infarction or cardiovascular-related death (10, 20). These patients may also experience increased readmissions and repetitive work up including angiography due to persistence of symptoms (21). Notably, patients with MINOCA have similar clinical outcomes as those with myocardial infarction with obstructive CAD (22). Therefore, though INOCA and MINOCA are not necessarily specific to isolated microvascular dysfunction, CMD likely contributes at least in part to a large proportion of cases. The more specific diagnosis of MVA will be useful in better defining the prevalence of CMD in patients without obstructive CAD.

In addition to contributing to disease in patients without obstructive coronary lesions, CMD is also closely related to obstructive CAD. Indeed, many of the risk factors for CMD and CAD overlap, and coronary endothelial dysfunction often precedes atherosclerosis and obstructive CAD (23). With reduced coronary flow secondary to CMD, wall shear stress decreases, which can contribute to endothelial dysfunction of the more proximal coronary vasculature and promote atherosclerotic plaque formation (23). Once obstructive atherosclerotic plaques form, reduced perfusion at the distal microvasculature results in structural remodeling and alterations in vascular tone (23). CMD may also clinically manifest in patients that present with acute myocardial infarction and undergo percutaneous intervention, but do not have the expected restoration of myocardial perfusion, a clinical entity described as “no reflow” (24). Reduced coronary blood flow after ischemic insult may propagate myocardial injury and impair recovery (10).

In addition, CMD has been implicated in the pathogenesis of heart failure with preserved ejection fraction (HFpEF). In a prospective study of patients without flow-limiting CAD or reduced left ventricular ejection fraction, decreased CFR was independently associated with impaired diastolic function and a greater than fivefold increased risk of hospitalization for HFpEF (25). Other studies of patients with HFpEF suggest that at least 70% of these patients have CMD based on CFR or IMR (26–28). Reduced CFR was also independently associated with increased NH₂-terminal pro-brain natriuretic peptide (NTproBNP) and right ventricular free wall strain, both markers of heart failure severity (26). Many of these studies are limited by the measurement of CFR that is less reliable than IMR for measurement of CMD, though there is a growing trend in measuring the more specific IMR. Among patients with type 2 diabetes, microvascular disease in other tissue beds including nephropathy, retinopathy, and neuropathy was associated with increased risk of incident heart failure independent of CAD (29). These findings all substantiate the relationship between CMD and heart failure, particularly HFpEF. In addition, CMD has been implicated in other heart failure disease entities, including Takotsubo cardiomyopathy, dilated cardiomyopathy, and hypertrophic cardiomyopathy (10, 30). The nuanced relationship between CMD with reduced coronary blood flow and heart failure has been comprehensively reviewed elsewhere (30). Importantly, across multiple heart failure disease entities, reduced coronary blood flow that is in part secondary to reduced endothelium-dependent vasodilation further exacerbates contractile dysfunction leading to a “vicious cycle” of injury (30).

In summary, insults to the endothelium that contribute to CMD and impair normal endothelium-dependent vasomotor function have important clinical consequences, particularly in myocardial ischemia with and without coronary artery obstruction and nonischemic cardiomyopathies, highlighting the importance of understanding mechanisms by which these insults occur. Additional clinical studies that utilize invasive and noninvasive imaging modalities that are specific to CMD are emerging and will be important in better understanding the complete clinical impact that CMD contributes to cardiovascular disease.

MECHANISMS OF CORONARY MICROVASCULAR ENDOTHELIAL AND ARTERIOLAR DYSFUNCTION

The normal physiological mechanisms by which microvascular vasomotor tone is regulated as described earlier depends on intact endothelium. However, clinical factors such as diabetes, hypertension, hyperlipidemia, obesity, and aging can contribute to endothelial injury and consequently lead to arteriolar vasomotor dysfunction. The following sections will highlight recent advances in the understanding of the mechanisms by which inflammation, oxidative stress, insulin resistance, hyperlipidemia, hypertension, shear stress, endothelial cell senescence, and tissue ischemia dysregulate coronary endothelial homeostasis and arteriolar vasomotor function (Fig. 2).

Figure 2.

Mechanisms of endothelium-dependent vasomotor dysfunction by clinical condition. Overlapping mechanisms across clinical conditions indicated in appropriate overlapping space. ADAM17, a disintegrin and metalloprotease 17; AGE/RAGE, advanced glycation end products/receptor for advanced glycation end products; eNOS, endothelial nitric oxide synthase; JNK2, c-Jun NH₂-terminal kinase; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; SK, small-conductance calcium-activated potassium channels.

Inflammation and Oxidative Stress

Major sources of endothelial injury and subsequent dysfunction are inflammation and oxidative stress, which often coincide and exacerbate one another. The association of inflammation and endothelial injury was highlighted in a proteomic analysis of 174 women with CMD as assessed by coronary flow velocity reserve, in which the proinflammatory TNF-α/IL-6/C-reactive protein (CRP) pathway was significantly associated with CMD (31, 32). Furthermore, endothelial dysfunction as measured by contractile response to acetylcholine is more severe in coronary artery segments with greater macrophage infiltration (33), underscoring the relationship between increased inflammation and coronary endothelial injury.

Proinflammatory cytokines and other factors contribute to the pathogenesis of coronary endothelial dysfunction. For instance, administration of TNF-α, a proinflammatory cytokine, to rodents reduces endothelial-dependent vascular relaxation responses to acetylcholine and bradykinin, with reduced eNOS expression (34–36). The effect of TNF-α may be mediated in part by ceramide, a second messenger of TNF-α that stimulates superoxide production (36), by xanthine oxidase, which generates reactive oxygen species (ROS), and by c-Jun NH₂-terminal kinases, which are stress-activated protein kinases (37). Furthermore, pretreatment of coronary arteries with antioxidants such as superoxide scavengers or superoxide dismutase (SOD) largely prevented the blunting of endothelium-dependent vasodilation by TNF-α (36, 38), indicating that inflammation-induced endothelial dysfunction may be secondary to increased oxidative stress. The relationship between oxidative stress and inflammation is further substantiated by a murine study of ischemia-reperfusion injury where endothelium-dependent vasodilation is blunted in the setting of increased TNF-α expression, which is restored by not only antibodies that neutralize TNF-α, but also inhibitors of xanthine oxidase and superoxide scavengers (39). The deleterious effects of TNF-α on endothelial function are mitigated by inflammatory cytokines such as IL-10 in noncoronary vasculature (35, 40, 41), though studies are needed to confirm whether IL-10 produces similar benefits to endothelial function in the coronary microvasculature.

Elevated C-reactive protein (CRP), a proinflammatory marker, was shown to be associated with impaired coronary endothelial function in patients with angiographically normal coronary arteries based on acetylcholine-induced changes in coronary blood flow (42). This study is informative but limited given that elevated CRP is a marker of increased inflammation in a variety of clinical conditions, and endothelial dysfunction in this setting may not necessarily be caused by CRP itself. However, animal studies have demonstrated deleterious effects of CRP administration to coronary microvascular function (43, 44). The effect of CRP on endothelium-dependent dilation in coronary arterioles may be mediated by activation of NADPH oxidase, which catalyzes free radical production, and p38 kinase, an upstream activator of NADPH oxidase, as demonstrated in vitro in porcine coronary arterioles (43). Another important inflammatory marker related to coronary endothelial dysfunction is IL-6, which along with TNF-α was shown to be an independent predictor of coronary endothelial dysfunction in hypertensive patients as measured by coronary vascular resistance (45). As demonstrated in a murine model, IL-6 impairs coronary endothelium-dependent vasomotor function synergistically with TNF-α, in part by impairing EDHF-mediated dilation (46, 47).

