Diabetes Volume 65, February 2016 331

Joost C. van den Born,1 Hans-Peter Hammes,2 Wolfgang Greffrath,3 Harry van Goor,1 and Jan-Luuk Hillebrands,1 on behalf of the DFG GRK International Research Training Group 1874 Diabetic Microvascular Complications (DIAMICOM)

Gasotransmitters in Vascular Complications of

Diabetes 2016;65:331–345 | DOI: 10.2337/db15-1003

In the past decades three gaseous signaling molecules— are important risk factors for vascular dis- so-called gasotransmitters—have been identified: nitric eases, with a two- to fourfold increased risk when com- oxide (NO), carbon monoxide (CO), and hydrogen sulfide pared with individuals without diabetes (3). These vascular (H2S). These gasotransmitters are endogenously pro- complications are divided into microvascular (retinopathy, duced by different enzymes in various cell types and neuropathy, and nephropathy) and macrovascular (cerebro- play an important role in physiology and disease. Despite vascular, coronary artery, and peripheral arterial diseases) fi their speci c functions, all gasotransmitters share the complications with respective clinical symptoms (Fig. 1). DIABETES IN PERSPECTIVES capacity to reduce oxidative stress, induce angiogenesis, Although the pathophysiology of type 1 and type 2 diabetes and promote vasorelaxation. In patients with diabetes, a is different, the proposed underlying mechanism leading to lower bioavailability of the different gasotransmitters is vascular complications seems to be similar and is thought to observed when compared with healthy individuals. As be related to endothelial dysfunction (4,5) and the associ- yet, it is unknown whether this reduction precedes or ated formation of reactive oxygen species (ROS). Chronic results from diabetes. The increased risk for vascular disease in patients with diabetes, in combination with promotes multiple biochemical pathways to the extensive clinical, financial, and societal burden, calls overproduce ROS, either through mitochondrial overpro- for action to either prevent or improve the treatment of duction or through enzymatic responses to high glucose (6). vascular complications. In this Perspective, we present a is the main cause of blindness in concise overview of the current data on the bioavailability adults. The worldwide prevalence is approximately 35% of gasotransmitters in diabetes and their potential role in in patients with diabetes (7). The essentials of diabetic the development and progression of diabetes-associated retinopathy can be best characterized by the combina- microvascular (retinopathy, neuropathy, and nephropa- tion of increased vessel permeability and progressive thy) and macrovascular (cerebrovascular, coronary ar- vascular occlusion. Although the clinical diagnosis of tery, and peripheral arterial diseases) complications. retinopathy is still made by the changes in small (early) Gasotransmitters appear to have both inhibitory and and larger (later) vessels, it has become clear that almost stimulatory effects in the course of vascular disease de- every cell type in the retina can be subject to damage by velopment. This Perspective concludes with a discussion complex metabolic changes, induced by chronic hyper- on gasotransmitter-based interventions as a therapeutic glycemia (8–11). option. Polyneuropathy is defined as a diffuse and bilateral disturbance of functions or pathological changes in multi- ple peripheral nerves. Diabetic peripheral polyneuropathy DIABETES AND ITS COMPLICATIONS is very frequent in the course of diabetes and even in Diabetes is characterized by hyperglycemia and , affecting up to 50% of all patients with resistance or deficiency. Diabetes is a top 10 cause of diabetes (12–14). However, although being frequent and death worldwide; its prevalence is increasing and currently severe, it is inadequately treated in most patients—77% estimated to be 9% among adults (1,2). Both type 1 and of those with chronic painful report

1Department of Pathology and Medical Biology, Division of Pathology, University Corresponding author: Jan-Luuk Hillebrands, [email protected]. Medical Center Groningen, University of Groningen, Groningen, the Netherlands Received 20 July 2015 and accepted 1 October 2015. 25th Medical Department, Medical Faculty Mannheim, University of Heidelberg, © 2016 by the American Diabetes Association. Readers may use this article as Mannheim, Germany long as the work is properly cited, the use is educational and not for profit, and 3Department of Neurophysiology, Centre for Biomedicine and Medical Technology the work is not altered. Mannheim, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany See accompanying article, p. 346. 332 Gasotransmitters in Diabetic Diabetes Volume 65, February 2016

