Oxidative Stress and New Pathogenetic Mechanisms in Endothelial Dysfunction: Potential Diagnostic Biomarkers and Therapeutic Targets

Cardiovascular diseases (CVD), including heart and pathological circulatory conditions, are the world’s leading cause of mortality and morbidity. Endothelial dysfunction involved in CVD pathogenesis is a trigger, or consequence, of oxidative stress and inflammation. Endothelial dysfunction is defined as a diminished production/availability of nitric oxide, with or without an imbalance between endothelium-derived contracting, and relaxing factors associated with a pro-inflammatory and prothrombotic status. Endothelial dysfunction-induced phenotypic changes include up-regulated expression of adhesion molecules and increased chemokine secretion, leukocyte adherence, cell permeability, low-density lipoprotein oxidation, platelet activation, and vascular smooth muscle cell proliferation and migration. Inflammation-induced oxidative stress results in an increased accumulation of reactive oxygen species (ROS), mainly derived from mitochondria. Excessive ROS production causes oxidation of macromolecules inducing cell apoptosis mediated by cytochrome-c release. Oxidation of mitochondrial cardiolipin loosens cytochrome-c binding, thus, favoring its cytosolic release and activation of the apoptotic cascade. Oxidative stress increases vascular permeability, promotes leukocyte adhesion, and induces alterations in endothelial signal transduction and redox-regulated transcription factors. Identification of new endothelial dysfunction-related oxidative stress markers represents a research goal for better prevention and therapy of CVD. New-generation therapeutic approaches based on carriers, gene therapy, cardiolipin stabilizer, and enzyme inhibitors have proved useful in clinical practice to counteract endothelial dysfunction. Experimental studies are in continuous development to discover new personalized treatments. Gene regulatory mechanisms, implicated in endothelial dysfunction, represent potential new targets for developing drugs able to prevent and counteract CVD-related endothelial dysfunction. Nevertheless, many challenges remain to overcome before these technologies and personalized therapeutic strategies can be used in CVD management.

2.1. NADPH Oxidase (NOX) Activity and ROS Production As previously reported, ROS plays a pivotal role in CVD, including hypertension, atherosclerosis, diabetes, cardiac hypertrophy, and heart failure. However, physiological ROS production is essential for the maintenance of normal vascular homeostasis [ 64 ]. In the vasculature, several enzyme systems, differentially localized and expressed, contribute to ROS formation. These include the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, endothelial nitric oxide (NO) synthase, enzymes of the respiratory chain, cytochrome P450 monooxygenase, and xanthine oxidase [ 64 ]. Among these systems, NADPH oxidase seems to play a central role in orchestrating the activation and dysfunction of other enzymes. It is the main source of ROS in the vessel wall [ 64 , 65 ]. In general, it is accepted that under physiologic conditions, vascular NADPH oxidases have a relatively low level of constitutive activity that exerts an important role in the cardiovascular system homeostasis [ 64 , 65 , 66 ]. However, enzyme activity can be increased both acutely and chronically in response to stimuli such as cytokines, growth factors, hyperlipidemia, and high glucose, which can impair vascular functions and cause CVD [ 64 ]. Different isoforms named Nox1, Nox2, Nox4, and Nox5 are expressed in the human vasculature. Nox4 is the predominant isoform in endothelial cells and mainly produces H 2 O 2 , which might explain the functional differences compared to other Nox isoforms producing superoxide anion (O2-) [ 67 ]. The specific contribution of each Nox isoform in cardiovascular physiology and disease remains unclear. Several studies on animal models have been performed to clarify the role of Nox isoforms in CVD. For example, Nox1 overexpression in the media has been reported in increased neo-intima formation after vascular injury [ 64 ]. Moreover, Nox1 and Nox5 are proven to induce O2- formation and favor human vascular smooth muscle cells (VSMC) proliferation [ 68 ]. Nox5 can contribute to the oxidative stress in human coronary artery disease [ 67 ]. The upregulation of Nox1 and Nox2 in vascular cells