, Ali H. Eid 9,10,*1 Biomedical Research Center, College of Medicine, and Department of Biomedical Sciences, College of Health Sciences, Qatar University, P.O. Box 2713, Doha, Qatar
2 Faculty of Medicine, American University of Beirut, 11-0236 Beirut, Lebanon
3 Department of Health Sciences, Clinical Pharmacology and Oncology Section, University of Florence, 35630 Florence, Italy
4 Skolkovo Innovative Center, Institute for Atherosclerosis Research, 121609 Moscow, Russia
5 Laboratory of Angiopathology, Institute of General Pathology and Pathophysiology, 125315 Moscow, Russia
6 Faculty of Pharmacy, Al Alamein International University, 35016 Al Alamein, Egypt
7 Department of Medical Laboratory Sciences, College of Health Sciences and Sharjah Institute for Medical Research, University of Sharjah, 27272 Sharjah, United Arab Emirates
8 Department of Biomedical Sciences, University of Sassari, 07100 Sassari, Italy
9 Department of Basic Medical Sciences, College of Medicine, QU Health, Qatar University, P.O. Box 2713 Doha, Qatar
10 Biomedical and Pharmaceutical Research Unit, QU Health, Qatar University, P.O. Box 2713 Doha, Qatar
†These authors contributed equally.
Academic Editor: Giordano Pula
Abstract
Cardiovascular disease (CVD) is a major cause of mortality worldwide. A better understanding of the mechanisms underlying CVD is key for better management or prevention. Oxidative stress has been strongly implicated in the pathogenesis of CVD. Indeed, several studies demonstrated that reactive oxygen species (ROS), via different mechanisms, can lead to endothelial cell (EC) dysfunction, a major player in the etiology of several CVDs. ROS appears to modulate a plethora of EC biological processes that are critical for the integrity of the endothelial function. This review seeks to dissect the role of oxidative stress-induced endothelial dysfunction in CVD development, with emphasis on the underlying mechanisms and pathways. Special attention is given to ROS-induced reduction of NO bioavailability, ROS-induced inflammation, and ROS-induced mitochondrial dysfunction. A better understanding and appraisal of these pathways may be essential to attenuate oxidative stress or reverse EC dysfunction, and hence, reduce CVD burden.
Keywords
- endothelial cell dysfunction
- oxidative stress
- reactive oxygen species (ROS)
- cardiovascular diseases (CVDs)
- nitric oxide
- eNOS uncoupling
- inflammation
- mitochondrial dysfunction
Cardiovascular disease (CVD) remains the number one killer disease where it claims 17.9 million lives each year accounting for 31% of the total deaths in the world. Several risk factors such as sedentary lifestyle, tobacco smoking, air pollution, an unhealthy diet, physical inactivity, and obesity contribute to the increasing incidence of CVDs [1, 2]. Importantly, most of these risk factors are modifiable [3], providing a means of preventive and therapeutic interventions. CVD risk factors could manifest as elevated blood pressure (hypertension), increased blood glucose levels (diabetes mellitus), in addition to overweight, abnormal blood lipids (dyslipidemia), and obesity [3, 4, 5]. Of note, inflammation has been recently recognized as a critical CVD risk factor [6, 7, 8, 9]. Relatedly, oxidative stress has been implicated, at least in part, in the incidence and pathogenesis of several CVDs [10] and as such, this review will highlight the involvement of oxidative stress in the pathogenesis of CVDs.
Despite extensive efforts to curb their incidence and progression, CVDs continue to cause increasing economic and health burden of across the globe [1, 10, 11]. Consequently, there is an immediate need to find new therapeutic routes of CVDs in order to reduce their burden. Hence, understanding the role of oxidative stress in relation to the progression of different CVDs is a step in this direction.