Figure 1—Schematic overview of diabetes-associated microvascular (retinopathy, nephropathy, and neuropathy) and macrovascular (cerebrovascular, coronary artery, and peripheral arterial diseases) complications and their clinical long-term manifestations. TIA, transient ischemic attack.

persistent pain over 5 years (15). Experimental studies atherosclerotic plaque formation. As opposed to stable suggest the importance of neurovascular vasodilation in plaques, vulnerable, nonstable plaques are prone to (16); however, the mechanisms remain rupture, causing downstream ischemic events such as poorly understood, which may explain the current lack of transient ischemic attack and stroke (Fig. 1). adequate treatment in (diabetic) neuropathic pain. (DN) is one of the leading causes GASOTRANSMITTERS IN MICROVASCULAR of end-stage renal disease in the Western world, occurring DISEASE in ;30% of patients with type 1 and type 2 diabetes and Nitric Oxide and Microvascular Complications of accounting for about 40% of new cases of end-stage renal Diabetes disease based on U.S. data (17). At the structural level, the Nitric oxide (NO) was first recognized as an endothelium- glomeruli are often affected as evidenced by basement derived relaxing factor (19). It is endogenously formed membrane thickening, mesangial lesions (Kimmelstiel- from its substrate L-arginine by three different nitric ox- Wilson lesions), and nodular sclerosis. Clinically, DN is ide synthase (NOS) enzymes. Endothelial NOS (eNOS) is accompanied by and chronic renal failure. In predominantly associated with vascular tone. Inducible addition, arterioles are often affected. The mechanisms NOS (iNOS), although also present in the vascular system, leading to renal changes include the metabolic defect, is mainly active in the immune system under conditions nonenzymatic glycation of proteins, and hemodynamic of oxidative stress. It functions as a promoter of inflam- changes, such as hypertension leading to glomerular hy- mation. Neuronal NOS (nNOS), present in neurons and pertrophy (18). skeletal muscle cells, is important for neuronal cell-cell Macrovascular complications are characterized by the interactions (20). NO acts as a vasodilator and inhibits development of atherosclerosis in arteries throughout the platelet aggregation and stabilizes atherosclerotic plaques body. Atherosclerosis results from a proinflammatory (21). In humans, NO-dependent vasodilatation is im- state starting with endothelial dysfunction and culminat- paired in patients with type 2 diabetes, and lower eNOS ing in the narrowing of the arterial lumen as a result of expression and reduced NO production are the suggested diabetes.diabetesjournals.org van den Born and Associates 333 underlying cause (22,23). Blockade of NOS causes insulin AGEs, aminoguanidine reduces vascular cell damage in resistance in a rat model, indicating that in this model several animal models (33,34). Zheng et al. (30) found loss of NO synthesis precedes type 2 diabetes (24). In that nitrosative stress was reduced in the retinae of 2 2 different animal models for diabetes, lower bioavailability iNOS / mice, together with an inhibition of vasoregres- of NO is observed. Reduced NO production was found in sion and retinal thinning. However, the essential role of spontaneous type 1 diabetic BioBreeding rats (25) as well iNOS for the development of diabetic retinopathy seems as streptozotocin (STZ)-induced in male not to be the case for other NOS isoforms, as deletion of Sprague-Dawley rats (26). In mouse models of diet-in- eNOS exacerbates diabetic retinopathy (35). In STZ-in- 2 2 duced obesity and type 2 diabetes, NO bioavailability is duced type 1 diabetes, eNOS / mice developed more reduced, leading to endothelial dysfunction and impaired severe retinopathy compared with wild-type diabetic con- 2 2 NO-mediated vasodilatation (27,28). In contrast to these trol mice. The worsened phenotype in these eNOS / protective effects of NO, iNOS-produced NO seems to mice was accompanied by increased iNOS expression, fur- play an important role in inducing nitrosative stress ther suggesting an important role for iNOS in the devel- 2 2 and inflammation, also in the course of diabetes. Thus, opment of diabetic retinopathy. However, eNOS / mice NO seems to play a dual role in the development and suffer from higher blood pressure, so the worsened retinal progression of diabetes as well as in the development of phenotype can partly be explained by hypertensive injury. vascular dysfunction (29). In essence, NO appears to have a dual role (i.e., protective Effects of NO depletion and supplementation on the and noxious effects) in the diabetic retina, as schemati- development of microvascular complications have been cally shown in Fig. 2A. primarily studied in experimental models as summarized Neuropathy in Table 1 and discussed below. Until now, NO was the best-characterized gasotransmitter Retinopathy contributing to nociception and pain. Its downstream In the retina, iNOS is sensitive to hyperglycemia and targets within the peripheral nervous system (PNS) responsible for overproduction of NO (30,31). The result- include cyclic guanosine monophosphate (cGMP) pro- ing surplus of NO is either quenched by advanced glyca- duction by activation of soluble guanylyl cyclase (sGC) tion end products (AGEs) or leads, through the reaction and phosphorylation of membrane receptors and chan- with superoxide, to the formation of peroxynitrite with nels by cGMP-dependent protein kinases (36)—mecha- subsequent nitrosylation of proteins, lipids, and DNA. NO nisms usually associated with increased nociception. production is important in inflammatory signaling, and Consistently, different members of the large family of inflammation is thought to be important in incipient di- transient receptor potential channels, several of which abetic retinopathy (32). Increased reactive nitrogen spe- are known as nociceptive sensor molecules such as cies (RNS) has been observed in diabetic rat retinae and in TRPV1 and TRPA1, are activated by NO via cysteine S- vitro, and these changes were corrigible by aminoguani- nitrosylation (37). In contrast, several mechanisms were dine, an inhibitor of NO synthases (33). As an inhibitor of identified that may induce antinociception and analgesia

