Antioxidants and Atherosclerosis: Mechanistic Aspects

Atherosclerosis is a chronic inflammatory disease which is a major cause of coronary heart disease and stroke in humans. It is characterized by intimal plaques and cholesterol accumulation in arterial walls. The side effects of currently prescribed synthetic drugs and their high cost in the treatment of atherosclerosis has prompted the use of alternative herbal medicines, dietary supplements, and antioxidants associated with fewer adverse effects for the treatment of atherosclerosis. This article aims to present the activity mechanisms of antioxidants on atherosclerosis along with a review of the most prevalent medicinal plants employed against this multifactorial disease. The wide-ranging information in this review article was obtained from scientific databases including PubMed, Web of Science, Scopus, Science Direct and Google Scholar. Natural and synthetic antioxidants have a crucial role in the prevention and treatment of atherosclerosis through different mechanisms. These include: The inhibition of low density lipoprotein (LDL) oxidation, the reduction of reactive oxygen species (ROS) generation, the inhibition of cytokine secretion, the prevention of atherosclerotic plaque formation and platelet aggregation, the preclusion of mononuclear cell infiltration, the improvement of endothelial dysfunction and vasodilation, the augmentation of nitric oxide (NO) bioavailability, the modulation of the expression of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on endothelial cells, and the suppression of foam cell formation.

is performed by cytokines and oxidized lipids. Cytokines such as tumor necrosis factor (TNF-α), interleukin-1,-4, and -6 (IL-1, IL-4, IL-6) and interferon gamma (IFN-γ) induce the expression of leukocyte and monocyte adhesion molecules, especially vascular cell adhesion molecule-1 (VCAM), intercellular adhesion molecule-1 (ICAM) and E-selectin, on the endothelial surface [26][27][28]30]. Monocytes and T lymphocytes accumulate in the vascular wall intima mediated by these adhesion molecules [30]. Specific chemokines cause smooth muscle cell migration from media to intima and then cellular proliferation. Monocytes in the sub-endothelial space are differentiated into macrophages through chemotactic proteins, such as monocyte chemotactic protein-1 (MCP-1), macrophage colony-stimulating factor (M-CSF), and IL-8 [30][31][32]. Mononuclear phagocytosis occurs in the foam cell formation stage, and macrophages recognize and uptake Ox-LDL molecules via "scavenger" receptors (SRs). Finally, macrophages become foam cells, and the aggregation of yellow foam cells on the arterial walls leads to development of fatty streaks. The demise of foam cells occurs by programmed cell death or apoptosis during the developing atherosclerotic lesion. A necrotic core is formed as the result of apoptotic foam cell death, and this acts as a depot for cellular debris and lipids [32][33][34][35].
A fibrous atherosclerotic plaque cap is created during the migration of smooth muscle cells from media to intima vascular layers and the induction of extracellular matrix production in atheroma formation. The atherosclerotic plaque cap consists of collagen-rich fiber tissues, smooth muscle cells (SMC), macrophages, and T lymphocytes. Atheromatous lesions are developed by tissue macrophages and decreased blood flow in the vessels [34][35][36], and atherosclerotic plaque formation is mediated by components of the fibrous cap. Macrophages and T lymphocytes secrete metalloproteinase and TNF-α in the margins of the developing plaque in order to lyse the fibrous cap extracellular matrix and inhibit collagen synthesis in the SMC, respectively. The lysis of the extracellular matrix leads to the destruction of the fibrous cap, and the thrombogenic contents are exposed to the blood stream initiating the coagulation process, blood clot formation, the adhesion of platelets, and thrombus formation, which may completely block the arteries [37][38][39]. Figure 1 depicts the main atherosclerotic events.
Biomolecules 2019, 9, x FOR PEER REVIEW 3 of 20 activation of endothelial cells is performed by cytokines and oxidized lipids. Cytokines such as tumor necrosis factor (TNF-α), interleukin-1,-4, and -6 (IL-1, IL-4, IL-6) and interferon gamma (IFN-γ) induce the expression of leukocyte and monocyte adhesion molecules, especially vascular cell adhesion molecule-1 (VCAM), intercellular adhesion molecule-1 (ICAM) and E-selectin, on the endothelial surface [26][27][28]30]. Monocytes and T lymphocytes accumulate in the vascular wall intima mediated by these adhesion molecules [30]. Specific chemokines cause smooth muscle cell migration from media to intima and then cellular proliferation. Monocytes in the sub-endothelial space are differentiated into macrophages through chemotactic proteins, such as monocyte chemotactic protein-1 (MCP-1), macrophage colony-stimulating factor (M-CSF), and IL-8 [30][31][32]. Mononuclear phagocytosis occurs in the foam cell formation stage, and macrophages recognize and uptake Ox-LDL molecules via "scavenger" receptors (SRs). Finally, macrophages become foam cells, and the aggregation of yellow foam cells on the arterial walls leads to development of fatty streaks. The demise of foam cells occurs by programmed cell death or apoptosis during the developing atherosclerotic lesion. A necrotic core is formed as the result of apoptotic foam cell death, and this acts as a depot for cellular debris and lipids [32][33][34][35].
A fibrous atherosclerotic plaque cap is created during the migration of smooth muscle cells from media to intima vascular layers and the induction of extracellular matrix production in atheroma formation. The atherosclerotic plaque cap consists of collagen-rich fiber tissues, smooth muscle cells (SMC), macrophages, and T lymphocytes. Atheromatous lesions are developed by tissue macrophages and decreased blood flow in the vessels [34][35][36], and atherosclerotic plaque formation is mediated by components of the fibrous cap. Macrophages and T lymphocytes secrete metalloproteinase and TNF-α in the margins of the developing plaque in order to lyse the fibrous cap extracellular matrix and inhibit collagen synthesis in the SMC, respectively. The lysis of the extracellular matrix leads to the destruction of the fibrous cap, and the thrombogenic contents are exposed to the blood stream initiating the coagulation process, blood clot formation, the adhesion of platelets, and thrombus formation, which may completely block the arteries [37][38][39]. Figure 1 depicts the main atherosclerotic events.