As highlighted in the earlier studies, increased inflammation often disrupts endothelial function by means of increased oxidative stress. The specific mechanisms by which oxidative stress impairs endothelial function will now be discussed. Foremost, oxidative stress can induce coronary endothelial dysfunction via uncoupling of eNOS, in which NAPDH consumption and oxygen reduction are uncoupled from oxidation of l-arginine and formation of NO. Uncoupled eNOS results in decreased NO production, increased free radical generation, and subsequent oxidation of BH₄ to BH₃ radical that further diminishes normal eNOS activity (48). In addition, superoxide anions react with NO, resulting in the formation of peroxynitrite, reduced NO bioavailability, and blunted endothelium-dependent vasodilatory responses (48, 49). Furthermore, peroxynitrate propagates oxidative injury, damaging proteins, lipids, and DNA (50), with specific injury to the activity and function of prostacyclin synthase and eNOS (51, 52) (Fig. 3). ROS may also inactivate eNOS via S-glutathionylation, further contributing to endothelial dysfunction (53).

Figure 3.

Effects of oxidative stress on normal endothelial nitric oxide synthase activity. Normal endothelial nitric oxide synthase (eNOS) activity involves the conversion of l-arginine to l-citrulline and nitric oxide (NO), with substrates nicotinamide adenine dinucleotide phosphate (NADPH) and oxygen (O₂). Tetrahydrobiopterin (BH₄) is an important cofactor in this enzymatic reaction. Nitric oxide then diffuses to the vascular smooth muscle cell (SMC), resulting in vasodilation. Multiple risk factors lead to endothelial cell oxidative stress, which disrupts normal eNOS activity via eNOS uncoupling, superoxide production, peroxynitrate production, eNOS inhibition, decreased BH₄, and decreased NO bioavailability, which ultimately impairs endothelium-dependent vasodilation. Created with BioRender.com and published with permission.

Several enzymes within endothelial cells produce ROS that can contribute to endothelium-dependent vasomotor dysfunction. NADPH oxidases (NOX), which produce superoxide from oxygen using NADPH as an electron donor, are activated in a variety of clinical conditions, including hypertension, diabetes, and hypercholesterolemia (50). These enzymes are found within both endothelial cells and vascular SMCs (54). There are several maladaptive activators of vascular NOX that disrupt physiological redox balance resulting in endothelial dysfunction, including excessive angiotensin II, thrombin, TNF-α, hyperglycemia, oxidized LDL, and shear stress (55). In a canine model of CHF, endothelium-dependent relaxation via ACh was impaired in failing hearts, which corresponded to increased superoxide generation and increased NOX2 expression (56). Administration of the antioxidant apocynin augmented endothelium-dependent relaxation in this setting (56). Other studies have demonstrated that NADPH oxidase expression is inversely correlated with endothelium-dependent relaxation (41, 57, 58), and associated with eNOS uncoupling and endothelial dysfunction (59, 60).

Another important endothelial enzyme that may increase oxidative stress is xanthine oxidase (XO), which is activated by angiotensin II among other factors, and donates electrons to oxygen to produce superoxide and hydrogen peroxide (54, 61). Similar to NAPDH oxidases, increased vascular XO activity is inversely related to endothelium-dependent vasodilation (58). Furthermore, xanthine oxidase activity is known to contribute to endothelial dysfunction in ischemia-reperfusion injury, with XO inhibition restoring endothelium-dependent vasodilation in a variety of settings (39, 40, 62).

Finally, the mitochondria are a source of endothelial ROS production, which when dysregulated can promote oxidative stress and endothelial cell injury. The mitochondrial respiratory chain generates hydrogen peroxide and superoxide, which when overgenerated can cause endothelial damage (63, 64). Coronary endothelial cells from diabetic mice have increased concentrations of mitochondrial ROS that impairs endothelium-dependent vasodilation and these effects are improved with mitochondrial ROS inhibition (65, 66).

Importantly, ROS do have an important physiological role in maintaining healthy endothelium-dependent vasomotor function. For instance, hydrogen peroxide, which is released by coronary endothelial cells in response to shear stress and by contracting myocardium in the setting of increased myocardial oxygen demand, induces arteriolar vasodilation via both endothelium-dependent and independent pathways (67–69). This hydrogen peroxide-mediated vasodilation in coronary arterioles occurs in a NO-independent manner. Hydrogen peroxide production in these settings is thought to compensate for impaired NO-mediated vasodilation with myocardial injury (69, 70), yet overproduction of hydrogen peroxide is maladaptive with proapoptotic, proinflammatory, and proatherogenic effects (71). ROS have other important physiological roles in maintaining endothelium-dependent vasomotor function. NOX knockdown mice have blunted endothelium-dependent vasodilation and reduced NO synthesis (72). Furthermore, endogenous ROS increase AMPK-mediated coronary endothelial eNOS activation, NO synthesis, and endothelium-dependent vasodilation (73). Though increased NOX expression improves endothelium-dependent coronary vasodilation in the short term, prolonged increased NOX and ROS expression inactivates mitochondrial SOD2 and increases mitochondrial ROS resulting in decreased endothelium-dependent vasodilation and decreased endothelial cell proliferation (64). These studies highlight the important role that ROS play in healthy endothelial function, and the deleterious effects of redox dysregulation on endothelium-dependent arteriolar function.

In summary, inflammation and oxidative stress are interrelated and contribute to endothelium-dependent vasomotor dysfunction. TNF-α and IL6 are important proinflammatory cytokines that drive inflammation and oxidative stress in the coronary endothelium in disease states. eNOS uncoupling, peroxynitrite-driven cellular injury, and ROS production by NAPDH oxidases, xanthine oxidase, and mitochondria impair endothelium-dependent vasomotor function, though ROS such as hydrogen peroxide also have important physiological functions. Additional studies are needed that better define optimal redox balance in coronary microcirculation in healthy and diseased states, particularly given the central role that oxidative stress plays across disease processes and the lack of therapies that successfully target oxidative stress, an area that will be discussed later in this review.

Insulin Resistance and Hyperglycemia

Coronary endothelial dysfunction in the setting of diabetes is well established. In patients with diabetes, endothelium-dependent vasodilation is impaired even in the absence of angiographic coronary artery disease (74), and the degree of dysfunction increases with severity of insulin resistance and glucose intolerance (75). The mechanisms by which hyperglycemia and insulin resistance impair endothelial cell activity are multifaceted and will be discussed in this section.

Several of the mechanisms of endothelial dysfunction in diabetes are shared with general pathogenetic pathways that result from chronic diabetes, including formation of advanced glycation end products (AGEs), activation of protein kinase C (PKC), and disturbances in polyol pathways (76). AGEs are formed in the setting of hyperglycemia in which intracellular glucose-derived precursors interact with amino groups of proteins. With increased AGE production, there is increased binding to AGE receptors (RAGE), which are expressed on inflammatory T cells, resulting in increased cytokine production and ROS generation, among other effects. These effects have been demonstrated in animal and human studies. In a murine model of type 2 diabetes, diabetic mice had increased expression of RAGE and decreased endothelium-dependent vasodilation to ACh, an effect that was partially reversed with administration of soluble RAGE that binds to AGE and prevents binding to RAGE (77). With this model, the investigators also demonstrated that AGE/RAGE signaling is involved in increased expression of TNF-α and oxidative stress markers including NOX-2 (77). In another study, in vitro treatment of human coronary artery endothelial cells (HCAEC) with AGEs resulted in reduced eNOS mRNA and protein levels, reduced NO production, increased NOX activity and superoxide levels, and decreased catalase and SOD activities (78). Increased inflammation secondary to AGE signaling may be amplified by NF-κB and TNF-α signaling via activation of IKK-β, a required component of NF-κB activation that is increased in the setting of insulin resistance (79). IL-6 also contributes to inflammatory injury to coronary endothelium in the setting of diabetes (46).

