Hydrogen sulfide and vascular regulation – An update

Graphical abstract

Abstract

Background

Hydrogen sulfide (H₂S) is considered to be the third gasotransmitter after carbon monoxide (CO) and nitric oxide (NO). It plays an important role in the regulation of vascular homeostasis. Vascular remodeling have has proved to be related to the impaired H₂S generation.

Aim of Review

This study aimed to summarize and discuss current data about the function of H₂S in vascular physiology and pathophysiology as well as the underlying mechanisms.

Key Scientific Concepts of Review

Endogenous hydrogen sulfide (H₂S) as a third gasotransmitter is primarily generated by the enzymatic pathways and regulated by several metabolic pathways. H₂S as a physiologic vascular regulator, inhibits proliferation, regulates its apoptosis and autophagy of vascular cells and controls the vascular tone. Accumulating evidence shows that the downregulation of H₂S pathway is involved in the pathogenesis of a variety of vascular diseases, such as hypertension, atherosclerosis and pulmonary hypertension. Alternatively, H₂S supplementation may greatly help to prevent the progression of the vascular diseases by regulating vascular tone, inhibiting vascular inflammation, protecting against oxidative stress and proliferation, and modulating vascular cell apoptosis, which has been verified in animal and cell experiments and even in the clinical investigation. Besides, H₂S system and angiotensin-converting enzyme (ACE) inhibitors play a vital role in alleviating ischemic heart disease and left ventricular dysfunction. Notably, sulfhydryl-containing ACEI inhibitor zofenopril is superior to other ACE inhibitors due to its capability of H₂S releasing, in addition to ACE inhibition. The design and application of novel H₂S donors have significant clinical implications in the treatment of vascular-related diseases. However, further research regarding the role of H₂S in vascular physiology and pathophysiology is required.

Introduction

Hydrogen sulfide (H₂S) was discovered to be the third gasotransmitter after nitric oxide (NO) and carbon monoxide (CO). This novel gaseous molecule has been proved to be widely involved in the regulation of various systems in human body [1]. Moreover, H₂S has attracted great attention in regulating the structure and function of blood vessels. Many researchers have shown that H₂S exerts vital effects on vascular cellular processes, such as inflammation, apoptosis, cell cycle, cytoprotection, and mitochondrial metabolic function and biogenesis [2].

In the vasculature, H₂S modulates vascular tension, suppresses the proliferation, and exerts a bidirectional effect on apoptosis and autophagy of vascular smooth muscle cells (VSMCs). Furthermore, the development of many vascular remodeling-associated diseases, including hypertension, atherosclerosis and pulmonary hypertension has been proved to be related to the impaired H₂S generation. In addition, H₂S and the use of zofenopril, one of the ACE inhibitors that can promote the release of H₂S, in cardiovascular diseases are also gradually being valued. Therefore, the understanding how H₂S is endogenously generated, as well as the regulation of blood vessels by H₂S under physiological and pathological conditions, may elucidate the pathogenesis of vascular diseases and uncover new promising targets for the prevention and treatment of vascular diseases.

Endogenous H₂S generation and metabolism

The generation of endogenous H₂S is mostly catalyzed by enzymes, while only a small part is produced by non-enzymatic pathways [3], [4]. The enzymes that catalyze H₂S production mainly include cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), 3-mercaptopyruvate sulfur transferase (3-MST) and cysteine aminotransferase (CAT) [5], [6]. CBS and CSE are the primary enzymes involved in H₂S production [7] that catalyze the substrate L- cysteine with tissue specificity. CBS is abundant in the brain, liver, and kidney, with small amount of expression in the uterine artery, mesenteric artery, and carotid ball [8]. CSE predominantly catalyzes synthesis of H₂S in the liver, ileum, portal vein, thoracic aorta and non-vasculature [2], [9]. Recently, 3-MST has been found to catalyze H₂S synthesis in the central and peripheral nervous systems, vascular endothelium and other tissues [10]. It catalyzes 3-mercaptopyruvate (3-MPT), which produces H₂S and pyruvate in vivo. Among these three enzymes, homocysteine is converted into cystathionine and cysteine in turn by sulfur transfer under the catalysis of CBS and CSE. Cysteine and thiols are catalyzed by CBS via β-substitution. Different from CBS, CSE catalyzes three kinds of reactions, including the α, β-cleavage of cysteine, the α, γ-cleavage of homocysteine, and the γ-substitution of homocysteine through a second mole of homocysteine. As a sulfurtransferase, 3-MST is responsible for transferring sulfur from mercaptopyruvate to an active cysteine site, and then forms MST-SSH, a persulfide intermediate. Except for thioredoxin, a variety of small molecules such as dihydrolipoic acid, homocysteine, cysteine and glutathione (GSH) release H₂S by receiving the persulfide group in the presence of reductant [11]. Opposite to the enzymatic reaction, the non-enzymatic reaction of H₂S generation is partial for cysteine as a substrate and is catalyzed by coordinated activities of VitB₆ and iron. Non-enzymatic production of H₂S occurs in the spleen, heart, lung, muscles, bone marrow and plasma, especially in RBCs [12]. The aortic H₂S production rate is reported to be 5.8 ± 1.7 pmol s⁻¹ mg protein^-1[13]. In addition, various arteries demonstrate different production rates of H₂S. The H₂S production rate in the caudal artery, the mesenteric artery, the pulmonary artery and the thoracic aorta was 8.12 ± 0.85, 6.17 ± 0.56, 5.31 ± 0.70 and 4.06 ± 0.28 pmol s⁻¹ mg wet tissue^-1, respectively [14].

