REVIEW URRENT C OPINION
Hydrogen sulfide: role in vascular physiology and pathology Kim M. Holwerda a, S. Ananth Karumanchi b, and A. Titia Lely c
Purpose of review Hydrogen sulfide (H2S), a colorless gas that is endogenously generated in mammals from cysteine, has important biological functions. Within the vasculature it regulates vessel tone and outgrowth of new vessels. This review summarizes recent literature on H2S signaling in the vasculature and its therapeutic potential in vascular disorders Recent findings H2S is able to induce vasorelaxation via ATP-sensitive potassium channels in vascular smooth muscle cells. Large-conductance calcium-dependent Kþ-channels and Kv7 voltage-gated Kþ-channels are also involved in H2S signaling. Vascular endothelial growth factor is the key downstream mediator that is involved in H2S induced angiogenesis. By having both direct effects on its receptor and increasing the bioavailability of vascular endothelial growth factor, H2S is proangiogenic. H2S-based therapies in vascular diseases are an expanding area of research. The applications of several compounds, such as natural donors and synthetic slow release compounds, have been extensively studied in vascular diseases such as hypertension, ischemia–reperfusion disorders and preeclampsia. Summary H2S has a key role in vascular homeostasis during physiology and in pathological states. H2S-based therapies may have a role in several vascular diseases. Keywords angiogenesis, hydrogen sulfide, vascular disease, vasodilation
INTRODUCTION Hydrogen sulfide (H2S) is a small, water-soluble, freely permeable endogenous gasotransmitter. In humans and other mammals, H2S is produced by two pyridoxal-50 -phosphate-dependent enzymes, cystathionine-b-synthase (CBS) and cystathionineg-lyase (CSE), from the amino acid L-cysteine [1]. H2S is also produced from L-cysteine in an additional pathway by 3-mercaptopyruvate sulfurtransferase (3-MST) in combination with cysteine aminotransferase [2]. An important physiological function of H2S in the vasculature is the regulation of vascular tone. CSE knockout mice show age-dependent hypertension and loss of endothelium-dependent vasorelaxation [3]. Within the vessel wall, H2S can be derived from both endothelial cells and vascular smooth muscle cells (VSMCs) [2]. By triggering cholinergic activity, and a subsequent increase of intracellular calcium, CSE acutely produces H2S in endothelial cells [3]. In VSMCs, H2S induces vasorelaxation partly by directly opening of potassium-ATP (KATP) channels [1]. www.co-nephrolhypertens.com
Cai et al. [4] were the first to report the proangiogenic effect of H2S in vitro; they demonstrated that H2S stimulated cell proliferation, migration and formation of tube-like structures. Since then, several groups have showed that H2S also acts as a proangiogenic factor in vivo [5,6]. The proangiogenic effects of H2S are associated with an increase in vascular endothelial growth factor (VEGF) expression and activation of its receptor [6].
a Division of Pathology, Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands, bDepartments of Medicine, Obstetrics and Gynecology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA and cDivision of Women and Baby, Department of Obstetrics and Gynecology, University Medical Center Utrecht, Utrecht, the Netherlands
Correspondence to Kim M. Holwerda, Department of Pathology and Medical Biology, Hanzeplein 1, PO Box 30001, 9713 GZ Groningen, the Netherlands. Tel: +31 50 3614179; fax: +31 50 3619107; e-mail: [email protected] Curr Opin Nephrol Hypertens 2015, 24:170–176 DOI:10.1097/MNH.0000000000000096 Volume 24 Number 2 March 2015
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Hydrogen sulfide in vascular physiology Holwerda et al.
KEY POINTS
significant hyperpolarization in VSMCs in a dosedependent manner [8 ]. Blocking IKca and SKca channels abolished this effect of H2S, indicating that endothelium-derived H2S hyperpolarizes endothelial cells via activation of these channels. Subsequently, hyperpolarization in adjacent VSMCs is induced (Fig. 1a) [8 ]. Although studies are contradictory, largeconductance calcium-activated Kþ channels (BKca channels) also seem to be involved in H2S signaling. H2S inhibits BKca channels in nonvascular cell types, whereas H2S-induced vasodilation of rat aortic rings is not affected by blocking BKca channels [1,9]. By blocking BKca channels in rat mesenteric arteries, H2S-induced vasorelaxation is inhibited [10]. Recently, the same group discovered that H2S is able to increase intracellular calcium sparks in VSMCs [11]. Calcium sparks occur in a spatially and temporally manner when calcium is released from the endoplasmatic reticulum via RyR channels [12]. After endothelial cell disruption, or inhibition of BKca or RyR channels, the effect of H2S to increase calcium sparks and induce vasorelaxation is impaired. This indicates that H2S increases calcium sparks in VSMCs via BKca channels in the endothelium. Within VSMCs, calcium-sparks activate BKca channels, which induce vasorelaxation (Fig. 1a) [11]. This mechanism is another way that endothelium-derived H2S contributes to VSMC hyperpolarization and vasodilation. Finally, H2S is able to induce vasorelaxation via stimulation of Kv7 voltage-gated Kþ channels in VSMCs, which are important in stabilizing the membrane potential. Ex vivo the Kv7 channel blocker XE991 can block H2S-mediated dilation of both rodent aorta and mesenteric artery [13]. More recently, it was confirmed that Kv7 channels are pharmacological targets for H2S. In particular, the Kv7.4 subunit of this channel is involved in the vasorelaxing effect of H2S (Fig. 1a) [14]. &&
H2S is an endothelial-derived hyperpolarization factor. In addition to the KATP channels, signaling of H2S acts through several other ion channels such as BKca, IKca and SKca channels and Kv7 channels. The main pathway involved in H2S-induced angiogenesis is the VEGF pathway. In addition to increasing bioavailability of VEGF, H2S can also directly act on its receptor. Vasorelaxation and angiogenesis induced by H2S take place by both nitric oxide-dependent and nitric oxideindependent mechanisms. Promising therapeutic strategies using several H2S-based therapies are currently explored for vascular diseases.
In the current review, we will summarize more recent studies (2013–2014) on the vascular functions of H2S. In addition, we will give an overview of the role of H2S in the activation of ion channels, its proangiogenic mechanisms and the interaction between H2S and nitric oxide. Finally, we will discuss the possible therapeutic role of H2S in hypertension, ischemia–reperfusion disorders and preeclampsia.
ACTIVATION OF ION CHANNELS BY HYDROGEN SULFIDE H2S is able to activate ion channels in both VSMCs and endothelial cells, as shown in Fig. 1a. In 2011, the ability of H2S to activate ion channels by S-sulfhydration was discovered [7]. S-sulfhydration is a covalent change of a cysteine residue, yielding a persulfide moiety (-SSH). In the Kir6.1 subunit of KATP channels, H2S can sulfhydrate cysteine-43 leading to activation of the channel. Furthermore, H2S induces vasorelaxation via intermediate-conductance and small-conductance calcium-dependent Kþ channels (IKca and SKca channels) in endothelial cells. It is suggested that the activation of IKca channels by H2S is also due to sulfhydration [7]. Because H2S is able to stimulate IKca and SKca channels in an endothelial-dependent manner, it was speculated for years that H2S might act as an endothelial-derived hyperpolarization factor (EDHF). However, just a year ago, solid evidence showed that H2S indeed is an EDHF [8 ]. A characteristic of an EDHF is that it is produced by endothelial cells and influences the contractility of VSMCs by inducing hyperpolarization. By using advanced techniques it was found that H2S yielded &&
&&
SIGNALING PATHWAYS INVOLVED IN THE PROANGIOGENIC ACTIONS OF HYDROGEN SULFIDE H2S is also a profound proangiogenic agent. Angiogenesis is a process recognized by the sprouting of new blood vessels from preexisting blood vessels. VEGF is an important mediator of angiogenesis and the majority of its biological effects are mediated by the vascular endothelial growth factor receptor 2 (VEGFR2) [15]. The ability of H2S or H2S-releasing compounds to increase VEGF production was recently confirmed by several groups [16,17,18 ,19]. We demonstrated that exogenous administration of H2S increased both circulating
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(a)
Muscarinic stimulation
P
eNOS
p38 H2S Akt CSE 3MST
IKca
SKca
NO
BKca
K+ K+ K+ Hyperpolarization (+) (+)
EC K+
CBS CSE
NO
H2S
PDE
ER
BKca Ca2+
GC
VSMC
cGMP
Kv7
PKG
KATP
-SSH Kir6.1
K+
K+
Vasorelaxation SPRC
(b)
VEGFR2
3MST CSE
ENOS
P
p38 Akt
PLC ins P3
ER
NO
H2S
s••s
2+
STAT3
Ca
VEGF
STAT3
GC
VEGF
PDE
cGMP
KATP
VEGF promotor
MAPK
?