Oxidative stress can result from the over-production or accumulation of free
radical reactive species such as the oxygen reactive species (ROS), nitrogen
reactive species (RNS), and reactive sulfur species (RSS) [12]. Inside the cell,
oxidative stress is under a tight control [13]. Very low levels of the free
radical reactive species are normally produced as by-products of cellular
metabolic processes [14] where several of these species, such as hydrogen
peroxide (H
Among the reactive species, ROS gets the lion’s share of investigations,
apparently because it contributes to physiological signaling and the maintenance
of cellular redox state. ROS, which include molecules such as H
Accumulating evidence strongly implicates oxidative stress in the pathogenesis of CVDs, including hypertension, atherosclerosis, aortic aneurysms and vascular restenosis [27, 28, 29]. In fact, oxidative stress-induced alterations of endothelial cells (ECs) or vascular smooth muscle cells (VSMCs) are among the critical factors that regulate blood pressure [30]. Activation of NOXs, which are expressed by ECs and VSMCs [27], leads to excessive generation of ROS [31] and, in extension, oxidative stress. This status is permissive for the onset of some CVDs by inducing EC dysfunction and inflammation, depressing the levels of nitric oxide (NO), promoting VSMC proliferation, migration and deposition of extracellular matrix (ECM) proteins, as well as altering vascular response and vasotone (Fig. 1) [27, 32, 33, 34]. It is not surprising, then, that inhibiting ROS generation, via antioxidants, reduces blood pressure in rodent animal models [29].
Fig. 1.Mechanism of oxidative stress-induced endothelial dysfunction leading to CVD. Oxidative stress, high levels of ROS for example, can induce inflammation, mitochondrial dysfunction, and eNOS uncoupling and decreased NO bioavailability. These in turn contribute to endothelial dysfunction by increasing ECs adhesion to monocytes, elevating the rates of apoptotic cell death, impairing ECs angiogenic potential, among others. Dysfunctional ECs can contribute to CVD directly by increased monocyte adhesion in atherosclerosis for example, or indirectly by enhancing the phenotypic switching of VSMCs. VSMCs switch into the synthetic de-differentiated phenotype that contributes to dysregulation of vasotone and development of CVD. In this scenario, ROS-induced mitochondrial dysfunction leads to the production of additional ROS, by altered mitochondrial metabolism or NOXs for example, causing the exacerbation of the endothelial dysfunction. ROS, reactive oxygen species; eNOS, endothelial nitric oxide synthase; NO, nitric oxide; ECs, endothelial cells; CVD, Cardiovascular disease; VSMCs, vascular smooth muscle cells; NOXs, NADPH oxidases.
Obviously, endothelial dysfunction by itself may not be sufficient to elicit all the pathological aspects of CVD. That is mainly because VSMCs and perivascular adipose tissue contribute to vascular homeostasis by virtue of their ability to produce vasoactive compounds such as adipokines, ROS, and NO [35]. Given the variety of cells and processes involved, this review will focus on oxidative stress-elicited vascular alterations, specifically those that can induce endothelial dysfunction, contributing to the pathogenesis of CVD. EC dysfunction is reversible, making approaches that can reverse it attractive avenues in the management of CVDs [36]. Heightened oxidative stress can cause EC dysfunction in several ways. ROS can compromise endothelium-dependent vasorelaxation, induce apoptosis of ECs, increase ECs adhesion to monocytes, or modify ECs angiogenesis potential (Fig. 1) [37]. Below, we discuss the major mechanism of oxidative stress-induced EC dysfunction, namely ROS-induced reduction of NO bioavailability, ROS-induced inflammation, and ROS-induced mitochondrial dysfunction.
Excessive ROS production through NOX activation or other oxidative stress generating systems inside the cell, like monoamine oxidase, xanthine oxidase, cyclooxygenase, lipoxygenase, or mitochondrial metabolism, can result in a state of oxidative stress [31]. This stress can lead to vascular damage by targeting a repertoire of vascular processes. Indeed, augmented ROS depresses levels of NO, induces monocyte invasion, elevates lipid peroxidation, promotes phenotype switching of VSMCs, induces EC dysfunction, precipitates inflammation as well as alters vascular responses and vasotone (Fig. 1) [27, 32, 33]. Collectively, evidence greatly implicates oxidative stress in the development of CVD including hypertension, atherosclerosis, heart failure, atrial fibrillation, aortic aneurysms and vascular restenosis [10, 27, 28, 29].
In the case of hypertension, the evidence includes the fact that oxidative
stress of ECs or VSMCs is among the factors that regulate blood pressure [30].