has been demonstrated to contribute to atherosclerosis and vascular diseases [ 67 ]. The association between endothelial dysfunction and Nox2 has been studied in several conditions such as dyslipidemia, obesity, smoking, hypertension, and aging [ 68 ]. Nox2 levels were found increased in the aortas of apolipoprotein E (ApoE)−/− atherosclerotic mice, whereas mice overexpressing Nox2 showed endothelial activation [ 64 , 68 ]. The relationship between Nox2 and aging has been investigated in ApoE−/− mice, which showed an upregulation of Nox2 in atherosclerotic plaque [ 68 ]. Endothelial-specific overexpression of Nox2 in transgenic mice has been reported to enhance angiotensin II-induced endothelial dysfunction contributing to vascular remodeling and hypertension [ 69 ]. However, in humans, the genetic deficiency of Nox2 is associated with enhanced endothelium-dependent flow-mediated vasorelaxation, decreased markers of vascular aging and oxidative stress [ 68 ]. In coronary arteries, patients with a congenital heart disease were found to have enhanced superoxide production in association with the upregulation of Nox2 [ 70 ]. Recent studies have put attention on the major physiologic and pathological roles of Nox4. Nox4 has been reported to contribute to oxidative stress, but evidence from Nox4−/− mice suggests that endogenous Nox4 has a vasoprotective function during ischemic or inflammatory stress [ 64 ]. Some animal studies showed that a genetic deficiency of Nox4 was associated with endothelial dysfunction and increased atherosclerosis burden [ 68 ]. However, Nox4−/− mice were protected from oxidative stress, blood–brain barrier leakage, and neuronal apoptosis in a stroke model [ 71 ]. In another model of cardiac-specific deletion of Nox4, cardiac hypertrophy, fibrosis, and apoptosis after pressure overload was reduced, suggesting different cell and dose-dependent responses of Nox4 in CVD [ 67 ]. It has also been postulated that increased activity of Nox4 or a putative “uncoupling” of Nox4 may switch its activity from H 2 O 2 to O2- formation explaining its deleterious effects on vascular functions [ 65 ].

Glutathione peroxidase (GPx) is a selenium-containing antioxidant enzyme that reduces hydrogen peroxide and lipid peroxides into water and lipid alcohols, respectively, and in turn, oxidizes glutathione into glutathione disulfide (GSSG). In the absence of adequate GPx activity or glutathione levels, hydrogen peroxide and lipid peroxides are not detoxified and may be converted to hydroxyl radicals and lipid peroxyl radicals [ 74 ]. In humans, four isoforms of GPxs are known, each with the selenocysteine at the active site. The most abundant cellular form of GPx is GPx-1. GPx-1 deficiency has been linked to the development of endothelial dysfunction, inflammation, and neointimal formation [ 77 ]. The role of GPx-1 has been demonstrated in the normalization of VSMC proliferation rate through the inhibition of matrix metalloproteinase 9 (MMP9), and the consequent restoring of the endothelial function [ 73 ]. A more aggressive atherosclerosis has been reported in double knockout (ApoE−/−) (GPx-1−/−) mice compared to (ApoE−/−) controls, with a marked increase in ROS levels and a decrease in NO bioavailability, which was associated with increased protein nitration within the atherosclerotic lesions [ 78 ].

Thioredoxin system consists of both thioredoxin (Trx) and thioredoxin reductase (TrxR) that can reduce hydrogen peroxide and target proteins. The two mammalian isoforms Trx (Trx1 and Trx2) and three isoenzymes of TrxR (Trx1-3) are broadly expressed and can be found intra- and extracellularly. The thioredoxin system may effectively regenerate proteins that were inactivated by oxidative stress [ 74 ]. Trx acts by reducing ROS as well as controlling apoptosis and inflammatory response. A pivotal role of Trx has been reported in the regulation of endothelial cell survival, protecting the cells from laminar shear stress, and inhibiting the activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) consequent to tumor necrosis factor alpha (TNF-α) exposure [ 3 ]. Trx was also found in medial smooth muscle cells of coronary arteries of healthy humans, while a diffused expression pattern was evident in atherosclerotic arteries [ 73 ]. The system was also reported to exert an essential role in regulating metabolic processes, insulin signaling, blood pressure regulation, and inflammation [ 73 ].

