Hydrogen sulfide in the cardiovascular system: A small molecule with promising therapeutic potential

2.1 Chronotropic and Inotropic Effects of H₂S

Depending on H₂S concentration a negative [21] or positive [22] chronotropic effect was registered in rats. The negative chronotropic effect of H₂S fixed in SA nodes in rabbits was blocked by inhibitor of K_ATP channels glibenclamide (20μM) proving the involvement of K_ATP channels in mediating this effect [23]. A decrease in heart rate by H₂S reduces energy consumption and contributes to lowering cardiac work load due to the reduced contract force or the required energy of muscular contractions, therefore the negative chronotropic effect is especially beneficial in case of angina. It was shown that NaHS (donor of H₂S) improve arrhythmia associated with I/R injury. Single-channel recording on isolated cardiac myocytes demonstrated that 40μM NaHS increased the open probability of K_ATP channels [24]. On the other hand, heart rate was almost unchanged after administration of H₂S at low concentrations in rats, although their blood pressure was substantially lowered under the same treatment [25]. Pacemaker cells action potentials in rabbits did not alter after blockade of endogenous H₂S production by propargylglycine (PPG) - an inhibitor of endogenous H₂S synthesis. This suggests that H₂S has no notable chronotropic effect at low concentration [26].

In in vivo and in vitro studies, a negative inotropic effect of H₂S was revealed in rats, which may be due to the blocking of voltage-operated Ca^2 + channels and by inhibiting the adenylate cyclase, producing cyclic AMP, an important secondary messenger, regulating contractility of cardiac myocytes [15]. It was shown that H₂S induces negative inotropic effect in the isolated rat hearts during irreversible ischemia and reperfusion injury (I/R injury), lowering central venous pressure, thereby protecting the heart from damage [27]. A similar effect was registered in vivo in murine model after NaHS reperfusion [28].

The opening of K_ATP channels in the myocardium was proven to play a key role in the realization of the negative inotropic effect of H₂S, because glibenclamide, a classic blocker of these channels, inhibited this effect. This is consistent with the negative inotropic effect of other activators of K_ATP channels, which cause hyperpolarization of the cell membrane [26]. However, in other studies NaHS did not have a significant effect on the contractility of isolated rat ventricular cardiomyocytes in vitro. In these isolated cardiomyocytes, a negative inotropic effect was recorded for sodium nitroprusside (NO donor) and L-arginine while a positive inotropic effect for isoproterenol (a β-adrenergic receptor agonist). Both the negative effect of NO and the positive effect of isoproterenol were attenuated by NaHS. The physiological significance of this role of NaHS in counteracting both positive and negative inotropic effects on the heart need to be clarified [29].

2.2 Cardioprotection

There are numerous experimental evidences that H₂S protects the myocardium from damage during arrhythmia, cardiac hypertrophy, myocardial fibrosis, myocardial infarction, ischemia-reperfusion and heart failure thereby exhibiting a cardioprotective effect [30].

Enhanced H₂S levels in myocardium, whether by increased endogenous H₂S generation or by exogenous H₂S supplementation have been found to prevent ischemic injury protecting the heart. The mechanisms of cardioprotective effect of hydrogen sulfide are not fully understood, however, the molecular mechanisms providing vasodilation, antioxidation, antiapoptosis, anti-inflammation and cellular metabolism alterations have been elucidated [3].

In vitro and in vivo studies prove the cardioprotective role of H₂S, which results in the reduction of myocardial damage in ischemia/reperfusion [31]. In a rodent model of myocardial infarction, it was shown that administration of H₂S decreases mortality and reduces the size of the necrosis. Apparently, the vasodilating effect of H₂S causes an increase in coronary blood flow in ischemic diseases and reduces cellular damage. In addition, there is evidence that H₂S stimulates angiogenesis, the formation of new blood vessels, enhancing the migration of endothelial cells, that also has a cardioprotective effect [15].

Administration of exogenous L-cysteine reduced the size of myocardial infarction in ischemic heart disease due to an increase in endogenous production of H₂S catalyzed by CSE, since inhibition of CSE activity by propargylglycine (PPG) eliminated this effect of L-cysteine [15]. It has been shown that H₂S activates K_ATP channels in mitochondria and sarcolemma of cardiomyocytes, which underlies the cardioprotective effect. The H₂S donor NaHS can promote vasodilation of the coronary arteries increasing the volume of coronary blood flow during ischemia and decreasing cellular damage [12].

Exogenous administration of H₂S, endogenous overexpression of CSE and modulation of H₂S content proved to be therapeutically justified in ischemic heart failure [32, 33]. Successful therapeutic effect of H₂S has been demonstrated in models of ischemic lesions. Both preconditioning and postconditioning with free H₂S releasing compounds (NaHS, Na₂S and GYY4137) in myocardial ischemia/reperfusion caused a decrease in the infarction zone. The mechanism of such protection includes the activation of the antioxidant system and the involvement of anti-apoptotic and anti-inflammatory signaling pathways [34–36].

3.1 Vascular tone and arterial pressure regulation

Hydrogen sulfide and its donors have long been known as substances that promote vascular relaxation, alleviate hypoxic pulmonary hypertension, reduce the adhesion of leukocytes to the vascular wall, reduce vascular restenosis and have an anti-inflammatory effect [26, 38].