PKG
K+
Angiogenesis
EC
FIGURE 1. Schematic illustration of mechanisms by which H2S induces vasorelaxation (a) and angiogenesis (b). Arrowheaded lines represent activation, bar-headed lines inhibition and dashed lines diffusion from one cell type to another. 3-MST, 3-mercaptopyruvate sulfurtransferase; BKca, large conductance calcium-dependent potassium-channels; CBS, cystathionine-bsynthase; cGMP, cyclic guanosine monophosphate; CSE, cystathionine-g-lyase; eNOS, endothelial nitric oxide-synthase; ER, endoplasmic reticulum; H2S, hydrogen sulfide; IKca, intermediate conductance calcium-dependent potassium-channels; InsP3, inositol trisphosphate; KATP, potassium ATP-channels; Kv7, voltage-gated potassium-channel; MAPK, mitogen-activated protein kinase; PDE, phosphodiesterase; PLC, phospholipase C; SKca, small-conductance calcium-dependent potassium-channels; SPRC, S-propargyl-cysteine; STAT3, signal transducer and activator of transcription 3; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2.
and local (kidney) VEGF production and ameliorates hypertension and proteinuria in a rat model of ‘antiangiogenic state’ [19]. Agents that release H2S or modulate endogenous H2S, such as diallyl trisulfide (derived from garlic), SG-1002, and S-propargylcysteine, increase local VEGF production in animal models for ischemia [16,17,18 ]. The mechanisms via which H2S influences the VEGF-signaling pathway, and consequently exerts its proangiogenic &
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effect, are not completely understood. In 2009, it was suggested that H2S induces angiogenesis by stimulating KATP channels in endothelial cells, which in turn activate MAPK pathways, inducing angiogenesis [5]. Since then, additional mechanisms have been explored, as schematically illustrated in Fig. 1b. An interesting interaction between H2S and VEGFR2 is reported by Tao et al. [20 ] who found &
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that H2S, in its anion form HS – , is able to break the disulfide (S–S) bond between Cys1045 and Cys1024 within the VEGFR2. When a mutant form of VEGFR2 is used, which is not able to form the S–S bond, H2S is not able to activate the receptor and induce endothelial cell migration. Moreover, H2S is able to break the S–S bond and induce endothelial cell migration in the absence of VEGF protein, confirming a direct activation of the receptor by H2S [20 ]. Interestingly, another group showed that H2S-induced vasodilation is impaired after blocking the VEGFR2 ex vivo [19]. This effect is in line with the fact that H2S is able to directly activate the VEGFR2. However, a positive-feedback mechanism in which the VEGFR2 stimulates H2S production via CSE activation is also described [21]. Subsequently, increased H2S in cultured endothelial cells stimulates PLC to produce InsP3, leading to a release of intraluminal calcium and the induction of angiogenesis (Fig. 1b) [21]. H2S also promotes angiogenesis via (indirect) activation of the transcription factor STAT3, which in turn activates the VEGF promoter in the endothelial cell nucleus (Fig. 1b) [18 ]. This mechanism was demonstrated using exogenous S-propargyl-cysteine, a compound that modulates endogenous H2S by inducing CSE activity. In both in-vitro and in-vivo settings, S-propargyl-cysteine promotes angiogenesis [18 ] by increasing H2S, S-propargylcysteine induced phosphorylation of the transcription factor STAT3, a potential target of angiogenesismediated therapy. A direct effect of H2S on STAT3 was excluded, but the authors found evidence that increased H2S production activates the VEGFR2 that enables STAT3 to translocate to the nucleus wherein it activates downstream promoters such as the VEGF promoter (Fig. 1b) [18 ]. Finally, apart from upregulation of VEGF expression, data suggest that H2S is able to release VEGF from cells, as demonstrated in cultured podocytes [19]. The study did not unravel the mechanism through which H2S releases VEGF, but there are several possibilities. The ability of H2S to influence matrix metalloproteinases, which are known to modulate VEGF release from the inside of the cell, could be a possible mechanism. Although, studies showing the effect of H2S on matrix metalloproteinase-2 and matrix metalloproteinase-9 expression are conflicting [22–24]. It is unknown whether H2S is able to release VEGF from other cell types such as endothelial cells or VSMCs (Fig. 1b).
endothelium-derived relaxing factor in the vasculature. By stimulating soluble guanylyl cyclase in VSMCs, leading to increased cyclic GMP production, it subsequently induces vasorelaxation. H2S is able to interact with the nitric oxide-signaling pathway in several ways. For example, H2S promotes vasorelaxation and angiogenesis by inhibiting phosphodiesterase, an enzyme breaking down cyclic GMP [25,26]. Furthermore, H2S increases nitric oxide synthase (NOS) expression in endothelial cells and stimulates the reduction from nitrite to nitric oxide in ischemic tissue [27]. Conversely, nitric oxide is able to regulate endogenous H2S production by increasing both CSE activity and expression [1]. It was reported that H2S and nitric oxide are mutually dependent in controlling vascular function [26]. However, recently it was shown in vitro that in addition to a nitric oxide-dependent mechanism, H2S also exerts proangiogenic functions in a nitric oxide-independent manner [28]. Moreover, it was shown that depletion of H2S in the kidney resulted in a reduction of nitric oxide metabolites but this was not true the other way around; reduction of H2S was not observed after nitric oxide depletion [29]. Therefore, a mutual dependency between the two gases appears at least to be absent in renal tissue. One of the actions involved in the crosstalk between nitric oxide and H2S is phosphorylation of endothelial nitric oxide synthase (eNOS) by H2S [26]. This finding was confirmed in vitro by Altaany et al. [28] showing that H2S is able to increase eNOS phosphorylation via phosphorylation of p38 and Akt and subsequently increasing nitric oxide production. An interesting in-vivo study in 2014 evaluated this mechanism as well. Authors observed increased phosphorylation at the inhibitory site (eNOST495) and decreased phosphorylation at the activating site (eNOST1177) in CSE knockout mice. These alterations in eNOS phosphorylation were accompanied by reduced nitric oxide bioavailability [30 ].
THE CROSSTALK BETWEEN HYDROGEN SULFIDE AND NITRIC OXIDE
Hypertension
&
&
&
&
H2S shares structure and functions with the gasotransmitter nitric oxide, the most potent
&
THERAPEUTIC STUDIES OF HYDROGEN SULFIDE IN VASCULAR DISORDERS Although H2S has been implicated in several vascular disorders, we will focus in this section on the therapeutic studies in hypertension, acute kidney injury and preeclampsia.
