Role of Free Radical-induced Oxidative Stress in the Pathogenesis of Coronary Artery Disease

Coronary artery disease (CAD) is a common heart disease. The disease has many victims, especially in advanced societies and old ages. Coronary arteries disease develops when the coronary arteries become too narrow that limit blood supply to the heart. In the advanced case, if blood supply to a coronary artery is completely blocked, the blood supply to the part of the heart muscle will stop that results in myocardial infarction (1-2).


The Mechanism of Plaque Formation in Vessels and Subsequent Damage to coronary Cells
The primary event following the development of atherosclerosis is endothelial damage, which leads to the penetration and accumulation of LDL in the subendothelial space. LDL accumulated in pathological states during the oxidation process is converted to oxidized LDL (ox-LDL). These modified lipoproteins increase the expression of vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) on endothelial cells, leading to the recruitment of leukocytes in the subendothelial space. These inflammatory cells migrate into intima by the interaction of chemotaxis factors such as monocyte chemoattractant protein-1 (MCP-1), eotaxin, and INF-γ. The monocytes that enter this space swallow deformed lipoproteins. These fat-laden macrophages are called foam cells and the appearance of these foam cells in the arterial wall is characteristic of the primary atherosclerotic lesion. Lymphocytes and monocytes migrated to intima, together with foam cells, release a variety of cytokines that lead to inflammation and ROS production. Growth factors released by these cells, as well as stimulation of smooth muscle cell migration by oxygen free radicals and collagen deposition, lead to the development of atheromatous plaque. The important thing in this process is that oxygen free radicals make smooth muscle cells express scavenger receptors and be converted into foam cells. ROSs also release matrix metalloproteinases (MMPs), which destroys the wall of fibers in atheromatous plaque and the endothelial basement membrane, leading to the physical breakdown of the plaque and in turn vascular obstruction (21).

Reactive Oxygen Species
There are many ROSs that are produced by different reactions in the body ( Figure 1) and play central roles in vascular physiology ( Figure 2) and pathophysiology, the most important of which include nitric oxide (NO), superoxide (O -2 ), hydrogen peroxide (H 2 O 2 ), and peroxynitrite (ONOO -). NO is usually produced by endothelial nitric oxide synthase (eNOS) in the vascular endothelium, but inducible nitric oxide synthase (iNOS) is expressed in macrophages and smooth muscle cells under inflammatory conditions. NO is a critical mediator of endothelium-dependent vasodilation and may also play a role in plaque aggregation and maintain balance between smooth muscle cell growth and differentiation. Superoxide is produced by the reduction of an oxygen electron by various types of oxidases. When O -2 is produced along with NO, they react rapidly and form a highly reactive molecule (ONOO -). ONOOis an important mediator for lipid peroxidation and protein nitration, including LDL oxidation. It has significant proatherogenic effects. In the absence of NO, O -2 is rapidly converted to ROS and more stable H 2 O 2 by the superoxide dismutase enzyme, which is then converted to H 2 O by catalase or glutathione peroxidase. The effects of O -2 and H 2 O 2 on vascular function are critically dependent on their production rate. When formed in small amounts intracellularly, they can act as secondary intracellular messengers and pathways that result in the growth of vascular smooth muscle cells (VSMCs) and fibroblasts. High ROS concentrations can cause DNA damage, significant toxicity, or even apoptosis, which has been proved in endothelial cells and smooth muscle cells (22,23).