Consistently, the use of antioxidants to inhibit ROS generation has been shown to
reduce blood pressure in rodent animal models [29]. Further evidence includes the
fact that ROS production is enhanced in animal models of experimental as well as
genetic hypertension [29, 38, 39, 40, 41]. Also, when ROS generation is dampened by NOX
inhibitors, xanthine oxidase inhibitors, free radical-scavenging antioxidants, or
SOD mimetics, then blood pressure will drop and hypertension will not develop in
rodent models of hypertension [23, 24, 38, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53]. In human patients, there is a
clear increase of ROS generation during essential hypertension, renovascular
hypertension, and other kinds of hypertension such as malignant hypertension and
salt-sensitive hypertension [30, 44, 48, 53, 54, 55, 56]. Also, in human population-based
observational studies, there is an inverse relation between blood pressure and
the levels of blood antioxidants. Similarly, clinical studies in hypertensive
patients have revealed a negative correlation between diastolic blood pressure
and the levels of NO and antioxidant enzymes [53, 57]. On the other hand, there
is a positive correlation between high blood pressure and an elevation of tissue
concentrations of O
The role of EC dysfunction in the pathogenesis of atherosclerosis has been well-established. Indeed, EC dysfunction facilitates the leakage of low-density lipoprotein (LDL) molecules into the sub-endothelial cell layer (intima) where they accumulate and then become oxidized in a mechanism that largely depend on ROS. Oxidized LDL (ox-LDL) then induces dysfunctional ECs to produce cell adhesion molecules, such as VCAM-1 and ICAM-1, which attract inflammatory leukocytes into the sub-endothelial space [62, 63]. These inflammatory cells release interleukins and other pro-inflammatory cytokines which eventually potentiate proliferation and migration of VSMCs, as well as increase matrix and lipid deposition, leading to the formation of an atherosclerotic plaque [32, 63, 64, 65, 66]. Importantly, atherosclerotic plaques usually advance more readily in the presence of dysfunctional ECs that have lost their NO production ability. This is not surprising as atherosclerosis is an inflammatory disease and NO has potent anti-inflammatory properties [32, 66].
One of the key players in oxidative stress-precipitated atherosclerotic disease
is Nox2. Indeed, expression of Nox2 has been found to be upregulated in
macrophages and aortic ECs of apolipoprotein E (ApoE) knockout mice just before
the incidence of atherosclerotic lesions [67]. Importantly, a direct evidence
linking the incidence of these early lesions to reduced NO bioavailability and
incresed O
Evidence that implicates oxidative stress in the development of heart failure encompasses different experimental as well as clinical studies that correlated an increase in ROS generation with the incidence of heart failure [10, 68, 69, 70]. Similarly, there is evidence for the involvement of ROS in other CVDs [10, 71]. Noteworthy, it is suggested that the common risk factors for CVDs like diabetes mellitus, smoking, aging, and others progress, at least in part, through further generation of ROS, thus leading to the exacerbation of oxidative stress and the pathogenesis of CVDs [71, 72].