Antioxidant Defense Mechanisms
An antioxidant is a molecule that is capable of "neutralizing" the oxidation of ROS before they react with cellular biomolecules and change their structure or function [39,40]. Antioxidant defense has two levels: a. Primary defense mechanism: This defense mechanism inhibits oxidative damage directly by scavenging free radicals before they can damage intracellular biomolecules. Endogenous enzymes play an important role in this step [9,41,42]. Primary defense mechanisms are summarized into four steps shown below.
Superoxide dismutase (SOD) converts the superoxide radical generating hydrogen peroxide (H 2 O 2 ): H 2 O 2 is then transformed by the enzymes catalase and glutathione peroxidase (GPx) into water and molecular oxygen: Glutathione peroxidase is an enzyme that catalyzes the reduction of H 2 O 2 to water utilizing Glutathione (GSH): Glutathione disulfide (GSSG) is reduced back to GSH by GSH reductase: Secondary defense mechanism (chain-breaking defense): Vitamin C, vitamin E, and uric acid scavenge free radicals as a part of the secondary defense system. Additionally, nuclear enzymes which participate in DNA repair can be considered as a secondary defense system against oxidative damage caused by oxygen free radicals [9,41].

Antioxidant Defense Mechanisms
An antioxidant is a molecule that is capable of "neutralizing" the oxidation of ROS before they react with cellular biomolecules and change their structure or function [39,40]. Antioxidant defense has two levels: a. Primary defense mechanism: This defense mechanism inhibits oxidative damage directly by scavenging free radicals before they can damage intracellular biomolecules. Endogenous enzymes play an important role in this step [9,41,42]. Primary defense mechanisms are summarized into four steps shown below.
Superoxide dismutase (SOD) converts the superoxide radical generating hydrogen peroxide (H2O2): H2O2 is then transformed by the enzymes catalase and glutathione peroxidase (GPx) into water and molecular oxygen: Glutathione peroxidase is an enzyme that catalyzes the reduction of H2O2 to water utilizing Glutathione (GSH): Glutathione disulfide (GSSG) is reduced back to GSH by GSH reductase: b. Secondary defense mechanism (chain-breaking defense): Vitamin C, vitamin E, and uric acid scavenge free radicals as a part of the secondary defense system. Additionally, nuclear enzymes which participate in DNA repair can be considered as a secondary defense system against oxidative damage caused by oxygen free radicals [9,41].   Overall, antioxidants may be categorized into two groups: Enzymatic and non-enzymatic antioxidants. Endogenous enzymatic antioxidants include superoxide dismutase (SOD), catalase (Cat), glutathione peroxidase (GPx), and thioredoxin reductase (TrxR) [9,39,[43][44][45]. Endogenous non-enzymatic antioxidants include glutathione (GSH), uric acid, bilirubin, coenzyme Q (CoQ)/CoQH2) and lipoic acid [46].
Examples of exogenous non-enzymatic antioxidants are represented by α-tocopherol (vitamin E), ascorbic acid (vitamin C), B vitamins, carotenoids, and polyphenols. [9]. Data concerning the important enzymatic, non-enzymatic and synthetic antioxidants and their mechanisms of actions are summarized in Table 1.