Table 1—Effect of NO in diabetic microvascular disease Model Intervention ↑ / ↓ Outcome References Retinopathy Mouse: STZ-induced L-NAME, iNOS2/2 ↓ Reduced diabetic leukostasis and 31 diabetes blood-retinal barrier permeability eNOS2/2 ↓ Increased and accelerated 35 retinopathy features Rat: STZ-induced Molsidomine ↑ Prevented diabetes-induced 61 diabetes vascular injury Neuropathy Human: type 2 diabetes NO donors (glyceryl trinitrate, ↑ Reduced neuropathic pain 39,40 isosorbide dinitrate) Mouse: STZ-induced iNOS2/2 ↓ Improved nerve conduction velocities 45 diabetes and lessened neuropathy Nephropathy Mouse: Leprdb/db eNOS2/2 ↓ Increased glomerular injury, 48 proteinuria, and renal insufficiency Mouse: STZ-induced eNOS2/2 ↓ Increased vascular damage 49 diabetes and renal insufficiency

Rat: OLETF NOS cofactor BH4 ↑ Reduced glomerular injury 51 and proteinuria L-NAME ↓ Increased glomerular injury, 50 inflammation, and proteinuria ↑ indicates increased NO; ↓ indicates reduced NO. OLETF, Otsuka Long-Evans Tokushima Fatty. 334 Gasotransmitters in Diabetic Angiopathy Diabetes Volume 65, February 2016