Endothelial cells line the interior surface of blood and lymphatic vessels. They serve as the mechanical barrier between the circulating blood and VSMCs. As of recent, ECs are no longer thought of as an inert entity. Indeed, ECs have been shown instead to have both sensory and effector regulatory abilities as well as both metabolic and synthetic functions [73]. Indeed, ECs have been found to be involved in key homeostatic, immune, and inflammatory processes along the cardiovascular network [71, 73]. Endothelial cells are now acknowledged as major players in diverse physiological and metabolic functions including the control of thrombosis and thrombolysis, blood clotting system, platelet and leukocyte interaction with the wall of the vessel, formation and growth of blood vessels (angiogenesis), redox balance, the orchestration of acute and chronic inflammation and regulation of vascular tone [74, 75]. For example, ECs can control the tone of the underlying VSMCs by secreting various relaxing and contracting factors. Indeed, ECs secrete numerous vasodilator molecules including NO, prostacyclin, H2S, and EDHFs (Endothelium-Derived Hyperpolarizing Factor) or vasoconstrictive molecules such as thromboxane A2, endothelin-1, and PDGF, effectively contributing to the regulation of vascular tone [11, 35, 73, 76]. As such, endothelium functions are regulated and maintained by multiple cell surface receptors where the activation of a set of which can induce ECs to release vasoactive agents that modulate VSMCs proliferation and vascular tone [11, 77]. Local as well as circulating signals can stimulate the vascular endothelium to release either vasodilators or vasoconstrictors; depending on the receptor activated. Imbalance of the released vasoactive agents can cause an increase in ROS generation which can then lead to endothelial dysfunction and eventually CVDs, including hypertension and atherosclerosis [11, 44, 78, 79]. Given the versatility of functions carried out by ECs, the vascular endothelium can be viewed as an extended and distributed organ with a dynamic and adaptable interface. Furthermore, at the single cell level, ECs can act as integrators of the local pathophysiological microenvironment; hence any dysfunction of the integrators, even broadly, would encompass implications for CVDs incidence and progression [75]. Overall, it is not surprising that endothelial dysfunction can predict the progression of anatomically overt vascular diseases and prominently correlate with the progression of CVDs including hypertension and atherosclerosis [11, 80].
Consistent with the above discussion, endothelial dysfunction has been acknowledged not only as a pathological state of the endothelium, but also as a predictor of various CVDs or even mortality [35, 71, 81, 82]. Notably, endothelial dysfunction is considered as the hallmark of hypertension [79]. As well, endothelial dysfunction is the earliest observable change in the chronology of an atherosclerotic lesion [75]. Indeed, the ability to measure endothelial dysfunction in patients, through measurement of acetylcholine-dependent dilation or flow-mediated dilation (FMD), makes EC dysfunction a measurable as well as early predictor of CVDs [35]. In accordance, impaired endothelial function has been demonstrated in patients with CVDs including peripheral arterial occlusive disease, coronary artery disease, or heart failure [83].
Endothelial dysfunction is mainly caused by an imbalance of the production and the bioavailability of vasodilating versus vasoconstricting agents [75]. It culminates in impaired endothelium-dependent relaxation of vessels mostly due to decreased vascular bioavailability of NO [84]. In this context, the definition of endothelial dysfunction can be expanded to include all of the maladaptive alterations in the functional phenotype of ECs that can be correlated with a CVD [75]. The imbalance in the bioavailability of vasodilators versus vasoconstrictors can be due to impaired production of different vasoactive agents released by ECs, VSMCs, or perivascular adipose tissue. This imbalance can result in an altered endothelial cell phenotype characterized by being vasoconstrictor, pro-inflammatory, pro-atherothrombotic, and pro-proliferative; and this EC phenotype leads to impaired regulation of blood flow and/or vascular tone. Collectively, this EC state may be referred to as endothelial dysfunction (Fig. 1) [35]. Overall, endothelial dysfunction can be considered a hallmark of vascular injury in most CVDs.
The pathophysiological events that can drive endothelial dysfunction are diverse
and include hypercholesterolemia (e.g., oxidatively modified lipoproteins),
metabolic syndrome (e.g., advanced glycation end products, ROS, adipokines),
hypertension (e.g., angiotensin-II, ROS), aging (e.g., advanced glycation end
products, cell senescence), proinflammatory cytokines (e.g., Interleukin-1
(IL-1), Tumor Necrosis Factor-
Of particular interest to this review, endothelial dysfunction is associated with increased vascular ROS production, oxidative stress, and vascular inflammation in patients with hypertension [53]. In this regard, ROS levels increase in isolated arteries exposed to high pressure in vitro [85] leading to endothelial dysfunction [86]. Furthermore, short-term increases in blood pressure can also disrupt endothelial function and enhance oxidative stress in vivo [83, 87, 88, 89]. Also, ROS contribute to EC dysfunction in experimental and clinical atherosclerosis [37, 71]. To add, excessive ROS production damages endothelial cells, especially at the terminal arteries, and causes the modification of intracellular endothelial redox homeostasis. In fact, many studies have demonstrated the notion that elevated production of ROS contributes to the oxidative alterations in the arterial wall, leading to alteration of the intracellular redox homeostasis and cellular damage [90, 91]. Finally, increased levels of oxidative stress can cause EC dysfunction in several ways as has been discussed (Fig. 1) [37].