Transcription factors are the core of intracellular signaling, as they integrate multiple inputs from the environment, such as ROS and NO, and translate them into coordinated cellular responses [ 83 ]. Among transcription factors, nuclear factor kappa B (NF-κB) or nuclear factor-2 erythroid related factor-2 (Nrf2) have been primarily studied in different experimental models (Stefanie Kohlgrüber Antioxid Redox Signal. 2017). NF-κB is a transcription factor, involved in pro-inflammatory cytokine production and endothelial dysfunction [ 3 , 84 ]. The herbal medicinal product Phytodolor ® (STW 1) and its components have shown anti-inflammatory properties on lipopolysaccharide (LPS)-activated human macrophages by inhibiting the translocation of the subunit p65 of NF-κB into the nucleus [ 85 ]. IMM-H007 (H007) is a small molecule compound with anti-inflammatory properties regulating endothelium inflammation [ 86 ]. It has been reported that H007 significantly reduced monocyte adhesion and transendothelial migration by inhibiting TNFα-mediated inhibitor of nuclear factor kappa B (Iκ-Bα) degradation and NF-κB nuclear translocation, as well as preventing JNK/c-Jun phosphorylation [ 86 ]. Bromodomain and extraterminal (BET) proteins are essential for the expression of a subset of NF-kB-induced inflammatory genes. BET mimics, including JQ1+, prevent binding of BETs to acetylated histones and down-regulate the expression of selected genes. JQ1+ decreased NF-kB p65 recruitment to native interleukin-6 (IL-6) and interleukin-8 (IL-8) promoters, proliferation, and migration of primary human pulmonary microvascular endothelial cells [ 87 ]. Nrf2 is a redox-regulated transcription factor in the regulation of antioxidant defense systems [ 84 ]. Nrf2 interacts with antioxidant response element sequences of genes coding for antioxidant enzymes, including γ-glutamyl cysteine ligase, NAD(P)H quinone oxidoreductase-1, glutathione S-transferase, heme oxygenase-1, uridine diphosphate glucuronosyltransferase, superoxide dismutase, catalase, and glutathione peroxidase-1. These enzymes play a crucial role in protecting the endothelium from ROS induced-endothelial dysfunction [ 88 ]. It has been reported that Nrf2-driven free radical detoxification pathways exert an important vasoprotective function against aging, atherosclerosis, hypertension, diabetes, ischemia, and smoking-related cardiovascular diseases [ 89 ]. Nrf2 activators can act directly, preventing the interaction of Nrf2 with the Nrf2-binding site of Kelch-ECH-associated protein-1 (KEAP1), like the case of ML334, or they can favor the transcription of Nrf2-targeted antioxidant genes (berberine) or increasing Nrf2 half-life (MG-132) [ 88 ]. Pharmacological activation of Nrf2 with sulforaphane (SFN) proved to be effective in endothelium-dependent vasodilation and H 2 O 2 -induced relaxation in vascular beds of aging rats. The pharmacological activation of Nrf2 improved age-related impairment of endothelium-dependent and ROS-induced vasodilation in different rat and human vascular districts by up-regulating Nrf2-related genes and decreasing oxidative stress [ 90 ]. Hypoxia-inducible factor-1α (HIF-1α) is a hypoxia-specific transcription factor-induced at the transcriptional, post-transcriptional, and posttranslational levels by hypoxia and can be used to restrict gene expression to ischemic areas [ 91 ]. It is implicated in the development of atherosclerosis, acting on endothelial cells (ECs), VSMC, and macrophages [ 92 ]. Prolyl hydroxylase (PHD) inhibitors, stabilizers of hypoxia-inducible factors (HIFs), have been used to treat acute organ injuries such as renal ischemia-reperfusion, myocardial infarction, and, in some contexts, chronic kidney disease [ 93 ]. Enarodustat (PHD inhibitor) proved to reduce cardiac hypertrophy and fibrosis in a unilateral urinary obstruction (UUO) model in mice [ 94 ]. Moreover, enarodustat counteracted kidney fibrosis reducing pro-inflammatory cytokine expression, apoptosis, and improving capillary density in a nephrectomy model [ 93 ].