Hyperglycemia also stimulates do novo synthesis of the second messenger diacylglycerol (DAG) from glycolytic intermediates resulting in increased PKC activation, which also contributes to coronary endothelial dysfunction in the setting of diabetes (76). Coronary vessels isolated in rats with type 1 diabetes had elevated protein expression of PKC subtypes α, β₁, and β₂, which corresponded to increased vasoconstriction in the setting of endothelin treatment that was mitigated with PKC inhibition (80). Furthermore, elevated PKC expression in endothelial cells impairs insulin-induced eNOS stimulation (81), an effect that may mediated by activation of G protein-coupled receptor kinases (GRKs), which negatively regulate the insulin-mediated Akt/eNOS pathway (82). In addition, PKC activation may contribute to increased oxidative stress resulting in endothelial injury. For instance, in porcine coronary microvessels, activation of PKCβ2 impaired NO-mediated vasodilation via production of superoxide from XO and JNK signaling via Rho kinase activation (83).

Another effect of hyperglycemia in blood vessels is disturbance in polyol pathways due to metabolism of intracellular glucose or glycolytic metabolites to sorbitol and to fructose, which uses NADPH as a cofactor, depriving the cell of stores that are required for glutathione reductase to regenerate reduced glutathione (GSH), an important antioxidant within cells (76). Abnormal polyol pathways have been linked to endothelium-dependent vasomotor dysfunction (84, 85). Aldose reductase, which catalyzes the reduction of glucose to sorbitol and marks the first step in the polyol pathway of glucose metabolism, has increased activity in the setting of diabetes and is associated with endothelial dysfunction (86, 87).

Beyond hyperglycemia, insulin resistance also contributes to endothelial dysfunction, particularly via impairment of the phosphatidylinositol-3-kinase (PI3K) pathway. Under normal conditions, insulin binds to the insulin receptor that activates two different pathways, including the insulin receptor substrate 1 (IRS-1)/PI3K/Akt signaling pathway that activates eNOS signaling and increases NO production, as well as the MAPK signaling pathway that promotes endothelin-1 secretion (88, 89). In the setting of insulin resistance, the PI3K pathway is impaired and the MAPK pathway is activated, resulting in decreased NO and increased endothelin production (90, 91).

Other studies have highlighted additional various mechanisms by which diabetes contributes to coronary microvascular dysfunction. In a porcine model of chronic myocardial ischemia, coronary arterioles from diabetic pigs had impaired endothelium-dependent vasodilatory responses to ADP, substance P, and VEGF, with intact vasodilatory response to endothelium-independent nitroprusside (92). These impairments in microvessel reactivity were markedly improved in pigs that received insulin treatment, indicating the importance of glycemic control in preserving endothelium-dependent vasomotor function (93). The maladaptive effects of diabetes on the coronary microvasculature is not limited to the endothelium, with coronary microvessels from human patients with diabetes expressing increased phosphorylated myosin light chain (MLC) that correlates with impaired vascular smooth muscle relaxation (94). Studies of atrial samples from patients with diabetes before and after cardioplegia and cardiopulmonary bypass further underscore the effect of diabetes on endothelial dysfunction. Baseline coronary endothelium-dependent arteriolar dilation is impaired in patients with uncontrolled diabetes compared with those with controlled diabetes or no diabetes (95). Cardioplegia and cardiopulmonary bypass results in impaired endothelium-dependent vasodilation in all three groups that is significantly worse in patients with uncontrolled diabetes, an effect that could be mediated by increased PKC signaling (95).

Studies on calcium sensitive potassium channels have also highlighted the role of diabetes in mediating endothelial dysfunction. For instance, in a study investigating small-conductance calcium-activated potassium (SK) channels expressed in endothelial cells, which are largely responsible for EDHF-mediated coronary arteriolar relaxation, diabetes was associated with decreased relaxation responses to ADP and substance P, decreased endothelial SK currents, and decreased hyperpolarization responses with administration of a selective SK channel activator NS309 compared with control (96). Treatment of diabetic mice with mitochondrial ROS inhibitors improved endothelium-dependent relaxation responses to ADP and NS309, with improved endothelial SK currents (66). Furthermore, NS309 administration improved endothelium-dependent relaxation responses to ADP and substance P after cardioplegic-hypoxia/reoxygenation injury, and this effect was maintained though less pronounced in vessels from patients with diabetes compared with control patients (97). However, SK protein expression in human coronary artery endothelial cells were similar between control and diabetes groups, indicating that diabetes may impair EDHF-mediated SK channel function at a posttranslational level (96, 97). Diabetes also impairs signaling to large-conductance calcium-sensitive BK channels, which are expressed on vascular SMCs to elicit vasodilation, via several mechanisms, both endothelium independent and dependent. One important endothelium-dependent mechanism by which diabetes impairs BK signaling is via disruption of transient receptor potential vanilloid subtype 1 (TPRV1) channels, which are expressed on coronary endothelial cells. These channels couple myocardial metabolism and blood flow in a nitric oxide-dependent and BK channel-dependent manner (98). TPRV1 channels have reduced expression and activity in diabetic mice and in swine with metabolic syndrome (99, 100).

Diabetes also promotes epigenetic modifications that may affect endothelial function. In particular, studies have demonstrated that diabetes induces DNA hypomethylation and histone acetylation on the p66Shc promoter, corresponding to increased p66Shc expression and subsequent ROS production, apoptosis, and reduced NO bioavailability (101–103). These changes persist in the setting of diabetes even with intensive glycemic control. These epigenetics studies have been demonstrated in myocardial tissue (103), but additional studies will be needed to confirm whether diabetes produces similar epigenetic modifications localized to coronary endothelial cells.

In summary, diabetes results in coronary endothelium-dependent vasomotor dysfunction due to maladaptive AGE/RAGE signaling, PKC signaling, disrupted polyol pathways, and dysregulated PI3K and ion channel signaling. Additional studies on the reversibility of these changes with glycemic control will be important and clinically relevant, and further investigations into the epigenetic disruptions that occur with hyperglycemia and insulin resistance will provide further insight into how to better target CMD in the setting of diabetes.

Hyperlipidemia and Obesity

Studies of microvascular function in the setting of hyperlipidemia, obesity, and metabolic syndrome have highlighted the deleterious effects of these conditions on endothelium-dependent vasomotor function. Hyperlipidemia is a major risk factor for coronary microvascular dysfunction, with elevated total cholesterol and LDL-C levels correlating with CMD as measured by CFR and IMR (104, 105). Endothelium-dependent arteriolar vasodilation is compromised in the setting of hyperlipidemia, because in part to elevate oxidized LDL levels that impair eNOS expression and NO activity (106, 107). These effects could be mediated, in part, by hypercholesterolemia-mediated suppression of inwardly rectifying potassium Kir2.1 channels, which may mediate flow-induced vasodilation via eNOS activation under physiological conditions (108–110), though the role of Kir in flow-induced vasodilation is not widely accepted. Increased LDL may also reduce uptake of l-arginine into endothelial cells and contribute to increased production of superoxide (111). In addition, JNK2 is a known mediator of coronary endothelial dysfunction and is associated with hypercholesterolemia-induced endothelial injury (37, 112). JNK contributes to decreased eNOS expression, NO release from endothelial cells, decreased SOD expression, and increased superoxide and peroxynitrite production (37, 112). The effects of hyperlipidemia on coronary microvascular vasomotor function change over time, with a longitudinal study of hypercholesterolemic swine demonstrating that from 2.5 to 15 mo of follow-up, mechanisms of coronary vasomotor dysfunction shifted from early impairment of endothelium-dependent vasodilation via bradykinin to late enhancements of vasoconstriction responses to endothelin (113). These late effects were due to increased endothelin B receptor-mediated vasoconstriction (113).