After synthesis by transsulfuration from L-cysteine, various metabolic pathways participate in the regulation of H₂S concentration in the cell. Significant pathways for H₂S metabolism include oxidation by sulfide quinone oxidoreductase (SQR) and persulfide dioxgenase (ETHE1) in the mitochondrion and methylation by cysteine dioxygenase (CDO) in the cytoplasm [15]. Sulfide is oxidized in the mitochondrion by SQR to generate persulfide. Persulfide is further oxidized to sulfite by ETHE1, and sulfite is finally oxidized by rhodanese or sulfite oxidase. After ubiquinone captures electrons released in the SQR reaction, the electrons are transferred to complex III in the electron transport chain [16]. In addition to the above oxidation pathway metabolism, Olson et al [17] proved that superoxide dismutase (SOD) also oxidizes H₂S to produce polysulfides. Methemoglobin and molecules containing metallo or disulfides such as oxidized glutathione may also eliminate H₂S [3], [18].

Physiological regulation of blood vessels by H₂S

H₂S on vascular tone

H₂S has a bidirectional regulatory effect on vascular tone. H₂S can not only relax blood vessels, but also contract blood vessels [19]. A study published in Science [20] showed that the activation of CSE by calcium-calmodulin (CaM) under physiological conditions is the main mechanism of H₂S production in the vascular system. Mutant mice lacking CSE displayed lower levels of H₂S, with abnormally elevated blood pressure and loss of endothelium-dependent vasodilatory function. These findings directly prove the significance of H₂S for the maintenance of vascular function. Intriguingly, the vasodilation of H₂S on the portal vein and the ileum was notable stronger than that on the thoracic aorta [21]. In addition, compared with H₂S, hydrogen polysulfides (H₂Sn) tended to contain more sulfane sulfur atoms which have a relaxing effect and ultimately lowered blood pressure [22], [23].

H₂S also has vasoconstrictive effects under certain conditions. NaHS contracts VSMCs at concentrations between 5 × 10⁻⁶ M and 10⁻⁴ M [24]. A study by Ping reported similar results [25]. NaHS at concentrations ranging from 10 to 300 μM induced coronary artery constriction in rats. Therefore, the regulation of H₂S on vascular tone is bidirectional.

The mechanisms underlying H₂S-induced vasodilation are not fully understood. The effects of vasodilation have been attributed to iron channels that are activated by H₂S according to previous studies [26]. It is suggested that H₂S exerts a vasorelaxant effect via opening ATP-sensitive potassium channels (K_ATP channels) in VSMCs [27]. H₂S mediates a new type of protein post-translational modification that is sensitive to redox, namely sulfhydration. [28]. More specifically, H₂S causes sulfhydration of cysteine-43 (C43) in Kir6.1 (a subunit of K_ATP channel), resulting in a decrease in the capacity of Kir 6.1 binding to ATP, while the capacity of Kir 6.1 binding to PIP₂ is enhanced. This event eventually causes K_ATP channels to open and VSMCs to relax [29]. Excepting the K_ATP channel, growing evidence demonstrates that calcium-activated potassium channels (K_ca channels) are also activated by H₂S [30], [31]. H₂S increases smooth muscle Ca²⁺ spark activity to activate endothelial large-conductance calcium-activated potassium channels (BK_Ca channel) [32]. Transient receptor potential cation channel V4 (TRPV4) is also modified by H₂S through sulfhydration. This is followed by the activation and the opening of TRPV4-dependent Ca²⁺ internal flow and the endothelial BK_Ca channel and results in vasodilation [33]. In addition, the SK_2.3 channel which acts as an α-subunit isoform of the SK_Ca channel is activated by H₂S through S-sulfhydration [34]. Moreover, the activation of voltage sensitive potassium channels (K_V channels) and Kv7.4 voltage-gated potassium channels which are predominantly expressed in VSMCs are seen as targets for H₂S action on vascular tone [35], [36]. Recent reports have also demonstrated that H₂S caused S-sulfhydration of L-type Ca²⁺ channels, leading to a decrease in intracellular Ca²⁺ concentration ([Ca²⁺] _i) [37].

Whether H₂S participates in the regulation of the cyclic guanosine monophosphate (cGMP) pathway remains controversy. A compelling amount of evidence indicates that H₂S exerts a vasodilative effect through the activation of endothelial nitric oxide synthase (eNOS) and the inhibition of cGMP degradation [38], [39], [40]. There are several primary mechanisms thought to participate: (1) H₂S directly reacts with NO to produce nitroxyl (HNO), thereby activating the HNO– transient receptor potential ankyrin 1 (TRPA1)–calcitonin gene-related peptide (CGRP) pathway to regulate vascular tone [41]. (2) H₂S inhibits the activity of phosphodiesterase 5 (PDE5) by reducing cGMP degradation and promoting cGMP signaling, followed by the activation of cGMP-dependent protein kinase (PKG) to phosphorylate the vasodilator-stimulated phosphoprotein (VASP), eventually resulting in vasodilation [42]. In addition, Sun et al. [43] believed that H₂S sulfhydrated associated PDE5A dimerization to exert the vasorelaxant function. (3) H₂S may alleviate oxidative stress, resulting in increased eNOS coupling by phosphorylation of eNOS^S1177 [44], [45]. (4) The reaction of soluble guanylyl cyclases (sGCs) to NO can be enhanced by H₂S [40], [46]. It might be related to the reduction of sGC heme Fe by H₂S, so as to facilitate NO-regulated cellular signaling processes [47]. However, there is disagreement over the role of H₂S. For instance, Wang [48] et al. suggested that H₂S did not rely on cGMP pathway to exert vasodilation, although vasodilation was strengthened by specific sGC inhibitors (ODQ and NS-2028). Similarly, NaHS-induced relaxation was unaffected by ODQ in rat coronary arteries [49]. Taken together, the vasorelaxation of H₂S varied very widely in different species and cell types. This might explain the conflicting results [46].

The vasodilation of H₂S was also related to the suppression of mitochondrial complexes I and III. It was shown that NaHS (100–1000 μM) suppressed mitochondrial electron transport to exert a vasodilation effect in rat mesenteric arterioles. This effect was inhibited by complex I and complex III inhibitors [30].