It was found that intravenous bolus administration of hydrogen sulfide solution caused a dose-dependent decrease in blood pressure in rats [11] and administration of hydrogen sulfide inhalations in patients with arterial hypertension contributed to a decrease in blood pressure [39]. The donor of hydrogen sulfide (NaHS) also caused relaxation of various vessels in vitro: aorta, renal, mesenteric arteries, portal vein, etc. Despite the known role of endothelium in the vascular tone regulation, its removal did not significantly affect the H₂S action on vascular smooth muscle cells (VSMC) [11], pointing a direct effect of H₂S on VSMC through their inherent regulatory mechanisms. It was proven that both endothelium and VSMC can produce H₂S, while H₂S may exert vasodilator properties that are endothelium-independent [15]. The relaxing effect of H₂S on VSMC is mainly associated with the opening of K_ATP channels [40].

However, it was shown that the vascular effects of H₂S depends not only on activation of K_ATP-channels, because glibenclamide only partially attenuated NaHS-induced vasorelaxation. Recent research has demonstrated that vasodilatory response is largely inhibited by Kv7-blockers linopirdine and XE-991 pointing the involvement of these channels in the effects of H₂S. Vascular Kv7 channels are abundantly expressed in the VSMC and activation of vascular Kv7 channels mainly contributes to the vasorelaxing responses evoked by NaHS in both rat aortic tissue and human aortic cells [41].

Over the past decade an important role for Kv7 voltage-gated potassium channels in the regulation of the excitability of smooth muscle cells has been demonstrated. It was suggested that the downregulation of these Kv7 channels may contribute to the high blood pressure in hypertension. Therefore, the pharmacological increase of the activity of the remaining Kv7 channels may be a novel therapeutic approach for the management of hypertension [42].

The role of H₂S in the pathogenesis of hypertension in spontaneously hypertensive rats (SHR) has been studied. Hypertension in animals developed spontaneously, with a decrease in H₂S production and CSE expression in the aortic tissues and a decrease in the H₂S content in blood plasma [43]. Administration of NaHS for 5 weeks delayed the progression of hypertension in SHR and partially reversed hypertension-induced vascular remodeling and collagen accumulation [44].

In mice with a genetic deletion of CSE, the production of H₂S in the cardiovascular system was substantially (but not completely) blocked. Due to a lack of endogenous H₂S, arterial hypertension was registered at the age of 8 weeks, but it was successfully prevented by injection of exogenous H₂S. The development of hypertension in CSE knockout mice was due to severe impairment of endothelial-dependent vasodilation of small resistive arteries. Hydrogen sulfide acts on both endothelial cells and VSMC, causing vascular relaxation. In CSE knockout mice, this chain is disrupted due to lack of CSE [45].

Thus, H₂S is an endogenous gaseous modulator of vascular contractile activity. Unlike vasorelaxation caused by NO and CO, H₂S-induced vasodilatation is not mediated by the involvement of the cGMP signaling pathway. At the same time, like NO and CO, H₂S can inhibit the proliferation of vascular smooth myocytes and accelerate apoptosis in vitro [46, 47]. This effect is realized through the activation of MAP kinase and caspase 3 [12].

NO is considered as an endothelial-derived relaxing factor (EDRF), however, in many vessels, vasodilation effect is only partially reduced in the presence of NOS inhibitors and upon eNOS knockout. It has been proposed that NO acts as an EDRF for large arteries while H₂S is an EDRF for small resistance arteries [26]. It is believed that H₂S along with NO is an EDRF, causing hyperpolarization of the membrane potential due to the activation of K_ATP channels [46]. Based on the observation that physiological action of H₂S is mediated by K_ATP channel sulfhydration and activation, it was hypothesized that H₂S is a major if not predominant mediator of EDRF activity. It was experimentally proven that much if not most EDRF activity involves cGMP-independent hyperpolarization of blood vessels pointing that EDRF is mainly dependent upon an endothelial-derived hyperpolarizing factor (EDHF) whose activity is largely associated with H₂S [48].

It was found that exogenous H₂S demonstrates a biphasic effect on vascular tone: it acts as vasodilator at high concentrations of NaHS (>400μM) and as vasoconstrictor at low ones. It was shown that this biphasic effect is the result of high oxygen tension and the products of H₂S oxidation are responsible for vasoconstriction [49], since in the physiological range of O₂ tension low concentrations of NaHS causes vascular relaxation [50].

It has been hypothesized that tissue concentration of H₂S mostly determines its vascular effect. Based on the observation that endothelial denudation attenuates aortic rings constriction caused by NaHS (10–100μM), the constrictor effect of H₂S on vascular smooth muscle cells was proven as indirect, mediated by the production of endothelial-derived constrictors such as endothelin or by inhibition of endothelial-derived vasodilators such as NO [51]. At the same time at NaHS concentrations greater than 100 μM vascular relaxation was fixed in the same preparation.

3.2 H₂S as oxygen sensor

Convincing evidence has been obtained for the protective role of H₂S for many organs in mammals under hypoxia; however, the mechanisms by which H₂S acts as a hypoxia sensor and implements a regulatory response are still almost unclear. Experimental data evidence that the response of blood vessels to hypoxia in vertebrates is attenuated by inhibition of H₂S synthesis, and the level of H₂S in the blood vessel is regulated by the balance between the production of endogenous H₂S and its oxidation by available O₂ [52].

Like NO and CO, H₂S significantly contributes to the regulation of hypoxia-inducible factor HIF-1 functions under hypoxic contexts. In mammalian cells HIF-1 is the main regulator of hypoxia, which activates the transcription of more than 100 target genes in hypoxic conditions [53]. H₂S-mediated angiogenesis in hypoxia involves activation of HIF-1 [54]. Exogenous H₂S regulates HIF in a variety of ways. The carotid bodies activation is a sensitive and quick response to oxygen deficiency, which rapidly restores the overall oxygen supply. It has been proven that H₂S is an excitatory mediator in the sensing of hypoxia by carotid bodies [55, 56]. Upon contact with oxygen H₂S is rapidly converted to polysulfides or hydrogen peroxide. Whether polysulfides are involved in the H₂S-mediated response of carotid bodies to oxygen deficiency or in the HIF functions regulated by H₂S, need to be clarified [56].

