Role of neuronal nitric oxide synthase (NOS1)-derived nitric oxide (NO) in atherosclerosis progression

Abstract

Cardiovascular disease (CVD) is a slowly progressive vascular disease that remains asymptomatic for decades. The early atherosclerotic lesion may start its formation from infancy and slowly develop into advanced lesions throughout a lifetime 1 . CVD is the most common cause of death worldwide; about 17.5 million people die each year. An estimated 31% of death worldwide is due to CVD, out of which more than 75% of CVD death occurs in low income or middle-income countries 2 . One of the common pathophysiological conditions of various vascular diseases is the development of lipid-rich plaque or atherosclerosis. Atherosclerosis, considered as a chronic inflammatory disease of aggregated lipid droplets in the arterial intima, subsequently leading to foam cell formation and proinflammatory cytokine production by macrophages and vascular cells 3 . Although several hypotheses have been proposed for the development of atherosclerosis, high LDL-C concentration (greater than the physiological threshold of 10-20 mg/dL) in blood plasma is considered as the primary determinant for disease initiation 1 . Subintima retention of lipid crystals triggers the infiltration of monocytes, B-lymphocytes, neutrophils, endothelial cells, and resulting in chronic inflammation of the arterial wall 4 . Background and significance: 1. Macrophage and endothelial cell. One of the critical step in atherogenesis is the infiltration of monocytes into the subendothelial space of large arteries where they subsequent differentiation into tissue macrophages, which accounts for 75% of the total plaque size 5 . Under normal physiological conditions, the circulating monocytes ensure an effective influx and efflux of cholesterol in the peripheral blood by regulating its homeostasis machinery (scavenger receptors, ACAT1, ABCA1, and ABCG1). However, macrophage-dependent cholesterol handling is deregulated during hypercholesterolemia. The accumulated lipid molecules are engulfed by these macrophages leading to its transformation into fat-laden macrophages or foam cell formation, which represents a critical event in early atherogenesis 6 . Endothelial cells on the other hand provide a permeable barrier to a blood vessel and control the selective exchange of nutrients between blood plasma and artery wall. Stimulation of endothelial cells with pro-atherogenic factors or due to disturbed blood flow activates these cells leading to endothelial dysfunction and lipid retention within the subendothelial space. This further leads to activation of NFƙB transcription factor to increase the growth factors and cytokines secretions which in turn act as paracrine molecules for stimulation on neighboring macrophage and smooth muscle cell phenotype. Enhanced cytokine productions have a cytotoxic effect on these cells leading to cell apoptosis and necrosis. Thus, foam cell-derived necrotic core formation due to the death of endothelial cells, macrophages, SMCS, results in arterial narrowing and hardening, thus increasing the risk of heart attack and stroke. 2. Nitric oxide synthase (NOS). Nitric oxide synthase (NOS) is the class of enzymes that produce the smallest signaling molecule nitric oxide (NO), which acts as a key signaling molecule and plays a crucial role in immune defense the pathogenesis of inflammation, and neurotransmission. NOS utilizes L-arginine as a substrate and convert it to citrulline by using molecular oxygen, and cofactors nicotinamide-adenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) and release nitric oxide in the process 7 . There are three reported isoforms of NOS: nNOS (NOS1 or neuronal NOS), iNOS (NOS2 or inducible NOS), and eNOS (NOS3 or endothelial NOS) 8 . In vivo studies reveal significant expression of nitric oxide synthase (NOS) in high-fat diet feed apolipoprotein E knockout (ApoE-/-) mice 9 . In situ hybridization and immunocytochemistry analysis shows a higher expression of nNOS and iNOS expression, colocalized with markers for vascular smooth muscle cells and macrophages but not for endothelial cells in atherosclerotic plaque of ApoE-/- mice 9,10 . eNOS is predominantly expressed in endothelial cells and has a vasoprotective role in maintaining the homeostasis of endothelial cells. It regulates the blood pressure and assists in vascular toning 11 . Cholesterol loading results in a reduction in the bioactivity of eNOS derived nitric oxide (NO) leading to endothelial damage 12 . Under normal physiological conditions, nitric oxide regulates endothelial cells homeostasis, platelet and leukocyte interactions with the arterial wall, regulation of vascular tone and growth, and homeostasis of blood pressure. EC damage results in impaired endothelial NO production, leading to the enhanced thrombus formation, aberrant vessel tone, dysregulated VSMC growth, increased MCP1 production, and inflammatory cell recruitment. Thus, the outcomes of various studies concluded NO as a crucial modulator of vascular diseases with atheroprotective function. This emphasized strategies to enhance NO bioactivity in the treatment of atherosclerosis. Ex