One of the ROS subsets is represented by reactive nitrogen species (RNS), which includes peroxynitrite (ONOO−), dinitrogen trioxide (N 2 O 3 ), and the nitrosonium ion (NO + ). RNS determine post-translational modifications of proteins and nitrative stress with RNS-induced modifications, such as S-nitrosation and tyrosine nitration, which induce cellular dysfunction [ 99 ]. Different nitroproteins have been identified in plasma, but the ELISA for nitrated albumin remains the only clinically validated quantitative assay [ 100 ]. NO metabolites, NO synthase inhibitors, and N-acetyl-β-glucosaminidase (NAGase) are biomarkers capable of measuring endothelial dysfunction and oxidative stress in the early stages of impaired response to insulin [ 101 ]. NO metabolites and NAGase activity were elevated in glucose intolerance and type-2 diabetes (TD2) patients, while nitrotyrosine is higher only in the TD2 group [ 101 ]. Once formed, superoxide anions can inactivate NO, leading to the generation of ONOO- that is a potent oxidant [ 102 ]. ONOO- inhibits endothelium-dependent vasorelaxation, reducing the beneficial effects of NO on platelet aggregation and vascular smooth muscle cell proliferation and causing the oxidation of DNA and lipids; altogether, these factors are involved in the development of atherosclerosis [ 102 ]. Moreover, it has also been reported that aging increases nitrative stress in resistance arteries, evidenced by elevated ONOO- production in serum of aged mice [ 103 ]. ONOO- was responsible for the endothelium-dependent vasorelaxation dysfunction recovered by the treatment with FeTMPyP (ONOO- scavenger) [ 103 ].

Endothelial inflammation is indicated by elevated levels of soluble vascular cell adhesion molecule (sVCAM), soluble intercellular adhesion molecule (sICAM), E-selectin, along with ACE, von Willebrand factor (VWF), a disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS-13), C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), and other inflammatory cytokines [ 34 , 110 , 111 ]. The circulating non-transferrin-bound iron level has been found higher in diabetic patients. Besides, malondialdehyde plasma levels have been reported to be higher in both TD2 and obese groups and to be associated with higher levels of oxidized ascorbate [ 112 ]. E-selectin has found elevated in TD2 patients as well as CRP levels, which has been reported to be higher in TD2 and obese patients [ 112 ].

Endoglin (Eng) is a transmembrane glycoprotein and a co-receptor of transforming growth factor-beta (TGF-β) complex [ 115 ]. However, a soluble form of endoglin (sEng) can be produced by the proteolytic action of MMP-14. It has been found on endothelial cells, activated macrophages, activated monocytes, fibroblasts, and smooth muscle cells. It is involved in cardiovascular development, angiogenesis, and vascular remodeling [ 115 ]. It has been demonstrated that sEng levels increased both the expression of cell adhesion molecules and the number of rolling leukocytes, and that they impaired endothelial-dependent vascular function [ 116 ]. Moreover, sEng levels were higher in blood during endothelial injury or inflammation, but also in other vascular pathological conditions, such as atherosclerosis, hypercholesterolemia, hypertension, and T2D [ 117 ]. A clinical study conducted on 288 patients with T2D, hypertension, and healthy controls showed a significant correlation between endoglin and glycemia, glycated hemoglobin, systolic blood pressure, left ventricular hypertrophy and endothelial dysfunction [ 118 ]. Transgenic mice with high expression of human sEng showed arterial blood pressure higher than control, but a similar endothelium-dependent vascular function and expression of adhesion molecules [ 116 ]. In light of this evidence, further studies are needed to clarify better the role of Eng in the pathogenesis of endothelial dysfunction and CVD.

