Sulfane Sustains Vascular Health: Insights into Cystathionine γ–Lyase Function

Introduction

Hydrogen sulfide (H₂S) is widely known as a pungent toxic gaseous that has plagued humanity in various environmental conditions for centuries. However, H₂S is now recognized, along with nitric oxide (NO) and carbon monoxide (CO), to be an important biological gasotransmitter that were all previously believed to simply be environmental toxicants. ¹ Hydrogen sulfide can be produced in the mammalian body by three enzymes including cystathionine γ-lyase (CSE), cystathionine β synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST) from substrates cystathionine, homocysteine, cysteine and mercaptopyruvate. Recently, the physiological importance of H₂S in the cardiovascular system, particularly vascular growth and inflammatory regulation has been recognized; however, information regarding the importance of endogenous H₂S synthesis pathways and identification of critical enzymes has been less clear. ² In this issue of Circulation, two complementary manuscripts examining endogenous H₂S production and metabolism functions provide important insight into the role of CSE and H₂S bioavailability for vascular pathophysiological responses during preeclampsia and atherosclerosis. The first article by Wang et al ³ emphasizes the emergence of an important role for H₂S in regulating placental vasculature dysfunction during preeclampsia by altering placental growth factor (PIGF), soluble Flt-1 (sFlt-1) and soluble endoglin (sEng) levels. Whereas, the second article by Mani ⁴ and colleagues provides important insight into the role of endogenous H₂S production in modulating atherosclerosis, intimal proliferation, adhesion molecule expression (e.g. ICAM-1), oxidative stress, and lipid metabolism.

Preeclampsia

Preeclampsia is a pregnancy related vascular disorder characterized by hypertension, proteinuria, and peripheral edema. While the exact cause of preeclampsia is unknown, possible causes include systemic endothelial dysfunction and impaired vascular growth and remodeling in the placenta. ⁵ Human placenta expresses vascular endothelial growth factor (VEGF) and its receptor (flt-1). According to the angiogenic imbalance hypothesis, loss of VEGF activity causes preeclampsia due to sFlt-1 elevation, an endogenous inhibitor of VEGF. Evidence supports this hypothesis as maternal circulating levels of sFlt-1 and sEng (a cleavage product of TGF β-1) are elevated and PlGF levels low in women who develop preeclampsia ⁶. Continuously elevated levels of sFlt-1 and sEng ultimately lead to maternal endothelial dysfunction and impaired angiogenesis in the placenta.

VEGF and PlGF stimulate NO production ⁷ as does TGF β-1 to maintain vascular tone and endothelial function ⁸ and decreased activity of these mediators lead to decreased production of NO promoting endothelial dysfunction and compromised angiogenesis. Both NO/eNOS and CO have been implicated in preeclampsia, with CO/HO-1 critically regulating sFlt-1 and sEng levels ⁹. However, the role of H₂S production and metabolism during preeclampsia is poorly understood.

CSE and H₂S in preeclampsia

H₂S has potent effects on physiological responses such as angiogenesis, inflammation, vasodilation, and modulation of oxidative and redox stress. In regard to the angiogenic effect of H₂S, evidence suggests that H₂S promotes angiogenesis via stimulating PI3K/Akt or MAPK/ERK signaling pathways. ^10,11 Moreover, H₂S can alter angiogenic activity via crosstalk with NO through enzymatic (e.g. eNOS) or non-enzymatic pathways such as conversion of nitrite to NO. ^12,13

It is well known that among the three H₂S producing enzymes, CSE and CBS are predominantly present in human intrauterine tissue and placenta. However, the role of CSE/H₂S in placental abnormalities or preeclampsia has been unclear. The groundbreaking study by Wang et al lays the foundation for understanding the role of CSE/H₂S during preeclampsia. The authors found that H₂S levels are reduced in plasma of pregnant women with preeclampsia and that CSE enzyme expression is reduced in preeclamptic placental tissue. Additionally, they provide clear evidence that circulating PIGF levels are reduced in women with preeclampsia associated with dysregulation of CSE/H₂S signaling pathway. These findings are associated with CSE/H₂S mediated prevention of release of sFlt-1 and sEng. Importantly, animal studies inhibiting CSE activity in pregnant mice recapitulated key features of preeclampsia including hypertension, elevation of sFlt-1 and sEng, defective placental vascularization and arrest of fetal growth, which were reversed by exogenous H₂S therapy. Thus, this study shows that H₂S rescues placental vasculature abnormalities, ameliorates hypertension and fetal growth restriction in the mouse placenta. These discoveries open new possibilities for preeclampsia diagnosis and therapeutics.

Although the authors in this article have shown compelling evidence of a crucial role for CSE/H₂S during preeclampsia, many questions await further study such as: why is CSE/CSE expression reduced during preeclampsia and what are the effects on alternative H₂S generating enzymes such as CBS and 3-MST? What is the precise concentration of H₂S needed to maintain placental vascular health and are different biochemical pools of H₂S (e.g. free, acid labile and bound sulfane sulfur) altered during preeclampsia? Utilization of analytical measurement techniques could address these important questions and would provide key information necessary to move therapeutic studies forward. ^14,15 Thus, further study is needed to understand the regulation of expression and activity, as well as substrate bioavailability that may also contribute to H₂S regulation of preeclampsia. Lastly, there could be interaction with other factors, such as NO, CO and VEGF, that are known to be involved in preeclampsia pathophysiology. It is possible that interactions between these metabolic mediators could collectively participate in pathological mechanisms that wait further investigation.