Nitric oxide signaling is critical for metabolic and vascular health including normal EC function. It is a key vasodilator produced by ECs and it regulates vascular tone and has anti-inflammatory, antioxidant, and antithrombotic effects [92]. It can prevent aggregation of platelets and multiplication of VSMCs [29]. It is also known to regulate metabolic homeostasis. Of note, endothelial dysfunction is evident in endothelial cells that do not produce sufficient amounts of NO and, therefore, are not capable of inducing suitable vasodilation of the vasculature. Also, NO release by ECs in the vasculature can be measured in patients as an alteration of FMD, and this is highly correlated with the extent of endothelial damage in patients. In agreement, NO release by endothelial cells is the chief cause of FMD [93]. Lastly, dysregulation of NO production or its signaling responses can be correlated with cardio-metabolic disorders [92].
Four distinct isoforms of nitric oxide synthase (NOS) are responsible for NO
production namely: NOS 1 or neuronal NOS (nNOS), NOS 2 or inducible NOS (iNOS),
NOS 3 or endothelial NOS (eNOS), and NOS 4 or mitochondrial NOS (mtNOS). NOS
converts its substrate, L-arginine, into L-citrulline in the presence of O
Fig. 2.Physiological Nitric Oxide (NO) signaling pathway
leading to vascular relaxation. NO is generated in ECs mainly due to the
catalytic activity of eNOS. eNOS converts L-Arginine into L-Citrulline and
releasing NO, in the presence of O
One manifestation of ROS-induced endothelial dysfunction is a decrease in NO
bioavailability which leads to a vasoconstrictive, proinflammatory, proliferative
and thrombotic status; that is EC dysfunction [44, 93]. Reduction of NO
bioavailability, and hence induction of endothelial dysfunction, can take place
in the following ways: (1) inactivation of NO via its reaction with
O
Fig. 3. Major mechanisms leading to reduction of NO bioavailability
contributing to induction of endothelial dysfunction. The dashed arrows indicate
pathways that lead to a decrease in NO levels. (a) and (b) A reduction in the
levels of eNOS substrate (L-Arginine) or cofactor (BH4). (c) Inactivation of NO
through reaction with O
ROS can directly interact with NO, effectively reducing the bioavailable NO
levels, to produce ONOO
Fig. 4.Oxidative stress-induced uncoupling of eNOS leading to
endothelial cell dysfunction. Oxidative stress, mainly ROS, can reduce the
levels of the eNOS cofactor BH4 by converting it into BH3• radical
leading to eNOS uncoupling. Uncoupled eNOS transfers electrons to O
It should be noted that there is crosstalk between the different ROS generating
pathways, leading to amplified generation of ROS in a manner that leads to a
feed-forward vicious cycle of ROS generation [35, 104, 105, 106]. For example, it is
well established that mitochondria and NOX, mitochondria and eNOS, NOS and
xanthine oxidase, or eNOS and NOX can crosstalk to enhance ROS generation in what
has become to be recognized as “ROS-induced ROS release”. This crosstalk is
required to sustain oxidative stress and the consequent pathogenesis of CVDs [25, 35, 104, 105, 106, 107]. Using bovine aortic ECs, Doughan et al. [108],
demonstrated that angiotensin II can activate PKC which induces NOX to produce
O
Fig. 5.Cross-talk between vascular endothelial dysfunction and
mitochondrial damage during angiotensin II signaling. Signaling of Ang II
through its receptor activates PKC as one of its downstream targets. Ang II
signaling leads to mitochondrial dysfunction due to the increased PKC-dependent
activation of NOXs which produce O
Overall, ROS, mainly O
Inflammation is a major contributor to vascular health and the pathogenesis of
CVDs. Indeed, inflammation is a risk factor of several CVDs [6]. Elevated levels
of several cytokines like IL-1, IL-6, IL-17A, and TNF-
It is well recognized that ROS can activate NF-
Fig. 6.Mechanisms of oxidative stress-induced inflammation
leading to endothelial dysfunction. ROS can cause the activation of the IKK
complex through oxidation of IKK
Oxidative stress and high levels of ROS result in oxidation of numerous proteins [117]. The oxidation of proteins can result in the secretion of inflammatory mediators, such as the inflammatory signaling molecule, peroxiredoxin-2. In fact, peroxiredoxin-2 is thought of as a link between oxidative stress and inflammation (Fig. 6) [118].