The impact of epigenetics in CVD is recently emerging as an essential regulatory role at a different level, from pathophysiology to therapy. Epigenetic studies in CVD were undertaken due to its prominent role in inflammation and vascular involvement and revealed a significant number of modifications affecting the development and progression of CVD [ 127 ]. Furthermore, epigenetics contributes to cardiovascular risk factors such as smoking, diabetes, hypertension, and age [ 127 ]. Epigenetics refers to all heritable changes in gene regulation without altering DNA sequencing. The primary epigenetic mechanism of human cells includes DNA methylation, posttranslational histone modifications, and non-coding RNA (i.e., long non-coding RNAs and microRNAs) [ 127 ]. Growing evidence suggests that endothelial response to blood flow is modulated through epigenetic mechanisms such as histone modification, chromatin remodeling, and microRNAs [ 128 ]. 4.1. DNA Methylation DNA methylation has been studied extensively and represents a well-understood epigenetic mechanism. During this process, cytosine (C) residues preceding guanosine (G) in the DNA sequence are methylated. CpG-islands are short interspersed DNA sequences with clusters of CG sequences. The abnormal methylation of CpG islands in the promoter region of genes leads to a silencing of genetic information and, finally, to alter cell function [ 129 ]. For instance, hyper- and hypomethylation can result in suppression and stimulation of transcription in human endothelial cells, in response to a disturbed flow [ 128 ]. As previously described, p66(Shc) upregulation is implicated in hyperglycemia-induced endothelial dysfunction and aging [ 47 , 48 , 49 ]. Its overexpression was reported to be epigenetically regulated by promoter CpG hypomethylation and nonderepressible, 5-induced, histone three acetylation [ 48 ]. Gene silencing of p66(Shc) has been proven to restore endothelium-dependent vasorelaxation, counteracting cell apoptosis by preventing cytochrome c release, caspase 3 activity, and cleavage of poly (ADP-ribose) polymerase in vitro and in vivo, suggesting p66(Shc) as a potential therapeutic target in endothelial dysfunction [ 48 ].

Recently, the regulatory role of small and long non-coding RNAs has been highlighted. MiRNAs are central orchestrators of these networks [ 131 ]. MiRNAs are small non-coding RNAs of approximately 22 bp in length. In general, miRNAs downregulate target gene expression [ 132 ]. miRNAs’ biosynthesis is a complex process that starts in the nucleus and is completed in the cytoplasm of the cell [ 132 ]. Several miRNAs have been proposed to regulate vascular function and the development of atherosclerosis from different pre-clinical and clinical studies [ 133 ]. MiRNAs mediate the adaptation of endothelial cells subjected to shear stress, thereby transforming the mechanical stimuli into intracellular signals [ 128 ]. MiRNAs are involved in the regulation of physiological and pathological processes, such as systemic arterial hypertension, atherosclerosis, T2D, and obesity [ 134 ]. Experimental evidence showed that dysregulation of microRNAs (miRNAs) has a role in vascular aging [ 135 ]. However, the molecular mechanisms of miRNAs are not still well understood [ 135 ]. In systemic arterial hypertension (SAH), miRNAs play a significant role in the regulation of key signaling pathways that lead to the hyperactivation of the RAAS, endothelial dysfunction, inflammation, proliferation, and phenotypic changes in smooth muscle cells [ 134 ]. Normally, vascular endothelial growth factor (VEGF) is involved in angiogenesis and blood pressure regulation. Some studies demonstrated that VEGF signaling is modified in SAH. Different miRNAs were involved in the regulation of VEGF signaling [ 136 ]. In particular, miR-126 is highly expressed in cardiac endothelium. It contributes to angiogenesis and the maintenance of vascular integrity, by targeting the phosphatidylinositol 3′-kinase (PI3K) regulatory subunit p85 β, which leads to modulation of the PI3K/protein kinase B (Akt) signaling pathway in endothelial progenitor cells [ 137 ]. Moreover, it has been demonstrated that miR-126 inhibits sprouty related EVH1 domain containing 1 (SPRED-1), another negative regulator of angiogenesis that acts by inhibiting the VEGF signaling pathway [ 138 ]. Transgenic mice lacking miR-126 expression showed developmental abnormalities such as vascular leakage, hemorrhaging, and partial embryonic lethality, likely