Often clinically coinciding with hyperlipidemia, obesity is also associated with impairment of endothelial function (114). Studies on the effects of obesity on endothelium-dependent vasodilatory responses are conflicting. For instance, coronary microvessels from obese rats have an increased dilation response to acetylcholine and reduced constriction response to endothelin, with similar vasodilatory responses to endothelial-independent sodium nitroprusside (SNP), compared with vessels in lean rats (115). In a study of human coronary microvessels collected from patients undergoing cardiac surgery, obesity was associated with impaired dilation to both bradykinin and SNP in normotensive patients, but in the setting of hypertension, increasing body mass index (BMI) was correlated with bradykinin and SNP-induced vasodilation (116), obscuring changes in endothelial function but highlighting increased NO sensitivity of arterioles in the setting of obesity. These findings suggest augmented endothelial-vasodilatory responses in the setting of obesity. The relationship of increased endothelial-dependent vasodilation in the setting of obesity may be supported by the finding of increased serum nitric oxide levels in morbidly obese patients after weight reduction surgery (117). Increased vasodilatory reactions in coronary microvessels in the setting of obesity is thought to be due to increased cardiac output and intravascular volume in the setting of obesity (116), though increased blood flow may still not meet the elevated metabolic demands of obesity (118). Female sex hormones may interact with the relationship between obesity and coronary blood flow, given that obese menopausal women were found to have significantly reduced myocardial blood flow compared with obese premenopausal women, who had high myocardial blood flow compared with lean subjects (118).

Time course may also play a role in the effect of obesity on endothelial function, highlighted by a study on coronary vascular function in Zucker obese rats by Oltman et al. In this study, ACh-induced relaxation in coronary microvessels was similar in obese and lean rats at age 8–12 wk, diminished at age 16–24 wk, and nearly abolished at age 28–36 wk, whereas SNP-induced relaxation was unchanged despite age or presence of obesity (119). Similarly, adipose-expressed ADAM17, a disintegrin and metalloproteinase that regulates soluble TNF levels, contributes to increased disruption of coronary endothelium-dependent vasodilation in older patients with obesity compared with older lean or younger patients with obesity (120). These findings may indicate that obesity may impair coronary endothelial function with increased age or time progression. These longitudinal studies on the effects of obesity on coronary endothelial function also provide insight into discrepant effects of obesity on endothelial-dependent vasomotor function. Although coronary microvessels may initially adapt to increased metabolic demands and hemodynamic changes in the setting of obesity, these adaptive responses may diminish over time and fail to meet metabolic demands, further counteracted by comorbid conditions that are associated with long-standing obesity such as hyperlipidemia and insulin resistance/diabetes (121).

Obesity-related endothelial injury is mediated in part by oxidative stress. In obese rodents, endothelial-dependent vasodilation is improved with incubation with superoxide scavengers and XO inhibitors, suggesting that increased superoxide production may mediate obesity-related endothelial injury (37, 119). Furthermore, obese swine have impaired coronary endothelial function, with increased ROS markers including superoxide, nitrotyrosine, and NOX (122). Similar to changes that occur in the setting of diabetes, obesity is associated with increased epigenetic modification and gene expression of p66Shc that contributes to ROS production (123, 124). These changes have been confirmed in peripheral microvessels but would be an interesting area of study in the coronary microvessels.

Adipose-derived inflammatory factors, termed “adipokines,” are thought to play a major role in endothelial injury in the setting of obesity. Examples of adipokines include adiponectin, leptin, and resistin, as well other common anti-inflammatory cytokines such as TNF-α and IL-6 (118). The local effects of adipose tissue are highlighted by findings that epicardial adipose tissue in patients with coronary artery disease carry inflammatory cell infiltrates and express proinflammatory IL-6 and TNF-α (125), which may mediate local inflammatory effects to the coronary microvasculature (126). Dysregulation of adipokines may be mediated by hypoxia inducible factor 1α (HIF-1α) activation in adipocytes secondary to inadequate perfusion of large adipose tissue stores, which promotes increased expression of leptin, resistin, TNF, and IL-6 (118, 127). Increased leptin released by perivascular adipose tissue disrupted coronary endothelial-dependent vasodilation to bradykinin via PKC-β signaling in pigs with metabolic syndrome (128). Resistin mediates oxidative stress via increased NOX2 and NOX4 expression, as well as impaired mitochondrial respiratory chain function (129, 130), reduces bradykinin-induced vasodilation in porcine coronary arteries in vitro (131), and reduces eNOS levels in HCAEC in vitro (130). Adiponectin, which is thought to have a beneficial effect on endothelial function by increasing NO bioavailability, restoring Ach-induced vasodilation, and restoring eNOS coupling, is reduced in the setting of obesity with reduction of its vasodilating effects (118, 132–135). Another adipokine increased in obesity, chemerin, impairs NO signaling and Akt-dependent signaling in human endothelial cells in vitro and increases vascular oxidative stress (136). The effects of TNFα and IL6 on the endothelium were previously discussed, though CD40L, a member of the TNF ligand family, has also been implicated as a mediator of obesity-associated vascular inflammation, oxidative stress, and endothelial dysfunction (137, 138).

Another important mediator of endothelial dysfunction in obesity is arginase, which regulates NO synthesis by competing with l-arginine as a substrate for eNOS, and is increased in obese animal models (139, 140). Arginase is expressed in vascular SMCs and endothelial cells, and is known to contribute to reduced NO production in coronary microvessels (141, 142). Furthermore, arginase inhibition in peripheral vascular microvessels improves endothelium-dependent vasodilation in patients with coronary artery disease and diabetes (142). In fact, endothelial cell-specific arginase-1 knockout mice were protected from obesity-induced endothelial injury as measured by vasodilatory responses to ACh (143).

Though not necessarily specific to hyperlipidemia or diabetes alone, large animal models that reflect multiple disease states more accurately reflect clinically relevant comorbidities. In a swine model of diabetes, hyperlipidemia, and CKD, porcine coronary microvessels had impaired endothelium-dependent vasomotor function secondary to decreased NO bioavailability (144). In a separate swine model of high-fat diet-induced hyperlipidemia and streptozotocin-induced diabetes, porcine coronary vessels had increased responsiveness to endothelin mediated by increased endothelin B (ET-B) receptor activity (113). Furthermore, combined disease processes may have synergistic effects on endothelial dysfunction. Zhang et al. (145) and others assessed the interaction between diet and renovascular hypertension in swine and found decreased endothelial NOS expression only in obese/high-fat diet pigs with comorbid hypertension, suggesting a synergistic effect. Interestingly, the decreased endothelial NOS expression was accompanied by increased oxidative stress again only in the obese-hypertensive swine (145). Indeed, oxidative stress is a common pathway by which comorbidities lead to endothelial dysfunction, making targeted therapies that target this pathway an enticing treatment strategy, but as will be discussed later, additional investigations are needed in this area. Hypertension also has a synergistic effect with hyperlipidemia in impairing coronary endothelium-dependent vasodilation (146), though the mechanisms of this synergism have yet to be investigated in detail. Unfortunately, most studies that consider multiple disease processes generally do not compare combined disease processes with individual ones. Additional studies that compare separate disease entities with combined disease processes would be helpful to further understand how different disease processes interact to affect endothelial function.