Figure 2—Beneficial and deleterious effects of gasotransmitters in the development of microvascular complications in diabetes: retinop- athy (A), neuropathy (B), and (glomerular) nephropathy (C). In these schematic representations of the three target organs, gasotransmitters are depicted in green when having beneficial effects and depicted in red when having deleterious effects on the development of micro- vascular complications. Gasotransmitters may have different properties as indicated by numbers 1–14 in the panels and explanatory text. As indicated in number 8, NO and CO might activate the cGMP pathway via sGC (e.g., via phosphorylation by protein kinases, release of transmitters, synaptic plasticity). and increase efficacy of analgesic compounds. In the cen- suggested to play a role in the induction of peripheral tral nervous system, NO interacts with the descending diabetic neuropathy and neuropathic pain via induction inhibitory control mechanisms of nociception (38). In pa- of RNS (44), including protein nitrosylation, lipid perox- tients with type 2 diabetes suffering from painful diabetic idation, DNA damage, and cell death (29). Hyperglycemia neuropathy, treatment with the NO donors glyceryl trini- activates iNOS and therefore generally increased nitrosa- trate (39) and isosorbide dinitrate (40) significantly im- tive stress in the PNS (44,45). Absence of iNOS reduced proved pain symptoms, indicative of the beneficial action nitrosative stress in peripheral nerve fibers displaying of NO in diabetic neuropathy. Similar effects were ob- normal nerve conduction velocities; diabetic neuropathy 2 2 served when locally applying a NO-releasing cutaneous was also less severe in diabetic iNOS / mice than in patch (41). These effects may, however, also be indirectly diabetic wild-type mice. Thus, diabetic neuropathy de- explained by variations in local microcirculation: transient pends on nitrosative stress induced in axons and Schwann changes in sciatic nerve microcirculation were observed in cells by NO produced from iNOS. In contrast, nNOS is response to NO in animals with STZ-induced diabetes required for maintaining PNS function and nerve fiber developing diabetic neuropathy (42). In STZ-induced di- density and contributes to a lesser extent to the develop- abetic rats, NOS activity is increased in primary sensory ment of diabetic polyneuropathy (45). In summary, NO neurons (43). The potent oxidant peroxynitrite, a product may play pivotal direct and indirect roles in the progres- of a superoxide anion radical reaction with NO, was sion of diabetic neuropathy, presumably by impairing diabetes.diabetesjournals.org van den Born and Associates 335 microcirculation in PNS at pathophysiological levels and to biliverdin, iron, and CO by HO. Three different iso- contributing to oxidative stress and inflammation and formsofHOexist;theinducibleform,HO-1,andthe tissue injury (29), as schematically shown in Fig. 2B. constitutive isoforms, HO-2 and HO-3. HO-1 and HO-2 are physiologically active, whereas the role of HO-3 in hu- Nephropathy man physiology remains unclear (55). CO has numerous There is still controversy regarding whether the genera- physiological functions, including vasodilation and inhibi- tion of NO is enhanced or decreased in DN. In the early fi tion of platelet aggregation. In skeletal muscle biopsies and stages of DN, Chiarelli et al. (46) found signi cantly circulating leukocytes from patients with type 2 diabetes, higher concentrations of NO end products (nitrite/nitrate) mRNA expression of HO-1 was dramatically decreased in the serum of DN patients with microalbuminuria compared with age-matched control subjects without dia- compared with the serum of healthy individuals. How- betes (56,57). In STZ-induced type 1 diabetic rats, a de- ever, an association as such does not imply causality per creased vasorelaxant function of CO was demonstrated, se. This excess of NO can indicate an upregulated inflam- despite higher HO-1 expression levels (58). In Zucker di- matory response by iNOS or a (protective) compensatory abetic fatty (ZDF) rats, CO production was decreased in response against renal injury, mediated by eNOS. In ex- aortic tissue compared with that in nondiabetic controls. perimental STZ-induced type 1 diabetes, renal NO pro- Increasing HO-1 activity with cobalt protoporphyrin duction is decreased in the early phase of the disease (47). resulted in higher levels of CO, lower glucose levels, and Deficiency of eNOS results in accelerated nephropathy in increased insulin sensitivity (59). These data are in favor of diabetic mice (48,49), also supporting a protective role for reduced vascular risk in the presence of higher CO levels, NO in DN (50). Supplementation of tetrahydrobiopterin which might be mediated via effects on insulin sensitivity (BH ), a cofactor of NOS, reduced proteinuria and renal 4 (60). Taken together, reduced bioavailability of CO in the damage in type 2 diabetic rats (51). Taken together, NO diabetic state is accompanied by and a production is clearly modulated in DN, and decrements in reduction of endothelial health, indicating a potential role its expression point to a contributing role for this gaso- for the HO-1/CO pathway in the development of diabetes transmitter in DN. Scavenging of ROS positively influ- and its associated complications. The effects of CO deple- ences the redox status and may mechanistically underlie tion and supplementation in diabetic mice and rats on the these findings. The modes of action of NO in the devel- development of microvascular complications are summa- opment of DN are schematically shown in Fig. 2C. rized in Table 2 and will be discussed below. Summary Retinopathy As discussed above, NO demonstrates both protective and Oxidative stress in the diabetic retina promotes the damaging properties in the development of microvascular activation of HOs (61). In the diabetic retina, HO-1 is disease. The producing enzyme seems to play a major role predominantly found in glial cells, in particular in Müller in the contrasting actions of NO: eNOS- and nNOS-derived cells, and to some extent in the microvasculature (62). In NO exerts the vast majority of their positive effects via vitro, HO-1 overexpression protects retinal endothelial upregulation of the production of cGMP by activation