3.3 H₂S and angiogenesis

The proliferation and migration of endothelial cells in response to a stimulus is extremely important in embryogenesis, angiogenesis, wound healing, tissue ischemia, and various inflammatory diseases. While VSMC proliferation is inhibited by hydrogen sulfide, the proliferation and migration of vascular endothelial cells (EC) either in culture or in the blood vessel walls is stimulated by H₂S. Cultured human umbilical vein endothelial cells (HUVECs) and bEnd3 microvascular endothelial cells had a higher proliferative and migratory activity, as well as wound healing ability after treatment with NaHS proving stimulatory effect of H₂S on ECs [57, 58].

Endogenous sulfide production is also important for the endothelial cell’s migration and growth. CSE knockout in human umbilical vein endotheliocytes and mouse aortic endotheliocytes caused inhibition of the proliferation rate, while CSE overexpression led to its increase [49]. In a model of ischemia of the hind limb in rats, it was demonstrated that four-week administration of NaHS notably increased the collateral vessels growth, enhanced capillary density, and intensified peripheral blood flow in the ischemic limb compared to the control [59]. H₂S-producing enzymes are closely related to basic cellular metabolism, including amino acid synthesis and redox balance, which can also affect cell proliferation and migration [49].

3.4 Atherosclerosis and H₂S

Atherosclerosis is a complex process that includes, along with other disorders, endothelial dysfunction, and vascular inflammation. Numerous studies indicate a considerable role of H₂S in the pathogenesis of atherosclerosis, in its progression and in processes of ischemic vascular remodeling and tissue damage under ischemia-reperfusion [60, 61].

It was shown that H₂S is endogenously produced by macrophages and CSE production of H₂S in macrophages is stimulated by an inflammatory endotoxin lipopolysaccharide (LPS). Moreover, proatherogenic oxidized low-density lipoproteins (oxLDL) inducing foam cell formation in macrophages were inhibited by NaHS [62].

Knocking out of CSE or CBS, followed by persistent endogenous deficiency of H₂S, accelerates atherosclerosis. It was fixed that in ApoE^-/-CSE^-/- and ApoE^-/-CBS^-/- mice early stage of atherosclerosis develops even without diet manipulations [63].

Vascular homeostasis and function are largely regulated by blood flow. Vasorelaxation, vascular remodeling, and susceptibility to atherosclerotic plaque formation are dependent on wall shear stress. Disturbed flow in branch points, vessel curvatures, and bifurcations stimulates an atherosusceptible endothelial phenotype with decreased NO generation, enhanced oxidant stress, and elevated expression of proinflammatory genes. It was shown that CSE plays an important role in vascular remodeling induced by blood flow. Disturbed flow in conduit vessels stimulates CSE expression and sulfane sulfur production. The elevated CSE expression correlates with recruitment of macrophages to such areas, which is possibly realizes through a nuclear factor NF-κB dependent pathway. CSE knockout mice exhibits a complex change of vascular remodeling under disturbed flow [64, 65].

In native endothelial cells endogenous H₂S is mainly generated by CSE, which expression and activity are strictly regulated by shear stress and inflammation [66]. It was revealed an inverse correlation between circulating L-cystathionine levels, H₂S levels and endothelial function both in mice and humans, pointing L-cystathionine potential applicability as vascular disease biomarker. Moreover, it was shown that CSE expression in situ and in vitro is negatively regulated by blood shear stress, thereby at sites of low or disturbed blood flow the expression of this enzyme is elevated. Such flow conditions are favorable for atherosclerotic plaques formation, therefore the enhanced CSE expression at these sites seems to be protective [66].

Exogenous H₂S reduces the expression of the osteopontin gene, thereby reducing vascular calcification, which is usually recorded not only in atherosclerosis, but also in variety of diseases, including diabetes mellitus, hypertension, chronic renal failure, arterial stenosis and aging [67].

4.1 H₂S and hemostasis

It was demonstrated that H₂S exhibits an antithrombotic effect, inhibiting various stages of platelet activation (adhesion, secretion, and aggregation) and the process of thrombus formation [69, 70]. Platelet aggregation caused by various agonists: ADP, arachidonic acid, collagen, adrenaline, and thrombin, was dose-dependently attenuated by NaHS [68], and the most pronounced inhibitory effect was noted for thrombin-activated platelets. The adhesive properties of fibrinogen and collagen are modified by H₂S, and this modification impairs platelet adhesion [70].

Other researchers have found a weak inhibitory effect of H₂S at high concentrations on the human platelet aggregation and adhesion [71].

The revealed inhibitory effect of hydrogen sulfide does not depend on either NO synthesis or involvement of K_ATP channels or activation of adenylate cyclase or guanylate cyclase [68]. It has been suggested that the alteration of platelet functions may be due to thiol-disulfide reactions [70] or may be associated with presence of thiol group. Hydrogen sulfide can modify the main proteins of the hemostatic system (such as fibrinogen, thrombin, and plasminogen) causing the notable changes both in the process of coagulation and fibrinolysis [72].

In murine model, H₂S donor GYY4137 significantly prolonged the time of venular thrombus formation. pointing GYY4137 ability to regulate thrombogenesis by influencing the processes of platelet activation, adhesion, and aggregation [73]. A significant acceleration of arteriolar and venular thrombolysis by GYY4137 in comparison with control (DMSO) was shown, thrombus stability was reduced by GYY4137 contributing to endogenous thrombolysis in mice [74].