Superoxide Dismutase (SOD)
Superoxide dismutase neutralizes superoxide, thus preventing peroxynitrite formation and the reduction of transition-metal ions [10]. SOD catalyzes the superoxide anion radical to yield H 2 O 2 and O2. It can also decrease atherosclerotic lesion size by reducing levels of F2-isoprostanes and isofurans in the aorta as well as through the inhibition of any involvement of MCP-1and VCAM-1 [23,47].

Catalase
Catalase exists in peroxisomes and converts hydrogen peroxide (H 2 O 2 ) that is formed by the dismutation of superoxide to H 2 O [10,48,49]. What is more, this enzyme also induces a decline in vascular smooth muscle cell (VSMC) proliferation [48,49].

Glutathione Peroxidase (GPx)
GPx is a selenocysteine enzyme that reduces peroxides, especially lipid hydroperoxides, to the corresponding alcohols [10]. The most important mechanisms of GPx in the prevention and treatment of atherosclerosis stem from the inhibition of H 2 O 2 -mediated expression of MCP-1 and VCAM-1, in addition to an anti-inflammatory action [10,50].

Thioredoxin Reductase
The mechanism the of action of thioreductase arises from redox regulation in signaling and cell survival, increasing nitric oxide (NO) bioavailability and decreasing oxidative stress and any resultant lesions [51][52][53].

Endogenous Non-Enzymatic Antioxidants
Endogenous non-enzymatic antioxidants are small molecules found either intracellularly or extracellularly in a lipid or aqueous environment. Examples include glutathione, coenzyme Q, bilirubin, uric acid, and lipoic acid [23].

Glutathione
Glutathione is an important small water-soluble tripeptide antioxidant present in cells [54,55], and it is a co-factor for antioxidant enzymes, such as GPx. A major action of glutathione in atherosclerosis is the modulation of the size of atherosclerotic lesions in the aortic arch by scavenging hydroxide (OH), hypochlorous acid (HOCl), and peroxynitrite (ONOO − ) [54].

Coenzyme Q (CoQ)
Coenzyme Q is present in cellular membranes. It is a lipophilic antioxidant with anti-inflammatory properties, and coenzyme Q10 is the main form found in humans. CoQ inhibits lipid and protein oxidation and reduces the conversion of α-tocopheroxyl radical to α-tocopherol. It is capable of scavenging peroxyl radicals, thereby improving endothelial function [56][57][58][59][60].

Uric Acid
Uric acid is the end-product of purine catabolism. There are two immediate sequential precursors-hypoxanthine followed by xanthine-the conversions of which are both catalyzed by xanthine oxidase. Mechanistically, uric acid increases cytokine production, scavenges OH as well as HOCl, and incites inflammatory responses, SMC proliferation, endothelial dysfunction, and plaque instability [68].

Lipoic Acid
Lipoic acid is synthesized by mitochondria. It is a cofactor for mitochondrial α-ketoacid dehydrogenases (e.g., the pyruvate dehydrogenase complex), and it inhibits atherosclerotic lesion development [69]. It is also a scavenger of ONOO-, HOCl, and peroxyl radicals. Other key actions of lipoic acid comprise an attenuation of endothelial dysfunction, a decrease in inflammatory markers, and an increase in endothelial nitric oxide synthase (eNOS) activity [70][71][72].
In several animal models, vitamin E has preventative effects against atherosclerosis by: Scavenging free radicals in VSMC, diminishing the oxidation of LDL by the inhibition of Cluster Differentiating 36 (CD36) and Scavenger receptor class B type I (SR-BI) expression in VSMC, reducing VSMC proliferation via the inhibition of protein kinase C (PKC), preventing foam cell formation, lessening the secretion of cytokines and extracellular matrix in VSMC, preventing mononuclear cell infiltration, lessening inflammation, curtailing the destabilization of fibrous plaque, inhibiting the apoptosis of VSMC, modulating signal transduction and gene expression in VSMC, increasing the expression of connective tissue growth factor (CTGF) in VSMC (cell lines), preventing endothelial dysfunction related to cholesterol, modulating endothelial cells and the expression of adhesion molecules such as VCAM-1 and ICAM-1 on endothelial cells, preventing lysophosphatidylcholine (LPC)-induced endothelial dysfunction and the preservation of endothelial NO release, modulating monocytes, macrophages, T cells and mast cells, enhancing the expression of cytosolic phospholipase A2 (PLA2), cyclooxygenase, and vasodilating prostacyclin (PGI2) in endothelial cells, inhibiting thrombin formation, and reducing leukotriene synthesis [73][74][75][76][77][78][79][80][81][82].
In several clinical studies, vitamin E revealed contrasting findings. In a study examining the effect of 50 mg·day −1 synthetic vitamin E in a population with coronary heart disease, the results showed no effect on major cardiovascular events [83]. Another study showed 300 mg day −1 synthetic vitamin E had no effect on cardiovascular disease, including the rate of non-fatal myocardial infarction in patients with previous myocardial infarction [84]. Additionally, vitamin E did not significantly decrease the incidence of cardiovascular disease such as stroke [85]. In the Cambridge heart antioxidant study (CHAOS), vitamin E supplementation failed to have an impact on cardiovascular outcomes in patients at high risk of cardiovascular events [86].