Cellular and Enzymatic Sources of ROS in the Vessel Wall
Oxidative stress reflects the imbalance between ROS production and the ability of a biological system to neutralize and inhibit its toxic mediators or repair damage. Any disturbance and disruption of the normal state of oxidation-reduction through the production of  peroxides and free radicals results in toxic effects and damage to all intracellular components and structures, including proteins, lipids, and DNA (24)(25)(26)(27).
Various ROSs are produced by enzymatic or chemical reactions. NO is produced in endothelial cells by activating eNOS during normal vascular function. Vasodilator hormones increase intracellular Ca 2+ , which leads to increased eNOS activity and NO release. Physical forces, such as shear stress, activate eNOS through protein kinase A or Akt-dependent phosphorylation. The pathophysiological expression of iNOS in macrophages and VSMCs increases the level of cytokines and consequently local inflammation. This, in turn, results in NO production in the absence of further stimuli. In addition, eNOS is deactivated and O -2 is produced more than NO 3 under some conditions. Therefore, depending on the surrounding environment, NOS enzymes are potentially important sources of NO and O -2 (28). Almost all types of vascular cells produce O -2 and H 2 O 2 (29). In addition to mitochondrial sources of ROS, O -2 or H 2 O 2 can be produced by many enzymes (Figure 2). Cytochrome P450 and membrane-associated NADPH oxidases are thought to be two important sources of ROS in normal vessels (30,31). The homologous cytochrome P450 isoenzyme with CYP 2C9 has been identified in coronary arteries and shown to produce O -2 in response to bradykinin (32). NADPH oxidases, which are structurally similar to neutrophil NADPH oxidases but produce less O -2 over a longer period of time, have been identified in vascular cells. Endothelial enzymes, VSMCs, and fibroblasts are not the same but have subunits and unique regulatory mechanisms. One important aspect of ROS generation by VSMC (Vascular smooth muscle cells) NADPH oxidase is that it occurs mainly intracellularly and makes it ideally suited for modifying signal transduction pathways and gene expression (29).
The activity of NADPH oxidases can be modulated by vasodilator hormones affecting the vascular wall and G-protein 1-rac (33). Angiotensin II (Ang II), tumor necrosis factor-α (TNF)-α, thrombin, and plateletderived growth factor all enhance oxidase activity and elevate intracellular O -2 and H 2 O 2 levels in VSMCs. Ang II and ceramide lactoside activate endothelial cells, whereas fibroblasts increase O -2 production in response to Ang II, TNF-α, interleukin-1, and platelet-activating factor. Physical pressures such as cellular stretching, laminar shear stress, and oscillatory flow distribution occurring at the branch points are also potent activators of O -2 production in endothelial cells. There are two major mechanisms by which the hormones and physical forces activate NADPH oxidase: 1. Rapid mechanism by which the expression of the enzyme is activated by phosphorylation, GTPase activity, and the production of related second messengers (34) and 2. long-term mechanism which works when enzyme rate-limiting subunits are overexpressed and consequently higher levels of the enzyme are likely to be activated (35).
Macrophages may be the major source of vascular O -2 in the case of diseases. They oxidize LDL by activating various enzymes. Neutrophils and monocytes may also secrete myeloperoxidase, which seems to initiate lipid peroxidation (36). Two potential candidates for the initiation of myeloperoxidase-dependent lipid peroxidation include tyrosyl radical and nitrogen dioxide (NO 2 ). The removal of myeloperoxidase markedly reduces the formation of F2-isoprostanes (F2iPs), indicators of in vivo lipid peroxidation in an experimental model of peritonitis (37).  (40)(41)(42). ROS regulates several general classes of genes, including adhesion molecules and chemotactic factors, antioxidant enzymes, and vasoactive substances. Some of these are clearly considered adaptive responses, such as the induction of superoxide dismutase and catalase by H 2 O 2 . There is a specific relationship between severe regulation of adhesion molecules (VCAM-1, ICAMs) and chemotactic molecules (MCP-1) by oxidant-sensitive mechanisms with vascular pathology. These molecules reinforce adhesion and migration of monocytes into the vessel wall. In contrast, transcriptional induction of adhesion molecules by cytokines is inhibited by NO independently through cyclic guanosine monophosphate. These mechanisms are incorporated into the normal vascular wall by suppressing the expression of the adhesion molecule and stimulate its expression in vasculopathies or vessels (43,44

Conclusion
Free radical-induced oxidative stress plays a key role in the pathogenesis of cardiovascular diseases. Modulation of free radical production is an important modality in the reduction or treatment of cardiovascular diseases.