NAC: N-acetylcysteine; Pts: patients; M: male; F: female; YOA: years of age; RP: Raynaud’s phenomenon; SSc: systemic sclerosis; mg: milligram; kg: kilogram; h: hour; Ref: references; i.v.: intravenous infusion; DU: digital ulcer; tid: three times a day; AAA: abdominal aortic aneurysm; b.m.: body mass; b.w.: body weight; CKD: chronic kidney disease; GSH: glutathione. NAC has been reported to protect against coronary artery diseases (CAD), myocardial infarction (AMI), myocardial injuries, and cardiomyopathy ^{[15][16][17][18][19][20]}[15,16,17,18,19,20]. In patients with cardiac surgery, NAC decreases diabetes-associated cardiovascular, cardiopulmonary bypass, and cardiac surgery complications, including early reperfusion injury, pump-induced inflammatory response, and myocardial stress ^[21][22][23][21,22,23]. However, a systematic review has shown that NAC has no significant efficacy in improving major adverse outcomes, including mortality, acute renal failure, HF, length of stay and/or outcomes of care in intensive care unit, arrhythmia, and AMI, in patients following cardiac surgery [24]. Multiple clinical studies have demonstrated that NAC treatment effectively reduces the risk of atrial fibrillation (AF), a common arrhythmia post cardiac surgery ^{[25][26][27][28][29]}[25,26,27,28,29], although one study reveals that a high dose of oral NAC treatment had no benefit on postoperative AF [30]. In addition, NAC has been reported to improve HF [31] however, no beneficial effect was observed in patients with doxorubicin-induced cardiomyopathy ^[32][33][32,33]. Animal studies have shown that NAC improves peripheral vascular diseases (PVD), with reduced muscular fatigue ^[34][40], restoration of redox balance and calcium retention capacity, as well as the suppression of reactive oxygen species (ROS) production in mice with hind limb ischemia ^[35][41]. NAC prevented excessive intracellular and extracellular ROS formation in mice with limb ischemia and enhanced the recovery of ischemic limb blood flow and function, in association with a selective increase in circulating endothelial progenitor cell (EPC) numbers (a group of cells critical for endothelial and vascular function) ^[36][37][42,43]. NAC treatment also attenuated C-reactive protein-induced ROS production in EPCs and apoptosis in vitro ^[38][44]. Animal studies also demonstrated that treating low-density lipoproteins (LDL) receptor deficient (LDLR KO) mice on a high-fat diet (HFD) with NAC in drinking water for 4 months significantly decreased ROS production and partially reversed the effects of hyperlipidemia on EPC populations ^[39][45]. Another animal study has revealed that NAC treatment effectively attenuated atherosclerosis progression following particulate matter (PM) exposure in LDLR KO mice with HFD, prevented excessive ROS generation, and reduced the levels of circulating oxidized LDL (ox-LDL) and inflammatory cytokines ^[40][46]. Preclinical animal studies have also shown that NAC normalized serum TNF-α level that was resistant to etanercept or infliximab, and improved HF in rats with cardiac injury ^[41][42][43][47,48,49]. A systematic review has demonstrated that NAC significantly decreased diabetes-associated cardiovascular complications including ischemia and non-ischemia cardiac damage through inhibition of oxidative stress in various animal models [23].

