Hydrogen sulfide: a gaseous signaling molecule modulates tissue homeostasis: implications in ophthalmic diseases

Hydrogen sulfide (H2S) serves as a gasotransmitter in the regulation of organ development and maintenance of homeostasis in tissues. Its abnormal levels are associated with multiple human diseases, such as neurodegenerative disease, myocardial injury, and ophthalmic diseases. Excessive exposure to H2S could lead to cellular toxicity, orchestrate pathological process, and increase the risk of various diseases. Interestingly, under physiological status, H2S plays a critical role in maintaining cellular physiology and limiting damages to tissues. In mammalian species, the generation of H2S is catalyzed by cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CSE), 3-mercapto-methylthio pyruvate aminotransferase (3MST) and cysteine aminotransferase (CAT). These enzymes are found inside the mammalian eyeballs at different locations. Their aberrant expression and the accumulation of substrates and intermediates can change the level of H2S by orders of magnitude, causing abnormal structures or functions in the eyes. Detailed investigations have demonstrated that H2S donors’ administration could regulate intraocular pressure, protect retinal cells, inhibit oxidative stress and alleviate inflammation by modulating the function of intra or extracellular proteins in ocular tissues. Thus, several slow-releasing H2S donors have been shown to be promising drugs for treating multiple diseases. In this review, we discuss the biological function of H2S metabolism and its application in ophthalmic diseases.


Introduction
Hydrogen sulfide (H 2 S) was identified by Carl Wilhelm Scheele through chemical analysis in the 17th century. However, it has long been believed that this gas emanated from the sewer system is related to a series of a special type of eye diseases occurred in sewer workers. This disease is associated with painful inflammation, secondary bacterial invasion and even blindness. Like nitric oxide (NO) and carbon monoxide, endogenously produced H 2 S is now known as another gaseous signaling molecule that affects the structure and function of proteins by participating in their short-lived covalent reactions 1 . This gasotransmitter can easily diffuse across cell membranes and does not need a specific mechanism for their degradation and reuptake. In human, the concentration of H 2 S in tissues can be at μM ranges for maintaining the physiological cellular functions. Its levels can differ according to age, tissues and measuring methods 2,3 . For example, the H 2 S concentration in the peripheral blood is generally 30-300 μM 4 , while the physiological concentration of H 2 S in the brain is up to three times of that in serum 5,6 . The H 2 S gas/water coefficient of distribution is 0.39, which can be affected by pH 2,7 . In comparison to healthy individuals, the H 2 S concentration in the serum of asthmatic patients can reach to 600 μM 8 .
The oxidation products of H 2 S include persulfide, sulfite, thiosulfate and sulfate 9 . When the concentrations of H 2 S in tissues or cells are high, H 2 S is considered as a toxic substance and its oxidation products may cause cytotoxic effects through inhibiting mitochondrial cytochrome C oxidase and disrupting cell energy production, leading to tissue inflammation or DNA damage 10 . However, when it is generated at physiological rates or at low concentrations, it has entirely different effects on biological processes such as cellular division, DNA repair and metabolism, modulation of protein kinase, regulation of cell cycle and organization of cytoskeletal framework 11 . Recent investigations have found that the potential regulatory role of H 2 S is to add cysteine, a thiol group in proteins (aka S-sulfhydration, or persulfide formation) 12 . This modification critically changes the physiological actions and pathological status of proteins in response to inflammation or oxidation by generating a -SSH group. The persulfides have better reactivity than corresponding thiols and can readily react with electrophiles. Persulfidation of proteins such as K ATP contributes to various H 2 S-induced biochemical reactions 13 . When H 2 S is produced at low levels through enzymatical degradation of cysor homocysteine, it is critical in maintaining the functions of nervous system and vascular system 14,15 . Exogenously administrated H 2 S has been found to extend the lifespan of worms, relieve inflammation and promote reparation of injured tissues 16 . In views of the potential value of H 2 S in body systems and its presence in mammalian eyes 17 , this review focuses on the role of H 2 S in the common ophthalmic diseases and the underlying mechanisms, hoping to provide therapeutic strategy for ophthalmic diseases. Detailed analysis on the crosstalk between ocular tissues and H 2 S generation pathway will pave the road for understanding the pathogenesis of multiple ophthalmic diseases and optimize the application of H 2 S donor for treatment.