due to miR-126 interaction with pro-angiogenic growth factors [ 138 ]. Endothelial cells transfected with a miR-126 inhibitor showed increased inflammation and TNF-α-induced VCAM-1 expression [ 139 ]. MiR-15b and miR-16 are also involved in the regulation of angiogenesis and vascular function. Both miRNAs targets VEGF mRNA and are downregulated in hypoxic conditions [ 140 ]. Along with VEGF, FGFs are also powerful promoters of angiogenesis [ 141 ]. In the plasma of hypertensive patients, up-regulation of miR-505 associates to FGF18 inhibition, thus representing a potential biomarker of pre-hypertension [ 142 ]. MiR-17-3p and miR-31 have been reported to be involved in vascular inflammation through the expression of adhesion molecules VCAM-1, ICAM-1, and E-selectin, and the increase of oxidative stress and NO depletion [ 143 , 144 ]. Instead, miR-155 affects endothelium-dependent vasodilation by reducing eNOS expression [ 145 ] and adaptive neovascularization [ 146 ]. MiR-19a has been reported to display anti-proliferative properties in endothelial cells by inhibiting cyclin D1 mRNA [ 147 ]; instead, miR-19b counteracts the TNF-α-induced endothelial cell apoptosis [ 148 ]. A possible role in endothelial cell apoptosis has been hypothesized for Let-7g, miR-21, and miR-223 [ 149 , 150 ]. Moreover, in an experimental rat model, a higher level of miR-223 causes hypertension-induced heart failure [ 151 ]. Several miRNAs, such as miR-155, miR-146a/b, miR-132/122 cluster, and miR-483-3p, have been demonstrated to be involved in RAAS-mediated cardiovascular inflammation [ 152 ]. MiR-145, miR-27a/b, and miR-483-3p inhibit ACE expression [ 153 ]. Instead, miR-143/145 clustering is downregulated in the presence of a high level of ACE in SAH [ 154 ]. Moreover, miR-483-3p and miR-155 have been reported to be reduced when angiotensin II level is high [ 155 , 156 ]. In SAH, arterial structural remodeling occurs, leading to lumen reduction [ 157 ]. The key event in this process is VSMC proliferation and migration following arterial damage [ 158 ]. Several miRNAs participate in the regulation of VSMC phenotype. In particular, miR-221 seems to reduce the contractile phenotype of VSMCs in response to platelet-derived growth factor [ 159 ]. Some studies reported that higher levels of miR-221 and miR-222 correlated with SAH [ 160 ]. Normally, the miR-143/145 cluster is highly expressed in VSMCs and regulates the differentiation of stem/progenitor cells. The downregulation of miR-143/145 cluster, in SAH, may influence the contractile phenotype of VSMCs [ 161 , 162 ]. MiR-133 has been reported to be a negative regulator of proliferation [ 163 ] as well as miR-365

Several factors appear to affect endothelial dysfunction, and different strategies have been proposed to change these factors and counteract it. Other than conventional drug therapies, lifestyle and dietary interventions are majorly encouraged, especially in age-related vascular endothelial dysfunction [ 198 ]. For example, regular exercise and caloric restriction can exert beneficial effects increasing NO bioavailability, eNOS, and SOD activity, as well as reducing oxidative stress, cholesterol, and systolic blood pressure. Besides, several ‘anti-aging’ nutraceutical compounds have been recently proposed to counteract oxidative stress and senescence [ 40 ]. For example, both omega 3 polyunsaturated fatty acids (ω-3 PUFA) and extra virgin olive oil (EVOO) have been reported to exert numerous metabolic and cardiovascular benefits in the elderly due to their high content in antioxidant compounds [ 199 , 200 , 201 , 202 ]. In old rats, the treatment prevented aging-induced endothelial dysfunction, oxidative stress, inflammation, and vascular insulin resistance through activation of the PI3K/Akt pathway and decreasing the response to the vasoconstrictor angiotensin II [ 202 ]. Moreover, the Citrus flavonoid naringenin (NAR) has been demonstrated to exert anti-senescence effects, and, when administered to old mice, it reduced ROS production in myocardial tissue, inflammation, and vascular remodeling [ 203 ]. NAR beneficial effects are attributable to its similarity to the natural structure of SIRT1 activator resveratrol, an enzyme involved in many physiological functions and reported to be depleted in endothelial dysfunction [ 203 ]. An experimental study on Moringa oleifera (MOI) suggested a vascular protective effect of MOI seeds against the aging-related vascular dysfunction through increased Akt signaling, endothelial NO synthase activation and downregulation of arginase-1, improving the endothelial-dependent relaxation of mesenteric arteries [ 204 ]. In aged mice, the restoration with the NAD + booster nicotinamide mononucleotide (NMN) of the depleted NAD + levels demonstrated vasoprotective effects [ 171 , 205 ]. NMN increased endothelium-dependent vasodilation and counteracted oxidative stress [ 171 ]. Unfortunately, current conventional and antioxidant drugs are not entirely effective in reversing endothelial dysfunction. Therefore, besides standard therapies, different new approaches have recently been proposed in endothelial dysfunction and CVD therapy. 