Accumulating evidence from H₂S studies demonstrates that H₂S derived from perivascular adipose tissue (PVAT) also exerts a critical effect in the regulation of vascular tension [33], [50]. PVAT exerts predominantly anti-contractile effects, which is induced by adipocyte-derived relaxing factor (ADRF) [51], [52]. Schleifenbaum et al. [53] suggested that H₂S could be an ADRF to regulate vascular tone. The mechanism of H₂S as ADRF could relate to activate K_ATP and (or) voltage-sensitive K_CNQ potassium channels [54], [55]. Importantly, the findings from Kohn et al. [55] suggest that with technical progress, future studies on the vascular H₂S/K_CNQ pathways make it possible to relieve vascular dysfunction.

In summary, H₂S-induced vasorelaxation takes place via the activation of iron channels, the interactions with NO-cGMP signaling, the inhibition of mitochondrial complexes I and III, and H₂S as an ADRF. However, under certain conditions, H₂S has vasoconstrictive effects which appear to involve the activation of Na⁺-K⁺-2Cl^-–co-transporters and voltage-gated calcium ion channels by H₂S [24]. Additionally, Ping et al. [25] suggested that the activation of the Rho kinase signaling pathway by H₂S may participates in the contraction of rat coronary arteries.

Effects of H₂S on proliferation and apoptosis of vascular smooth muscle cells

Accumulating evidence implicates H₂S as an inhibitor of VSMC proliferation. It was shown that the VSMC proliferation rate in CSE knockout mice was dramatically increased. However, endogenous H₂S significantly inhibited the proliferation of smooth muscle cell (SMC) in CSE knockout mice [56]. Similarly, NaHS, a commonly used H₂S donor, dose-dependently suppressed the proliferation of VSMCs [57]. The potential mechanisms for H₂S-induced proliferation are as follows: Du et al. [57] demonstrated that H₂S suppressed the activity of mitogen-activated protein kinase (MAPK), which might be responsible for H₂S-inhibited VSMC proliferation. Furthermore, endogenous CSE/H₂S pathway can inhibit the cascade conduction of MAPK/thioredoxin interacting protein (TXNIP) signals [58], thereby protecting endothelial function. In addition, H₂S dramatically inhibited the transcription and expression of Brg1 gene, reduced the recruitment of Brg1 in the promoter region of proliferating genes (pcna, ntf3 and PDGFα) and consequently inhibited the proliferation of VSMCs [59]. On the other hand, H₂S not only decreased the expression of insulin-like growth factor-1 receptor (IGF-1R), but also modified IGF-1R through sulfhydration to prevent IGF-1 binding, ultimately inhibiting VSMC proliferation [60]. Recently, Wang et al. [61] demonstrated that calcium-sensing receptor (CaSR) increased endogenous generation of H₂S via calcium-CaM signal pathways, ultimately inhibiting the proliferation of VSMCs. Therefore, several genes, molecules, and signaling pathways (such as MAPK/TXNIP signals, Brg1, ERK1/2, IGF-1R and CaSR) have been identified in the regulation by H₂S, and contribute to the suppression of VSMC proliferation.

H₂S can promote or inhibit vascular cell apoptosis. Several studies agree with the view that H₂S promotes apoptosis. Studies [62], [63] have demonstrated that H₂S can activate the ERK/caspase 3 pathway and promote the apoptosis of human aorta smooth muscle cell (HASMC). CSE overexpression or exogenous H₂S supplementation promotes apoptosis via stimulating extracellular regulated protein kinases (ERK) 1/2, p38 MAPK, and p21 ^Cip/WAK-1 but suppressing cyclin D1 [56], [62]. In contrast, several studies suggest that H₂S inhibits apoptosis. H₂S decreased the elevated ratio of Bcl2-associated x (Bax)/B-cell lymphoma-2 (Bcl-2) and the activity of caspase-3, thus inhibiting apoptosis caused by high glucose [64]. It was also shown that NaHS suppressed apoptosis by reducing the expression of caspase-12, C/EBP homologous protein (CHOP), and glucose-regulated protein 78 (GRP78) which are related to endoplasmic reticulum stress (ERS), thus protecting vascular endothelial function [65]. Therefore, the regulation of apoptosis by H₂S is bidirectional. It can promote and inhibit apoptosis under different pathological conditions.

Effect of H₂S on vascular autophagy

Autophagy is essential for homeostasis in processes including cell development and differentiation, regulation of cell longevity and programmed cell death, degradation of invading pathogens, and provision of antigens to the immune system [66]. Pathogens, abnormal proteins and organelles are engulfed by autophagosomes and undergo lysosomal degradation [67], [68]. H₂S is reported to either promote or inhibit autophagy depending on the different pathological process [69], [70]. NaHS was shown to activate mitophagy in rat aortic endothelial cells (RAECs) [71]. Mechanistically, NaHS facilitates Parkin recruited by PTEN induced putative kinase 1 (PINK1), and then ubiquitylates mitofusin 2 (Mfn2), leading to the upregulation of mitophagy [71]. However, several studies showed that both supplementation of H₂S and the overexpression of its synthetases mitigated mitophagy [72]. H₂S inhibited adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway, which is closely associated with autophagy [73]. On the other hand, the ratio of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II to LC3-I is commonly used as an indicator of autophagy. Expression of LC3A I/II was significantly decreased with supplementation of H₂S (30 μM) [72]. NaHS could also inhibit the excessive autophagy of vascular endothelial cells by suppressing nuclear factor erythroid-2-related factor 2 (Nrf2)- reactive oxygen species (ROS) -AMPK signaling pathway [74]. Taken together, there are still different opinions of vascular autophagy regulation by H₂S. A variety of pathological conditions likely contribute to the differences in the effect that have been observed.

