Hydrogen sulfide (H₂S) is an identified and recognized gasotransmitter after nitric oxide and carbon oxide. As endogenous methionine catalysis production, H₂S major generates from homocysteine trans-sulfide metabolism. Cystathionine β synthase (CBS), cystathionine γ lyase (CSE), cysteine aminotransferase (CAT), and 3-mercaptopyruvate sulfur transferase (3-MST) are major synthetases of H₂S. In cardiovascular tissues, CSE and 3-MST are expressed, and CBS to a much lesser extent. There is no accurate experimental evidence of dominant CSE protein expression in cardiovascular tissues. However, in mouse liver (most endogenous H₂S generation organ), CSE protein exceeds CBS protein by about 60-fold [1], and CSE mRNA is also about 4.8-fold comparison to CBS and 48.9-fold to 3-MST [2]. Therefore, H₂S biosynthetic activity might be sourced from CSE. More and more studies highlight that CSE/H₂S protects against cardiovascular diseases, including atherosclerosis in particular.

Atherosclerosis is one of most prevalent cardiovascular diseases worldwide, characterized as chronic inflammation and lipid accumulation in the large arteries. Atherosclerotic plaque rupture or erosion causes the formation of thrombosis or blood clot, resulting in acute events, such as myocardial infraction, or ischemic stroke, contributing to a major mortality rate in the case of human diseases [3]. Although acute coronary syndrome (ACS) or sudden deaths in younger individuals or women patients are usually triggered by plaque erosion, even superficial erosion ^[4][5][4,5], most cardiovascular events occur due to the plaque rupture. Therefore, plaque stability is crucial for preventing cardiovascular events. Some risk factors (such as hyperhomocysteinemia, hypercholesteremia etc.) promote necrotic core formation due to efferocytosis impairment (causing a reduction of apoptotic foam cells clearance), collagen synthesis decrease, and protease (e.g., matrix metallopeptidase) expression/activity elevation, triggering the thickness of fibrotic cap, local acute or chronic inflammation response, intraplaque hemorrhage, and plaque calcification [6], contributing to the pathophysiological modulation of plaque stability.

2. Intraplaque Cell Types and Interactions in Plaque Stability

Plaque stability is mediated by many complicated factors. In 1989, Muller JE and colleagues encapsulated a hypothetic concept of “vulnerable atherosclerotic plaque”, characterized as collagen exposure, or large necrotic core, based on studies of autopsy and angiographic data from myocardial infraction or sudden cardiac death ^[7][11]. In 2000, Virmani R. groups showed a “thin” fibrous cap (<65 μm) plaque was “vulnerable” ^[8][12]. However, some in vivo human image clinical trials demonstrated that “vulnerable plaque” did not inevitably rupture or cause thrombotic events ^[9][10][11][13,14,15]. Although these studies are contradictory, they still imply some “vulnerable” characteristics: such as large necrotic core, more macrophages, VSMC apoptosis and necrosis, reduced the collagen level and active inflammation ^[12][16]. In human atherosclerotic plaque, approximately 14 different cell populations were identified, including endothelial cells, VSMCs, myeloid cells, and immunocytes (T cells, B cells and mast cells) ^[13][17]. Animal and human evidence has demonstrated that each intraplaque cell population plays a critical role in plaque development and stability.