3. NAC and ROS

Excessive ROS plays a critical role in the development and progression of atherosclerosis. Many atherosclerogenic risk factors, including hypertension, diabetes, smoking, and dyslipidemia, increase ROS production, trigger inflammatory response, alter vascular function, and promote the growth of vascular smooth muscle ^[44][45][46][50,51,52]. NAC has been reported to prevent atherosclerosis formation in various animal models. Treatment of hypercholesterolemic rabbits with NAC significantly decreased the gelatinase expression, gelatinolytic activity, and matrix metalloproteinases (MMP)-9 expression in foam cells ^[47][53]. Similarly, it has been reported that treating atherosclerotic rabbits with NAC for 8 weeks significantly attenuated atherosclerotic formation, in association with a reduction in blood ox-LDL, MMP-9, MMP-2, and expression of MMP mRNA ^[48][54]. Using human THP-1 cells treated with phorbol 12-myristate 13-acetate for 48 h, followed by ox-LDL incubation for 4 days to induce foam cell formation, NAC treatment significantly reduced ROS production and ox-LDL uptake, leading to an inhibition of foam cell formation via a down-regulation of CD36 expression ^[49][55]. Another study reports that the treatment of apolipoprotein E knockout (ApoE KO) mice with NAC orally for 8 weeks significantly attenuated the progression of atherosclerosis, with decreased plaque collagen content and nitrotyrosine expression, probably via a reduction in oxidative stress ^[50][56]. In addition, aortic fatty streak plaque was effectively prevented in ApoE KO mice when treated with NAC via intraperitoneal injection for 8 weeks, with a reduction in aortic wall superoxide production ^[51][57]. A study shows that NAC treatment in drinking water for 12 weeks suppressed atherosclerotic development in ApoE KO mice with streptozotocin-induced type-1 diabetes, in association with improved GSH-dependent methylglyoxal elimination, decreased oxidative stress, and the restoration of phosphorylated Akt (p-Akt)/phosphorylated endothelial nitric-oxide synthase (p-eNOS) pathways in aortas ^[52][58]. Native LDL per se is not atherogenic, and ox-LDL is one of the key components in hyperlipidemic states and a potent source of ROS ^[39][53][54][45,64,65]. NAC could inhibit the in vitro oxidation of LDL and prevent the depletion of antioxidant vitamins ^[55][66]. One study has shown that NAC treatment also effectively attenuated the in vivo biotransformation of human native LDL to ox-LDL in a mouse model ^[56][60]. In a small study with 10 patients with CAD and hyperlipidemia, NAC treatment for 7 days significantly decreased the serum ox-LDL level, while there was no significant change in the serum ox-LDL level in patients with placebo ^[56][60]. These data suggest that NAC decreases ROS levels through multiple mechanisms, including an inhibition of the in vivo biotransformation of native LDL to ox-LDL.

4. NAC, Inflammation, and Macrophages

Inflammation is closely related to the development and progression of CVDs, especially atherosclerosis. Myeloid cells-mediated innate immune responses significantly contribute to chronic vascular inflammation ^[57][67]. It has been reported that treatment of human umbilical vein endothelial cells (HUVEC) with NAC effectively blocks the interleukin (IL)-4-induced expression of vascular cell adhesion molecule-1, which stimulates the adhesion of lymphocytes and monocytes to the surface of the vascular endothelium during the early phase of atherosclerosis development ^[58][59][68,69]. IL-6 is known to increase inflammation and the development of vascular diseases, including atherosclerosis. NAC treatment inhibits the production of IL-6 in acetoacetate-treated human U937 monocytes ^[60][70]. Lysophosphatidylcholine is produced from the hydrolysis of phosphatidylcholine by secretory phospholipase A2 (sPLA2) and has proinflammatory and proatherogenic effects on the vasculature ^[61][71]. NAC treatment significantly reduced TNF-α-induced expressions of group V sPLA2 (sPLA2-V) mRNA and protein in HUVEC ^[62][72]. Intraperitoneal injection NAC significantly attenuated balloon-induced neointimal formation in the carotid artery in rats via the inhibition of NF-κB activity in the medial smooth muscle cells ^[63][73]. Treatment of hyperlipidemic rabbits with a combination of the anti-inflammatory drug colchicine with fenofibrate or NAC for 7 weeks significantly reduced aortic atherosclerotic plaque. However, the atherosclerotic burden was significantly lower in the hyperlipidemic rabbits treated with a combination of colchicine with NAC compared with that of colchicine plus fenofibrate. Serum IL-6 levels were also significantly decreased in animals treated with colchicine plus NAC ^[64][63]. Macrophages are one of the important sources for inflammatory cytokines ^[65][74] and play a critical role in the pathogenesis of atherosclerosis ^[66][75]. An increase in macrophage polarization to proinflammatory macrophages (M1), or a decrease to anti-inflammatory macrophages (M2), increases the level of inflammation and promotes atherosclerotic progression ^[67][76]. Thus, the M1/M2 ratio is an important determinant for the direction of inflammatory response ^[68][77]. Macrophages are also important for the stability of atherosclerotic plaques ^[69][78]. The data from a study using aging LDLR^−/− mice showed that inflammatory markers (CRP, MCP-1, and IL-6) were significantly increased, while the anti-inflammatory cytokine IL-10 was significantly decreased in aging LDLR^−/− mice, in association with a significantly increased aortic ROS level and an increased M1/M2 ratio, largely due to decreased M2 population in the aorta. Further studies using bone marrow transplants with GFP-labeled bone marrow cells showed that the increased M1/M2 ratio in the aorta of aging LDLR^−/− mice was predominantly due to decreased M2 polarization, without a significant change in M1 polarization. NAC treatment effectively prevented changes in the expressions of pro-inflammatory and anti-inflammatory cytokines, the ROS level, and macrophage polarization in the aorta of aging LDLR^−/− mice. Interestingly, NAC treatment has no effect on the migration of monocytes from circulation into the aorta in aging LDLR^−/− mice or on M1 population in the aorta ^[70][59].