of cells from high glucose and oxidative/nitrosative stress sGC. However, iNOS-produced NO is involved in inflam- (63). In a STZ-induced type 1 diabetes model in rats, HO-1 matory signaling and is an important contributor to the upregulation by hemin resulted in protection against the development of diabetic angiopathy. In addition, the pres- development of diabetic retinopathy (64). This protection ence of ROS is important in the actions of NO. An excess of is reflected by the downregulation of p53, vascular endothe- NO in the presence of abundant ROS (superoxide) pro- lial growth factor (VEGF), and HIF-1a and a reduction of duction leads to the formation of peroxynitrite with subsequent nitrosylation of proteins, lipids, and DNA. diabetes-induced apoptosis in retinal ganglion cells (RGCs). On the contrary, HO-1–derived CO is proangiogenic (65), There is also increasing evidence for harmful effects of fl NO in protein tyrosine nitration (52). Protein nitration is a and angiogenesis, causing increased retinal blood ow, is a posttranslational modification that takes place in the com- key factor in the development of diabetic retinopathy (66). bined presence of oxidative stress and NO, which is the case This implies that the proangiogenic effects of CO may actu- in disease conditions such as diabetes (53). Given the data ally aggravate diabetic retinopathy. The effects of CO in the A available, we conclude that NO plays a dual role in the diabeticretinaareschematicallyshowninFig.2 . progression and maintenance of diabetic microvascular Neuropathy complications, which is mostly driven by the expression In the case of diabetic neuropathy, CO acts as a pain- of its producing enzymes (NOS) and the presence of ROS. modulating second messenger within the nervous system Carbon Monoxide and Microvascular Complications of (67). The activation of HO/CO signaling reduced symp- Diabetes toms of neuropathic pain, presumably by the activation of Carbon monoxide (CO), the second gasotransmitter, is anti-inflammatory and antioxidant mechanisms (68). CO produced by the different heme oxygenase (HO) enzymes exerts antinociceptive effects and increases the anti- as a product of heme metabolism (54). Heme is converted allodynic and antihyperalgesic efficacy of morphine in 336 Gasotransmitters in Diabetic Angiopathy Diabetes Volume 65, February 2016 chronic inflammation and neuropathic pain (69)—the lat- heme proteins such as hemoglobin. Due to the high levels ter strictly dependant on NO produced by nNOS and iNOS. of CO bound to hemoglobin (forming carboxyhemoglobin), Furthermore, CO relieves neuropathic pain symptoms by oxygen is not able to bind to that particular hemoglobin reducing the expression of iNOS and nNOS as well as by molecule, and disrupted oxygen transport develops. However, reducing the activation of spinal microglia (70). Interestingly, endogenous production of CO by HO enzymes obviously the constitutive isoform HO-2 is coexpressed with NOS in does not result in toxic levels. In contrast to NO, the exact the PNS and central nervous system (67), and CO, similar to working mechanism and molecular targets of CO are mostly NO, is also capable of activating the proalgesic cGMP protein unknown. Nevertheless, one of the known pathways is that kinase pathway (70). In fact, there is a close interaction be- CO is able to increase cGMP production by activation of sGC, tween the CO and NO systems in thecourseofneuropathic albeit with lower affinity than NO. Moreover, CO is able to pain, suggesting that they might act as cotransmitters in bind to complex IV (cytochrome c oxidase) of the mitochon- neuronal signaling transmission (67). In nociception, the drial electron transport chain and thereby regulate ROS more stable CO may set basal activity by tonic background production. In summary, CO is mainly protective in diabetic stimulation and NO may transiently amplify nociceptive vascular disease via inhibition of ROS formation, via in- signaling. Substances increasing endogenous CO (e.g., CO- teraction with NO, and via the sGC/cGMP pathway. releasing molecules [CORMs] or HO inducers alone or in combination with analgesics) may be useful for the treat- Hydrogen Sulfide and Microvascular Complications ment of (diabetic) neuropathic pain. The effects of CO in of Diabetes fi diabetic neuropathy are depicted in Fig. 2B. Hydrogen sul de (H2S) is the third gasotransmitter and was recognized as such in the 1990s (76,77). It is endogenously Nephropathy produced by three different enzymes. The pyridoxal-59- In the kidney, HO-1 and HO-2 are important in cytopro- phosphate (PLP)–dependent enzymes cystathionine b-synthase tection and serve as physiologic regulators of heme- (CBS) and cystathionine g-lyase (CSE) are the two major dependent protein synthesis during which CO is produced. H2S-producing enzymes. The third H2S-producing enzyme Inducers of the HO pathway (like hemin) are protective is 3-mercaptopyruvate sulfurtransferase (3MST). The main fl against renal in ammation and ameliorate DN in type 2 substrates for H S production are homocysteine and cyste- diabetic ZDF rats and STZ-induced type 1 diabetic rats (71– 2 ine. 3MST produces H Sfrom3-mercaptopyruvate,whichis 73). The antioxidant effect of HO-1 is believed to play a role 2 produced by the enzymes cysteine aminotransferase and in renal protection in diabetic rats (74). The opposite, de- D-amino acid oxidase from L-cysteine and D-cysteine, respec- ficiency of HO-2, results in higher superoxide anion levels tively (78). H S is a physiologically active compound and is and increased renal dysfunction after STZ-induced diabetes 2 called endothelium-derived hyperpolarizing factor (79,80); it (75). Enhanced production of CO seems to be beneficial for causes vasodilatation but also acts as scavenger of ROS and the kidney in DN, suggesting possibilities for therapeutic stimulating angiogenesis (81). Renal CSE and CBS expression intervention. The effects of CO in DN are shown in Fig. 2C. and H2S production are markedly lowered in spontaneous Summary diabetic Ins2Akita mice(82).Innonobesediabeticmice,an- Besides the beneficial effects of CO, high CO concentra- other mouse model of type 1 diabetes, it was also shown tions are toxic because of the high affinity of CO to bind that diabetic mice had lower H2Slevelscomparedwith