H2S is involved in the regulation of blood pressure. Exogenous (slow-) releasers of H2S such as sodium hydrosulfate (NaHS) and GYY4137 have effectively
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Clinical nephrology Table 1. Recent advances in H2S-based therapeutic compounds in (animal models for) vascular disease Condition
Compound
Animal models
Clinical applications
Hypertension
Sodium thiosulfate (STS)
Lowers blood pressure in a rat model for angiotensin induced hypertension
Used for calciphylaxis
Aryl isothiocyanates
Inhibits ex-vivo vasoconstriction via Kv7-channels
Arylthioamides
Inhibits ex-vivo vasoconstriction, and lowers blood pressure in spontaneous hypertensive rats
Zofenopril
Lowers blood pressure in spontaneous hypertensive rats by releasing H2S and independent of ACE inhibition
NaHS
Exerts antioxidant activity and has cytoprotective effect through antinecrotic mechanisms and protects against mortality in a mice model for renal ischemia reperfusion injury
D-cysteine
Increases renal levels of the sulfate sulphur pool and protects against ischemia-reperfusion injury in mice
Sodium thiosulfate
Protects against kidney damage and proteinuria in a rat model for angiotensin induced hypertension
NaHS
Lowers blood pressure, proteinuria and circulating sFlt1 levels in a rat model with sFlt1-overexpression
GYY4137
Inhibits circulating sFlt1 and soluble endoglin levels in a mice model with a preeclamptic phenotype
(Ischemic) kidney disease
Preeclampsia
Used as ACE-inhibitor
Used for calciphylaxis
ACE, angiotensin-converting enzyme; H2S, hydrogen sulfide; NaHS, sodium hydrosulfate; sFlt1, soluble FMS-like tyrosine kinase-1.
decreased blood pressure in several animal models for disease [3,31,32]. However, until now, no effective H2S-based therapies are available in the clinic. Intensive research is in progress concerning various compounds that release H2S or increase its endogenous production (Table 1). An interesting rediscovered compound is thiosulfate, a major H2S metabolite. There is evidence that thiosulfate can be an endogenous source for H2S signaling [33]. In the clinical setting, sodium thiosulfate is used as a therapy for calciphylaxis. Snijder et al. [34] were the first to show that sodium thiosulfate can act as a therapeutic agent in an angiotensin II-induced model for hypertension and associated hypertensive heart disease. In the past 2 years, Martelli et al. [35] evaluated the H2S-releasing properties and vascular effects of arylthioamides and aryl isothiocyanates. They found 174
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that the arylthioamide p-hydroxybenzothioamide slowly released H2S in the presence of endogenous biomolecules such as L-cysteine. Furthermore, the arylthioamide inhibited vasoconstriction of rat aortic rings ex vivo, induced hyperpolarization in human VSMCs and lowered SBP in normotensive rats [35]. The same group showed that isothiocyanates also have vasorelaxing effects ex vivo, which is mediated via Kv7 channels [36]. Inhibitors of the angiotensin-converting enzyme (ACE) are widely used as antihypertensive drugs. It was observed that zofenopril had extra beneficial effects in patients compared with other ACE inhibitors. The beneficial effect can probably be explained by the potential of zofenopril to release H2S from its sulfhydryl moiety. Indeed, it was found that zofenopril had additional vascular effects, which were independently from ACE-inhibition but related to H2S release [37]. Volume 24 Number 2 March 2015
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Ischemia–reperfusion and other kidney diseases Several studies have been published on the direct renal protective effect of H2S, possibly independent of blood pressure (Table 1). At first, the antioxidant role of endogenous H2S derived from CSE was investigated in renal ischemia reperfusion injury. CSE knockout mice suffered from increased kidney damage and mortality after renal ischemia compared with wild types, an effect that was rescued by H2S (administered as NaHS). Both in-vivo and in-vitro data revealed the antioxidant activity of H2S and its cytoprotective effect through antinecrotic mechanisms [38]. Furthermore, the compound D-cysteine was found to have a protective role in a mouse model with renal ischemia– reperfusion injury [39]. Via 3MST and D-amino acid oxidase, H2S can be produced from D-cysteine. In the kidney, D-cysteine seems to be an important source of H2S. Interestingly, it was found that exogenous D-cysteine increased the renal levels of the sulfane sulphur pool, which is the major physiologically H2S pool. Other renal protective effects of D-cysteine, such as ameliorating renal fibrosis and glomerulosclerosis, have not been investigated so far. In a model of severe kidney damage caused by chronic infusion of angiotensin II, both H2S (administered as NaHS) and sodium thiosulfate attenuated hypertension, proteinuria, tubular damage and renal fibroses [40]. In another hypertensive rat model, (sFlt1-overexpression induced by adenovirus); exogenous H2S (using NaHS) had therapeutic effects as well [19]. Simultaneously to lowering blood pressure in this model, H2S reduced proteinuria and kidney damage. Indeed, the blood pressure lowering effects may be the primary mechanism of H2S to protect the kidney. However, we also showed that H2S therapy increased the expression of VEGF mRNA in the kidney [19]. As reduced local production of renal VEGF induces massive proteinuria and kidney damage [41], H2S might also provide blood pressure-independent renal protection.
Preeclampsia High circulating levels of sFlt1 are related to maternal symptoms of the gestational disease preeclampsia; a severe syndrome that is characterized by both hypertension and de-novo proteinuria in the second half of pregnancy. Placentas from preeclamptic patients show downregulation of both CBS and CSE and circulating plasma H2S is impaired in patients suffering from preeclampsia [42–44]. It is not surprising that H2S-related therapy is emerging in this field as well (Table 1).
Within cultured human endothelial cells, knock down of CSE expression by silencing RNA resulted in increased sFlt1 and soluble endoglin (sEng), the latter is another antiangiogenic factor related to preeclampsia. Overexpression of CSE in the same cells with an adenovirus resulted in decreased sFlt1 and sEng [44]. Furthermore, in an assay wherein invitro tube formation is inhibited by both recombinant sFlt1 and preeclamptic serum, H2S is able to increase tube formation [19,44]. In vivo, the effect of H2S on angiogenic factors was also observed [19]. After treatment with NaHS, free plasma sFlt1 levels were obviously reduced, whereas levels of free VEGF were increased [19]. Apparently, H2S is able to shift the angiogenic balance from antiangiogenic to proangiogenic. Furthermore, inhibiting CSE expression by using DL-propargylglycine (PAG) in pregnant mice induced a preeclampsia-like phenotype [44]. After treatment with the slow H2S-releaser GYY4137, the mice were rescued from the preeclampsia-like phenotype [44]. These two studies imply that H2S can act as a therapeutic agent in preeclampsia.
CONCLUSION H2S is an endogenous gas with important physiological functions. Within the vasculature, main functions of H2S are vasodilation and promoting new vessel growth. Based on the discussed in-vitro and in-vivo rodent data, H2S is aligned to have therapeutic potential in diseases such as hypertension, renal ischemia–reperfusion disorders and hypertension. Therefore, several compounds such as the H2S-donor NaHS, synthetic slow releasers such as aryl isothiocyanates, arylthioamides and GYY4137, and also clinical available compounds such STS and zofenopril, are in the running to become H2S-based therapies for vascular disease. Acknowledgements None. Financial support and sponsorship This work was supported by a Mandema Stipendium (to A.T.L.) from the University Medical Center Groningen. Exploration. S.A.K. is an investigator of the Howard Hughes Medical Institute. Conflicts of interest S.A.K. is a coinventor on patents related to angiogenic biomarkers, has financial interest in Aggamin Therapeutics, is a consultant to Siemens Diagnostics, and receives research funding from Thermofisher.