Generation of H 2 S in ocular tissues
In mammalian cells, the generation of H 2 S is dependent on four major enzymes: cystathionine-γ-synthase (CSE), cystathionine-β-lyase (CBS), 3-mercapto-methylthio pyruvate aminotransferase (3MST) and cysteine aminotransferase (CAT) 18,19 . Generally, the generation of H 2 S relies on the desulfurization of cysteine or homocysteine by the two pyridoxial 5-phosphate (PLP)-dependent enzymes, CSE and CBS 7 . Of note, the distribution of these enzymes for H 2 S production shows tissue specificity. For example, CBS is the main enzyme for H 2 S generation in the central nervous system 20 . CSE is the major enzyme for H 2 S production in the vasculature system, liver, and kidney [21][22][23] . The presence of these H 2 S-productive enzymes are proved in ocular tissues, especially in the retina [24][25][26] . According to recent studies, H 2 S can also be produced from D-cysteine, catalyzed by D-Amino acid oxidase (DAO) and 3MST 27 .
Endogenously production of H 2 S is discovered in various tissues of bovine eye, including cornea, aqueous humor, iris, ciliary muscle, lens, choroid and retina, except vitreous humor. The highest production of endogenous H 2 S was detected in cornea and retina 17 . CBS is most highly expressed in the cornea, conjunctiva, and iris, while much lower amount be found in retina and optic nerve, relatively lower amount in lens, but absent in the vitreous humor. CBS expression remains high in anterior segments throughout the lifespan, and it has a trend of agedependent increase in retina 24 . CSE is characterized in retina of amphibians and mammals, where its activity can be traced 25 . 3MST/CAT pathway is the dominating way to produce H 2 S in mammalian retina as both 3MST and CAT are located in the retinal neurons, which is increased at low concentrations of Ca 2+ that achieved in brightness 26 . Deficiency of H 2 S or its substrates are found to be related to ectopialentis, myopic, cataract 28 , optic atrophy, and retinal detachment [29][30][31] (Fig. 1).

Reduction of intraocular pressure (IOP)
High IOP is the major cause for optic neuropathy in patients of glaucoma, which damages the retinal neurons and optic nerve heads 32 . Stable IOP depends on the balance of aqueous humor (AH) generation in the ciliary body and AH outflow in the chamber angle, especially in trabecular meshwork 33 . The outflow facility could be increased by cyclic adenosine monophosphate (cAMP) administration in anterior chamber for the maintenance of IOP 34 . H 2 S-releasing compounds could act on adenylyl cyclase and ATPsensitive potassium channels (K ATP ) channels in eyes, thus increase cAMP concentrations in porcine ocular anterior segments and help mediate the outflow of AH 35 . Ex vivo study has indicated that H 2 S participates in the phosphodiesterase (PDE) inhibition and enhancement of intramitochondrial cAMP levels, which stimulates protein kinase A (PKA) to instruct bioenergetic effects 36 . The inhibition of PDE activity by H 2 S is a relevant factor to cumulative cAMP and cyclic guanosine monophosphate (cGMP). Meanwhile, elevated intraocular cGMP level is related to reducing the trabecular meshwork cell volume and promoting outflow of AH 37 . H 2 S-producing donors such as GYY4137, are well-investigated for stabilizing IOP, as their administration upregulate the intraocular glutathione (GSH) expression with increased cGMP levels 38,39 .