Pathophysiological regulation of H₂S on blood vessels

H₂S and hypertension

Treating hypertension which is defined as ≥ 140/90 mmHg with chronically increased blood pressure remains a great challenge. Several clinical studies showed a close correlation between hypertension and reduction of H₂S. The reduction of endogenous H₂S synthesis and H₂S-dependent vasodilation led to a microvascular dysfunction in hypertensive patients [75]. Notably, CBS, CSE and 3-MST as the H₂S generating enzymes, were markedly decreased in humans with hypertension [76], suggesting that H₂S generation pathway may be involved in the pathogenesis of hypertension. Similar results have also been shown in animal research. For instance, a decreased endogenous H₂S content in the aorta was observed in the development and progression of spontaneously hypertensive rats (SHRs) [77]. The use of DL-propargylglycine (PPG), a CSE inhibitor, dramatically elevated the level of basal blood pressure in WKY rats and promoted vascular remodeling, demonstrating that a sufficient H₂S level is necessary for the maintenance of basal blood pressure [78]. Similar to that of SHRs, it was shown that CBS/H₂S pathway was down-regulated in salt-sensitive Dahl rats [79].

Extensive evidence shows that H₂S exerts a crucial effect on blood pressure regulation in pathological cases. For instance, studies by Sun et al. [80] suggested that NaHS lowered tail artery pressure in SHRs. Similarly, it was shown that H₂S delayed the shift from prehypertensive to hypertensive status in SHRs [81]. Notably, H₂S improved endothelial function in renovascular hypertensive rats and ameliorated the damaged endothelium-dependent contraction (EDC) and endothelium-dependent relaxation (EDR) [82], [83]. Furthermore, the H₂S donor alleviated hypertension, reversed aortic remodeling, and inhibited the renin–angiotensin–aldosterone (RAS) system in renal tissue of Dahl rats [79]. These experimental results demonstrate that H₂S dramatically suppressed the elevation in blood pressure in two animal models.

Many scholars have discussed the protective effect of H₂S on hypertension and its potential mechanisms. Previous studies [82], [83] showed that the H₂S donor NaHS significantly suppressed the activation of NOD-like receptors (NLRP3), inflammasomes, and oxidative stress in SHRs. Moreover, the amelioration of excessive EDC of H₂S was associated with the inhibition of the bone morphogenetic protein 4 (BMP4) and its downstream signal molecules [84]. NaHS can also protect renal artery endothelial cells and improve endothelial function through the activation of the peroxisome proliferator-activated receptor δ (PPARδ) signaling [85]. In addition to improvements in the vascular endothelium, NaHS also regulated immune function by reducing the expression of connexin 40 (Cx40)/connexin 43 (Cx43) T lymphocytes in SHRs, and reversed changes in multiple T lymphocyte subtypes in SHRs [86], which may explain the anti-inflammatory effect of H₂S. Ion channels are considered as key targets for H₂S depressurization. A report from Sun et al. [80] suggested that the K_ATP channel is activated by H₂S and causes vasodilation. Furthermore, H₂S may activate K_ATP channel by inhibiting Forkhead box O1 (FOXO1) and Forkhead box O3a (FOXO3a) phosphorylation, subsequently inducing their nuclear binding to SUR2B and Kir6.1. In addition to the regulation of the K_ATP channel, H₂S can also activate the TRP vanilloid 1(TRPV1) ion channel through S-sulfhydration, increasing the sensitivity of carotid sinus pressure receptors in SHRs [87]. TRPA1 channels were also activated by H₂S, inducing the release of CGRP and promoting vasodilation [88], [89]. On the other hand, H₂S also inhibited the pathological state of SHRs by regulating the RAS system. H₂S reduced the expression of RAS-related mRNA (Ren, Atp6ap2, Agt, Ace, and Agtr1a) in the kidneys of SHRs, which blocked the RAS system and exerted a vasomotor effect [81]. Finally, an underlying H₂S mechanism may be related to the inhibition of collagen deposition. H₂S dose-dependently inhibited MAPK activation induced by angiotensin II in SHRs and down-regulated the affinity of angiotensin II type 1 (AT1), ultimately inhibiting vascular remodeling and collagen deposition in SHRs [90]. Furthermore, reduced collagen deposition by H₂S may be related to the suppression of transforming growth factor-β /Smad signaling pathway [91].

The mechanism by which H₂S regulates blood pressure in high-salt Dahl rats may be as follows. Liang et al. [92] showed that H₂S reduced the oxidative stress response in the paraventricular nucleus of high-salt Dahl rats, attenuated sympathetic activity, and promoted the secretion of anti-inflammatory factors, thus inhibiting the inflammatory response. H₂S may also regulate blood pressure by the inactivation of epithelial sodium channels (ENaC). Reabsorption of sodium by the ENaC promotes the progress of salt-sensitive hypertension. It was shown that H₂S completely blocked abnormal activation of ENaC caused by excessive H₂O₂. H₂O₂ increased sodium reabsorption by up-regulating phosphatidylinositol 3, 4, 5-trisphosphate. H₂S can significantly inhibit PTEN inactivation caused by H₂O₂, thereby reducing oxidative stress [93].

To summarize, the mechanisms by which H₂S inhibits hypertension are complicated, including the reduction of oxidative stress and inflammation, the modulation of immune function and ion channels, and the inhibition of collagen deposition and vascular remodeling.

H₂S and atherosclerosis

Atherosclerosis (AS) is a chronic, complicated and progressive pathological process of large and medium-sized arteries. Several studies have shown that H₂S deficiency is related to the pathogenesis of AS. For example, Gao et al. [94] suggested that H₂S deficiency may predispose stable coronary artery disease (CAD) patients to vulnerable plaque rupture. As reported in many clinical studies, Wang et al. [95] found disorders of the vascular CSE/H₂S pathway in apolipoprotein E (ApoE)-knockout mice. Another study from Meng et al. [96] also demonstrated that decreased endogenous H₂S generation accelerated AS in CSE-knockout mice. Accumulating evidence [97], [98] has shown that endogenous H₂S produced by CSE in blood vessels has an anti-AS effect. Unstable plaques generated by AS are prone to rupture and have the risk of infarction [99]. In ApoE-knockout mice, H₂S stabilizes atherosclerotic plaques and suppresses lipid deposition [100], [101].