Table 2—Effect of CO in diabetic microvascular disease Model Intervention ↑ / ↓ Outcome References Retinopathy Rat: STZ-induced HO inhibitor SnPP ↓ Prevented diabetes-induced vascular injury 61 diabetes Hemin ↑ Maintained RGCs and reduced ROS in retina 64 Neuropathy Mouse CORM-2, CORM-3, ↑ Reduced neuropathic pain 70 HO-inducer CoPP Rat Hemin, CORM-2 ↑ Reduced neuropathic pain, inflammation, 68 and ROS/RNS Nephropathy Mouse: STZ-induced HO-22/2 ↓ Enhanced renal injury and loss of renal function 75 diabetes HO inducer CoPP ↑ Reduced glomerular injury and renal 75 insufficiency Rat: STZ-induced HO inducers hemin, CoPP ↑ Improved renal injury, inflammation, ROS, and 72–74 diabetes renal function HO inhibitors SnMP, CrMP ↓ Enhanced renal injury and renal function and 72,73 counteracted the protective effects of hemin Rat: ZDF Hemin ↑ Improved renal injury, inflammation, and 71 renal function HO inhibitor SnMP ↓ Enhanced renal injury and renal insufficiency 71 ↑ indicates increased CO; ↓ indicates reduced CO. diabetes.diabetesjournals.org van den Born and Associates 337