5. NAC and Atherosclerosis and CAD

Atherosclerosis and related CAD are a leading cause of mortality and morbidity in the world ^[71][79]. A multicenter clinical study, NAC in acute MI (NACIAM), with 112 patients, has shown that a combination of intravenous NAC treatment with a low dose of nitroglycerin (NTG) significantly shrinks the infarction size in patients with acute ST elevation MI undergoing primary percutaneous coronary intervention (PCI) ^[72][34]. Two more small studies (28 and 30 patients each) have reported that the combination of NAC with NTG and streptokinase reduced the levels of oxidative stress and plasma malondialdehyde (MDA), and improved left-ventricular function in patients with acute MI ^[73][74][35,36]. Similar results have been reported when NAC supplemented cold-blood cardioplegia ^[75][37] or when NAC was added to crystalloid cardioplegia in patients with CAD following a coronary artery bypass graft (CABG) ^[76][38]. NAC was also shown to potentiate the effects of NTG on the treatment of patients with unstable angina pectoris and other CAD patients ^[77][78][39,80]. A review of data from clinical studies has shown that NAC has cardioprotective effects in patients who had ischemic heart disease and underwent CABG and PCI ^[79][81]. However, a recent systematic review of 29 clinical trials with 2486 participants and a meta-analysis with 578 patients have demonstrated that NAC treatment does not significantly reduce major adverse events in patients undergoing cardiac surgery, including AMI and mortality ^[24][80][24,82]. Animal studies have shown that the intraperitoneal injection of NAC prevented nonthyroidal illness syndrome-related thyroid hormone derangement and preserved cardiac function in male rats with acute ischemic myocardial injury via the restoration of the redox balance ^[81][84]. Intravenous injection of NAC decreased oxidative stress, infarct area, and apoptosis in a rat cardiac ischemia-reperfusion injury model ^[82][85]. However, intracoronary administration of NAC in a pig model that simulated a catheter-based reperfusion model for the therapy of acute ST-elevated MI (STEMI) showed no significant effect on reducing the infarction size ^[83][86]. A recent study, using an aging LDLR^−/− mouse model with a regular diet, has demonstrated that NAC treatment significantly decreased aortic ROS levels and inflammatory cytokines in the serum and aortas of aging LDLR^−/− mice. The same study has also shown that early and adequate NAC treatment could effectively attenuate atherosclerosis progression in aging LDLR^−/− mice without extreme hyperlipidemia ^[70][59].

6. NAC and Lipid Metabolism

The disruption of the lipoprotein metabolism plays an important role in the development and progression of atherosclerosis ^[84][87]. Pretreatment with NAC significantly inhibited the differentiation of murine 3T3-L1 preadipocytes into adipocytes and decreased intracellular fat accumulation and the expressions of obesity-related proteins, including monoamine oxidase A, heat-shock protein 70, aminoacylase-1, and transketolase ^[85][88]. Similarly, NAC attenuates lipid accumulation and mitogen-activated protein kinases phosphorylation in murine embryonic fibroblasts during adipogenic differentiation ^[86][89]. Lipoprotein (Lp)(a) binds to apoprotein (a) and is considered an independent risk factor for premature atherosclerosis ^[87][90]. It has been shown that treating the serum from patients with a high concentration of Lp(a) with a high concentration of NAC (8 mg /mL or above) in vitro leads to dissociation of Lp(a) from apoprotein ^[88][91]. A small and yet significant reduction in Lp(a) concentration was observed in 12 subjects with a high serum Lp(a) level (87 mg/dL) following 6 weeks of NAC treatment ^[88][91]. However, another small clinical study of seven subjects with a median Lp(a) concentration of 14.3 mg/dL has demonstrated that NAC treatment for 6 weeks had no significant effect on plasma Lp(a) levels ^[88][91]. Similarly, no significant effect of NAC treatment on serum Lp(a) levels were found in 11 subjects with serum Lp(a) levels over 0.3 g/L ^[89][92]. An animal study has shown that treating LDLR KO mice on a HFD with NAC for 2 months or 4 months has no significant effect on the blood lipid profile, including triglycerides (TG), LDL, high-density lipoprotein (HDL), total cholesterol (TC), and non-HDL cholesterol ^[56][60]. Similarly, no significant effect of NAC treatment (250 mg/day, twice a day for 1 week) on the lipid profile was observed in human subjects with CAD and hyperlipidemia ^[56][60].