Key mechanisms for the anti-AS effect of H₂S include anti-oxidative stress, anti-inflammatory effect, and regulation of ion channels [102] to protect the vascular endothelium. Intriguingly, it was reported that vascular CSE/H₂S, as the target of estrogen, was involved in the mechanism by which estrogen protected against AS [103]. The detailed mechanism is as follows.

First, H₂S attenuates oxidative stress to protect against AS. It induces S-sulfhydration of glutathione peroxidase 1 (GPx1) to prompt glutathione synthesis, resulting in alleviating lipid peroxidation and improving antioxidant capacities [104]. Several studies [105], [106] further found that H₂S may induce Nrf2 to dissociate from kelch-like ec-associated protein 1 (Keap1) by sulfhydration of Cys151 in Keap1, enhancing nuclear translocation of Nrf2 and thereby exerting antioxidant stress and cardiovascular protection. Moreover, translocation of Nrf2 further stimulated its downstream molecules, including the NADPH quinoneoxidoreductase 1 (NQO1), thus preventing the release of inflammatory cytokines [107]. H₂S was found to attenuate atherosclerotic lesions by blocking oxidative modification of low density lipoprotein (LDL) and elevating antioxidative ability [108]. A recent study shows that H₂S-induced antioxidant stress is also related to its elimination of oxidized hemoglobin (Hb) and inhibition of the interaction between Hb and lipid in AS [109]. Through the regulation of above molecules, H₂S exerts a critical role in prevention of collagen deposition and protection of vascular function.

Secondly, H₂S attenuates inflammation to protect against AS. Inactivation of nuclear factor kappa-B (NF-κB) caused by H₂S reduces the expression of inflammatory factor intercellular cell adhesion molecule-1 (ICAM-1), which may be an important reason for H₂S to maintain the stability of AS plaques [95]. Moreover, Du et al. [110] found that H₂S modified cysteine 38 in p65 via sulfhydration, which was responsible for NF-κB inactivation. Recent studies also showed that the anti-inflammatory effect of H₂S might suppress TXNIP, an activator of NLRP3, which inhibited excessive production of interleukin 18 (IL‐18) and interleukin 1β (IL‐1β) [111]. Additionally, H₂S was identified as an agonist of histone deacetylase Sirtuin-1 (SIRT-1). H₂S directly induced deacetylation of SIRT-1 and its target proteins (P53, P65, and sterol response element-binding protein), alleviating inflammation in the endothelium and macrophages, inhibiting macrophage cholesterol uptake in ApoE knockout mice, and eventually reducing the formation of AS plaques [112]. Furthermore, it is worth noting that the activation of matrix metalloproteinases (MMPs) was involved in AS. As a member of MMPs, MMP9 is considered to be a critical factor causing instability of AS plaques [113]. Studies have found that H₂S reduced MMP9 activity by inhibiting activator protein 1 (AP-1) nuclear translocation, thus alleviating the inflammatory reaction of AS [100].

Thirdly, the interactions between NO and H₂S may also be one of the anti-AS mechanisms. Specifically, H₂S upregulates the expression of inducible nitric oxide synthase (iNOS) protein and promotes NO production. [114].

Fourthly, H₂S has an anti-apoptotic effect. Studies showed that H₂S increased the stability of plaques in ApoE knockout mice by inhibiting caspase-3/9 activity and lipoprotein receptor-1 (Lox-1) [100].

Additionally, there are other mechanisms that mediate the anti-AS effect of H₂S. H₂S donors can reduce the level of adrenomedullin (ADM) and increase the level of atrial natriuretic peptide (ANP) in AS rats, thus antagonizing the formation of AS [115]. Mani et al. [96] proposed that H₂S plays an anti-AS effect, which may inhibit intimal proliferation and adhesion molecule expression. Recently, a study also showed that NaHS notably activated angiotensin converting enzyme 2 (ACE2)-related pathways, so as to promote the transformation from pro-atherosclerosis to anti- atherosclerosis [116].

In conclusion, H₂S retarded the development of AS by a variety of molecular mechanisms that include anti-oxidative stress, anti-inflammation, anti-apoptosis, and interactions with NO.

H₂S and pulmonary hypertension

Abnormal vascular remodeling and increased pulmonary artery pressure that results in right ventricular (RV) hypertrophy and heart failure are characteristic pathological features of pulmonary hypertension (PH). PH consists of hypoxic pulmonary hypertension (HPH) and PH caused by high pulmonary blood flow and so on. Acute or chronic hypoxic stimulation leads to the progression of HPH, which is typically characterized by PH and increased pulmonary vascular resistance. It was shown that both the expression of CSE and its activity were inhibited in lung tissues during HPH [117]. In another model of PH, endogenous H₂S pathway was also downregulated in rat PH models caused by high pulmonary blood flow [118]. In addition, Feng et al. [119] suggested that the contents of H₂S in lung tissues and serum of rats in the monocrotaline (MCT)-induced PH group were obviously inhibited, and CSE expression was dramatically co-downregulated.

However, a clinical study demonstrated that H₂S at 500 μM induced an average dilation of 42.3% from the pre-constricted tension in dissected human arterial rings. In addition, H₂S at 500 μM also induced an average reduction of 17.73% in pulmonary artery pressure [120]. This effect was also seen in animal models. For instance, H₂S donors reduced pulmonary artery pressure and alleviated structural remodeling of pulmonary vessels during HPH [117]. In addition, exogenous H₂S restored H₂S contents in plasma, alleviating pulmonary artery remodeling caused by HPH.

The mechanisms by which H₂S protects against PH include but are not restricted to anti-inflammation [121], anti-endoplasmic reticulum stress (ERS) [122], induction of apoptosis [123], anti-proliferation [124], [125] and upregulation of the CO/HO pathway [126]. The detailed mechanisms are as follows.