5.1. Nanotechnologies for Drug Delivery Systems Nano therapies based on nanoparticles are very promising for the treatment of cardiovascular diseases. Nano-medicine is an advanced version of a conventional drug that includes the development and application of nanomaterials and nanotechnologies [ 59 ]. Nano-medicine is highly specific for targeted drug delivery, improves drug bioavailability, minimizes associated side effects, and reduces costs [ 59 ]. A broad spectrum of nanoparticles ranging from liposomes and niosomes to polymers, lipid and organic polymer hybrids and precursors, carbon nanotubes, quantum dots, metal, metal oxides including nanoparticles and biological molecules are used for vascular diseases [ 206 ]. An advanced application of nano-medicine may be described by the enhanced therapeutic efficacy of statins against CAD [ 207 ]. Broz et al. reported using a vesicle system to deliver high-dose statin, reducing toxicity in other tissues. Vesicles were loaded with pravastatin and surface-functionalized with an oligonucleotide with a high affinity for inflammatory macrophages [ 208 ]. Instead, Leuschner et al. demonstrated the efficacy of nanoparticle-assisted systemic delivery of a short interfering RNA (siRNA) silencing C-C chemokine receptor type 2 (CCR2), a chemokine receptor for monocyte recruitment, that significantly decreased plaque burdens [ 209 ]. As described above, synthetic siRNA can effectively inhibit its target gene, but it cannot pass through the cell membrane because of its large size and negative charge [ 210 ]. Therefore, the delivery of siRNA into cells represents a significant challenge in clinical application, and nanoparticles may provide a solution. The use of liposomes may go over this problem by extending the circulatory half-lives of drugs and improving their vascular endothelium deposition. For example, liposomal glucocorticoid therapy reported by Lobatto et al. demonstrated a significant reduction of inflammation in atherosclerotic plaques [ 211 ]. Moreover, the use of light-activated dextran-coated iron-oxide nanoparticles, in an apolipoprotein E (ApoE) knockout mouse model, has been reported to be effective in reducing inflammation through macrophage ablating, without causing significant skin toxicity [ 212 ]. Dextran-coated iron-oxide nanoparticles, loaded with phototoxic agents, induced macrophage death within atherosclerotic plaques once irradiated [ 213 ]. PARP, PTPase, ROCK, geranylgeranyltransferase, and

Tempol, an SOD mimetic, has proven effective in diabetes-associated microvascular complications and in restoring endothelial vasorelaxation in alloxan-induced diabetic rabbits [ 232 ]. Ebselen, a lipid-soluble seleno-organic compound that mimics the activity of GPx1, has reported being effective in counteracting atherosclerosis of diabetic ApoE KO mice [ 256 ]. Ebselen analog, diphenyl diselenide, has been reported to be effective against hypercholesterolemic LDL receptor KO mice, reducing atherosclerotic lesions, oxidative stress, and inflammation and favoring vascular function [ 257 ]. The same promising results are reported for the use of Trx mimetics. The administration of recombinant human Trx (rhTRX) has proven effective in counteracting hypertension in aged wild-type mice [ 258 ]. It has been reported that the supplementation of Trx using thioredoxin mimetic peptides (TMP) counteracted ROS production and restored the VEGF-A-induced migration, proliferation, survival of hyperglycemic ECs [ 259 ]. As previously described, the pivotal role of p66Shc in mitochondria dysfunction and the inverse relationship between p66Shc and SIRT1 activity, which in turn reduces oxidative stress and endothelial dysfunction [ 260 ], have suggested the use of SIRT mimetics as a strategy to counteract endothelial dysfunction and CVD progression [ 88 ].