In summary, hyperlipidemia mediates coronary endothelial dysfunction secondary to elevated oxidized LDL levels, JNK2 signaling, and increased endothelin receptor activity. Obesity is associated with increased adipokine signaling, increased ADAM17 activity, increased arginase levels, and oxidative stress, all of which impair endothelium-dependent vasomotor function. Time course appears to play an important role in mechanistic disruptions of endothelial function in the setting of hyperlipidemia and obesity, and additional longitudinal studies will be needed to clarify these changes.

Hypertension and Shear Stress

Sustained hypertension impairs endothelial vasomotor function via several mechanisms. Coronary vessels from swine with hypertension have decreased vasodilatory responses to bradykinin and substance P, and decreased levels of SOD, catalase, and antioxidant vitamin E compared with control animals (146). These effects are amplified in swine with comorbid hyperlipidemia, suggesting a synergistic effect on endothelial injury (146). These effects could be secondary to increased NOX activity, which oxidizes BH₄ and uncouples eNOS, thereby propagating oxidative injury and reducing NO production (147). In addition, S-glutathionylation of eNOS in endothelial cells, which occurs in the setting of oxidative stress, is increased in hypertensive vessels and is associated with loss of nitric oxide and impaired endothelium-dependent vasodilation (53). Oxidative stress may also be increased in the setting of hypertension via a number of other mechanisms. For instance, angiotensin II has been demonstrated to play a critical role in hypertension-induced oxidative stress and endothelial injury. Angiotensin II, which is elevated is many patients with hypertension and is thought to play important roles in hypertension-related disease processes, increases vascular expression of NOX1 and NOX2 and promotes ROS production, thereby increasing inflammation, adverse vascular remodeling, and endothelial dysfunction (148, 149). Angiotensin II also stimulates mitochondrial superoxide and depletes SOD2 in bovine coronary arteries, with direct effects on arteriolar NO-sensitivity due to guanylate cyclase depletion (150). Another mediator of hypertension-induced oxidative stress may be aldosterone, which activates mineralocorticoid receptors that activate NOX4 in endothelial cells and reduces coronary blood flow (151–153). In addition, prolonged exposures to high blood pressure promotes PKC-dependent NOX pathways in a rat model of hypertension, which is associated with impaired endothelium-dependent vasodilation that is restored with NOX and PKC inhibitors (154). Of note, PKC pathways may also be involved in aldosterone-mediated coronary microvascular injury (153).

Like obesity, hypertension is associated with upregulation of arginase-mediated endothelium-dependent vasomotor dysfunction. Arginase activity in the heart is higher in spontaneously hypertensive rats, an effect that is mitigated with blood pressure control (155). Zhang et al. (156) demonstrated these effects in a porcine model of hypertension, in which coronary arterioles from hypertensive pigs were found to have increased arginase expression and activity, and an attenuated dilatory response to adenosine compared with nonhypertensive pigs, with normal responses to SNP in both groups. Furthermore, adenosine-stimulated NO release was reduced in the hypertensive group (156). These deleterious effects were partially restored by inhibition of arginase or incubation with l-arginine (156), again highlighting the effect of arginase as a competitive inhibitor of the l-arginine-eNOS enzymatic reaction.

Salusin-β, a vasoactive peptide expressed in vascular endothelial and SMCs, may also play a role in hypertension-induced endothelial injury. Coronary arteries from hypertensive rats have increased salusin-β expression compared with those from nonhypertensive rats, and knockdown of salusin-β decreased arterial blood pressure and improved endothelium-dependent vasodilation via ACh and vascular remodeling (157, 158). Hypertensive rats also had decreased coronary eNOS activity and expression, decreased NO production, increased NOX activity, and increased ROS production, which were all mitigated in salusin-β knockdown mice (157, 158).

Hypertension may also lead to endothelial dysfunction by impairing EDHF production and EDH activity in the coronary vasculature. A rat study of coronary vessels from hypertensive rats demonstrated impaired relaxation responses that were due in part to EDH reduction (159). These changes may be mediated by reduced expression of SK channel expression in the setting of hypertension, among other mechanisms (3, 160).

An important mechanism by which hypertension may lead to endothelial dysfunction is through decreased wall shear stress. Shear stress, which is a measure of the biomechanical force acting in the direction of blood flow at the vessel wall, is determined by fluid viscosity, vessel geometry, and blood flow (161). Endothelial mechanoreceptors are sensitive to shear stress forces, which stimulate generally vasculoprotective changes including adaptive remodeling, redox balancing, and vasodilation with increased eNOS expression (161–163). In patients with hypertension, there is a chronic decrease in shear stress (164). Lower shear stress is associated with increased endothelial ROS and reduced endothelium-dependent vasodilation (165, 166).

Microvascular remodeling that occurs in the coronary microvasculature in the setting of hypertension has a demonstrated genetic component. Arteriolar remodeling is associated with modulation of several genes, many of which are blood pressure independent, including telomere elongation helicase 1, calcium-dependent phospholipase A₂, Dnaja4, and reticulocalbin 2, the latter two involved with profibrotic DNA loci (167). Blood pressure-independent genetic changes that occur in hypertension, like glycemic-independent changes that occur in diabetes, highlight the importance of genetic-focused research to better understand how coronary microvascular injury occurs and to develop disease targeted therapies.

In summary, hypertension causes endothelial injury via eNOS S-glutathionylation, increased angiotensin II and aldosterone activity, PKC-dependent NOX activation, increased arginase activity, salusin-β upregulation, and decreased shear stress. In addition, microvascular remodeling in hypertension may be driven at a genetic level further dysregulating coronary microvascular vasomotor tone. Additional studies are necessary that investigate how duration of hypertension affects these pathways, and what impacts do early and late blood pressure control have on the reversibility of hypertension-mediated CMD.

Aging and Senescence

Aging and endothelial cell senescence have deleterious impacts on normal endothelium-dependent vasomotor function. Cellular senescence is the process by which cells stop division, undergo structural alterations such as flattening and enlarged polypoid nucleus, and exhibit altered effects on angiogenesis and cellular production (168). These changes include dysregulation of microvascular tone. For instance, endothelial senescence contributes to disrupted microvascular vasodilatory responses secondary to increased endothelin activity and decreased NO availability (168–170).

Redox dysregulation contributes to endothelial dysfunction in the setting of aging and endothelial senescence. For instance, aged rats have elevated nitrotyrosine, a marker of ROS, and decreased SOD expression, with corresponding impairments in coronary endothelium-dependent vasodilation (171, 172). In addition, older age is associated with increases in vascular superoxide production and downregulation of transcription factor Nrf2 that results in decreased expression of several Nrf2 targets including GGC, NQO1, HO-1, all of which play important roles in mitigating oxidative injury (173). Studies of coronary arterial endothelial cells in vitro indicate increased NF-κB levels with cellular senescence that is associated with increased oxidative stress (174). Older age may also amplify oxidative injury brought on by elevated blood pressure via increased xanthine oxidase and eNOS uncoupling (175).

Interestingly, aldose reductase and increased AGE/RAGE signaling may play a role in aging-related endothelial dysfunction independent of presence of diabetes (87, 172). AGE/RAGE signaling is known to be increased in the vasculature with aging (176–178). In nondiabetic rats without dietary interventions, older age was associated with impaired endothelium-dependent relaxation to acetylcholine that improved with aldose reductase inhibitors or soluble RAGE (87). Other studies demonstrating the deleterious effects of AGE/RAGE signaling in the coronary microvasculature have been previously discussed (77, 78).

Changes in calcium signaling within senescent endothelial cells also contribute to disrupted function. Boerman et al. (2) determined that older mice aged 24–26 mo had decreased frequency and duration of active endothelial calcium signaling compared with younger mice aged 3–6 mo. These changes were associated with 40% fewer endothelial internal elastic lamina fenestrations per square millimeter in older versus younger mice (2). These reductions in calcium signaling likely contribute to reduced hyperpolarization responses to SK and BK channels with increased age that are important for EDH and EDHF signaling as well as eNOS activation (179).