7. NAC and Homocysteine

An increased level of blood homocysteine is arguably considered a risk factor for atherosclerosis through increased oxidative stress, endothelial dysfunction, and thrombosis formation ^[90][93]. It has been reported that treating human subjects with NAC significantly reduced blood homocysteine levels by 45% over the placebo control ^[89][92]. The NAC treatment of patients with chronic renal failure led to a 16% reduction in plasma homocysteine levels ^[91][94]. Intravenous administration of NAC significantly decreased the level of plasma homocysteine in healthy subjects ^[92][95]. Data from controlled trials in middle-aged male subjects with unmedicated hyperlipidemia, with or without smoking, has shown that oral NAC treatment significantly reduced the level of total plasma homocysteine, regardless of lipid or smoking status ^[93][96]. However, Miner and colleagues have reported that treating cardiac transplant recipients with NAC had no significant impact on plasma homocysteine levels or brachial endothelial function ^[94][97].

8. Effects of NAC on Endothelial Cells

Endothelial dysfunction has been considered the first step of atherosclerosis development ^[95][98]. It was reported that treating endothelial cells from porcine pulmonary arteries with NAC significantly increased intracellular glutathione levels and partially prevented TNF-α-induced endothelial dysfunctions ^[96][99]. NAC also attenuated aortic endothelial damage in ApoE KO mice with streptozotocin-induced diabetes and HFD in association with increased levels of pAkt and -p-eNOS in aorta, as well as NO in serum ^[52][58]. Treatment of human aortic endothelial cells (HAEC) with NAC significantly attenuated TNF-α-induced ROS production and the DNA-binding activities of activator protein-1 and NF-κB, as well as p65 Ser276 phosphorylation ^[97][100]. Long-term treatment of endothelial cells (EC) from arterial segments of patients with severe CAD with NAC delayed senescence of diseased EC via the catalytic subunit of telomerase activation and transient telomere stabilization ^[98][101]. Intra-arterial infusion of NAC in healthy human subjects at a rate to achieve a blood concentration of 1 mM potentiated the effects of NTG on vasodilation and enhanced the biotransformation to an endothelium-derived relaxing factor equivalent nitrosothiol ^[99][102]. Intracoronary infusion of NAC in patients with or without coronary atherosclerosis significantly potentiated acetylcholine-induced coronary and femoral vasodilation and SNP-induced coronary vasodilation ^[100][103].

9. Mechanisms for the Actions of NAC on Atherosclerosis

The mechanisms for the effects of NAC on ROS generation, inflammation and atherosclerosis are very complex and have not been fully defined. Traditionally, NAC is considered to function as an antioxidant through a reduction in disulfide bonds or the scavenging of ROS or replenishing intracellular GSH stores ^[101][104]. However, in many settings and situations, the mechanisms of actions of NAC have remained unclear. Accumulating data has supported the concept that NAC is more like an anti-inflammatory agent with immunomodulatory properties, through its ability to attenuate the activation of oxidant-sensitive pathways, including the NF-κB and p38 mitogen-activated protein kinase (MAPK) signaling pathways, and subsequent reductions in pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 ^{[5][102][103]}[5,105,106]. Due to the complex nature of atherosclerosis pathogenesis, the mechanisms for the effects of NAC on atherosclerosis are also complex, including (but not limited to) (1) modifications of lipid metabolism, (2) inhibition of the expressions of gelatinase, MMP-2, and MMP-9, (3) blocking the in vivo biotransformation of native LDL to ox-LDL, and directly suppressing ROS production from ox-LDL, (4) increasing intracellular glutathione levels, and thus protecting endothelial function, (5) attenuation of NF-κB and p38-MAPK signaling, thus decreasing inflammatory cytokine production, (6) the preservation of circulating endothelial cell progenitor cells, (7) reduction of homocysteine levels, (8) attenuation of endothelial senescence and damage, while enhancing endothelial function through multiple mechanisms, including activation of Akt signaling and eNOS, and (9) the preservation of M2 polarization in the hyperlipidemic condition, thus reducing inflammation and oxidative stress, as shown in Figure 1.