Blood clotting time was prolonged in presence of NaHS (0.01–100μM), the fibrin polymerization velocity was decreased, and the fibrinolysis was stimulated in human plasma. These results point the potential anticoagulant properties of H₂S in in vitro study and suggest the ability of H₂S to be a powerful agent for thrombosis prevention in cases of pathology with enhanced procoagulant plasma activity. However, the exact mechanisms of hydrogen sulfide influence on process of hemostasis and thrombosis need to be clarified. As a possible mechanism one can consider the involvement of H₂S in the plasma proteins S-sulfhydration. Sensitivity to H₂S action was revealed for fibrinogen which function in clotting cascade is leading among other plasma proteins. Due to the complex nature of hemostasis the effects of H₂S on various elements of coagulation system seems to be manifold because of its pleiotropic character [75].

4.2 Erythropoiesis and hemorrhage

Both H₂S-generating enzymes CBS and CSE were found to be active in the blood. The endogenous source of hydrogen sulfide are endothelial cells that secrete these enzymes [75]. Another source of H₂S in the blood are erythrocytes, which can generate it non-enzymatically from elemental sulfur or inorganic polysulfides. This way of H₂S production is stimulated by hyperglycemia and increased oxidative stress [13, 14]. The needed amounts of reducible sulfur as well as other essential components of this non-enzymatic pathway are present in blood in vivo. [26]. Enzymatic synthesis of H₂S was also revealed in erythrocytes, the key enzyme of endogenous H₂S generation in rat erythrocytes is 3-mercaptopyruvate sulfurtransferase (MPST), H₂S production in red blood cells by L-cysteine pathway is markedly lower [76].

In experiments in vitro a positive effect of H₂S on the renal erythropoietin (EPO) generating during hypoxia, but not normoxia was demonstrated. Then in murine model it was elucidated the effect of H₂S on in vivo EPO production by the kidneys. Apparently, H₂S has a significant effect on erythropoiesis and the production of erythropoietin by the kidneys. The important role of H₂S in oxygen sensing during erythropoiesis as well as the involvement of the HIF pathway in regulation of EPO production by H₂S was stressed by these findings. Significant lowering of hemoglobin, EPO, CBS, and NFκB-p65 levels during hypoxia was registered under knocking out one of the three major H₂S-generating enzymes compared to wild-type mice. This effect was reversible and attenuated upon supplementation of exogenous H₂S. This phenomenon was also reversed during normoxia by the upregulation of hemoglobin and a variety of HIF-regulated genes in comparison with wild-type mice [77, 78].

The indirect confirmation of H₂S role in maintaining the normal count of erythrocytes has been received in clinical studies demonstrated that content of thiosulfate in the urine in patients with chronic renal failure and anemia was significantly lower than in non-anemic patients with chronic renal failure [77].

Published data concerning the role of hydrogen sulfide in blood loss are quite contradictory. There is experimental evidence that H₂S can reversibly reduce the tissues metabolic requirements under massive blood loss causing insufficient oxygen supply. In rodent model with controlled hemorrhage (60% of the total blood volume), the 24 h survival rate in bled animals after hemorrhage was no more than 23%, while it was increased up to 75% after administration of exogenous H₂S by inhalation of gaseous H₂S or intravenous infusion of NaHS under the same experimental conditions. The animals survived after H₂S supplementation demonstrated normal behavior and their respiration analysis confirmed stable metabolism during and after hemorrhage [79]. Conversely, in another study, it was demonstrated that in rats with hemorrhagic shock, heart rate and blood pressure recovered faster, tissue damage was minimized in the presence of PPG, which inhibits H₂S synthesis, thereby indicating a negative role of hydrogen sulfide in this process [80].

4.3 H₂S and erythrocyte microrheological properties

Blood rheology is a key determinant of blood flow and tissue perfusion. Red blood cells (RBC) flowing through narrow capillaries which lumen is comparable or smaller than cellular diameter, need to be deformable to supply oxygen to the tissue [81]. Thus, RBC are highly deformable in norm, and this rheological property significantly contributes to providing blood flow in the microcirculation [82]. Another important cellular determinant of apparent blood viscosity is RBC aggregability –the tendency of erythrocytes to join forming reversible aggregates (so called “rouleaux” because of their similarity to a stack of coins) under low shear flow or in stasis. In norm such aggregates are dispersed by enhancing shear forces, while RBC may reunite under flow slowing or in stasis, thus the size of RBC aggregates is inversely proportional to the magnitude of shear forces. Therefore, the RBC aggregation affects the in vivo fluidity of blood, obstructing low-shear microvascular blood flow [83]. Unfavorable changes in RBC microrheological properties (deformability and aggregability) in pathology may affect blood viscosity and oxygen supply to the tissues, which in turn may impact blood flow and disease progression [84]. Since RBC are actively involved in the metabolism and scavenge of hydrogen sulfide [13, 76], it can be assumed that the functional properties of erythrocytes may be influenced by the alterations of this gasotransmitter content.

Information on the possible effect of gaseous molecules on the functional properties of erythrocytes (including their microrheological characteristics) in published data is very scarce. While the assessment of the effect of NO on blood rheology was presented in published data to some extent [85, 86], the studies of the hydrogen sulfide influence on the microrheological properties of erythrocytes and the blood flowing properties were undertaken recently and are presented in few publications. Our research group has demonstrated beneficial alterations of human’s RBC microrheological properties in vitro in presence of H₂S as well as after NO treatment: extent of aggregation was notably reduced, and deformability was moderately but statistically increased. It was registered a dose-dependent effect of NO and H₂S donors (sodium nitroprusside and NaHS) on the microrheological properties of the erythrocytes separated by age. The extent of RBC aggregation was lowered in the presence of both gasotransmitters, and this decrease was most notable for “old” cells [87]. Further study of possible mechanisms of the hydrogen sulfide effect on the aggregability and deformability of erythrocytes suggests that there is a direct (cGMP-independent) pathway of gasotransmitters action on the membrane viscoelastic properties of red blood cells [88] and a crosstalk between NO and H₂S in case of their combined supplementation [89]. In patients with type 2 diabetes mellitus positive effect of the exogenous H₂S on the microrheological characteristics of RBC was attenuated compared with healthy control [90].