3. Cystathionine γ Lyase/Hydrogen Sulfide Role in Atherosclerotic Plaque Stability

As a gasotransmitter, H₂S can easily penetrate the biological membranes due to its lipophilic characteristics. In the body fluid, approximately 1/2 H₂S remains undissociated form, and 2/3 exists as HS- ion form at equilibrium with H₂S. Recent studies also show that most H₂S exists in the body as protein binding form ^[14][55]. Thus, binding H₂S, HS-, and free H₂S maintain a dynamic equilibrium in the body. Once a damage stimulus is received, such as inflammation or reactive oxygen species (ROS), the metabolic activity is increased in cells, and the pH values of local microenvironment are reduced, which can accelerate free H₂S generation from HS-. Due to the reduction feature of H₂S, the constant consumption caused a decrease of local H₂S level, which led to up-regulation of CSE/H₂S system (such as acute inflammation ^[15][56]) at compensatory phase, or down-regulation of CSE/H₂S system at uncompensated period. This may be an interpretation of CSE/H₂S reduction in many cardiovascular diseases ^[15][56]. More and more research has shown that CSE/H₂S play a protective role in atherogenesis ^[16][57]. In human and mouse atherosclerotic plaque, CSE protein expression was downregulated ^[17][18][7,58]. Global CSE knockout exacerbated plaque growth by promoting inflammation and oxidative stress ^[19][59]. Oppositely, H₂S donors attenuated atherogenesis by inhibiting endothelial inflammation and foam cell formation ^[17][20][21][7,8,9]. For the mechanism, CSE/H₂S may modulate VSMC phenotype switch, macrophage polarization, and lipid-phage, endothelia inflammation and monocytes adhesion, and atherothrombosis response, attributed to the genesis and development of plaque ^[16][22][57,60]. Recently, Xiong Q and colleagues showed that H₂S enhanced plaque stability by increasing fibrous cap thickness and collagen content, reducing plaque VSMC apoptosis and expression of the collagen-degrading enzyme matrix metallopeptidase-9 (MMP-9) in ApoE knockout mouse model ^[23][10]. Here, rwesearchers discuss the effect of endogenous CSE/H₂S in plaque stability, regarding essential risk factors and intraplaque cell population functions.

3.1. H

₂

S Reduced Hypercholesterolemia

Heightening blood cholesterol is a critical process for atherosclerosis initiation and growth. Lowing lipid therapy is the still first-line atherosclerosis therapeutic selection, and it effectively reduces the morbidity and mortality of cardiovascular events. In healthy adult volunteers, circulatory H₂S level positively correlated with blood HDL-cholesterol level (r = 0.49), but negatively correlated with LDL/HDL ratio (r = −0.39) ^[24][61]. ReseaOurchers previous researchstudy also showed that H₂S donors lowered serum total cholesterol and LDL cholesterol ^[21][9], in part by increasing LDL receptor (LDLR) expression and enhancing LDL uptake activity ^[25][62]. In this mechanism, firstly, H₂S sulfhydrates Kelch-like ECH-associated protein 1 (Keap-1) at Cys151 site, then promotes nuclear erythroid 2-related factor 2 (Nrf-2) dissociation and nuclear translocation, and up-regulates LDL-related protein 1 (LRP-1) expression in hepatocytes ^[26][63]. Secondly, H₂S activates the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)-sterol regulatory element binding proteins 2 (SREBP-2) signaling pathway and inhibits proprotein convertase subtilisin/kexin type 9 (PCSK9), causing LDLR protein elevation ^[25][62], which is not dependent on liver X receptor α (LXRα) ^[27][64].

3.2. H

₂

S Attenuates Hyperhomocysteinemia

Hyperhomocysteinemia is an independent risk factor for atherosclerosis. Some studies also supported that high blood total homocysteine (Hcy) level correlated with cardiovascular events. However, lowering homocysteine therapy by B type vitamin and/or folic acid by enhancing Hcy re-methylation do not effectively reduce morbidity and mortality of cardiovascular events, but just slightly attenuate stroke mortality for those presenting with hypertension and hyperhomocysteinemia ^[28][65]. H₂S is a product of homocysteine trans-sulfur catalysis. H₂S can increase CSE catalysis Hcy activity or methylenetetrahydrofolate reductase activity by sulfhydration, resulting in the reduction of serum Hcy, then attenuate Hcy-induced macrophages infiltration in the plaque and plaque area ^[29][30][66,67]. In vitro, H₂S also reduced Hcy-induced reactive oxygen species (ROS) and endoplasmic reticulum stress ^[31][32][68,69]. These studies also provide a new therapeutic clue using H₂S donors, in order to prevent cardiovascular events in patients suffering from hyperhomocysteinemia.

3.3. H

₂

S Modulates VSMC Function in Pathogenesis of Plaque Stability

VSMC function contributes to the formation, growth/development, and even rupture of atherosclerotic plaque ^[33][20]. In the arterial wall, endogenous H₂S dominant derived from VSMCs ^[34][70]. Numerous evidence, direct and indirect, including in vivo and in vitro studies, demonstrated that the endogenous CSE/H₂S system may mediate VSMC proliferation, cell death (apoptosis, autophagy, ferroptosis), phage-like differentiation, and calcification, to participate in the pathophysiological process of plaque stability (Figure 1).