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22. Wang J, Zhang H, Su C, et al. Dexamethasone ameliorates H2S-induced acute lung injury by alleviating matrix metalloproteinase-2 and -9 expression. PLoS One 2014; 9:e94701. 23. Vacek TP, Qipshidze N, Tyagi SC. Hydrogen sulfide and sodium nitroprusside compete to activate/deactivate MMPs in bone tissue homogenates. Vasc Health Risk Manag 2013; 9:117–123. 24. Hawinkels LJAC, Zuidwijk K, Verspaget HW, et al. VEGF release by MMP-9 mediated heparan sulphate cleavage induces colorectal cancer angiogenesis. Eur J Cancer Oxf Engl 1990 2008; 44:1904–1913. 25. Bucci M, Papapetropoulos A, Vellecco V, et al. Hydrogen sulfide is an endogenous inhibitor of phosphodiesterase activity. Arterioscler Thromb Vasc Biol 2010; 30:1998–2004. 26. Coletta C, Papapetropoulos A, Erdelyi K, et al. Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and endothelium-dependent vasorelaxation. Proc Natl Acad Sci U S A 2012; 109:9161– 9166. 27. Bir SC, Kolluru GK, McCarthy P, et al. Hydrogen sulfide stimulates ischemic vascular remodeling through nitric oxide synthase and nitrite reduction activity regulating hypoxia-inducible factor-1a and vascular endothelial growth factor-dependent angiogenesis. J Am Heart Assoc 2012; 1:e004093. 28. Altaany Z, Moccia F, Munaron L, et al. Hydrogen sulfide and endothelial dysfunction: relationship with nitric oxide. Curr Med Chem 2014; 21:3646– 3661. 29. Wesseling S, Fledderus JO, Verhaar MC, Joles JA. Beneficial effects of diminished production of hydrogen sulfide or carbon monoxide on hypertension and renal injury induced by NO withdrawal. Br J Pharmacol 2014; doi: 10.1111/bph.12674. [Epub ahead of print] 30. King AL, Polhemus DJ, Bhushan S, et al. Hydrogen sulfide cytoprotective & signaling is endothelial nitric oxide synthase-nitric oxide dependent. Proc Natl Acad Sci U S A 2014; 111:3182–3187. This in-vivo study confirms the finding that eNOS can be phosphorylated by H2S. Interesting about this study is that specific phosphorylation sites were studied. In CSE knockout mice, an increased phosphorylation of the inhibitory site (eNOST495) was found, whereas there was decreased phosphorylation of the activating site (eNOST1177). These alterations were accompanied by an impaired nitric oxide bioavailability. 31. Lu M, Liu Y-H, Goh HS, et al. Hydrogen sulfide inhibits plasma renin activity. J Am Soc Nephrol JASN 2010; 21:993–1002. 32. Li L, Whiteman M, Guan YY, et al. Characterization of a novel, water-soluble hydrogen sulfide-releasing molecule (GYY4137): new insights into the biology of hydrogen sulfide. Circulation 2008; 117:2351–2360. 33. Olson KR, Deleon ER, Gao Y, et al. Thiosulfate: a readily accessible source of hydrogen sulfide in oxygen sensing. Am J Physiol Regul Integr Comp Physiol 2013; 305:R592–R603. 34. Snijder PM, Frenay AS, de Boer RA, et al. Exogenous administration of thiosulfate, a donor of hydrogen sulfide, attenuates Angiotensin II-induced hypertensive heart disease in rats. Br J Pharmacol 2014; doi: 10.1111/ bph.12825. [Epub ahead of print] 35. Martelli A, Testai L, Citi V, et al. Pharmacological characterization of the vascular effects of aryl isothiocyanates: is hydrogen sulfide the real player? Vascul Pharmacol 2014; 60:32–41. 36. Martelli A, Testai L, Citi V, et al. Arylthioamides as H2S donors: l-cysteineactivated releasing properties and vascular effects in vitro and in vivo. ACS Med Chem Lett 2013; 4:904–908. 37. Bucci M, Vellecco V, Cantalupo A, et al. Hydrogen sulfide accounts for the peripheral vascular effects of zofenopril independently of ACE inhibition. Cardiovasc Res 2014; 102:138–147. 38. Bos EM, Wang R, Snijder PM, et al. Cystathionine g-lyase protects against renal ischemia-reperfusion by modulating oxidative stress. J Am Soc Nephrol JASN 2013; 24:759–770. 39. Shibuya N, Koike S, Tanaka M, et al. A novel pathway for the production of hydrogen sulfide from D-cysteine in mammalian cells. Nat Commun 2013; 4:1366. 40. Snijder PM, Frenay AS, Koning AM, et al. Sodium thiosulfate attenuates angiotensin II-induced hypertension, proteinuria and renal damage. Nitric Oxide 2014; 42:87–98; doi: 10.1016/j.niox.2014.10.002. [Epub ahead of print] 41. Eremina V, Sood M, Haigh J, et al. Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. J Clin Invest 2003; 111:707–716. 42. Holwerda KM, Bos EM, Rajakumar A, et al. Hydrogen sulfide producing enzymes in pregnancy and preeclampsia. Placenta 2012; 33:518–521. 43. Cindrova-Davies T, Herrera EA, Niu Y, et al. Reduced cystathionine (-lyase and increased microRNA-21 expression are associated with increased vascular resistance in growth-restricted pregnancies: hydrogen sulfide as a placental vasodilator. Am J Pathol 2013; 182:1448–1458. 44. Wang K, Ahmad S, Cai M, et al. Dysregulation of hydrogen sulfide (H2S) producing enzyme cystathionine (-lyase (CSE) contributes to maternal hypertension and placental abnormalities in preeclampsia. Circulation 2013; 127:2514–2522.
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Hydrogen sulfide: role in vascular physiology and pathology Kim M. Holwerda a, S. Ananth Karumanchi b, and A. Titia Lely c
Purpose of review Hydrogen sulfide (H2S), a colorless gas that is endogenously generated in mammals from cysteine, has important biological functions. Within the vasculature it regulates vessel tone and outgrowth of new vessels. This review summarizes recent literature on H2S signaling in the vasculature and its therapeutic potential in vascular disorders Recent findings H2S is able to induce vasorelaxation via ATP-sensitive potassium channels in vascular smooth muscle cells. Large-conductance calcium-dependent Kþ-channels and Kv7 voltage-gated Kþ-channels are also involved in H2S signaling. Vascular endothelial growth factor is the key downstream mediator that is involved in H2S induced angiogenesis. By having both direct effects on its receptor and increasing the bioavailability of vascular endothelial growth factor, H2S is proangiogenic. H2S-based therapies in vascular diseases are an expanding area of research. The applications of several compounds, such as natural donors and synthetic slow release compounds, have been extensively studied in vascular diseases such as hypertension, ischemia–reperfusion disorders and preeclampsia. Summary H2S has a key role in vascular homeostasis during physiology and in pathological states. H2S-based therapies may have a role in several vascular diseases. Keywords angiogenesis, hydrogen sulfide, vascular disease, vasodilation
INTRODUCTION Hydrogen sulfide (H2S) is a small, water-soluble, freely permeable endogenous gasotransmitter. In humans and other mammals, H2S is produced by two pyridoxal-50 -phosphate-dependent enzymes, cystathionine-b-synthase (CBS) and cystathionineg-lyase (CSE), from the amino acid L-cysteine [1]. H2S is also produced from L-cysteine in an additional pathway by 3-mercaptopyruvate sulfurtransferase (3-MST) in combination with cysteine aminotransferase [2]. An important physiological function of H2S in the vasculature is the regulation of vascular tone. CSE knockout mice show age-dependent hypertension and loss of endothelium-dependent vasorelaxation [3]. Within the vessel wall, H2S can be derived from both endothelial cells and vascular smooth muscle cells (VSMCs) [2]. By triggering cholinergic activity, and a subsequent increase of intracellular calcium, CSE acutely produces H2S in endothelial cells [3]. In VSMCs, H2S induces vasorelaxation partly by directly opening of potassium-ATP (KATP) channels [1]. www.co-nephrolhypertens.com
Cai et al. [4] were the first to report the proangiogenic effect of H2S in vitro; they demonstrated that H2S stimulated cell proliferation, migration and formation of tube-like structures. Since then, several groups have showed that H2S also acts as a proangiogenic factor in vivo [5,6]. The proangiogenic effects of H2S are associated with an increase in vascular endothelial growth factor (VEGF) expression and activation of its receptor [6].
a Division of Pathology, Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands, bDepartments of Medicine, Obstetrics and Gynecology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA and cDivision of Women and Baby, Department of Obstetrics and Gynecology, University Medical Center Utrecht, Utrecht, the Netherlands
Correspondence to Kim M. Holwerda, Department of Pathology and Medical Biology, Hanzeplein 1, PO Box 30001, 9713 GZ Groningen, the Netherlands. Tel: +31 50 3614179; fax: +31 50 3619107; e-mail: [email protected] Curr Opin Nephrol Hypertens 2015, 24:170–176 DOI:10.1097/MNH.0000000000000096 Volume 24 Number 2 March 2015
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Hydrogen sulfide in vascular physiology Holwerda et al.