H 2 S donors also work on anterior uvea to relax iris smooth muscles 40 and thus lower IOP. On the other hand, norepinephrine released by intraocular degenerating sympathetic nerve terminals can cause a decrease in outflow facility with a subsequent elevated IOP in the long term, even though it may lead to an acute increase in outflow facility 41 . Increased levels of norepinephrine in AH during night is related to an increase rather than a decrease in IOP in rabbits 42 . H 2 S can reduce the release of norepinephrine from sympathetic nerves 43 , which contributes to stabilizing IOP.

Effect on ocular blood supply
Ischemia can cause glaucomatous damage accompany with or without an abnormal IOP. In vivo studies have revealed that inadequate blood supply can lead to optic nerve head atrophy and cell death in ganglions, which implies that abnormal ocular blood flow (OBF) necessarily affects metabolic processes to adapt to visual function needs 44 .
Several conflicting reports are published about the pharmacological reactions of H 2 S in vasculature of diverse organs in different species. It is reported that high concentrations of GYY4137 (1 mM) can significantly raise phenylephrine-induced tone in the ophthalmic arteries of rabbits 45 , but more evidences have proven that newly derived H 2 S donors exert vasodilator effects on precontracted posterior ciliary arteries (PCAs) 46,47 , which are crucial to OBF. Low concentrations of GYY4137 (100 nM-100 μM) may elicit relaxations in PCAs in the presence of phenylephrine induced tone via endogenous production of both prostanoids and H 2 S 47 . AP72 and AP67 show vasodilation effect on phenylephrine-induced PCAs in a concentration-dependent manner 46 . These effects are mainly dependent upon the action on K ATP channels by H 2 S. Taken together, these studies have established the role of H 2 S in modulating the OBF of glaucoma. Fig. 1 Generation of H 2 S. H 2 S generation is mainly controlled by four enzymes, CBS (major source of H 2 S production in central nervous system), CSE (major source of H 2 S production in vasculature system, liver and kidney), 3MST and CAT (major source of H 2 S production in retina). CSE and CAT are regulated by Ca 2+ . H 2 S is produced by these enzymes at steady-state low intracellular concentrations of Ca 2+ . Multiple ocular tissues showed the presence of endogenous H 2 S, including lens, iris, choroid, ciliary muscle, aqueous humor, cornea, and retina, except vitreous humor. The highest concentrations of endogenous H 2 S are detected in cornea and retina, of which the production differs in their major enzymes

Protection on neurons
The major features of glaucoma include progressive cell death of retinal ganglions and optic nerve damage 48 ,which are usually induced by loss of neurotrophic factors, intracellular and extracellular toxicity of glutamate, and neuro-inflammation [48][49][50][51][52] . In the nervous system, H 2 S functions as neurotransmitters 53 and possesses the ability to inhibit apoptosis and degradation of neurons 54 . H 2 S produced by astrocytes acts as a synaptic modulator and causes excitation to nearby neurons by controlling calcium ion influx of astrocytes 55 . For eyes, in vitro experiments have demonstrated that addition of H 2 S donors to the culture system effectively inhibits the release of sympathetic neurotransmission from isolated bovine irisciliary bodies 56 , and inhibits amino acid neurotransmission in isolated bovine retina 57 , which is mediated by its action on the K ATP channels or NO synthase. H 2 S can not only enhances the N-methyl-D-aspartate (NMDA) receptor-mediated responses in physiological concentrations 20 , but also modulates the over-activated NMDA receptors via the cAMP axis [58][59][60] . Aberrant metabolism or signaling pathways of H 2 S are found in various neurodegenerative diseases, such as declined levels of H 2 S in Alzheimer's patients 61 , impaired CSE transcription in Huntington's disease 62 , depleted sulfhydration in Parkinson's disease 63 , and increased H 2 S levels found in amyotrophic lateral sclerosis 64 . The fact that H 2 S modulates cell functions, protects neurons from apoptosis or oxidative stress are widely confirmed [65][66][67] . H 2 S is able to neutralize excess peroxynitrite (ONOO − ) or other free radicals, to antagonize lipid peroxidation and oxidation of thiols, and to reverse mitochondrial dysfunction 7 . It works as an anti-oxidant for eliminating the excessive glutamate together with glutathione 68 , as well as activating K ATP channels to combat oxidative glutamate toxicity 69 . H 2 S could inhibit the generation of reactive oxygen species (ROS) 70 and ameliorate the toxic effect of hypochlorous acid (HOCl) generated from myeloperioxidase (MPO) catalysis, thereby exerting anti-oxidant effects and protecting neuronal cells from cellular chlorinative damage 71 . H 2 S presents anti-apoptotic effect on the SH-SY5Y cell line in low concentrations by preserving mitochondrial functions, which is referred to suppressing cytochrome oxidase C and opening the mitochondrial K ATP channels 72 .