2 nondiabetic mice (83). In STZ-induced type 1 diabetic rats, levels, and the retinae of CBS+/ mice are characterized H2S levels were lower compared with age-matched nondia- by RGC loss, which is mediated by mitochondrial dysfunc- fi betic rats (84). However, CSE de ciency delayed the onset of tion (91). It is thus conceivable that H2S is neuroprotective STZ-induced type 1 diabetes, and diabetes was accompanied and there is indeed experimental proof for protective prop- by increased pancreatic H2S production without changes in erties of H2S in the retina, as evidenced by a decreased RGC pancreatic CSE of CBS protein expression (85). Another loss in H2S-pretreated animals after retinal ischemia/reper- player in the development of vascular complications is in- fusion (I/R) (92). Si et al. (93) investigated the effect of H2S sulin resistance. Insulin resistance is affected by H2Sina in experimental retinopathy of STZ-induced type 1 diabetic mouse model with high-fat diet–induced obesity. Interest- rats. They reported beneficial effects on neuronal dysfunction ingly, both the inhibition of H2SproductionbyDL-propar- (based on electroretinography) and retinal structure (i.e., in- gylglycine (PPG) and the treatment with slow-release H2S hibition of diabetes-induced retinal thickening and extracel- donor GYY4137 improved insulin resistance in these mice lular matrix proteins), while others clearly showed a link to (86). This unexpected beneficial effect of PPG could be improved endothelial function, such as tightened blood- explained by an upregulation of HO-1 resulting in higher retinal barrier and reduced vasoregression. H2Sisaknown CO levels, an effect of PPG that was recently described proangiogenic signaling molecule and can thereby also con- (87). In addition, this contradiction could be explained by tribute to enhanced angiogenesis in the diabetic retina. In the fact that PPG is an unspecific CSE inhibitor (based on line with this, increased levels of H2S were observed in vit- its cofactor PLP), thereby potentially inhibiting other PLP- reous body of patients with proliferative diabetic retinopathy dependent enzymes as well (88). In humans, diabetes is compared with patients with rhegmatogenous retinal de- associated with lower levels of H2S. In a small group of tachment (94). The effects of H2Sinthediabeticretina patients with type 2 diabetes, plasma H2Slevelswerere- are schematically shown in Fig. 2A. duced by 73% compared with those in healthy (lean) indi- Neuropathy viduals (89). Interestingly, obesity is correlated with lower H2S has mainly been reported to increase pain sensitivity levels of H2S compared with those in lean volunteers. Col- lectively, human and experimental diabetes are associated via several proposed modes of action (95). These include with reduced H S bioavailability, which might be related to sensitization of voltage-gated sodium and calcium chan- 2 – increased cardiovascular risk as observed in subjects with nels (95 97) and/or suppression of potassium channels. Furthermore, the pronociceptive transient receptor po- diabetes. The effects of H2S depletion and supplementation in diabetic mice and rats on the development of microvas- tential channels TRPV1 and TRPA1 (98,99), as well as cular complications are summarized in Table 3 and will be NMDA receptors, were suggested to be sensitized by fl discussed below. H2S. H2S displayed pronociceptive actions in in ammatory pain, both in STZ-induced type 1 diabetes and nondia- Retinopathy betic control animals. Interestingly, when treated with H2S has recently received attention in research on diabetic antagonists of the H2S-producingenzymes,painreduc- retinopathy as some H2S-related changes are compatible tion was much more pronounced in diabetic animals with a significant role of H2S in the development and prop- than it was in nondiabetic animals, indicative of an in- agation of diabetic retinopathy. Reduced H2S-mediated cell creased H2S sensitivity of the nociceptive system in rats protection supposedly plays a role in retinal diseases as CBS suffering from diabetes (100). Conversely, reduction of H2S expression is found in various eye compartments, including reduced the tactile allodynia developed in course of diabe- trans the retina, suggesting that the -sulfuration pathway is tes. The T-type voltage-gated calcium channel CaV3.2 is fi – present in the eye (90). Many CBS de ciency related eye sensitized by H2S, leading to increased pain sensitivity disorders are associated with increased homocysteine (101). An increased H2S tissue content and hyperactivity