KEY POINTS
significant hyperpolarization in VSMCs in a dosedependent manner [8 ]. Blocking IKca and SKca channels abolished this effect of H2S, indicating that endothelium-derived H2S hyperpolarizes endothelial cells via activation of these channels. Subsequently, hyperpolarization in adjacent VSMCs is induced (Fig. 1a) [8 ]. Although studies are contradictory, largeconductance calcium-activated Kþ channels (BKca channels) also seem to be involved in H2S signaling. H2S inhibits BKca channels in nonvascular cell types, whereas H2S-induced vasodilation of rat aortic rings is not affected by blocking BKca channels [1,9]. By blocking BKca channels in rat mesenteric arteries, H2S-induced vasorelaxation is inhibited [10]. Recently, the same group discovered that H2S is able to increase intracellular calcium sparks in VSMCs [11]. Calcium sparks occur in a spatially and temporally manner when calcium is released from the endoplasmatic reticulum via RyR channels [12]. After endothelial cell disruption, or inhibition of BKca or RyR channels, the effect of H2S to increase calcium sparks and induce vasorelaxation is impaired. This indicates that H2S increases calcium sparks in VSMCs via BKca channels in the endothelium. Within VSMCs, calcium-sparks activate BKca channels, which induce vasorelaxation (Fig. 1a) [11]. This mechanism is another way that endothelium-derived H2S contributes to VSMC hyperpolarization and vasodilation. Finally, H2S is able to induce vasorelaxation via stimulation of Kv7 voltage-gated Kþ channels in VSMCs, which are important in stabilizing the membrane potential. Ex vivo the Kv7 channel blocker XE991 can block H2S-mediated dilation of both rodent aorta and mesenteric artery [13]. More recently, it was confirmed that Kv7 channels are pharmacological targets for H2S. In particular, the Kv7.4 subunit of this channel is involved in the vasorelaxing effect of H2S (Fig. 1a) [14]. &&
H2S is an endothelial-derived hyperpolarization factor. In addition to the KATP channels, signaling of H2S acts through several other ion channels such as BKca, IKca and SKca channels and Kv7 channels. The main pathway involved in H2S-induced angiogenesis is the VEGF pathway. In addition to increasing bioavailability of VEGF, H2S can also directly act on its receptor. Vasorelaxation and angiogenesis induced by H2S take place by both nitric oxide-dependent and nitric oxideindependent mechanisms. Promising therapeutic strategies using several H2S-based therapies are currently explored for vascular diseases.
In the current review, we will summarize more recent studies (2013–2014) on the vascular functions of H2S. In addition, we will give an overview of the role of H2S in the activation of ion channels, its proangiogenic mechanisms and the interaction between H2S and nitric oxide. Finally, we will discuss the possible therapeutic role of H2S in hypertension, ischemia–reperfusion disorders and preeclampsia.
ACTIVATION OF ION CHANNELS BY HYDROGEN SULFIDE H2S is able to activate ion channels in both VSMCs and endothelial cells, as shown in Fig. 1a. In 2011, the ability of H2S to activate ion channels by S-sulfhydration was discovered [7]. S-sulfhydration is a covalent change of a cysteine residue, yielding a persulfide moiety (-SSH). In the Kir6.1 subunit of KATP channels, H2S can sulfhydrate cysteine-43 leading to activation of the channel. Furthermore, H2S induces vasorelaxation via intermediate-conductance and small-conductance calcium-dependent Kþ channels (IKca and SKca channels) in endothelial cells. It is suggested that the activation of IKca channels by H2S is also due to sulfhydration [7]. Because H2S is able to stimulate IKca and SKca channels in an endothelial-dependent manner, it was speculated for years that H2S might act as an endothelial-derived hyperpolarization factor (EDHF). However, just a year ago, solid evidence showed that H2S indeed is an EDHF [8 ]. A characteristic of an EDHF is that it is produced by endothelial cells and influences the contractility of VSMCs by inducing hyperpolarization. By using advanced techniques it was found that H2S yielded &&
&&
SIGNALING PATHWAYS INVOLVED IN THE PROANGIOGENIC ACTIONS OF HYDROGEN SULFIDE H2S is also a profound proangiogenic agent. Angiogenesis is a process recognized by the sprouting of new blood vessels from preexisting blood vessels. VEGF is an important mediator of angiogenesis and the majority of its biological effects are mediated by the vascular endothelial growth factor receptor 2 (VEGFR2) [15]. The ability of H2S or H2S-releasing compounds to increase VEGF production was recently confirmed by several groups [16,17,18 ,19]. We demonstrated that exogenous administration of H2S increased both circulating
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(a)
Muscarinic stimulation
P
eNOS
p38 H2S Akt CSE 3MST
IKca
SKca
NO
BKca
K+ K+ K+ Hyperpolarization (+) (+)
EC K+
CBS CSE
NO
H2S
PDE
ER
BKca Ca2+
GC
VSMC
cGMP
Kv7
PKG
KATP
-SSH Kir6.1
K+
K+
Vasorelaxation SPRC
(b)
VEGFR2
3MST CSE
ENOS
P
p38 Akt
PLC ins P3
ER
NO
H2S
s••s
2+
STAT3
Ca
VEGF
STAT3
GC
VEGF
PDE
cGMP
KATP
VEGF promotor
MAPK
?
PKG
K+
Angiogenesis
EC
FIGURE 1. Schematic illustration of mechanisms by which H2S induces vasorelaxation (a) and angiogenesis (b). Arrowheaded lines represent activation, bar-headed lines inhibition and dashed lines diffusion from one cell type to another. 3-MST, 3-mercaptopyruvate sulfurtransferase; BKca, large conductance calcium-dependent potassium-channels; CBS, cystathionine-bsynthase; cGMP, cyclic guanosine monophosphate; CSE, cystathionine-g-lyase; eNOS, endothelial nitric oxide-synthase; ER, endoplasmic reticulum; H2S, hydrogen sulfide; IKca, intermediate conductance calcium-dependent potassium-channels; InsP3, inositol trisphosphate; KATP, potassium ATP-channels; Kv7, voltage-gated potassium-channel; MAPK, mitogen-activated protein kinase; PDE, phosphodiesterase; PLC, phospholipase C; SKca, small-conductance calcium-dependent potassium-channels; SPRC, S-propargyl-cysteine; STAT3, signal transducer and activator of transcription 3; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2.
and local (kidney) VEGF production and ameliorates hypertension and proteinuria in a rat model of ‘antiangiogenic state’ [19]. Agents that release H2S or modulate endogenous H2S, such as diallyl trisulfide (derived from garlic), SG-1002, and S-propargylcysteine, increase local VEGF production in animal models for ischemia [16,17,18 ]. The mechanisms via which H2S influences the VEGF-signaling pathway, and consequently exerts its proangiogenic &
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effect, are not completely understood. In 2009, it was suggested that H2S induces angiogenesis by stimulating KATP channels in endothelial cells, which in turn activate MAPK pathways, inducing angiogenesis [5]. Since then, additional mechanisms have been explored, as schematically illustrated in Fig. 1b. An interesting interaction between H2S and VEGFR2 is reported by Tao et al. [20 ] who found &
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Hydrogen sulfide in vascular physiology Holwerda et al.