Referring to the anti-oxidant activity by H 2 S donors exerted on neurons, studies have found that H 2 S could increase the GSH concentration in neurons by enhancing the transporter of cysteine, cysteine/glutamate antiporter and γ-glutamyl cysteine synthetase (γ-GCS) 73,74 . γ-GCS and GSH synthetase act concertedly during the synthesis of GSH. Both enzymes can be regulated by Nrf2, which is also one potential targets of H 2 S 75 . The consequence of H 2 Sregulated Nrf2 pathway in neurons is to enhance the expression of glutathione-S-transferase (GST) and heme oxygenase (HO-1), the oxidative stress-related antioxidant enzymes 76 . ACS14 and ACS1, two donors of H 2 S, are confirmed to improve the intracellular GSH level and promote neuroprotective effects via opening K ATP channels 77 .
H 2 S could promote cell survival through effectively activating protein kinase C-α (PKC-α), inhibiting NF-κΒ signaling pathway, as well as upregulating Bcl-2 and X chromosome-linked inhibitor of apoptosis (XIAP) levels in RGC cells that pre-treated with glutamate (Glu) and buthionine sulfoximine (BSO) 76 . In comparison with glutamate treated RGC cells, addition of H 2 S enhances Akt phosphorylation and promotes cell viability in response to oxidative stress 76 . In a chronic ocular hypertension rat model, H 2 S is demonstrated to attenuate RGC apoptosis through balancing mitochondrial function, suppressing glial activation and downregulating the autophagy process 78 . Intracameral injection of NaHS to rats bearing glaucoma prohibits the loss of RGCs through recovering the levels of H 2 S in retina 79 . A long time release of H 2 S from GYY4137 combined with the in situ gel forming PLGA-based system, which lasts up to 72 h, has pointed to a great potential application in treating glaucoma 80 . (Fig. 2) H 2 S and diabetic retinopathy (DR) Reduction of the effects of advanced glycation end products (AGEs) in DR High glucose condition gives rise to the non-enzymatic condensation reaction between glucose and the amino terminus of protein, leads to the accumulation of AGEs' macromolecule, which has close relationship with the occurrence of DR 81,82 . AGEs can crosslink intracellular proteins to disturb their functions, and interfere normal metabolic pathways such as ATP production. AGEs destroys the inner blood-retinal barrier (BRB) in eye with subsequent oxidative stress reactions and inflammation 83,84 .
H 2 S promotes galactose metabolism to reduce AGEs generation in neuronal cells and prohibit excessive oxidative stress 85 . Mechanistically, H 2 S reduces ROS production and lipid peroxidation, while enhancing the expression of superoxide dismutase (SOD) and glutathione peroxidase (GPX), two endogenous antioxidant enzymes 86 . In addition, H 2 S could reverse high glucoseinduced increase in the expression of aldehyde oxidase 1 (AOX-1) and decrease in glutathione synthetase (GSS) level, ultimately to antagonize the AGEs-induced oxidative stress in cells 85 .