As discussed previously, increased ADAM17 activity may result in endothelium-dependent vasomotor dysfunction with increased age, particularly in the setting of obesity (120). Increased ADAM17 activity may be mediated by age-related declines in the expression of caveolin-1, an endothelial structural protein that inhibits ADAM17 activity (120). Another mediator of age-related endothelial dysfunction is Sirtuin 1 (Sirt1), an enzyme that deacetylates transcription factors in the nucleus. Sirt1 promotes eNOS expression and activity, NO production, and adaptive vascular remodeling (180). However, the expression of Sirt1 is decreased with aging, which impairs endothelial function. Mice aged 30 mo compared with aged 5–7 mo had decreased endothelial cell expression of Sirtuin 1, and treatment in old and young mice with a Sirtuin-1 inhibitor resulted in impaired endothelium-dependent vasodilation without disrupting endothelium-independent dilation (181). Sirt1 also inhibits oxidative stress, and decreased Sirt1 with aging may further impair endothelium-dependent vasomotor function (182). In addition, Sirt1 deacetylates p66Shc, which mitigates vascular ROS production (183, 184). These epigenetic changes have not yet been studied in the coronary microcirculation but may be an additional mechanism by which age-related decline of Sirt1 impairs coronary endothelial function.

In summary, endothelial cell senescence associated with aging is an important contributor to coronary endothelium-dependent vasomotor dysfunction. Decreased Nrf2 activity, increased AGE/RAGE activity, altered calcium signaling, increased ADAM17 activity, decreased sirtuin-1 expression, and increased oxidative stress all account for endothelial dysfunction in the setting of aging. Important future research directions in this area will be better defining time course of endothelial senescence, genetic, and molecular drivers of early endothelial senescence, particularly in human studies, and reversibility of these changes to better consider targeted therapies.

Tissue Ischemia

When the heart and other organs become ischemic due to myocardial infarction, stroke, or other processes, the endothelium becomes dysfunctional (185). This is especially evident if and when the tissue is reperfused such as during percutaneous coronary intervention (186) or coronary artery bypass surgery (187, 188). Endothelial dysfunction occurs even in nonischemic myocardial territories in the setting of acute myocardial infarction (189).

Endothelial injury secondary to ischemia-reperfusion injury is in part related to the release of ROS with subsequent eNOS uncoupling, impaired eNOS activity, and reduced BH₄ bioavailability, which may affect vasomotor regulation of the microcirculation as described elsewhere in this article (185, 190). Calcium overload also contributes to endothelial ischemia-reperfusion injury. Endothelial intracellular calcium rises with ischemic insults, and reoxygenation that accompanies reperfusion further increases intracellular calcium concentrations by depleting calcium from the endoplasmic reticulum that subsequently activates store-operated channels in the plasma membrane to increase calcium influx (185, 191–194). Though increases in endothelial calcium in physiological conditions typically stimulate NO and EDH release, the marked increase in intracellular calcium that occurs in the setting of ischemia-reperfusion causes endothelial injury secondary to endothelial myosin light chain kinase (MLCK) activation and increased gap formation, release of mitochondrial ROS and proapoptotic factors, and activation of proteolytic calpains (185, 195, 196). NADPH depletion also plays an important role in ischemia-mediated endothelial dysfunction. NADPH, a necessary substrate for eNOS activity, is markedly depleted in endothelial cells after ischemia-reperfusion that contributes to impaired NO production and endothelium-dependent vasorelaxation (197). Depletion of NADPH may be mediated by increased CD38 activity in the setting of ischemia-reperfusion (156). Thus, the endothelium plays a critical role in the setting of tissue ischemia and drives many of the microvascular disturbances that occur during myocardial infarction.

Coronary microvascular injury during myocardial ischemia-reperfusion injury may also be secondary to mechanical compression of the microvessels from interstitial edema, swelling and rupture of endothelial cells and capillaries, and physical obstruction secondary to plaque debris embolization (198–202). Endothelial cell destruction and/or dysregulation in this setting impair coronary vasomotor tone and may contribute to the “no reflow” phenomenon after myocardial ischemia-reperfusion. In addition, coronary atherosclerotic plaques contain substances that may be released spontaneously or with mechanical disruption such as during percutaneous coronary intervention and stenting, which may dysregulate coronary microvascular tone (202–205). These substances may include particulate debris that physically obstruct the coronary microcirculation causing microembolism and microinfarcts and thus promoting inflammation, as well as soluble substances that impair endothelial function (202). Studies using aspirated plasma from patients who underwent stenting of saphenous vein aortocoronary bypass grafts highlight these latter detrimental effects (204, 205). When injected into rat mesenteric arteries with intact or denuded endothelium, this plasma, which contains elevated levels of serotonin, thromboxane B₂, and TNF-α, induces a significant vasoconstrictor response (204, 205). The vasoconstrictor effect is largely mediated by serotonin, with TNF-α potentiating the vasoconstrictor response in an endothelium-dependent manner (204, 205), further substantiating the role of TNF-α in mediating endothelium-dependent vasomotor dysfunction. Endothelial damage occurs not only after percutaneous coronary intervention-related reperfusion, but also after coronary artery bypass grafting with cardiopulmonary bypass and cardioplegia (186, 187). Endothelium-dependent arteriolar dilation is significantly disrupted after reperfusion in this surgical setting, and interestingly potassium cardioplegia solution alone without reperfusion causes endothelial impairment (187, 206).

Cardioprotective strategies targeting microvasculature during ischemia-reperfusion are well summarized by Heusch, and include ischemic preconditioning by brief coronary occlusion/reperfusion cycles before sustained ischemia, ischemic postconditioning by similar cycles of occlusion/reperfusion but after a major ischemic insult, and remote ischemic conditioning by cycles of occlusion/reperfusion in a remote vascular bed from the coronary territory of interest, in addition to pharmacological therapy which will be discussed in a later section (198, 199). Clinical studies of these strategies have yielded somewhat disappointing results (198, 199), therefore underscoring the importance of better understanding detailed mechanisms of coronary microvascular injury in the setting of ischemia-reperfusion to better tailor effective treatment strategies for patients with myocardial ischemia.

In summary, tissue ischemia is an important contributor to coronary endothelium-dependent vasomotor dysfunction, particularly in the setting of ischemia-reperfusion injury. Increased ROS generation, calcium overload, and NADPH depletion all contribute to endothelial injury, in addition to interstitial edema and microvascular obstruction. In addition, atherosclerotic plaque disruption releases soluble substances that can exacerbate coronary endothelial dysfunction. Additional studies that relate these pathways to ischemic conditioning strategies will be important in better targeting ischemia-reperfusion injury to the coronary microvasculature.