Vitamin C
Vitamin C (ascorbate) is a water-soluble and ubiquitous antioxidant [7,23] with an ability to scavenge peroxyl radicals and HOCL [23,40], thus providing stability to the cell membrane. Fruit and vegetables, particularly citrus fruit, kiwi, cantaloupe, mango, strawberries, and peppers are rich sources of vitamin C [9].
It has various functions including: The improvement of nitric oxide-dependent vasomotor function [87], the enhancement of NOS activity (NO production) and the consequent augmentation of NO bioavailability, the improvement of endothelial dysfunction and vasodilation, the inhibition of cyclooxygenase, the diminishing of cell-cell adhesion [88], and the reduction of the chain-carrying α-tocopheroxyl radical to inhibit LDL peroxidation [89]. It also recycles other endogenous antioxidants, such as vitamin E [90]; discourages leukocyte aggregation and adhesion to the endothelium [9]; and scavenges ROS such as superoxide, hydroxyl radicals, peroxyl radicals, and many non-radicals, such as nitrosating agents and hydrochlorous acid [9].
A number of small-scale clinical studies have evaluated the effect of vitamin C on vascular health. The British Regional Heart Study demonstrated an inverse relation between plasma vitamin C concentration and endothelial dysfunction in men with no history of cardiovascular disease or diabetes [91]. Additionally, the European prospective investigation into cancer and nutrition (EPIC) Norfolk study showed the same results as the British Regional Heart Study in both men and women [92]. A large-scale study conducted over 20 years found that diets rich in vitamin C had no significant association with coronary heart disease [93].

B Vitamins
B vitamins have a fundamental role in the metabolism of essential amino acids, with a specific influence on homocysteine and the antioxidant, glutathione [9]. Other significant activities of B vitamins entail scavenging hydroxyl and lipid peroxyl radicals, improving endothelial function, and ameliorating the coupling of endothelial NO synthase through the essential cofactor, tetrahydrobiopterin [94,95].
In a clinical study intake of folate, hydroxocobalamin, and pyridoxine, supplements for eight weeks decreased serum homocysteine to a normal range in patients with venous thrombosis [96]. The vitamin intervention for stroke prevention (VISP) randomized controlled trial study demonstrated no significant effect of folate, hydroxocobalamin, and pyridoxine supplementation in decreasing incidence of coronary events or cardiovascular death [97]. The Cochrane systematic review reported no evidence to prevent cardiovascular events by using B vitamins [98].

Vitamin A and Carotenoids
Carotenoids are a large group of lipid soluble, colorful substances (yellow, orange, and red) such as α-carotene, β-carotene, β-cryptoxanthine, luteine and lycopene which occur extensively in fruit and vegetables [9].
A clinical study suggested an inverse relationship between the intake of β-carotene or retinol and risk of cardiovascular disease [101]. The US Preventative Task Force does not suggest β-carotene for the prevention of cardiovascular disease [102]. The Cochrane review on antioxidant consumption indicated that β-carotene and vitamin A significantly increase all-cause mortality [103].
The mechanistic effects of polyphenols involve: Suppressing ROS formation, scavenging ROS (both radical and non-radical oxygen), increasing the expression level of eNOS and the generation of NO or reducing NO oxidation by enhancing the intracellular free calcium concentration and by activating estrogen receptors in endothelial cells (ECs), blocking the action of xanthine oxidase and protein kinase C to prevent the production of the superoxide radical, and the protection of vascular endothelial cells and NO from oxidation. They also decrease redox-sensitive gene activation, preventing the expression of two major pro-angiogenic factors (vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2)) in smooth muscle cells, increase the production of major vasodilatory factors (NO, endothelium-derived hyperpolarizing factor (EDHF) as well as prostacyclin), inhibit angiogenesis (cell migration and proliferation of blood vessels), and also reduce platelet aggregation and hypertension [105][106][107][108][109][110].
Another important polyphenol that has received much attention is resveratrol (3, 5, 4 -trihydroxystilbene), a stilbene polyphenol, which occurs in grapes, red wine and Polygonum cuspidatum. Resveratrol has established antioxidant properties which include the inhibition of lipid oxidation, the regulation of vasodilator and vasoconstrictor production, the inhibition of platelet aggregation, and the inhibition of the transcription factors NF-κB (Nuclear Factor kappaB) and AP-1 (Activator Protein 1) through an interaction with upstream signaling pathways and/or by decreasing pro-inflammatory mediators (TNF-α, IL) [104,111].
Clinical studies such as the Zupthen Elderly study showed a significant inverse association between flavonoid intake and coronary heart disease after 5 years of consumption [112,113]. In addition, the Rotterdam study revealed a significant inverse relationship between total flavonoid intake from the diet with myocardial infarction incidence [114]. The consumption of cocoa or chocolate is inversely associated with carotid atherosclerosis [115].