Inhibition of oxidative stress and inflammation
Although the toxicity of H 2 S accounts for the pathogenesis of multiple diseases, H 2 S possesses versatile antiinflammatory effects in vivo or vitro.
High glucose levels disturb the electron transfer process of the cellular mitochondrial respiratory chain in diabetic patients, so that oxygen free radical O − and superoxide can be easily generated 87 . Excessive O − converts NO into ONOO − , which can irreversibly bind to cytochrome C and impair mitochondrial functions. In DR animal models, the enhanced level of intracellular oxygen species and its associated excessive lipid peroxidation can be suppressed by H 2 S 73,88 . One property of H 2 S in antiinflammation is to scavenge the pro-inflammatory oxidants, such as ONOO − , HOCl, superoxide and hydrogen peroxide 71,89 . Besides, the pro-inflammatory response can be shifted to anti-inflammation by H 2 S donors, as demonstrated to decrease the levels of TNF-α, IL-8 and IFN-γ, while increasing the levels of cyclooxygenase (COX)-2 and eicosanoids 90 . Similarly, GYY4137 has been reported to inhibit LPS-induced production of inflammatory mediators by macrophages, and to upregulate the release of anti-inflammatory cytokine, IL-10 91 . Such regulation on inflammatory cytokine production can be attributed to the suppressive function of H 2 S on NF-κB activation 91,92 .
The animal models of DR have showed that hyperglycemia-induced leukostasis is related with cell apoptosis and retinal capillary occlusion 93 . The effect of resolving inflammation by H 2 S relies on its role in mediating macrophage phagocytosis 94 and promoting the granulocytes survival through inhibition of p38 phosphorylation and caspase-3 cleavage 95 . It downregulates the expression of MPO in neutrophils, thereby alleviating some of their toxic actions 96 . Moreover, increased retinal expression of intercellular adhesion molecule-1 (ICAM-1) and leukocyte adhesion in vessels are observed in DR animal models 93 , but H 2 S could downregulate ICAM-1 expression in vascular endothelium under high glucose conditions 97 . The adhesion molecules, including lymphocyte function-associated antigen, P-selectin and ICAM-1, are indispensable in instructing immune cells to transmigrate across inflamed capillaries. Blockade of H 2 S synthetase abolishes the alleviation of inflammation, with increased adherence of leukocytes to vascular endothelium and their transmigration 98,99 .
Investigations on the regulation of H 2 S on myocardium in type 1 diabetic rat model has revealed that H 2 S interferes with the inducible NOS (iNOS)/NO system, inhibits iNOS activity and its catabolite mediated oxidative stress 100 . However, the anti-inflammatory function by H 2 S is not always achieved. In low dose, H 2 S donor inhibits the inflammatory response, while high doses of H 2 S donor achieves controversial results. Therefore, dosage is a switch to control the biphasic regulation of H 2 S donor on inflammation 101 , and the generation of H 2 S can be augmented by the appearance of inflammation 102 . The feedback mechanism of H 2 S in controlling the progression of inflammatory responses in DR remains unclear.

Protective effect on retinal neurons
During the pathological process of DR, neuron damage usually occurs earlier and accumulates into visible retinal vascular lesions 103 . ACS67, a H 2 S donor, can be used to prevent RGC apoptosis and reactive gliosis in Muller cells after ischemia-reperfusion or exposure to oxidative  104 . Also, administration of H 2 S donors recovers the expression of brain-derived neurotrophic factor (BDNF) and retinal synaptic vesicle protein in streptozotocin (STZ)induced diabetic rats, indicating that H 2 S might block neuronal degeneration of retinal in diabetic patients 105 . The neuroprotective effect of H 2 S in retina is also related to its regulation on the intracellular GSH content 104 .