vivo eNOS gene transfer to atherosclerotic rabbit shows improved relaxations of aortic rings, reduced adhesion molecule expression, and macrophage infiltration which leads to regression of atheroma in cholesterol-fed rabbits 13 . However, gene transfer of eNOS, but not along with inducible NOS (iNOS), fails to stop the atherosclerosis progression. Therefore, these observation highlights that nitric oxide has different intracellular effects depending upon its cell type source. ApoE-/- mice fed with cholesterol-enriched diet show activation of macrophages results in the upregulation of nNOS and iNOS expression with increased nitric oxide and peroxynitrite formation. Macrophage NO is derived from the high output inducible nitric oxide synthase (iNOS) pathway exerts its effect by the formation of lipid hydroperoxides, protein nitration, DNA damage, PARP activation and cellular cytotoxicity of cells and formation of the necrotic core. Further, nitric oxide strongly inhibits the migration of trapped macrophages within intima to the lumen of the blood vessel 14 . Genetic deletion of iNOS from ApoE-/- mice result in reduced lesion size due to the absence of iNOS-mediated LDL oxidation, reduced inflammatory cells infiltrate, and reduced NO-induced apoptosis, thus highlighting the pro-atherogenic role of iNOS in atherosclerotic plaque development (Detmers, Hernandez et al. 2000). The nNOS is expressed not only expressed in neurons but also present in endothelial cells and macrophages 15. Interestingly, nNOS expression remains undetectable in the standard functional artery; however, its expression increases drastically under the hyperlipidemia condition. A significant amount of nNOS expression can be detected in the neointima, endothelial cells, and macrophages of early atherosclerotic lesions in humans. Vascular nNOS contributes to the vasodilation and can partly compensate for the loss of eNOS in eNOS-/- mice. Stimulation of nNOS derived NO in macrophages increases its phagocytosis capacity 16 . Rapid nNOS derived NO and its stable derivative formation act as signaling agents in macrophages and endothelial cells for expression of cytokines (TNFα, IL1β, and IL6) that leads to endothelial dysfunction. Although studies have highlighted the anti-atherogenic role of vascular nNOS, however, the role of macrophage nNOS derived nitric oxide in foam cell formation and endothelial permeability is still needed to be elucidated. Therefore, we set out to examine the role of the macrophage nNOS in regulating foam cell formation and atherosclerosis development, as proposed in Figure 1. During our study we found the nNOS derived NO regulate endothelial permeability. Vascular endothelial cells form a protective barrier lining all blood vessels. Activation of vascular endothelial cells and macrophages during hypercholesterolemia increases the expression of functional CD40L as well as its receptor CD40 in vitro and within atherosclerotic plaques in humans. Both CD40-CD40L belonged to the TNF receptor superfamily and are expressed on mast cells, basophils, platelets, smooth muscle cells, endothelial cells, and macrophages. Clinical studies reveal that patients with hypercholesterolemia are frequently associated with the upregulation of CD40-CD40L signaling and are considered at high risk for the development of the cardiovascular disease. Ligation of CD40-CD40L dyad results in the activation of leukocyte adhesion molecule expression (VCAM1, ICAMI, and E-Selectin) and proinflammatory cytokine expression (TNFα, IL1β, and IL6). Thus, blocking the CD40-CD40L signaling could provide a novel means of atheroma stabilization by reducing the monocyte-endothelial interactions. Three objectives are proposed. 1. To explore the role of nNOS derived nitric oxide in lipid uptake by macrophages. In this aim, we will unravel the impact of nNOS-derived NO in regulating nLDL phagocytosis and uptake. We will also examine the effect on nLDL stimulation on macrophage signaling and phagocytic pathway activation by utilizing the specific nNOS inhibitor. 2. To determine the macrophage nNOS-mediated signaling involved in receptor and proinflammatory cytokine expression in response to OxLDL uptake. In this aim, we will test our hypothesis that macrophage nNOS-derived NO regulates the molecular signaling pathway. We will determine kinase or phosphatase activities upstream of the NFƙB transcription factor, which affects its nuclear translocation and its inflammatory responses. 3. To determine the extent of the impact of nNOS derived NO on CD40 /CD40L- mediated interaction between macrophage and endothelial cells and junction permeability. In this aim, we will test our hypothesis that macrophage nNOS-derived NO regulates CD40 expression in macrophages and CD40L expression in endothelial cells. We will check the effect of disruption of CD40/CD40L interaction in endothelial and macrophage by co-culture experiment in the presence and absence of a pharmacological inhibitor of nNOS. Also, by using the in-silico approach, we will screen the natural compound database for a lead molecule generation that could inhibit CD40-CD40L interaction and dampens disease progression.