5.1 Interactions and targets of H₂S in the cells

Biological effects of H₂S depends on its interactions with thiols, reactive oxygen and nitrogen species, oxidation products of macromolecules, metals and metalloproteins. S-sulfhydration (or sulfuration) of protein cysteine residues is widely accepted to be main molecular mechanism of H₂S signaling; it means conversion of thiol (–SH) to persulfide (perthiol, hydrodisulfide –SSH) groups. S-sulfhydration of many proteins including receptors, ion channels and enzymes mainly results in the increase in their activity, however, inhibitory effect of sulfhydration was also revealed. H₂S sulfhydrates protein thiol groups indirectly; this reaction should be initiated by previous oxidation of either thiol group or H₂S itself by reactive oxygen species such as hydrogen peroxide. In biological systems H₂S is readily oxidized to polysulfides containing 2–8 sulfur atoms, which are capable to sulfhydrate thiol groups of intact protein [91].

Since today there are certain technical problems with methods for accurately measuring the content of H₂S and its metabolites, it is not clear whether the observed regulatory effects are related to the action of free hydrogen sulfide or whether it is a “merit” of its polysulfide derivatives. Therefore, the statement that only the hydrogen sulfide molecule as such is capable to realize all the signaling and biological effects described in the literature can be considered too simplified [92]. Although H₂S is a short-lived molecule, numerous studies demonstrate its prolonged effect in mammals, which allows to hypothesize the physiological significance of hydrogen sulfide metabolites such as polysulfides, persulfides and other active forms of sulfur (RSS). In addition to the exogenous formation of inorganic polysulfides in a NaHS solution, the existence of endogenous inorganic polysulfides was also recorded [64, 93].

H₂S easily reacts with hemoproteins, the main mechanism of its toxicity is due to binding to cytochrome c oxidase. Binding to hemoglobin and myoglobin H₂S forms sulfhemoglobin and sulfmyoglobin, respectively. Besides the molecular basis of H₂S toxicity, interaction with hemoproteins may be also involved in some physiological or protective effects of H₂S [91].

The concept of gasotransmitters considers the specific properties of these gaseous signaling molecules, including their good permeability through cell membranes, interaction with hemoproteins, and their ability to regulate biological processes by activating certain signaling mechanisms. However, this concept does not consider the fact that the metabolic products of these gases (for example, oxidation products) often mediates biological functions to a greater extent than the molecules of these gases themselves. Oxidation of H₂S in real biological systems is an inevitable process, causing production of polysulfides and persulfides, which exhibit the same effects as H₂S [64]. Like H₂S polysulfides and persulfides can regulate various endothelial functions [61]. The cardioprotective role of sulfide and polysulfide was revealed on different models of cardiovascular diseases with tissue damage [94]. For instance, the risk reduction of cardiovascular disease associated with the consumption of garlic can be attributed to the protective effect of H₂S produced in erythrocytes from organic polysulfides of garlic [14].

5.2 Signaling pathways involved in the realization of the H₂S effect

Various cellular and molecular signals are involved in signaling pathways mediating the realization of hydrogen sulfide effects in the cardiovascular system. Ion channels, membrane and intracellular enzymes, various proteins, etc. may be the targets of H₂S action in the cell. It was found that the protein modification is one of the major mechanisms of H₂S action, H₂S is a strong reducing agent and can reduce double disulfide bonds. Another mechanism is the attaching of an additional sulfur atom to the thiol group. Such chemical modification of proteins leads to a change in their conformation and functional activity [26].

The activity of vascular enzymes such as eNOS, angiotensin converting enzyme, Na⁺ /K⁺ ATPase NADPH oxidase, and PDE₅ may be modulated by H₂S as well as activity of ion channels such as HCO^3 - and L-type Ca^2 + channels [95].

The mechanisms of H₂S cardioprotective effect in ischemia/reperfusion injury involve the inhibition of PDE₅ and prevention of cGMP breakdown. Increased cGMP and consequent cGMP-dependent protein kinase activation leads to upregulation of CSE levels causing H₂S production, triggering the activation of downstream effectors of ischemic preconditioning [96].

Like NO, H₂S binds to heme with high affinity; however, under physiological conditions, cyclic guanylate cyclase is not stimulated [40]. Guanylate cyclase inhibitors do not affect the ability of H₂S to relax blood vessels; therefore, the effect of H₂S does not depend on this enzyme [97].

The generally accepted concept of intercellular communication has undergone dramatic changes after the identification of mechanisms of gasotransmitters action. For instance, there are no intracellular vesicular stores for gasotransmitters, therefore these gaseous molecules must be synthesized as needed. It means that regulation of gasotransmitters action should be realized not by controlling the releasing of a gaseous molecule from its storage, but by alteration of their synthesizing enzymes activity. To reach intracellular targets gasotransmitters do not require specific binding to plasma membrane receptors, they simply diffuse into the cells through its membrane.