Multiple effects on retinal blood vessels
Dual role of BRB stability The dysfunction of BRB is a primary cause of retinal vascular lesions during DR pathogenesis. In DR development, retinal ischemia and hypoxia stimulate the expression of hypoxia inducible factor (HIF-1α) and trigger subsequent vascular endothelial growth factor (VEGF) signaling activation. The HIF-1α-VEGF-VEGFR2 signaling pathway is responsible for diabetes-induced BRB dysfunction and excessive angiogenesis 106 . In vivo experiments have shown that the reduced BRB permeability and decreased acellular capillaries in retinas of STZinduced diabetic rats after exogenous H 2 S treatment is accompanied by the reduction in VEGF content of vitreous and gene expression of VEGFR2, HIF-1α, as well as with increased expression of occludin 105 . Exogenous H 2 S administration is found to inhibit excessive deposition of laminin and collagen IVα3, in order to maintain the vascular integrity in the retinas of diabetic rats 107 . On the other hand, VEGF in intraocular tissues can stimulate endothelial cells to produce and release H 2 S 108 . At the onset of diabetes, H 2 S served as a protective factor against oxidative stress or nitrosative stress in the retina and vitreous humor, and it seems like H 2 S has a protective role on BRB in hyperglycemic condition. However, along with the progression of proliferative diabetic retinopathy (PDR), H 2 S may enhance the effect of VEGF on vascular endothelial cells, as well as the angiogenesis process [108][109][110] . Investigations on the level of H 2 S in the vitreous and plasma of PDR patients have revealed a much higher expression, indicating the potential effects of H 2 S in the pathogenesis of PDR 111 . As one of the main source to produce H 2 S in retina, 3-MST in hyperglycemic cells fail to convert 3-MP to H 2 S when the extracellular glucose concentration is elevated, and thus lost the ability of stimulating angiogenesis or cell proliferation, but the proangiogenic effect by exogenous H 2 S is not attenuated by hyperglycemia 112 . Moreover, the H 2 Sgenerating enzymes/H 2 S contributes to retinal neovascularization in ischemia-induced retinopathy 113 . These facts indicate that H 2 S may deteriorate retinal hemorrhage during the late stages of PDR.
Antithrombotic effect Besides inflammation and apoptosis, platelet adhesion is also involved in diabetes-induced retinal endothelial dysfunction 93 . The platelet adhesion to the injured diabetic endothelium takes part in ischemia and inflammation, both coagulation and fibrinolytic cascades in the vitreous are identified in DR 114 . Blood platelets tend to adhere to the vascular endothelium of DR rather than normal vessels 115 , which is involved in retinal capillaries occlusion and microvascular damage. H 2 S plays a potential role in reducing platelet aggregation, cell adhesion, and coagulantion [116][117][118][119] , it exerts antithrombotic effect through upregulation of NO synthesis, hydrolysis of disulfide bonds and the reduction of the calcium concentration in platelets [119][120][121] .