Sex Differences in Coronary Microvascular Endothelial Dysfunction

Several studies have explored the role that male and female sex have on microvascular endothelial dysfunction. Many of these studies focus on estrogen. In the vasculature including endothelial cells, estrogen can act on estrogen receptors that activate downstream kinases including PI3K/Akt and MAPK, which may lead to downstream effects such as increased eNOS, increased SOD, and decreased NOX expression (207, 208). It is known that in postmenopausal women, endothelial function is decreased secondary to reduced estrogen and progesterone levels (171). Estrogen and progesterone have been shown to have a protective and antioxidative role in endothelial injury (171, 209). In an in vitro setting of cultured human coronary artery endothelial cells, estrogen exposure results in increased eNOS and NO release from endothelial cells (210). Estrogen supplementation in ovariectomized rats upregulates eNOS activity and NO production and promotes flow-induced vasodilation in coronary arterioles (171, 211). However, the effects of estrogen on the coronary microvasculature may differ depending on clinical factors such as age and comorbidities. These disparate effects are highlighted by the studies on the effects of estrogen on coronary microcirculation in the setting of menopause. Menopause is known to result in a decline of endothelial function, largely secondary to estrogen deficiency, that is independent of age (171, 212), though clinical studies on hormone replacement therapy (HRT) suggest that estrogen supplementation may actually increase the risk of coronary heart disease (213, 214). Interestingly, HRT may be beneficial when administered shortly after menopause but detrimental with increasing age (212, 214). These disparate effects may be due to estrogen dependence on coupled eNOS to exert a vasodilatory effect, with increased oxidative stress and uncoupled NOS activity with increased age resulting in a vasoconstricting effect (214, 215). Indeed, estrogen therapy stimulates superoxide production in NOS-uncoupled coronary myocytes, an uncoupling that occurs with increasing age as previously discussed (215). Reviews by Somani et al. (212) and White et al. (214) explore these nuanced effects of estrogen on endothelial function in more detail.

Androgens also influence endothelial cell function. Androgens bind to androgen receptors present on endothelial cells and vascular SMCs, and can promote endothelium-dependent vasodilation through increases in intracellular calcium and Akt signaling pathways (208). Androgens can also be converted to estrogens via aromatase that can then act on estrogen receptors as described earlier. However, studies on the effect of androgen administration in men are conflicting, with some demonstrating improved endothelium-dependent relaxation with testosterone supplementation and others showing worsened NO availability and worsened hypertension, oxidative stress, and overall endothelial function with supplementation (208). These conflicting findings could be due to several factors, including type of hormone administered, dosing differences, age of study subjects, and unidentified pathways that may be affected depending on duration of androgen administration or deprivation. Studies are lacking on the effects of androgens on oxidative stress in the coronary circulation, though studies based on myocardial oxidative stress marker expression have disparate findings, with some indicating reduced oxidative stress activity via increased SOD and glutathione activities, and other showing the opposite (208, 216). These conflicting findings may also be due to differences in experimental animal models (castrated vs. intact animals, presence or absence of comorbidities, and age), differences in androgen agent and dosing, and genetic polymorphisms. Importantly, studies of androgens in women have demonstrated mostly deleterious effects including increased inflammation, oxidative stress, and increased circulating endothelin (208) (Fig. 4).

Figure 4.

Differential effects of sex hormones on endothelial cells in males and females. Estrogens bind to estrogen receptors, which have beneficial effects (green text) on the endothelium and promote endothelium-dependent vasodilation in both men and women. Androgens bind to androgen receptors, which have negative effects (red text) on endothelial function in women. In men, the effect of androgen activity is mixed (dotted arrows), with some studies demonstrating beneficial effects and others demonstrating deleterious effects. Androgens may be converted to estrogens via aromatase activity. [Ca]_i, intracellular calcium concentration; IK, intermediate-conductance calcium-activated potassium channels; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; SK, small-conductance calcium-activated potassium channels. Created with BioRender.com and published with permission.

There are also mechanistic differences in endothelium-dependent vasodilation in males and females, with females relying more on EDHF than NOS compared with males (217–220). Furthermore, in a mouse model of obesity and hyperlipidemia, endothelial dysfunction in females was secondary to impaired EDH-mediated relaxation, with associated decreases in small- (SK) and intermediate (IK) conductance calcium-sensitive potassium channels (218). These effects could be secondary to decreases in estrogen levels in premenopausal females in the setting of obesity and hyperlipidemia, given that estrogen increases transcription of SK and IK (218). In addition, in a rat model, microvessel wall shear stress was reduced and flow-induced dilation was increased in females compared with males (221).

An important study by Satoshi and others highlights a genetic basis by when microvascular dysfunction differs in men and women. In this study of 640 patients without obstructive coronary disease with measured CFR by invasive testing, single-nucleotide polymorphisms in MYH15, VEGFA, and NT5E genes were associated with microvascular dysfunction in men but not in women (222, 223). MYH15 and NTE5 are involved in vascular SMC tone and vascular calcification respectively, whereas VEGFA is involved in endothelial cell proliferation and migration (222, 223). Additional studies in this area are warranted to understand why these sex-specific genetic changes occur and how to target them pharmacologically.

To summarize, there are important differences in the mechanisms of microvascular endothelial dysfunction between males and females. Estrogens have known physiological benefits to endothelium-dependent vasomotor function, though HRT has had mixed results. Results from studies on androgens in microvascular endothelial function in men are also mixed. Importantly, genetic variations may exist that account for differences in coronary microvascular dysfunction between men and women. The efforts by funding agencies and journals to establish high standards of reproducibility regarding male and female sex in study design and reporting will likely aid in shedding light into the mechanisms by which sex influences endothelial vasomotor function in the setting of different disease processes.

THERAPEUTIC AGENTS FOR CORONARY MICROVASCULAR DISEASE

Given the clinical significance of CMD and a growing understanding of the mechanisms by which CMD occurs, targeted therapies to mitigate microvascular injury are of interest. This area of research is still in an early phase, as there are no randomized control trials for therapies specifically targeting CMD (10). Given that oxidative stress is a common mechanism of CMD in many of the disease processes discussed in this review, targeting oxidative stress would be an effective strategy to mitigate the effects of CMD in patients with multiple comorbidities. Unfortunately, antioxidant clinical trials to date have had disappointing results. A meta-analysis of 50 randomized control trials that investigated the effects of vitamin and antioxidant supplementation on cardiovascular disease found no reduction in the risk of major cardiovascular events with supplementation, even in several subgroup analyses including type of cardiovascular outcomes, dose of antioxidant, and type of antioxidant, a finding consistent with similar meta-analyses in this area (224). These meta-analyses generally focus on β-carotene, selenium, and vitamins A, C, and E, so it is important to consider other antioxidant therapies. Xanthine oxidase inhibitors (XOI), which are typically used clinically for treating hyperuricemia, have recently been shown in a large cohort study to reduce cardiovascular events in patients with comorbidities including hypertension, diabetes, and dyslipidemia, which was independent of baseline serum urate levels, though the observational study design makes it difficult to draw conclusions as to whether these effects are secondary to antioxidant changes or subsequent unmeasured decreases in urate levels (225). A meta-analysis of randomized control trials investigating the effect of XOI on cardiovascular events showed reduction in total cardiovascular events with XOI treatment, though again these effects could be secondary to urate reduction rather than antioxidant effects (226). These trials are by design looking at general cardiovascular events, and do not focus on or measure parameters of microvascular disease and coronary vasomotor tone. Disappointing results from antioxidant trials may be due to several factors. First, most of these studies have focused on over-the-counter vitamins with antioxidant activity that may not be the ideal targeted therapy or at the optimal dose to have a beneficial clinical effect. Studies of alternative antioxidants are lacking or very limited, and despite some positive results on XOI studies, it is difficult to distinguish whether cardiovascular benefit is from decreased urate levels or alternative mechanisms such as modulation of redox balance. Clinical studies using targeted antioxidants at the subcellular level (instead of global antioxidants) such as NOX inhibitors or mitochondrial ROS inhibitors could provide interesting insights into whether there is a role for antioxidant therapy in CVD. Recent studies from our laboratory demonstrated that ROS may have paradoxical effects on endothelial function and endothelial cell proliferation/angiogenesis depending on the source and localization of subcellular ROS (cytosolic vs. mitochondrial), levels and duration of ROS, and balance between subcellular ROS and antioxidant enzymes (64, 72, 73). Furthermore, the aforementioned mechanisms of coronary microvascular dysfunction occur over the course of years, and antioxidant therapy, which in most randomized trials was given for less than 5 years (224), may not show benefit until taken for an even longer time course or as a long-term preventative therapy (227), though it is still imprudent to recommend preventative antioxidant therapy given the lack of supporting data.