NADPH oxidase can also be a promising target for oxidative stress-induced endothelial dysfunction. Triazolopyrimidines are selective inhibitors of NADPH oxidase activity [ 88 ]. They inhibit NADPH oxidase-derived ROS in vitro and counteract atherosclerosis [ 268 ]. It has been reported that the pan-NOX inhibitors VAS compounds (VAS2870 and its analog VAS3947) demonstrated a strong antiplatelet effect, inhibiting platelet aggregation by blocking PKC downstream signaling. The same finding was also reported in an animal model in which VAS compounds prevented thrombus formation [ 269 ]. Moreover, VAS2870 has been reported to be effective in counteracting amylin-induced reduction of endothelial responses in rat mesenteric arteries [ 270 ]. A novel pan-NOX-inhibitor, APX-115, has proven to have significantly improved insulin resistance in diabetic mice, similarly to how GKT137831 positively affected oxidative stress [ 271 ]. GKT137831, an inhibitor of NOX1 and NOX4, is under clinical trial and has been reported to reduce oxidative stress and diabetic vasculopathy [ 272 ]. GKT137831 has been reported to protect lung tissue damage after ischemia and reperfusion injury in a murine model [ 273 ]. Nox2 inhibitors (CPP11G and CPP11H) has proven to counteract ROS production by inhibiting stress-responsive MAPK signaling and downstream AP-1 and NF-κB nuclear translocation in human ECs. Nox2 inhibition also improved hind-limb blood flow in mice, reducing ROS level and inflammation after TNF-α administration [ 274 ]. Besides, NOX-2 inhibitor gp91ds-tat, combined with the ROS scavenger N-acetyl-cysteine (NAC), has proven to be effective against platelet activation induced by prolonged exposure to oxLDL-associated oxidized phospholipids [ 275 ]. GLX351322, a selective inhibitor of NOX4, has been suggested to be effective in T2D [ 276 ], while Plumbagin, another Nox4 specific inhibitor, has been reported to counteract oxidative stress-induced endothelial dysfunction and preadipocyte apoptosis under hyperinsulinemic conditions [ 277 , 278 ]. Moreover, rutin, a glycoside of quercetin, protected from endothelial dysfunction through inhibition of NOX4 [ 279 ]. These inhibitors are able, in turn, to counteract mitochondrial dysfunction and atherogenesis [ 280 , 281 ].

Endothelial dysfunction is a primary step during atherogenesis, and defects in vascular integrity and homeostasis induce the progression of vascular diseases. It is becoming increasingly clear that the discovering of novel targets or specific and safe biomarkers represents a potential tool in cardiovascular research. Technological advances are promising, especially nanotechnology and gene-targeted therapies, and several attempts have been made to select appropriate targets and to design specific tools to counteract endothelial dysfunction. Several clinical trials to test the different pharmacological approaches described above, in various pathological conditions, are still ongoing. Although NOX inhibitors did not provide satisfying results in some previous clinical studies, some phase I trials are still in progress for the GKT137831 ( NCT04327089 ). The early intracoronary administration of the ROCK inhibitor Fasudil is under investigation during primary percutaneous coronary intervention in patients with ST-segment-Elevation Myocardial Infarction ( NCT03753269 ). Numerous randomized studies in progress evaluate the effects of dietary supplementation with MitoQ in several conditions, including diastolic dysfunction, peripheral arterial disease, hypertension, chronic kidney disease (CKD), heart failure with preserved ejection fraction ( NCT03586414 , NCT03506633 , NCT04334135 , NCT02364648 , NCT03960073 ). A phase 2 randomized trial examines the supplementation with nicotinamide riboside, which has SIRT mimetic effects, for the treatment of arterial stiffness and elevated systolic blood pressure in patients with moderate to severe CKD ( NCT04040959 ). One multicentric European phase 2 randomized trial is trying to assess the efficacy and safety of catheter mediated, adenovirus-mediated, endocardial gene transfer of the vascular endothelial growth factor-D (AdVEGF-D), to treat patients with refractory angina, to whom revascularization cannot be performed ( NCT03039751 ). The EXACT trial is an American multicentric open-label phase 1/2 trial that aims to treat refractory angina with the direct epicardial delivery to the ischemic myocardium of AdVEGF-All6A+. This replication-deficient adenovirus vector expresses a cDNA/genomic hybrid of human VEGF ( NCT04125732 ). Even though the outlook on future therapeutic strategies seems promising, many challenges remain to overcome before these new technologies can be used in everyday clinical practice.