Modulation of the retinal blood flow The altered retinal circulation of the diabetes is well documented, diabetic mice demonstrates reduced density of flowing deep vessels 122 . There may be both increased and decreased retinal blood flow in diabetic patients compared with healthy people, while no significant difference is observed in OBF between patients of nonproliferative DR and PDR 123 . Considering the possibility of ischemia and hypoxia induced by abnormal blood supply and vascular dysfunction, we notice that H 2 S has multiple effects on vessels. The application of H 2 S donors could protect blood vessels, regulate blood pressure and alleviate the inflammatory reactions in the vascular system 124 . H 2 S exhibits the dual vascular effects of vasoconstriction and vasodilation depends on the vascular district, the endothelium conditions, the H 2 S concentration and the method of precontraction 125 . Different from the increased cAMP production induced by H 2 S in brain cells 59 , H 2 S negatively modulates β-adrenoceptor function via suppressing the adenylyl cyclase activity in cardiac myocytes 126 . The adenyl cyclase/cAMP pathway is involved in H 2 S induced vasoconstriction 127 , but on the other hand, H 2 S can instruct vascular smooth muscle cells against excessive vascular contraction via affecting K ATP 9 . Moreover, H 2 S alleviates the contraction of vascular smooth muscle by reducing the concentration of intracellular calcium through acting on inositol 1,4,5triphosphate receptor 128 . While its variable effects on vasculature were still being discussed, the increased cGMP level due to the PDE inhibition, the affected NO/ cGMP pathway with activated endothelial nitric oxide synthase (eNOS) and COX-derived metabolic byproducts are all required for H 2 S-induced vasodilation 129,130 . In addition, the vasodilation induced by H 2 S is related to the promotion of prostaglandin generation 131 , angiotensin-converting enzyme inhibition 102 , as well as modulating the viability of anion exchangers to control intra-cellular pH value 132 . All these findings imply that H 2 S contributes to regulating retinal blood flow and is involved in the DR pathogenesis. (Fig. 3)

H 2 S and retinal degeneration Modulation and protection of retinal neurons
Several retinal degenerative diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD), are associated with aberrant function of retinal pigment epithelium (RPE) and photoreceptor cells, which are crucial to maintain accurate visual sense 133 .
One of the RPE features is the apical and basal membranes, where the apical parts envelop photoreceptor cell outer segment (POS) to remove and degrade them through phagocytosis 134 . With a circadian rhythm, the phagocytosis of POS distal tips is always triggered by light 135 . Disturbance of RPE phagocytic function leads to POS accumulation and inevitable photoreceptor degeneration. The deficiency of CBS activity and the accumulation of homocysteine in the retina may lead to abnormal RPE structure and functions, bringing about the development of AMD-like features 136 .
Another feature of RPE cells is melanogenesis for absorbing excess light and protect photoreceptors. Melanin dispersion toward the apical microvilli of the RPE correlates positively with the intracellular level of cAMP, while light suppresses cAMP synthesis in retina of mice 137 . It has been shown that cAMP stimulates melatonin synthesis 138 and elevated cAMP levels with its signaling system influences RPE migration 139 . Increased cAMP in the subretinal space can lead to entry of cAMP into RPE cells via organic anion transporters with consequent triggering of dark-specific physiological responses, the nonderivatized cAMP can activate pigment granule aggregation in isolated RPE sheets 140 . It is reported that H 2 S donors and its substrate could produce a time and dose-dependent increase in cAMP concentrations in rat RPE cells 141 , the process of which involves K ATP channels and the enzymes of CSE and CBS 141 .
The metabolic cascade of photoreceptor signal transduction is mediated by cGMP that synthesized by guanylyl cyclases in retinal neurons. The response is triggered when photopigments absorb light, with subsequent degradation of cGMP by PDE 142 . Activities of cAMPhydrolyzing and cGMP-hydrolyzing have been detected in homogenates of cultured pigment epithelia from rats 143 . It is reported that cGMP stimulates the absorption of subretinal fluid by activating the RPE cell pump 144 , which is consistent with the fact of decreased cGMP concentration in the retinal detachment cases 142 . As mentioned above, H 2 S participates in the inhibition of PDE activity and induction of cyclic nucleotides, and at least three forms of PDEs are present in human RPE cells 145 , we infer that the cumulative cAMP or cGMP instructed by H 2 S may help maintain physiological functions of RPE and photoreceptors.
Administration of H 2 S contributes to protecting retinal neurons from light-induced degeneration 26 . Chronic sustained light-induced damages in the macular area cause degeneration of RPE and photoreceptor cells. Long-term excessive light exposure can induce damage or death of photoreceptor cells by oxidative stress and intracellular calcium overload 146 . Calcium in relatively low level can activate the 3MST/CAT enzymes to produce H 2 S. In turn, H 2 S can prevent Ca 2+ influx in the photoreceptor cells by activating V-ATPase in horizontal cells and maintain the balance of intracellular calcium, so that H 2 S protects photoreceptor cells from retinal cell apoptosis and oxidative stress 26 . However, the regulation of Ca 2+ and the cytoprotective effect of endogenous H 2 S may fail when photoreceptor cells are under excessive light exposure.