Because of their high chemical reactivity both gasotransmitters (NO and H₂S) must be inactivated after random diffusion throughout cells. For this capture and neutralization, a suitable compound (such as glutathione) must be present in the cell in abundant concentrations. Nitric oxide typically realizes its targeted action by binding various forms of NO synthase generating NO with target proteins. Probably a similar mechanism of directed action of hydrogen sulfide on its specific targets also exists, but this is still unknown.

Apparently the signaling molecular mechanisms are the most unique feature of gasotransmitters. The molecule of classic messenger acts through an amplifying signal cascade, including long sequence of molecular interactions. Gasotransmitters act immediately, chemically modifying intracellular proteins, and thus quickly affecting cellular metabolism in a direct way [98]. It was shown that H₂S realizes its effects through mechanism like nitrosylation, forming covalent bonds with the SH-group of cysteines; this process was called sulfhydration. Sulfhydration is much more common compared to nitrosylation. While usually up to 5% of most proteins are nitrosylated, 10-25% of actin and β-tubulin are mainly sulfhydrated. Sulfhydration can affect the function of proteins differently from nitrosylation. During nitrosylation, the active SH groups of cysteines are covered, which usually leads to inactivation of proteins, although sometimes an activating effect was also recorded. Sulfhydration converts the SH-group into SSH-group, which is more chemically reactive and faster interacts with the cellular environment. For example, activity of glyceraldehyde-3-phosphate dehydrogenase increases by its sulfhydration up to 700% [50].

5.3 Crosstalk between gasotransmitters

The chemical and biological properties of the currently known gasotransmitters H₂S, NO, and CO are similar, all of them have common molecular targets and demonstrate similar cellular effects. They complete each other as well as compete in the regulation of biological functions. For instance, vasodilation is a common effect of these three gases at the tissue level, inhibition of oxidative phosphorylation is typical for all three gases at the cellular level, H₂S and NO act on cytochrome c oxidase, all of them bind to hemoglobin [26].

H₂S causes relaxation of vascular smooth muscle in synergy with NO [5], but H₂S can scavenge endothelial NO inducing vasoconstriction; H₂S inhibits the vasorelaxation effect of the NO donor sodium nitroprusside (SNP), in turn, SNP increases H₂S production and another NO donor, S-nitroso-N-acetylpenicillamine, up-regulates the expression of CSE [98].

Resent research showed that H₂S cardioprotective effect in case of ischemia/reperfusion injury is mediated by NO production through activation of eNOS by inducing the phosphorylation of its activation site. The angiogenic effect of H₂S is not revealed in eNOS knockout mice, proving the involvement of NO in this process. At the same time cooperative action between H₂S and NO is essential for angiogenesis because the suppression of CSE abolishes angiogenesis [99].

The effects of gasotransmitters can be mediated by their interaction with each other, they can interplay both at the level of their synthesis regulation and at the level of cellular targets. Hydrogen sulfide, for example, inhibits the activity of enzymes that synthesize NO [100], and the NO donor, sodium nitroprusside, enhances the expression of cystathionine-γ-lyase and cystathionine-β-synthase [25]. If the vasodilation effect of NO is realized in the aorta, the relaxation of the mesenteric arteries, which are related to resistive vessels and are more significant for the regulation of peripheral blood pressure, is mainly associated with H₂S. In addition, the mechanisms of action of H₂S and NO in the vessels are different. The effects of NO are mediated through the soluble form of guanylate cyclase and modulation of the K_Ca-channels, while H₂S acts through hyperpolarization, mainly involving by the activation of the K_ATP-channels [31]. NO, CO, and H₂S can activate high conductivity K_Ca-channels by means of various chemical modifications of the channel proteins. NO modifies sulfhydryl groups, CO modifies histidine residues, and H₂S reduces disulfide bonds [2].

It is known that inhibition of any of the H₂S-producing enzymes (CSE, CBS, or 3-MST) decreases the phosphorylation of eNOS at Ser1177 in response to shear stress, which indicates the fundamental role of H₂S metabolism in shear-dependent endothelial activation [101].

The data on the mutual influence of production and release of H₂S and NO are rather contradictory. Along with the data indicating that H₂S stimulates the production of NO by the endothelium [25], other studies have suggested that H₂S inhibits the activity of eNOS and blocks the effect of SNP (sodium nitroprusside) [97]. It was found that H₂S can modify the activity of phosphodiesterases, thereby affecting the level of cyclic nucleotides [102]. It was shown that NO increases the expression and activity of CSE and binds to the CSE circulating in the blood [97], and possibly to CBS [103]. At the same time, L-NAME can decrease the H₂S content by means of lowering the activation and expression of CSE [104].

In addition to the effect on enzymes, it was found that NO and H₂S can form nitrosothiol compounds, inhibiting the synthesis of thromboxane TxA₂, directly leading to cGMP-independent blockade of platelet activation [105]. Also, NO can be reduced by H₂S to nitroxyl (HNO), which presumably can independently act on cAMP and cGMP, possibly through the activation of SER-CA; however, at present it is technically impossible to measure the HNO content, and, consequently, to elucidate its role [106].

An increased content of free hydrogen sulfide in blood plasma may be a compensatory response to endothelial dysfunction and dysregulation of NO bioavailability [107]. Recent studies have shown that H₂S can affect the expression and functional activity of eNOS, promoting the reduction of the nitrite anion to NO, acting as an alternative pathway for regulating the bioavailability of NO [58]. In turn, NO can influence the level of H₂S in vascular tissues in two ways. It was found that NO increases the activity of CSE in vascular tissues [97]. Incubation of homogenate of aortic tissues with NO donor for 90 minutes resulted in a notable dose dependent rise of H₂S generation. This elevation of hydrogen sulfide production may be due to the stimulation of the H₂S-generating enzyme CSE by cGMP-dependent protein kinase which activity was increased by NO. In addition, NO can directly affect the CSE activity. The CSE protein in mammalian consists of 12 cysteines. It is not yet established if there are specific cysteine residues that can interact with NO; however, it is likely that NO is able to nitrosylate certain free SH- groups of CSE.