that H2S, in its anion form HS – , is able to break the disulfide (S–S) bond between Cys1045 and Cys1024 within the VEGFR2. When a mutant form of VEGFR2 is used, which is not able to form the S–S bond, H2S is not able to activate the receptor and induce endothelial cell migration. Moreover, H2S is able to break the S–S bond and induce endothelial cell migration in the absence of VEGF protein, confirming a direct activation of the receptor by H2S [20 ]. Interestingly, another group showed that H2S-induced vasodilation is impaired after blocking the VEGFR2 ex vivo [19]. This effect is in line with the fact that H2S is able to directly activate the VEGFR2. However, a positive-feedback mechanism in which the VEGFR2 stimulates H2S production via CSE activation is also described [21]. Subsequently, increased H2S in cultured endothelial cells stimulates PLC to produce InsP3, leading to a release of intraluminal calcium and the induction of angiogenesis (Fig. 1b) [21]. H2S also promotes angiogenesis via (indirect) activation of the transcription factor STAT3, which in turn activates the VEGF promoter in the endothelial cell nucleus (Fig. 1b) [18 ]. This mechanism was demonstrated using exogenous S-propargyl-cysteine, a compound that modulates endogenous H2S by inducing CSE activity. In both in-vitro and in-vivo settings, S-propargyl-cysteine promotes angiogenesis [18 ] by increasing H2S, S-propargylcysteine induced phosphorylation of the transcription factor STAT3, a potential target of angiogenesismediated therapy. A direct effect of H2S on STAT3 was excluded, but the authors found evidence that increased H2S production activates the VEGFR2 that enables STAT3 to translocate to the nucleus wherein it activates downstream promoters such as the VEGF promoter (Fig. 1b) [18 ]. Finally, apart from upregulation of VEGF expression, data suggest that H2S is able to release VEGF from cells, as demonstrated in cultured podocytes [19]. The study did not unravel the mechanism through which H2S releases VEGF, but there are several possibilities. The ability of H2S to influence matrix metalloproteinases, which are known to modulate VEGF release from the inside of the cell, could be a possible mechanism. Although, studies showing the effect of H2S on matrix metalloproteinase-2 and matrix metalloproteinase-9 expression are conflicting [22–24]. It is unknown whether H2S is able to release VEGF from other cell types such as endothelial cells or VSMCs (Fig. 1b).
endothelium-derived relaxing factor in the vasculature. By stimulating soluble guanylyl cyclase in VSMCs, leading to increased cyclic GMP production, it subsequently induces vasorelaxation. H2S is able to interact with the nitric oxide-signaling pathway in several ways. For example, H2S promotes vasorelaxation and angiogenesis by inhibiting phosphodiesterase, an enzyme breaking down cyclic GMP [25,26]. Furthermore, H2S increases nitric oxide synthase (NOS) expression in endothelial cells and stimulates the reduction from nitrite to nitric oxide in ischemic tissue [27]. Conversely, nitric oxide is able to regulate endogenous H2S production by increasing both CSE activity and expression [1]. It was reported that H2S and nitric oxide are mutually dependent in controlling vascular function [26]. However, recently it was shown in vitro that in addition to a nitric oxide-dependent mechanism, H2S also exerts proangiogenic functions in a nitric oxide-independent manner [28]. Moreover, it was shown that depletion of H2S in the kidney resulted in a reduction of nitric oxide metabolites but this was not true the other way around; reduction of H2S was not observed after nitric oxide depletion [29]. Therefore, a mutual dependency between the two gases appears at least to be absent in renal tissue. One of the actions involved in the crosstalk between nitric oxide and H2S is phosphorylation of endothelial nitric oxide synthase (eNOS) by H2S [26]. This finding was confirmed in vitro by Altaany et al. [28] showing that H2S is able to increase eNOS phosphorylation via phosphorylation of p38 and Akt and subsequently increasing nitric oxide production. An interesting in-vivo study in 2014 evaluated this mechanism as well. Authors observed increased phosphorylation at the inhibitory site (eNOST495) and decreased phosphorylation at the activating site (eNOST1177) in CSE knockout mice. These alterations in eNOS phosphorylation were accompanied by reduced nitric oxide bioavailability [30 ].
THE CROSSTALK BETWEEN HYDROGEN SULFIDE AND NITRIC OXIDE
Hypertension
&
&
&
&
H2S shares structure and functions with the gasotransmitter nitric oxide, the most potent
&
THERAPEUTIC STUDIES OF HYDROGEN SULFIDE IN VASCULAR DISORDERS Although H2S has been implicated in several vascular disorders, we will focus in this section on the therapeutic studies in hypertension, acute kidney injury and preeclampsia.
H2S is involved in the regulation of blood pressure. Exogenous (slow-) releasers of H2S such as sodium hydrosulfate (NaHS) and GYY4137 have effectively
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Clinical nephrology Table 1. Recent advances in H2S-based therapeutic compounds in (animal models for) vascular disease Condition
Compound
Animal models
Clinical applications
Hypertension
Sodium thiosulfate (STS)
Lowers blood pressure in a rat model for angiotensin induced hypertension
Used for calciphylaxis
Aryl isothiocyanates
Inhibits ex-vivo vasoconstriction via Kv7-channels
Arylthioamides
Inhibits ex-vivo vasoconstriction, and lowers blood pressure in spontaneous hypertensive rats
Zofenopril
Lowers blood pressure in spontaneous hypertensive rats by releasing H2S and independent of ACE inhibition
NaHS
Exerts antioxidant activity and has cytoprotective effect through antinecrotic mechanisms and protects against mortality in a mice model for renal ischemia reperfusion injury
D-cysteine
Increases renal levels of the sulfate sulphur pool and protects against ischemia-reperfusion injury in mice
Sodium thiosulfate
Protects against kidney damage and proteinuria in a rat model for angiotensin induced hypertension
NaHS
Lowers blood pressure, proteinuria and circulating sFlt1 levels in a rat model with sFlt1-overexpression
GYY4137
Inhibits circulating sFlt1 and soluble endoglin levels in a mice model with a preeclamptic phenotype
(Ischemic) kidney disease
Preeclampsia
Used as ACE-inhibitor
Used for calciphylaxis
ACE, angiotensin-converting enzyme; H2S, hydrogen sulfide; NaHS, sodium hydrosulfate; sFlt1, soluble FMS-like tyrosine kinase-1.