Potential in stem cell transplantation therapy
The RPE cells can modulate photoreceptor differentiation and retinal progenitor cells, which may play a role in the regulation of the retinal stem cell niche 147 . Transplantation of stem cell-derived RPE is proven to be effective in reversing retinal degeneration such as AMD 148,149 . MSCs are multipotent stem cells with selfrenewal abilities, immunoregulatory functions and multiple lineage differentiation potentials. In vitro expanded MSCs have been widely applied to treat many tissue injury, such as myocardial infarction 150 , skin wound 151 , organ transplantation 152 , autoimmune  156 . Studies have found that increased endogenous H 2 S level can block the hypoxia and serum deprivation-induced MSC apoptosis 157 , both the ERKs signaling pathways and the Akt signaling pathway are involved in the promotion of H 2 S on stem cell proliferation 158,159 . NaHS can prolong the survival of bone marrow mesenchymal stem cells (BMMSCs) and enhance their therapeutic effects for ischemic injury, also can improve the blood vessel integrity and prompt angiogenesis, with the upregulation of BDNF and VEGF expression 160 . In its regulation on stem cell differentiation, H 2 S is likely to affect neurogenesis by directly regulating Ca 2+ channels 161 , to initiate endothelial progenitor cell function and to enhance the angiogenesis process of wound sites in type 2 diabetic patients 162 .
Also, H 2 S is featured as one of the potential molecule for immunoregulation by MSCs. Deficiency of H 2 S attenuates the immunosuppressive function of MSCs on colitis in vivo, while supplementation of NaHS can restore the impaired therapeutic effects 163 . By the way, clinical H 2 S treatment is expected to improve long-term allograft survival in conjunction with immunosuppression for its positive effects on promoting organ survival against cold ischemia reperfusion injury 164 . Considering that NaHS pretreatment can enhance stem cells proliferation, promote the survival of therapeutically used stem cells and tissue cells via increased antioxidant defense 165 , H 2 S may be useful for the regeneration of retinal photoreceptors and RPE cells via transplantation strategies (Fig. 4).

Perspectives
Investigations in the past decade have provided new insights into the function of H 2 S during tissue damage and repair. In addition to its toxic effects, H 2 S is found to reduce intraocular pressure, inhibit inflammation and oxidative stress, promote stem cell-based regeneration, and restore the retinal microcirculation homeostasis. However, the exact therapeutic and pathological concentration of H 2 S remains elusive. Recently, novel H 2 S releasing drugs such as ATB-346 and ATB-352, have shown the efficacy in treating digestive diseases, with the promising application potential in various eye diseases. Due to the complexity of the BRB and the special anatomical structures of the eyes, the administrative routes of H 2 S should be carefully considered. Further investigation in this exciting field is expected to provide detailed information for better understanding the function of H 2 S in different types of eye diseases, and to design more effective and safe approaches for H 2 S application in clinical settings. Long-term excessive light exposure induces photoreceptor cell damage which is related to intracellular calcium overload. When the retinal photoreceptor cells are exposed under high intensity illumination, the cGMP-gated ion channels in membrane are shut down, with a cascade activity resulted in a relative low level of intracellular calcium. Such status facilitates H 2 S generation catalyzed by 3MST/ CAT enzymes, subsequently suppress Ca 2+ influx by activating V-ATPase. Exogenous H 2 S reduces the number of apoptotic retinal neurons after excessive light irradiation. Another approach to treating retinal degeneration disease is the stem cell-derived RPE-based therapy. Studies have found that H 2 S affects the immunoregulatory function of MSCs, and can enhance the proliferation and survival of the stem cells, thus improve their ability in tissue repair