First, H₂S antagonizes pulmonary vascular inflammation. Inflammation exerts a central effect on the pathogenesis of PH. Previous studies [122], [127] demonstrated that H₂S inhibited pro-inflammatory and oxidative stress. It was shown that H₂S alleviates pulmonary artery endothelial inflammation by inhibiting NF-κB signaling pathway [127]. Moreover, H₂S not only inhibits the NF-κB signaling pathway, but also alleviates ERS by inhibiting the expression of NADPH oxidase 4 (Nox4), as well as GRP78 and CHOP the ERS-related molecule markers [122], [65].

Secondly, H₂S induces PASMC apoptosis. The effect of H₂S on apoptosis is bidirectional, which can promote and inhibit apoptosis. However, Li et al. [123] suggested that H₂S induces apoptosis through inhibiting Bcl-2 and activating Fas signaling pathway of PASMCs in PH rats.

Thirdly, H₂S significantly inhibited the expression of proliferative cell nuclear antigen (PCNA) and urotensin II (U-II), which are critical molecules related to cell proliferation [128]. This anti-proliferative effect may be related to the up-regulation of cyclooxygenase-2(COX-2)/prostaglandin I₂ (PGI₂) signaling pathway [124], [125].

Fourthly, H₂S exerts the anti-oxidative stress effect in PH model. Oxidative stress is another important cause of elevated pulmonary arterial systolic pressure in humans. H₂S enhances the ratio of GSH/ oxidized glutathione (GSSG), which represents antioxidant capacity, by scavenging GSSG, thus exerting antioxidant capacity in HPH [129]. Moreover, the expression of collagen-promoting molecules connective tissue growth factor (CTGF) and MMP-13 were increased after the application of D, L-propargylglycine (PPG), whereas the expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) was significantly decreased. All of the above results indicate that H₂S alleviates oxidative stress injuries, thus inhibiting pulmonary vascular remodeling [130], [131].

Lastly, H₂S upregulates the CO/heme oxygenase (HO-1) pathway and is regulated by NO simultaneously in PH [132], [133]. The interaction between CO and H₂S potentially contributes to the pathogenesis of HPH. Zhang et al. demonstrated that H₂S might modulate the pathogenesis of HPH by activating HO-1 [126]. However, the mechanisms underlying H₂S through regulation of the CO/HO pathway in PASMCs remain unknown. Accumulating evidence [134] also demonstrates that defects of NO signaling possibly contribute to the progression of PH. The NO substrate, L-arginine, is known to upregulate CSE/H₂S signaling in PH caused by high blood flow [135]. Therefore, H₂S protects pulmonary vascular structure through the interaction with the other two gas molecules-NO and CO.

In summary, H₂S attenuates PH through several mechanisms, including anti-inflammation, induction of apoptosis, anti-proliferation, anti-oxidative stress, and regulating CO and NO signaling pathways.

H₂S and other cardiovascular diseases

Previous studies have confirmed that the abnormality of endogenous H₂S pathway may participate in the pathogenesis of ischemic heart disease (IHD) and left ventricular dysfunction [136]. Overexpression of CSE or supplementation of H₂S donors significantly improved cardiac function and structural lesions [137], [138], [139]. The following mechanisms might be involved in the protective effect of H₂S on the IHD and left ventricular dysfunction: 1) suppression of oxidative stress: H₂S increases the activity of antioxidant enzymes SOD, CAT and GSH in the cardiac tissues of mice with ischemia/reperfusion (IR) injury [140]. Furthermore, a 7-day treatment of H₂S donor Na₂S promoted the nuclear translocation of Nrf2, an important transcription factor that regulates antioxidant genes as an adaptive response to oxidative stress, in the hearts of mice with left coronary artery occlusion and reperfusion, which might contribute to the increase in the antioxidant enzymes [137]. Moreover, the upregulation of the rhythm gene Bmal1 expression was also involved in the antioxidant effects of H₂S in the ischemic cardiomyocyte H9c2 cells [141]. 2) inhibition of apoptosis and autophagy: H₂S reduced the proportion of apoptotic cells in the myocardium of mice with heart failure (HF) by increasing the expression of Bcl-2 and inhibiting the expression of Bax and caspase 3 [138]. In another study, H₂S alleviates autophagy of myocardial ischemia in SOD1 KO mice through the inhibition of S6 kinase (S6K) phosphorylation and AMPK phosphorylation [142]. 3) regulation of macrophage-related cardiac inflammatory response: H₂S promoted the infiltration of macrophages into the infarcted myocardium in both wild type and CSE-KO mice targeting on the macrophage integrin β1 and its downstream Src-FAK/Pyk2-Rac pathway [143]. Moreover, the polarization of infiltrated macrophage in the heart of mice with MI was also governed by H₂S. The results showed that H₂S donor NaHS promoted the number and the proportion of anti-inflammatory M2 macrophages in left ventricular tissue after MI by increasing mitochondrial biosynthesis and fatty acid oxidation [144]. 4) interaction with other bioactive molecules: In the previous studies, the interaction between H₂S and NO was involved in the vascular regulation [145]. Similarly, it is reported that H₂S enhanced endogenous NO generation by increasing the mRNA level of eNOS and nNOS and decreasing the mRNA level of iNOS in the heart tissues of myocardial IR rats [146]. 5) mitochondrial protection: H₂S maintains mitochondrial homeostasis by restoring the balance of Bcl-2/Bax and reducing mitochondrial-dependent apoptosis in HF rats [138], and improving mitochondrial respiration and ATP synthesis in isolated cardiac mitochondria from HF mice [137]. In addition, a blocker of mitoK_ATP channel 5-HD completely blocked the protective effect of H₂S donor on the isolated I/R rat heart, suggesting that the opening of mitoK_ATP channel might be involved in the regulatory effect of H₂S on the cardiac mitochondria [147].