Atherosclerosis

Atherosclerosis is a chronic and complex inflammatory process involving different cellular and molecular signaling pathways that leads to fat accumulation, plaque formation and stenosis of the arterial lumen. ² The gaseous mediators NO, CO and H₂S generated within the vasculature have been implicated in modulating vascular functions involved during atherogenesis. It is well known that dysregulation of NO production in the vasculature promotes atherosclerosis formation due to impaired L-arginine utilization, inactivity of eNOS or iNOS as well as decreased NO bioavailability. ¹⁶ Similarly, decreased production of CO due to reduced expression of HO-1 stimulates atherosclerotic plaque formation. ¹⁷ However, the role of H₂S in atherosclerosis is a novel finding that has only been explored within recent years. Past research suggested that CSE expression, activity, and subsequent H₂S production were reduced during balloon mediated injury and neointima development. ¹⁸ Importantly, H₂S can inhibit vascular smooth muscle cell proliferation, neointimal hyperplasia and atherosclerotic plaque size via inhibition of the MEK/ERK/caspase -3 signaling pathway. ¹⁹ However, the importance of endogenous enzyme synthesis mechanisms for H₂S production during atherogenesis remained unclear.

CSE and H₂S in atherosclerosis

In the study by Mani et al, the authors provide important new information regarding the role of CSE regulation of lipid metabolism and regulation of atherosclerosis formation. The authors revealed that genetic deficiency of CSE in mice increased plasma total and LDL cholesterol and atherosclerotic lesions using an atherogenic diet that were rectified by exogenous H₂S therapy. Importantly, hypertension due to CSE mutation did not significantly contribute to enhanced atherogenesis. It was also shown that CSE-ApoE double knockout mice had larger atherosclerotic lesion area that could be attenuated with exogenous H₂S.

With regard to possible molecular mechanisms, the authors found that CSE KO augments oxidative stress during atherosclerosis (i.e. lower levels of GSH & SOD and higher levels of MDA) that was corrected by exogenous antioxidant N -acetyl cysteine therapy. In addition, VSMC GPx and GR protein levels were reduced and ROS production was increased in CSE KO mice that was reversed by H₂S therapy indicating a role of CSE dependent H₂S in modulating redox stress defense during atherosclerosis. It was further observed that NF-κB mediated ICAM-1 expression was significantly increased in smooth muscle cells of CSE KO mice compared to WT mice, and that exogenous H₂S attenuated this response. Thus, this study clearly identified the importance of endogenous H₂S production via CSE in regulating atherogenesis highlighting the possibility of therapeutic approaches aimed at modulating enzyme expression or function during atherosclerosis.

While the authors provide clear ‘proof of concept’ for an important role of CSE/H₂S during atherosclerosis, many questions remain unknown. What are the levels of plasma and tissue H₂S that is needed to guard against atherogenic mechanisms? Is H₂S bioavailability differentially distributed in various biochemical forms throughout the vasculature itself thereby rendering regions of the vascular tree susceptible to atherogenesis? Additionally, studies focused on determining the function and regulation of CSE expression in different cell types involved in atherosclerosis are necessary to better understand the pathophysiological processes most affected by H₂S metabolism. Lastly, given the recent appreciation of H₂S – NO pathway interactions it is possible that novel crosstalk mechanisms may be involved in regulating inflammation during atherosclerosis. ²⁰

Conclusion

Results from these studies expand our understanding about the importance of CSE/H₂S signaling pathway in regulating vascular responses in diseases that primarily involve them. Together, these results begin to paint a picture whereby CSE and H₂S bioavailability regulate vascular health to control different pathological responses as illustrated in Figure 1. The notion that H₂S may be important for vascular health and function further suggests that therapeutic attempts to modulate synthesis enzyme pathways or bioavailability may be beneficial for diseases with a foundation of vascular pathology. However, additional detailed studies regarding H₂S bioavailability and its protective mechanisms are needed to better understand the role of this gaseous mediator. The realization of the CSE/H₂S synthesis paradigm for vascular pathologies provides important new information that could reveal novel therapeutic approaches.

Figure 1.

Role of CSE expression and H₂S production in vascular dysfunction. Dysregulated CSE enzyme function through either pharmacologic inhibition or genetic deletion reduces H₂S bioavailability. Reduced H₂S production contributes to preeclampsia pathophysiology by increasing sFlt-1 and sEng levels and decreasing placental PIGF levels (right side pathway). Gene targeted knockout of CSE leads to decreased levels of GSH and SOD, increased levels of ROS and increased NF-κB mediated ICAM-1 expression that exacerbates atherosclerotic plaque formation (left side pathway). Abbreviations- CSE: Cystathionine γ-lyase, H₂S: Hydrogen sulfide, sFlt-1: Soluble fms-like tyrosine kinase -1, sEng: Soluble endoglin, PIGF: Placental growth factor, NF-κB: Nuclear factor-kappaB, ICAM: intercellular adhesion molecule, GSH: Glutathione, SOD: Superoxide dismutase, ROS: Reactive oxygen species.