Regarding therapeutic strategies targeting other mechanisms of CMD, current therapies are focused on mitigating specific risk factors and controlling known disease processes. Trials that do include some measure of microvascular function are generally limited as they rely on CFR. However, consideration of these results are important as novel measurements more specific to CMD are more readily used. As previously discussed, glucose control is associated with improved coronary endothelium-dependent vasomotor function in patients (95). Some medications used in patients with diabetes may also improve coronary microvascular function. In particular, sodium-glucose cotransporter 2 (SGLT2) inhibitors, which have been shown to have beneficial effects on the heart independent of glycemic control, have been shown to improve coronary microvascular function in mice (228), and to reduce inflammation-mediated ROS and restore endothelial NO bioavailability in cardiac microvascular endothelial cells (10, 229). Therapies targeted at hyperlipidemia could also improve microvascular function. Statins, which have known antioxidant and anti-inflammatory effects, mitigate hypercholesterolemic coronary endothelium-dependent vasomotor dysfunction in swine (230), and have been shown to improve CFR in patients without obstructive CAD (231). However, a randomized trial that assessed the more specific IMR in women with INOCA found no improvement in microvascular function after 6 mo of therapy (232). Perhaps the short-term effects of statins may play less of a role regarding microvascular function than the long-term effects on hyperlipemia control. These discrepancies underscore the importance of studies that use novel measures of coronary microvascular function in future investigations of CMD-targeted therapies. A randomized clinical trial is currently underway to investigate the effects of PCSK9 inhibitors, another treatment for hyperlipidemia, on coronary microvascular function as measured by IMR (NCT04338165).

The effects of elevated angiotensin II on coronary ROS production and endothelial dysfunction have been discussed, so it is possible that angiotensin converting enzyme inhibitors (ACE-i) and angiotensin II receptor blockers (ARBs) may improve coronary endothelial function. Daily quinapril, an ACE-i, has been shown to improve endothelium-dependent vasomotor function after 6 mo of therapy in normotensive patients with coronary artery disease (233). Other trials have shown improved CFR in patients with INOCA on ACE-i therapy (10). Studies on microvascular function with ARB treatment have also had some positive results as measured not only by improved CFR (231), but also improved PET-determined microvascular flow reserve (234).

Other therapies have shown some benefit to the coronary microvasculature. Intracoronary injection with fasudil, a rho-kinase inhibitor, reduces ACh-induced myocardial ischemia and microvascular constriction (235, 236). Other therapies including β blockers, calcium channel blockers, nitrates, antianginal medications such as ivabradine and ranolazine, and phosphodiesterase type-5 inhibitors have all shown some promise as therapies for CMD based on improvements in CFR (10, 237, 238), but more focused studies with better parameters of microvascular function will be necessary to better understand these effects. Therefore, there are several therapies that may provide some benefit to coronary microvascular function, and improvements in measurements of microvascular function and a better understanding of the importance and means of targeting CMD should drive additional clinical trials and CMD-specific therapies.

LIMITATIONS OF PREVIOUS STUDIES AND AREAS OF FURTHER INVESTIGATION

Many of the studies examining the mechanisms of endothelium-dependent vasomotor function highlight important pathophysiological mechanisms of coronary microvascular disease but are lacking in reflecting complex but clinically relevant effects of multiple disease states and time. Animal studies and human in vitro studies that focus on one or two disease states are helpful in delineating mechanisms that are specific to particular individual comorbidities. However, studies such as those by Fulop et al. (116) highlight how mechanistic interactions between comorbidities can affect endothelial function differently than would be predicted by known mechanistic pathways of each particular disease. Large animal models with multiple disease states, such as in the case of metabolic syndrome that encompasses dyslipidemia, insulin resistance, and obesity, better capture the complex comorbid disease states that clinicians encounter in daily practice. Models of metabolic syndrome or multiple disease states should be investigated further to capture specific mechanisms by which endothelium-dependent vasomotor signaling is affected. Similar limitations of oversimplification are noted in many studies on this topic that do not account for sex-based differences in endothelium-dependent vasomotor dysregulation. As previously discussed, there are important differences in endothelial signaling between men and women, and efforts by funding agencies and journals to establish a high standard of rigor with regard to male and female sex in both study design and reporting will be important to achieve a more detailed understanding of these differences (239).

An additional limitation to the bulk of research on mechanisms of endothelial injury is that effects from a particular disease model are often captured at a single point in time, without longitudinal functional assessments. Studies like those by Sorop et al. (113) and Oltman et al. (119) provide important insights into how the impact of a given risk factor on endothelial function can evolve over time. Additional longitudinal studies on mechanisms of endothelial dysfunction would provide insight not only into how consequences of clinical factors such as diabetes, hypertension, hyperlipidemia, and obesity affect the endothelium over time, but also whether and for how long these changes are reversible. Understanding these relationships would provide better insight into therapeutic options for patients with sequelae of endothelial dysfunction.

As CMD is increasingly recognized as an important contributor to cardiovascular disease, exciting research contributions are growing at a rapid pace. Specific research areas that are driving the field forward are those which use novel measurements of coronary microvascular dysfunction such as IMR to investigate clinical implications of CMD and to assess treatment strategies (232, 240–242). This trend in the research field will provide more accurate assessments of and mechanistic insights into coronary endothelial-dependent vasomotor dysfunction. Genetic and epigenetic determinants of microvascular dysfunction are increasingly being considered in different vascular beds, and the few studies that investigate these important changes in the coronary microvasculature provide critical insights that will help translate to targeted therapies (167, 223). Future studies in this area may help to clarify and reconcile discrepant findings in different study models, and account for longitudinal changes in the effects of disease processes on endothelial function. Finally, as previously discussed, study models that account for multiple comorbidities simultaneously and separately help to push the field of CMD forward and provide clinically relevant information on patients that present with multiple comorbidities. Therefore, with advances in imaging modalities, understanding of pathophysiology, and innovative research designs in CMD, there is much to look forward to in the field of coronary microvascular endothelial function and vasomotor tone.

CONCLUSIONS

The coronary microvasculature involves several interrelated molecular pathways that are vital to maintaining homeostasis and preserving adequate blood flow. Dysregulation of coronary endothelial and arteriolar function contributes to negative cardiovascular outcomes. Several recent studies have highlighted the specific pathophysiological mechanisms by which inflammation, oxidative stress, hyperglycemia, hyperlipidemia, obesity, hypertension, aging, and tissue ischemia dysregulate microvasculature. Most insults to the endothelium have effects on redox signaling and nitric oxide availability, which disrupt normal endothelium-dependent vasomotor function. Sex-based differences in endothelial dysfunction are also significant and an ongoing topic of investigation. Future studies that elucidate mechanisms of coronary microvascular injury, particularly longitudinal effects and translational studies in more clinically relevant models will be necessary to develop therapies that target coronary microvascular disease in clinical practice.

GRANTS

This study was funded by National Heart, Lung, and Blood Institute Grants 1R01HL133624 (to M. R. Abid), R01HL46716 and R01HL128831-01A1 (to F. W. Sellke), and 1F32HL160063-01 (to S. A. Sabe).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.A.S., F.W.S., and M.A.R. conceived and designed research; S.A.S. and F.W.S. prepared figures; S.A.S. and F.W.S. drafted manuscript; S.A.S., F.W.S. , and M.A.R. edited and revised manuscript; S.A.S., J.F., F.W.S., and M.A.R. approved final version of manuscript.