Application of sulfhydryl group-containing angiotensin-converting enzyme (ACE) inhibitor in cardiovascular diseases

Angiotensin-converting enzyme (ACE) inhibitors are widely used as therapeutic agents in the treatment of cardiovascular diseases such as hypertension, IHD and left ventricular dysfunction in experimental studies and clinical trials [148], [149], [150]. The protective mechanisms of ACE inhibitors were mainly mediated by the inhibition of angiotension II generation and bradykinin degradation. For example, the mechanisms of cardioprotection in patients treated with ACE inhibitors might include the reduction in LV preload and afterload, suppression of sympathetic stimulation, restoration the balance of myocardial oxygen supply and demand, improvement in endogenous fibrinolysis, and alleviation of diastolic dysfunction, etc [151]. Compared with other ACE inhibitors, a sulfhydryl-group-containing ACE inhibitor zofenopril has been demonstrated to have a better clinical efficacy and safety in patients with hypertension, acute myocardial infarction (AMI) or CAD, particularly in high risk patients such as diabetes mellitus, in many clinical and preclinical studies such as SMILE series studies [152], [153], [154]. Borghi et al compared the difference in the efficacy between zofenopril and other ACE inhibitors in patients with AMI. The results showed that early administration of zofenopril in the patients ≥ 1 cardiovascular risk factor had a better prognosis and less risk of cardiovascular events than the administration of lisinopril and ramipril [153]. It has been reported that the peculiar protective effects of zofenopril including the capability of scavenging ROS, preventing of endothelial dysfunction, suppressing inflammatory response, promoting of NO generation and bioactivity, and regulating of cell apoptosis might be related to its sulfhydryl groups [151]. However, Bucci et al found that H₂S could be released from S-zofenoprilat, an active metabolite of S-zofenopril, in a cell-free assay and directly play a vasorelaxant effect in vitro. Also, the key H₂S-producing enzyme CSE expression in the vessel and the endothelial-dependent vasodilation in SHRs treated with S-zofenopril was recovered to normal level [155]. As well as the regulation of vessel function, H₂S was found to mediate the pro-angiogenic effect of zofenopril, supported by the fact that CSE inhibitor or CSE siRNA blocked the zofenopril-induced angiogenesis in vivo and in vitro [156]. In addition, CSE-dependent H₂S was also involved in the anti-inflammatory effect of zofenopril in IL-1β-induced endothelial inflammation model [157]. Interestingly, an increase in the H₂S and NO level in the myocardial tissue and plasma was found to be associated with the cardioprotective effect of zofenopril pretreated before I/R injury in mouse and pig I/R [158]. Therefore, although further studies are needed, the abovementioned studies suggest that the property of H₂S donor/generator might contribute to the superior clinical application of sulfhydrated ACE inhibitor zofenopril compared with other ACE inhibitors, which would open a new avenue for the treatment of cardiovascular diseases.

Conclusions

H₂S participates in the physiological and pathological regulation of vasculature. The mechanisms underlying H₂S-induced vasodilation are complex. H₂S induced vasorelaxation predominantly by activating iron channels, interacting with NO-cGMP signaling, inhibiting mitochondrial complex I and III, and acting as an ADRF. In addition, H₂S inhibits the proliferation of VSMCs in association with MAPK/ TXNIP, Brg1, ERK1/2, IGF-1R and CaSR signals. The regulation of H₂S on vascular cell apoptosis and autophagy is bidirectional. It can either promote or inhibit autophagy and apoptosis depending on the different pathological process (see Fig. 1, Fig. 2, Fig. 3, Fig. 4 and Table 1, Table 2, Table 3).

Fig. 1.

Generation and metabolism of endogenous H₂S.

Fig. 2.

Regulation of H₂S on hypertension. → means stimulating effect, whereas ⊥means inhibiting effect. P means phosphorylation.

Fig. 3.

Regulation of H₂S on atherosclerosis. → means stimulating effect, whereas ⊥ means inhibiting effect. –SSH means S- sulfhydrylation. Ace means acetylation. oxLDL, oxidized low-density lipoprotein; Ang II, angiotensin II; Ang (1–7), angiotensin (1–7).

Fig. 4.

Regulation of H₂S on pulmonary hypertension. → means stimulating effect, whereas ⊥ means inhibiting effect. \ means scavenging.

Table 1.

H₂S has a bidirectional regulation effect on vascular tone.

Action	Mechanisms	Models	H2S gas/donor application (concentration)	Refs.
Relaxation	Activation of KATP channel	Mesenteric artery VSMCs of rats	NaHS (100–300 μM)	[27], [29]
	Activation of KCa channel	Rat cerebral arteries	NaHS (10 and 100 μM)	[31]
	Activation of Ca2+ spark activity	Rat mesenteric small arteries	NaHS (10 μM)	[32]
	Activation of TRPV4 channel	Rat mesenteric small arteries	NaHS (1–1000 μM)	[33]
	Activation of BK channels	Rat mesenteric small arteries	NaHS (1–1000 μM)	[33]
	Activation of IKCa and SKCa channels	Mouse mesenteric arteries and aortas	NaHS (≥100 μM)	[34]
	Activation of Kv7 channels	Rat mesenteric small arteries	NaHS (100–3000 μM)	[30]
	Activation of Kv7.4 channels (subtype of Kv7)	Rat aortic rings	NaHS (1000 μM)	[35]
	Activation of KCNQ-type Kv channels	Rat and mouse aortas	NaHS (10–3000 μM)	[55]
	[37]
	Activation of HNO-TRPA1-CGRP pathway	Rat mesenteric arteries	Na2S (10 μM)	[41]
	Activation of cGMP-PKG-VASP pathway	Mouse aortic rings	NaHS (30 μM)	[42]
	Inhibition of sGC heme Fe	Mouse thoracic aorta	Na2S (50 µM)	[47]
Constriction	Activation of Na+-K+-2Cl-–co-transporters and voltage-gated calcium ion channels	Rat thoracic aortas	NaHS (5–100 μM)	[24]
	Activation of Ca2+ influx	Rat coronary arteries	NaHS (10–300 μM)	[25]

Table 2.

Effects of H₂S on proliferation and apoptosis of vascular smooth muscle cells.