Type of Antioxidants Action Mechanism Reference
Enzymatic endogenous

Effective Medicinal Plants on Atherosclerosis
Medicinal plants can be employed safely to prevent and treat atherosclerosis, mainly because of their tendency to produce fewer adverse effects [8]. They commonly possess antioxidant activity, representing a key underlying mechanism often linked to their phenolic constituents [120]-though their efficacy may also derive from combinations of other properties. The most effective medicinal plants used to prevent or treat atherosclerosis, along with their known mechanisms of action other than antioxidant activity, are listed in Table 2. Anti-lipid effects Nigella sativa [156] Cynara scolymus [157] * These plants all possess antioxidant activity which may inevitably contribute significantly to their overall effectiveness.

Reasons for Failing Antioxidant Strategies Related to Atherosclerosis in Humans
Several antioxidants have been tested with positive effects in different animal models for the potential treatment of atherosclerosis. However, studies in humans are either limited or have not disclosed positive effects. The reasons accounting for the failure of traditional antioxidant therapy in humans may be attributed to the following factors: 1.
Antioxidants should be utilized in the long term so that beneficial effects may be allowed an adequate period to emerge.

2.
Antioxidant treatment should ideally be instigated before full disease onset. 3.
The oxidant theory of atherogenesis is essentially a deficient and incomplete theory and does not incorporate effects of other pathways in atherogenesis.

5.
It is evident that the antioxidants that pass through the mitochondrial membrane, thus modifying mitochondrial oxidation, have superior effectiveness compared to traditional antioxidants. 6.
Combination antioxidant therapies may prove to be more effective overall because they may exploit any additional constituent mechanistic properties [16].
Diet plays an important role in the prevention of atherosclerosis and other cardiovascular diseases. In fact, the lifestyle habits, nutritional quality, and acquired eating patterns are effective on the risk of atherosclerosis [158,159]. The risks of atherosclerosis are decreased through an appropriate balance of nutrients. In fact, the balance of calorie intake and physical activity to keep a healthy body weight is very important. In this regard, the following diet and lifestyle have been recommended: -Intake a diet rich in vegetables, fruits (300 g/day of fruit or 400 g/day of vegetable consumption); whole-grain cereals (women 75 g/day, men 90 g/day); extra-virgin oil (≥4 tbsp/day); nuts (3e7 servings/week); a moderate consumption of fish and poultry (≥3 servings/week); a low intake of dairy products, red meat and sweets; and a moderate consumption of red wine for usual drinkers (≥7 glasses/week, average dietary fiber intake was higher than 30 g/day).

Conclusions
Atherosclerosis is a major cause of morbidity and mortality in the developed world. Due to the factor that ROS and the generation of oxidized LDL are leading contributors to the progression of atherosclerosis, dietary supplements and antioxidants with low adverse effects may well represent a good therapeutic strategy to prevent the progression of the disease. Natural and synthetic antioxidants facilitate atherosclerosis treatment through a variety of mechanisms, including the inhibition of LDL oxidation, the reduction of generated reactive oxygen species, the inhibition of cytokine secretion, the prevention of atherosclerotic plaque formation and platelet aggregation, the prevention of mononuclear cell infiltration, the improvement of endothelial dysfunction and vasodilation, the promotion of NO bioavailability, the modulation of the expression of adhesion molecules such as VCAM-1 and ICAM-1 on endothelial cells, and the suppression of foam cell formation.
It is not clear which of these different mechanisms of antioxidants action is more effective, but it seems that the use of multiple antioxidants is more effective target for antioxidant therapy.

Conflicts of Interest:
The authors declare that there are no conflicts of interest regarding the publication of this review.