decreased blood pressure in several animal models for disease [3,31,32]. However, until now, no effective H2S-based therapies are available in the clinic. Intensive research is in progress concerning various compounds that release H2S or increase its endogenous production (Table 1). An interesting rediscovered compound is thiosulfate, a major H2S metabolite. There is evidence that thiosulfate can be an endogenous source for H2S signaling [33]. In the clinical setting, sodium thiosulfate is used as a therapy for calciphylaxis. Snijder et al. [34] were the first to show that sodium thiosulfate can act as a therapeutic agent in an angiotensin II-induced model for hypertension and associated hypertensive heart disease. In the past 2 years, Martelli et al. [35] evaluated the H2S-releasing properties and vascular effects of arylthioamides and aryl isothiocyanates. They found 174
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that the arylthioamide p-hydroxybenzothioamide slowly released H2S in the presence of endogenous biomolecules such as L-cysteine. Furthermore, the arylthioamide inhibited vasoconstriction of rat aortic rings ex vivo, induced hyperpolarization in human VSMCs and lowered SBP in normotensive rats [35]. The same group showed that isothiocyanates also have vasorelaxing effects ex vivo, which is mediated via Kv7 channels [36]. Inhibitors of the angiotensin-converting enzyme (ACE) are widely used as antihypertensive drugs. It was observed that zofenopril had extra beneficial effects in patients compared with other ACE inhibitors. The beneficial effect can probably be explained by the potential of zofenopril to release H2S from its sulfhydryl moiety. Indeed, it was found that zofenopril had additional vascular effects, which were independently from ACE-inhibition but related to H2S release [37]. Volume 24 Number 2 March 2015
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Ischemia–reperfusion and other kidney diseases Several studies have been published on the direct renal protective effect of H2S, possibly independent of blood pressure (Table 1). At first, the antioxidant role of endogenous H2S derived from CSE was investigated in renal ischemia reperfusion injury. CSE knockout mice suffered from increased kidney damage and mortality after renal ischemia compared with wild types, an effect that was rescued by H2S (administered as NaHS). Both in-vivo and in-vitro data revealed the antioxidant activity of H2S and its cytoprotective effect through antinecrotic mechanisms [38]. Furthermore, the compound D-cysteine was found to have a protective role in a mouse model with renal ischemia– reperfusion injury [39]. Via 3MST and D-amino acid oxidase, H2S can be produced from D-cysteine. In the kidney, D-cysteine seems to be an important source of H2S. Interestingly, it was found that exogenous D-cysteine increased the renal levels of the sulfane sulphur pool, which is the major physiologically H2S pool. Other renal protective effects of D-cysteine, such as ameliorating renal fibrosis and glomerulosclerosis, have not been investigated so far. In a model of severe kidney damage caused by chronic infusion of angiotensin II, both H2S (administered as NaHS) and sodium thiosulfate attenuated hypertension, proteinuria, tubular damage and renal fibroses [40]. In another hypertensive rat model, (sFlt1-overexpression induced by adenovirus); exogenous H2S (using NaHS) had therapeutic effects as well [19]. Simultaneously to lowering blood pressure in this model, H2S reduced proteinuria and kidney damage. Indeed, the blood pressure lowering effects may be the primary mechanism of H2S to protect the kidney. However, we also showed that H2S therapy increased the expression of VEGF mRNA in the kidney [19]. As reduced local production of renal VEGF induces massive proteinuria and kidney damage [41], H2S might also provide blood pressure-independent renal protection.
Preeclampsia High circulating levels of sFlt1 are related to maternal symptoms of the gestational disease preeclampsia; a severe syndrome that is characterized by both hypertension and de-novo proteinuria in the second half of pregnancy. Placentas from preeclamptic patients show downregulation of both CBS and CSE and circulating plasma H2S is impaired in patients suffering from preeclampsia [42–44]. It is not surprising that H2S-related therapy is emerging in this field as well (Table 1).
Within cultured human endothelial cells, knock down of CSE expression by silencing RNA resulted in increased sFlt1 and soluble endoglin (sEng), the latter is another antiangiogenic factor related to preeclampsia. Overexpression of CSE in the same cells with an adenovirus resulted in decreased sFlt1 and sEng [44]. Furthermore, in an assay wherein invitro tube formation is inhibited by both recombinant sFlt1 and preeclamptic serum, H2S is able to increase tube formation [19,44]. In vivo, the effect of H2S on angiogenic factors was also observed [19]. After treatment with NaHS, free plasma sFlt1 levels were obviously reduced, whereas levels of free VEGF were increased [19]. Apparently, H2S is able to shift the angiogenic balance from antiangiogenic to proangiogenic. Furthermore, inhibiting CSE expression by using DL-propargylglycine (PAG) in pregnant mice induced a preeclampsia-like phenotype [44]. After treatment with the slow H2S-releaser GYY4137, the mice were rescued from the preeclampsia-like phenotype [44]. These two studies imply that H2S can act as a therapeutic agent in preeclampsia.
CONCLUSION H2S is an endogenous gas with important physiological functions. Within the vasculature, main functions of H2S are vasodilation and promoting new vessel growth. Based on the discussed in-vitro and in-vivo rodent data, H2S is aligned to have therapeutic potential in diseases such as hypertension, renal ischemia–reperfusion disorders and hypertension. Therefore, several compounds such as the H2S-donor NaHS, synthetic slow releasers such as aryl isothiocyanates, arylthioamides and GYY4137, and also clinical available compounds such STS and zofenopril, are in the running to become H2S-based therapies for vascular disease. Acknowledgements None. Financial support and sponsorship This work was supported by a Mandema Stipendium (to A.T.L.) from the University Medical Center Groningen. Exploration. S.A.K. is an investigator of the Howard Hughes Medical Institute. Conflicts of interest S.A.K. is a coinventor on patents related to angiogenic biomarkers, has financial interest in Aggamin Therapeutics, is a consultant to Siemens Diagnostics, and receives research funding from Thermofisher.
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REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Zhao W, Zhang J, Lu Y, Wang R. The vasorelaxant effect of H(2)S as a novel endogenous gaseous K(ATP) channel opener. EMBO J 2001; 20:6008– 6016. 2. Shibuya N, Mikami Y, Kimura Y, et al. Vascular endothelium expresses 3-mercaptopyruvate sulfurtransferase and produces hydrogen sulfide. J Biochem (Tokyo) 2009; 146:623–626. 3. Yang G, Wu L, Jiang B, et al. H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine gamma-lyase. Science 2008; 322:587–590. 4. Cai W-J, Wang M-J, Moore PK, et al. The novel proangiogenic effect of hydrogen sulfide is dependent on Akt phosphorylation. Cardiovasc Res 2007; 76:29–40. 5. Papapetropoulos A, Pyriochou A, Altaany Z, et al. Hydrogen sulfide is an endogenous stimulator of angiogenesis. Proc Natl Acad Sci U S A 2009; 106:21972–21977. 6. Wang M-J, Cai W-J, Li N, et al. The hydrogen sulfide donor NaHS promotes angiogenesis in a rat model of hind limb ischemia. Antioxid Redox Signal 2010; 12:1065–1077. 7. Mustafa AK, Sikka G, Gazi SK, et al. Hydrogen sulfide as endothelium-derived hyperpolarizing factor sulfhydrates potassium channels. Circ Res 2011; 109:1259–1268. 8. Tang G, Yang G, Jiang B, et al. H2S is an endothelium-derived hyperpolarizing && factor. Antioxid Redox Signal 2013; 19:1634–1646. This study confirms the longstanding hypothesis that H2S is an EDHF. By using advanced techniques it was found that H2S yielded significant hyperpolarization in VSMCs in a dose-dependent manner. Hyperpolarization is established through IKca- and SKca-channels. 9. Li Q, Sun B, Wang X, et al. A crucial role for hydrogen sulfide in oxygen sensing via modulating large conductance calcium-activated potassium channels. Antioxid Redox Signal 2010; 12:1179–1189. 10. Jackson-Weaver O, Paredes DA, Gonzalez Bosc LV, et al. Intermittent hypoxia in rats increases myogenic tone through loss of hydrogen sulfide activation of large-conductance Ca(2þ)-activated potassium channels. Circ Res 2011; 108:1439–1447. 11. Jackson-Weaver O, Osmond JM, Riddle MA, et al. Hydrogen sulfide dilates rat mesenteric arteries by activating endothelial large-conductance Ca2þ-activated Kþ channels and smooth muscle Ca2þ sparks. Am J Physiol Heart Circ Physiol 2013; 304:H1446–H1454. 12. Nelson MT, Cheng H, Rubart M, et al. Relaxation of arterial smooth muscle by calcium sparks. Science 1995; 270:633–637. 13. Ko¨hn C, Schleifenbaum J, Szija´rto´ IA, et al. Differential effects of cystathionineg-lyase-dependent vasodilatory H2S in periadventitial vasoregulation of rat and mouse aortas. PLoS One 2012; 7:e41951. 14. Martelli A, Testai L, Breschi MC, et al. Vasorelaxation by hydrogen sulphide involves activation of Kv7 potassium channels. Pharmacol Res Off J Ital Pharmacol Soc 2013; 70:27–34. 15. Chung AS, Ferrara N. Developmental and pathological angiogenesis. Annu Rev Cell Dev Biol 2011; 27:563–584. 16. Polhemus DJ, Kondo K, Bhushan S, et al. Hydrogen sulfide attenuates cardiac dysfunction after heart failure via induction of angiogenesis. Circ Heart Fail 2013; 6:1077–1086. 17. Kondo K, Bhushan S, King AL, et al. H2S protects against pressure overloadinduced heart failure via upregulation of endothelial nitric oxide synthase. Circulation 2013; 127:1116–1127. 18. Kan J, Guo W, Huang C, et al. S-propargyl-cysteine, a novel water-soluble & modulator of endogenous hydrogen sulfide, promotes angiogenesis through activation of signal transducer and activator of transcription 3. Antioxid Redox Signal 2014; 20:2303–2316. This study provides evidence that H2S can modulate the expression of VEGF by effecting its transcription. By activation of VEGFR2, STAT3 is able to translocate to the nucleus, where it activates the downstream VEGF promotor. 19. Holwerda KM, Burke SD, Faas MM, et al. Hydrogen sulfide attenuates sFlt1induced hypertension and renal damage by upregulating vascular endothelial growth factor. J Am Soc Nephrol JASN 2014; 25:717–725. 20. Tao B-B, Liu S-Y, Zhang C-C, et al. VEGFR2 functions as an H(2)S-targeting & receptor protein kinase with its novel Cys1045-Cys1024 disulfide bond serving as a specific molecular switch for hydrogen sulfide actions in vascular endothelial cells. Antioxid Redox Signal 2013; 19:448–464. This is an interesting study that describes the direct activation of the VEGF receptor by H2S via breaking a disulfide bond. 21. Potenza DM, Guerra G, Avanzato D, et al. Hydrogen sulphide triggers VEGFinduced intracellular Ca(2þ) signals in human endothelial cells but not in their immature progenitors. Cell Calcium 2014; 56:225–234.