The regulation of CSE expression is the second mechanism of NO-induced H₂S generation. Incubation of a cultured vascular smooth muscle cells with a NO donor for 6 hours considerably enhances the expression of CSE [97]. Other studies have also shown that the NO donor S-nitroso-N-acetylpenicillamine (SNAP) increases the CSE expression, another NO donor (SNP) enhances the CSE activity [108]. It was found that a lot of physiological and pathological processes are mediated by H₂S, polysulfides and their interaction with NO. The importance of interaction of H₂S and NO was highlighted in regulation of vascular tone and cardioprotection [99].

The interaction of CO and H₂S is not well understood; however, it was shown that CO is able to bind to both CBS and CSE, blocking their activity, and the affinity of CO binding to CBS is higher than that of NO [109].

Overall, the current evidence suggests that there is a system of interaction between gasotransmitters, which allows to regulate body functions at low concentrations of these gases due to their synergistic effect, when they are combined, because the total effect of interacting gaseous significantly exceeds the simple sum of their separate effects [71].

5.4 Biphasic effects of H₂S

Apparently, H₂S participates in numerous physiological and pathophysiological activities in cardiovascular system. However, numerous studies often provide conflicting data on the H₂S effect. This may be due to both different experimental conditions and various concentrations of hydrogen sulfide used to assess its effect.

Recently it was established that H₂S has a bell-shaped curve of dose-response, indicating its biphasic effect that means that lower concentrations of hydrogen sulfide exert notably different effects in comparison with the effects of H₂S seen at higher concentrations, moreover these effects sometimes are opposite [110, 111].

One of the most compelling examples of dual effects is the effect of H₂S on mitochondrial functions. A variety of effects of hydrogen sulfide in the mitochondria is well known. The direct donation of electrons into the electron transport chain of mitochondria is one of the effects of H₂S at low concentrations, another one is the supporting mitochondrial functions by inhibiting its cAMP phosphodiesterase. H₂S can also exert mitochondrial antioxidant effects and promote mitochondrial DNA repair directly interacting with its DNA repair enzymes. The activity of mitochondrial ATP synthase can be directly stimulated by H₂S through sulfhydration. At the same time, cellular respiration is blocked by high concentrations of H₂S due to the inhibition of cytochrome c oxidase, that is an essential element of the oxidative phosphorylation process within the cell normally binding oxygen. Inhibition of this enzyme impairs mitochondrial electron transport and ATP generation [110]. Other dual or uncertain effects of H₂S in cardiovascular system are summarized in Table 1.

6.1 Therapeutic relevance of hydrogen sulfide

The therapeutic relevance of hydrogen sulfide in treatment of cardiovascular diseases is related to its ability to regulate the peripheral blood circulation and heart functions disorders. It became evident that the state of so called “relative deficiency of H₂S” in certain cells, tissues and organs is linked to pathogenesis and progress of cardiovascular diseases [112]. Preclinical and clinical examination of various cardiovascular diseases, particularly myocardial ischemia/reperfusion injury and heart failure, have revealed that endogenous H₂S production is blunted in these pathological states contributing to the progression of disease [113].

One of the most accessible and informative indicators of this deficiency can be the H₂S content in the blood plasma which is dramatically decreased in cardiovascular diseases [39]. However, it should be noted that there is still an unresolved technical problem of accurate determination of hydrogen sulfide level, which leads to the marked variability in the baseline values reported by various research groups because the absolute levels are highly dependent on the method used [114, 115]. Therefore, more accurate and reliable indicators should be preferably used for diagnostics. As mentioned above, circulating L-cystathionine levels have recently been suggested as a potential biomarker of vascular disease in both mice and humans, based on its inverse correlation with H₂S levels and endothelial function [66].

Manipulation of H₂S levels has been tested in various disease models to achieve desired therapeutic effects demonstrating the high efficacy of this approach. However, pharmacological profile of H₂S is quite complex (often bell-shaped) which complicates its clinical application [113]. The using of precursors for endogenous H₂S synthesis (cysteine and homocysteine for CSE and CBS, 3-mercaptopyruvate for 3-MST, and a-ketoglutarate for CAT) is the most obvious method to rise H₂S levels in vivo. These three substrates provide elevation of H₂S levels and cause physiologic effects corresponding to increase of H₂S generating, such as organ protection [15], vascular smooth muscle relaxation, cell proliferation and angiogenesis [116], and bell-shaped effects on mitochondrial function [117]. In spite of association between the chronic cardiovascular pathologies and downregulation of these three H₂S-generating enzymes responsible for H₂S synthesis, the precise effects of these enzymes and their impact in cardiovascular homeostasis need to be clarified. Based on the location and activity of these enzymes in particular disease and responsiveness of certain tissues to H₂S therapy, effective therapeutics such as direct gene therapy or localized drug delivery may be proposed [118].

Another promising approach in therapeutic of cardiovascular disease is using of H₂S donors. The administration of H₂S donors may be considered as ideal treatment of cardiovascular diseases associated with deficiency of endogenous H₂S. In this case the main problem is delivery this donor at appropriate concentrations and/or rates to the desired point of application [119].