Action	Mechanisms	Cells/Models	H2S gas/donor application (concentration)	Refs.
Anti-proliferation	Inhibition of Brg1 transcription and expression by reducing the recruitment of Brg1 to the Pcna, Ntf3 and Pdgfα promoter regions	VSMCs	NaHS (1000 μM)	[59]
Anti-proliferation	Inhibition of the MAPK pathway	VSMC isolated from rat thoracic aortas	NaHS (50–500 μM)	[57]
Anti-proliferation	Inhibition of the MAPK/TXNIP cascade	HUVECs/CSE-KO mice	NaHS (56 µM/kg/d)	[58]
Anti-proliferation	Inhibition of the expression of IGF-1R and the binding of IGF-1 with IGF-1R via S-sulfhydration	SMCs isolated from mouse mesenteric arteries	NaHS (10–100 μM)	[60]
Inducing apoptosis/Anti-proliferation	Increasing ERK1/2, p21Cip/WAF-1, and decreasing cyclin D1 in SMCs-KO mice. Inhibition of proliferation-related genes CRL, HB-EGF and IB1 in CSE KO mice.	SMCs-KO mice/CSE-KO mice/HASMCs	H2S (100 μM)	[56], [62]
Inducing apoptosis	Activation of MAPKs and caspase-3	HASMCs	H2S (50–100 μM)	[63]
Inhibiting apoptosis	Activation of SOD activity Inhibition of ROS generation and MDA levels	HUVECs	NaHS (50 μM)	[64]
Inhibiting apoptosis	Inhibition of caspase-12, CHOP, GRP78	PAECs	NaHS (56 µM/kg/d)	[65]

Table 3.

Effect of H₂S on vascular autophagy.

Action	Mechanisms	Cells/Models	H2S gas/donor application (concentration)	Refs.
Promoting mitophagy	Activation of Parkin recruited by PINK1 and then ubiquitination of Mfn2	RAECs	NaHS (100 μM)	[71]
Inhibiting mitophagy	Phosphorylation of Akt and dephosphorylation of FoxO3a	MAECs	NaHS (30 μM)	[72]
Inhibiting autophagy	Dephosphorylation of AMPK and phosphorylation of mTOR	VSMCs isolated from rat thoracic aorta	NaHS (100 μM)	[73]
Inhibiting autophagy	Dephosphorylation of AMPK and activation of Nrf2	RAECs/db/db mice	NaHS (100 μM)	[74]

Recent experimental data provide evidence that H₂S can prevent vascular-related diseases, such as hypertension, atherosclerosis and PH. The underlying mechanisms may include the regulation of vascular tone, anti-inflammation, anti-oxidative stress, the inhibition of VSMC proliferation, and the modulation of VSMC apoptosis. Regulating H₂S level provides a novel therapeutic method against these vascular diseases. In addition, the application of H₂S system and ACE inhibitors in the treatment of cardiovascular diseases has gradually been paid attention. Notably, the effectiveness of zofenopril in clinical trials is significantly better than other ACE inhibitors due to its capability of H₂S releasing. Therefore, H₂S has important clinical implications. Further understanding of its protective role in cardiovascular system is needed.

Future studies should investigate the interaction amongst H₂S and other gaseous signaling molecules including NO and sulfur dioxide (SO₂). There remain many opportunities to explore its role in atherosclerosis, PH and hypertension. Of note, drugs targeting H₂S producing enzymes (CBS, CSE and 3-MST) merits further clinical research.

Conflict of Interest

The authors declare no conflict of interest.

Compliance with Ethics Requirements

This article does not contain any studies with human or animal subjects.

Biographies

Boyang Lv has been studying for a doctor's degree in pediatrics at Peking University since 2019. She now focuses on the regulation of sulfur-containing gaseous signal molecules for cardiovascular diseases.

Selena Chen is a master’s student in the Division of Biological Sciences at the University of California, San Diego, in the laboratory of Dr. Bryan Sun. Her research interests focus on understanding the genetic regulators of skin development and molecular pathways are disrupted during skin diseases. She was awarded her Bachelor of Science Degree in Biochemistry and Cellular Biology at the University of California, San Diego in 2018.

Chaoshu Tang is a professor at the Department of Physiology and Pathophysiology, Peking University Health Science Centre, China. He chaired a National “973” Key Basic Research Project. He has been engaged in scientific research and teaching of cardiovascular physiology and pathological mechanisms. Professor Tang has won the second prize of National Science and Technology Progress (1998); the first prize of Ministry (1991, 1997, 2003 and 2005); the second and third prize and many other awards.

Hongfang Jin is a professor of Pediatric Cardiology, Peking University First Hospital, China. She majors in endogenous hydrogen sulfide research. She is an Excellent Young Scholars of National Natural Science Foundation (NSFC) and an Excellent Young Scholar in National Youth Top-notch Talent Support Program of China. Professor Jin is the president of Basic Research Committee of Chinese Pediatric Cardiology Society; the vice-president of Pediatric Committee of Cardiovascular Society of Chinese Medical Doctor Association.

Junbao Du is a professor at the Department of Pediatric Cardiology, Peking University First Hospital, China. He majors in endogenous hydrogen sulfide research. Dr. Du is a Changjiang Scholar of China, Distinguished Young Scholars of NSFC and outstanding Young-middle Aged Expert Approved by Ministry of Health of China. He serves as a PI of Gasotransmitters and Cardiovascular Diseases Laboratory, Key Lab of Cardiovascular Sciences, the Ministry of Education, P. R. China. He is also the president of Pediatric Cardiology Committee of Cardiovascular Physicians Society of Chinese Medical Doctors Association and a committee member of Asian Pacific Pediatric Cardiology Society.

Yaqian Huang is an associate professor in the Department of Pediatrics at Peking University First Hospital, China. She had obtained her PhD degree in physiology from Peking University in 2013. Since then, she has been working at Peking University First Hospital. She is currently the vice president of Youth Committee of Chinese Pediatric Cardiology Society, Chinese Medical Association. Her research interests focus on sulfur-containing gaseous signal molecules.