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22. Wang J, Zhang H, Su C, et al. Dexamethasone ameliorates H2S-induced acute lung injury by alleviating matrix metalloproteinase-2 and -9 expression. PLoS One 2014; 9:e94701. 23. Vacek TP, Qipshidze N, Tyagi SC. Hydrogen sulfide and sodium nitroprusside compete to activate/deactivate MMPs in bone tissue homogenates. Vasc Health Risk Manag 2013; 9:117–123. 24. Hawinkels LJAC, Zuidwijk K, Verspaget HW, et al. VEGF release by MMP-9 mediated heparan sulphate cleavage induces colorectal cancer angiogenesis. Eur J Cancer Oxf Engl 1990 2008; 44:1904–1913. 25. Bucci M, Papapetropoulos A, Vellecco V, et al. Hydrogen sulfide is an endogenous inhibitor of phosphodiesterase activity. Arterioscler Thromb Vasc Biol 2010; 30:1998–2004. 26. Coletta C, Papapetropoulos A, Erdelyi K, et al. Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and endothelium-dependent vasorelaxation. Proc Natl Acad Sci U S A 2012; 109:9161– 9166. 27. Bir SC, Kolluru GK, McCarthy P, et al. Hydrogen sulfide stimulates ischemic vascular remodeling through nitric oxide synthase and nitrite reduction activity regulating hypoxia-inducible factor-1a and vascular endothelial growth factor-dependent angiogenesis. J Am Heart Assoc 2012; 1:e004093. 28. Altaany Z, Moccia F, Munaron L, et al. Hydrogen sulfide and endothelial dysfunction: relationship with nitric oxide. Curr Med Chem 2014; 21:3646– 3661. 29. Wesseling S, Fledderus JO, Verhaar MC, Joles JA. Beneficial effects of diminished production of hydrogen sulfide or carbon monoxide on hypertension and renal injury induced by NO withdrawal. Br J Pharmacol 2014; doi: 10.1111/bph.12674. [Epub ahead of print] 30. King AL, Polhemus DJ, Bhushan S, et al. Hydrogen sulfide cytoprotective & signaling is endothelial nitric oxide synthase-nitric oxide dependent. Proc Natl Acad Sci U S A 2014; 111:3182–3187. This in-vivo study confirms the finding that eNOS can be phosphorylated by H2S. Interesting about this study is that specific phosphorylation sites were studied. In CSE knockout mice, an increased phosphorylation of the inhibitory site (eNOST495) was found, whereas there was decreased phosphorylation of the activating site (eNOST1177). These alterations were accompanied by an impaired nitric oxide bioavailability. 31. Lu M, Liu Y-H, Goh HS, et al. Hydrogen sulfide inhibits plasma renin activity. J Am Soc Nephrol JASN 2010; 21:993–1002. 32. Li L, Whiteman M, Guan YY, et al. Characterization of a novel, water-soluble hydrogen sulfide-releasing molecule (GYY4137): new insights into the biology of hydrogen sulfide. Circulation 2008; 117:2351–2360. 33. Olson KR, Deleon ER, Gao Y, et al. Thiosulfate: a readily accessible source of hydrogen sulfide in oxygen sensing. Am J Physiol Regul Integr Comp Physiol 2013; 305:R592–R603. 34. Snijder PM, Frenay AS, de Boer RA, et al. Exogenous administration of thiosulfate, a donor of hydrogen sulfide, attenuates Angiotensin II-induced hypertensive heart disease in rats. Br J Pharmacol 2014; doi: 10.1111/ bph.12825. [Epub ahead of print] 35. Martelli A, Testai L, Citi V, et al. Pharmacological characterization of the vascular effects of aryl isothiocyanates: is hydrogen sulfide the real player? Vascul Pharmacol 2014; 60:32–41. 36. Martelli A, Testai L, Citi V, et al. Arylthioamides as H2S donors: l-cysteineactivated releasing properties and vascular effects in vitro and in vivo. ACS Med Chem Lett 2013; 4:904–908. 37. Bucci M, Vellecco V, Cantalupo A, et al. Hydrogen sulfide accounts for the peripheral vascular effects of zofenopril independently of ACE inhibition. Cardiovasc Res 2014; 102:138–147. 38. Bos EM, Wang R, Snijder PM, et al. Cystathionine g-lyase protects against renal ischemia-reperfusion by modulating oxidative stress. J Am Soc Nephrol JASN 2013; 24:759–770. 39. Shibuya N, Koike S, Tanaka M, et al. A novel pathway for the production of hydrogen sulfide from D-cysteine in mammalian cells. Nat Commun 2013; 4:1366. 40. Snijder PM, Frenay AS, Koning AM, et al. Sodium thiosulfate attenuates angiotensin II-induced hypertension, proteinuria and renal damage. Nitric Oxide 2014; 42:87–98; doi: 10.1016/j.niox.2014.10.002. [Epub ahead of print] 41. Eremina V, Sood M, Haigh J, et al. Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. J Clin Invest 2003; 111:707–716. 42. Holwerda KM, Bos EM, Rajakumar A, et al. Hydrogen sulfide producing enzymes in pregnancy and preeclampsia. Placenta 2012; 33:518–521. 43. Cindrova-Davies T, Herrera EA, Niu Y, et al. Reduced cystathionine (-lyase and increased microRNA-21 expression are associated with increased vascular resistance in growth-restricted pregnancies: hydrogen sulfide as a placental vasodilator. Am J Pathol 2013; 182:1448–1458. 44. Wang K, Ahmad S, Cai M, et al. Dysregulation of hydrogen sulfide (H2S) producing enzyme cystathionine (-lyase (CSE) contributes to maternal hypertension and placental abnormalities in preeclampsia. Circulation 2013; 127:2514–2522.
Volume 24 Number 2 March 2015
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