6.2 Naturally occurring and synthetic H₂S donors

A number of potential candidates for therapeutics have been proposed on base of various H₂S-releasing compounds. The peculiarity of hydrogen sulfide as a signaling molecule is dependence of its action on its concentration, the kind of target cells and disease type. H₂S is short-living molecule, this creates certain problems in the use of donors, as well as instant uncontrolled release of hydrogen sulfide by existing H₂S donors. Therefore, the needful task of translational medicine is the creation of compounds with desired properties, and the most important of them is the sustained controlled release of H₂S [121].

Sodium sulfide (Na₂S) and sodium hydrosulfide (NaHS) were the first inorganic donors of hydrogen sulfide used in the studies of the H₂S role in cardiovascular system. These simple H₂S donors provided excellent evidence of the importance of H₂S in physiological processes and diseases, but there were considerable limitations in their using as potential therapeutics. Administration of sulfide salts in vivo results in a rapid and largely uncontrollable surge in H₂S concentration in the circulation and tissue followed by a rapid decline. This pharmacokinetic profile is unsuitable for the treatment of chronic cardiovascular diseases, such as hypertension or heart failure, and may result in adverse or toxic effects [54].

Recently, the development of new H₂S donors has become a rapidly growing industry and many new donor types have been reported. Along with the general property of directly producing H₂S, these compounds are classified by the way of stimulation of hydrogen sulfide releasing. Release of H₂S may be triggered by various stimuli such as light, water, enzyme action, or other ones [3, 122–124].

To be clinically applicable, H₂S-donors should be slow-releasing, producing low and stable H₂S concentration. Naturally occurring H₂S donors are attractive in terms of commercial availability and clinical relevancy because of absence of toxicity compared to many synthetic donors. Synthetic donors that provide controlled H₂S release rates via structural modification with discrete byproducts are suitable for experimental investigations allowing to analyze the physiological roles of H₂S and, optimistically, clinically relevant H₂S-releasing prodrugs.

The main demands to H₂S donor candidates are water solubility, stability under storage, harmless of possible generating (if any) byproducts, specificity, and well-defined release mechanism (i.e., release only in response to a specific stimulus - nucleophile, a specific wavelength of light, or a specific enzyme) [5].

Among natural H₂S donors the more known are some organic polysulfide derivatives present in several species of the Alliaceae family (garlic) which act as slow H₂S -releasing compounds providing the positive effects in the cardiovascular system. H₂S is releasing by a complex chemical pathway, involving the cooperation of endogenous thiols [14].

Recently another natural compounds –isothiocyanates (ITCs), widely present in plants of the Brassicaceae family (such as broccoli, rocket salad, mustard, and horseradish), show benefits very similar to those attributed to H₂S-donors. It was experimentally shown that they improve hypertension and atherosclerosis in spontaneously hypertensive rats and attenuate the development of atherosclerosis and endothelial dysfunction in hypercholesterolemic rabbits like H₂S [125].

Abovementioned ITC function was used not only as an original H₂S-releasing moiety but also for the design of synthetic H₂S-donors and original “pharmacological hybrids” [126].

It was revealed that some ITCs release H₂S by means of reaction with L-cysteine. The L-cysteine-dependent release of H₂S from ITCs has been already reported for other H₂S donors (i.e., GYY4137 and diallyl disulfide) and has been demonstrated for other synthetic H₂S-releasing molecules (i.e., thiazolidinediones, arylthioamides, iminothioethers, mercaptopyruvate, and dithioates); this mechanism can involve a nucleophilic reaction of the thiol group of L-cysteine with the H₂S donor moiety; therefore these molecules considered as so called “smart H₂S donors”, since they can donate the gasotransmitter only in biological environments, i.e., in the presence of organic thiols [126].

A major advantage of the thiol-activated class of H₂S donors is that naturally occurring thiols provide a continuous source for H₂S release. However, patients with cardiovascular disease often have lowered levels of glutathione, which could potentially compromise the H₂S-releasing clinical relevance of thiol-activated donors.

Due to H₂S ability to engage multiple signaling pathways, and its multiorgan protective effects this gasotransmitter is considered as a promising agent for the treatment of cardiovascular diseases, which are regularly accompanied by multiple comorbidities. Several new classes of H₂S donors with controlled-release properties have been designed, synthesized, and successfully tested in various animal models of cardiovascular disease. One drawback in the development of these donors is the lack of in vivo pharmacokinetic studies and problem with oral bioavailability [127].

The application of novel conjugated compounds in a clinical setting proved to be effective for therapy of concomitant diseases, one of them H₂S releasing aspirin (ACS14) demonstrated not only protective effects within the cardiovascular system but provided decreased gastrointestinal side effects compared to native aspirin [71]. The development of H₂S-hybrid drugs, obtaining through the conjugation of suitable H₂S-releasing moieties to conventional drugs, is aimed to reduce possible adverse effects of the old drugs and/or improve their therapeutic impact.

Drugs acting on the renin-angiotensin system are potent candidates for the project of H₂S-hybrids because they are widely used for the pharmacological management of cardiovascular diseases, such as hypertension. For instance, hybrids of some sartans (antagonists of the AT1 receptor for angiotensin II) with DTT combined the antihypertensive effects due to AT1 receptor antagonism with additional H₂S-mediated vasorelaxing, antiplatelet and cardioprotective effects [96].

However, despite significant advances in basic research on the biomedical effects of H₂S over the past decades and the development of new H₂S donors, this approach has not yet found wide clinical application. Clinical trials to supplement H₂S in various human diseases have been limited [20].

The aim of future investigations is to elucidate the molecular mechanisms involved in action of endogenous or exogenous H₂S. The synthesis of novel stable donors with controlled H₂S release is one of the main directions of development of the gasotransmitters pharmacology. As the biological benefit of H₂S is deeper understood, its therapeutic effects in cardiovascular system can be utilized more efficiently in clinic [119, 120].