In another mechanism, oxidative stress contributes to the activation of NLRP3
(NOD-like receptor protein 3) inflammasome complex through several means [119, 120]. ROS produced by injured mitochondria can induce the activation of the NLRP3
inflammasome to produce IL-1
A major evidence of ROS-induced inflammation is that knock-out of ROS producing enzymes, like Nox, attenuates ROS-induced inflammation [25]. In addition, NOX activation is known to trigger inflammation [123]. Noteworthy, there is an interplay between oxidative stress and inflammation during EC dysfunction. For example, inflammation, once initiated by ROS, can in turn affect the activity of NOXs [124, 125].
Endothelial cells obtain much of their energy (around 75–99%) from glycolysis instead of oxidative mitochondrial metabolism [126]. Nevertheless, EC mitochondria remain physiologically relevant partly due to their ability to produce ROS. Mitochondrial ROS production can take place at complex I or complex III [127], and mitochondria-produced ROS are major contributors of oxidative stress and, hence, EC dysfunction [109]. In addition, mitochondrial ROS generation can be increased by other sources of ROS such as NOX-derived ROS, in a crosstalk scheme as discussed earlier [128]. In fact, this crosstalk is required for NOX-derived ROS to act on the endothelium [128].
ROS is not only produced by the mitochondria, but also the mitochondria is affected by ROS where excessive ROS has critical effects on mitochondria and may even lead to mitochondrial dysfunction. Importantly, mitochondrial damage can accelerate ROS-induced endothelial dysfunction. It is important to note that endothelial cells are not only exposed to endogenous oxidative species, but also to plenty of exogenous sources of reactive species that compound the ROS-induced endothelial dysfunction. For example, during atherosclerosis, activated neutrophils produce large amounts of ROS at areas of damaged endothelium [126].
Maintenance of Ca
Mitochondrial-generated ROS can also target and damage the mitochondrial
electron transport chain (ETC). Mitochondrial O
Relatedly, increased outer mitochondrial membrane permeability can enhance the release of mitochondrial constituents like ROS and mtDNA into the cytoplasm. This event can induce inflammation or apoptosis in several ways [135].
It is evident that ROS-induced mitochondrial damage contributes to EC dysfunction through several mechanisms. Further, exploration of these mechanisms can give insight into novel therapies of CVDs.
Imbalance of the oxidative state of the vasculature contributes to initiation and progression of CVDs. Moreover, normalization of this oxidative state has been shown to benefit vascular health by acting on VSMC, ECs, and other cells of the vasculature. In particular, the ensuing ROS-induced endothelial dysfunction has a multitude of clinical manifestations, in CVD and other diseases. Importantly, blockade of ROS generation or the use of antioxidants have been shown to be able to normalize the disrupted EC functions [136], and may thus prove vital for reversing EC dysfunction and ameliorating symptoms of CVD. Further exploration of the involved mechanisms may provide insight into newer therapies that can prevent or treat CVD.
Conceptualization—AHE; methodology—AS, KA, RA, AP, AO, AEY, GP, AHE; formal analysis—AS, KA, RA, AP, AO, AEY, GP, AHE; writing - original draft preparation—AS, KA, RA; writing - review and editing—AS, KA, RA, AP, AO, AEY, GP, AHE; supervision—AHE; project administration—GP, AHE; funding acquisition—GP. All authors have read and agreed to the published version of the manuscript.
Not applicable.
Not applicable.
This work has been made possible thanks to grants from the University of Sharjah (Seed 2001050151, collaborative 2101050160), fondo UNISS di Ateneo per la Ricerca 2020.
The authors declare no conflict of interest. GP (Gianfranco Pintus) is serving as one of the Editorial Board members of this journal. We declare that GP (Gianfranco Pintus) had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to GP (Giordano Pula).