Table 3—Effect of H2S in diabetic microvascular disease Model Intervention ↑ / ↓ Outcome References Retinopathy Mouse: STZ-induced CBS+/2 ↓ Increased loss of RGCs 91 diabetes Rat: STZ-induced NaHS ↑ Prevented diabetes-induced 93 diabetes vascular injury Neuropathy Rat: STZ-induced NaHS, L-Cysteine ↑ Increased neuropathic pain symptoms 100 diabetes CSE/CBS inhibitors PPG, ↓ Reduced neuropathic pain 96,100,101 b-cyanoalanine, hydroxylamine Akita Nephropathy Mouse: Ins2 H2S donor N-acetyl-cysteine ↑ Reduced ROS 82 Rat: STZ-induced NaHS ↑ Improved renal injury, inflammation, and 108,110 diabetes renal function and reduced ROS

↑ indicates increased H2S; ↓ indicates reduced H2S. 338 Gasotransmitters in Diabetic Angiopathy Diabetes Volume 65, February 2016

of CaV3.2 were observed in chemotherapy-induced neuro- partly via activation of KATP channels, and a rather pathic hyperalgesia and pain that could be reversed by newhypothesisistheinterferenceofH2Swiththe blocking H2S production (96). Painful peripheral diabetic cGMP pathway by inhibition of phosphodiesterase type “ neuropathy is accompanied by an enhancement of CaV3.2 5 activity, a mode of action comparable with natural T-type calcium channels and neuronal excitability. Thus, sildenafil” (112). ROS production is decreased by H2S CaV3.2 T-channels are thought to represent signal ampli- through direct interference with the mitochondrial res- fiers in peripheral sensory neurons, contributing to hyper- piration chain. It binds to cytochrome c oxidase, thereby excitability that ultimately leads to the development of directly inhibiting the formation of ROS. Another impor- pain in diabetic neuropathy (102). Conversely, antinocicep- tant effect of H2S in terms of diabetic angiopathy is tive effects also have been reported for H2S. These effects angiogenesis. The VEGF receptor 2 is the natural target were antagonized by the ATP-sensitive potassium (KATP) for H2S to achieve its proangiogenic effect (113). In di- channel blocker glibenclamide and by NOS inhibition abetic retinopathy, the development of new vessels re- fl (103). Inhalation of H2S reduced the development of neu- ects the severity. However, increased angiogenesis ropathic pain by reducing the resulting increase in inter- mightalsohavesomeprotectiveeffects,e.g.,angiogen- leukin-6 and chemokines, which was attributed to an esis of vaso nervorum in diabetic neuropathy. The effects inhibition of microglia activation in course of neuropathy of H2S on different ion channels are mainly important in diabetic neuropathy. Its interference with, for instance, (104). Furthermore, H2S functions as a neuroprotective agent by enhancing the production of glutathione, a major TRPV1, TRPA1, and CaV3.2 channels contributes to in- intracellular antioxidant that scavenges mitochondrial ROS creased nociception. Taken together, H2Sexertsdualef- fects in diabetic angiopathy, positive effects via its (105). Collectively, these data indicate that H2S displays both pro- and antinociceptive actions in diabetic neuropathy. The vasodilatory actions, and unwanted detrimental effects via different ion channels and angiogenesis. effects of H2S in diabetic neuropathy are depicted in Fig. 2B. Nephropathy GASOTRANSMITTERS IN MACROVASCULAR DISEASE In DN patients with atherosclerosis who are on dialysis, lower plasma levels of H2S were measured, which could Gasotransmitters have been studied in diabetes-associated macrovascular disease and therapeutically used in clinical indicate a loss of the supposed protective effects of H2S in these patients (106). This might be caused by endo- practice (NO only, CO and H2Shavenotyetbeenused).The thelial damage or downregulation via other pathways of effects of gasotransmitters depletion and supplementation in human and experimental diabetes on the development the enzymes producing H2S. Also, high urinary sulfate, as a proxy for H S, is significantly associated with a of endothelial function and macrovascular disease are sum- 2 fl slower decline in glomerular filtration rate in patients marized in Table 4 and brie y discussed below. In 1992, with type 1 diabetes and DN (107). In the experimental NO donor sodium nitroprusside (SNP) was used to mea- sure NO-dependent vasorelaxation in patients with type 1 setting, exogenous H2S reduces blood pressure and pre- ventstheprogressionofDNinspontaneouslyhyper- diabetes. SNP-stimulated vasodilatation was decreased in tensive rats (108). Renal protection via blood pressure patients without diabetes compared with subjects with di- reduction is also shown in angiotensin II–induced hy- abetes, indicating a lower NO sensitivity (114). In patients pertension and proteinuria in rats (109). Other studies with type 2 diabetes, the addition of NOS cofactor BH4 fl also suggest that H Sisakeymodulatorinrenal resulted in improved forearm blood ow, an effect that 2 fi NG remodeling and that its actions can be affected by the was nulli ed by NOS-inhibitor -monomethyl-L-arginine matrix metalloproteinase 9, which is shown to modu- (L-NMMA) (115). In patients with type 2 diabetes and late CBS and CSE (82). In STZ-induced type 1 diabetic coronary artery disease, treatment with NO substrate L-arginine and NOS cofactor BH4 protected against I/R en- mice, the administration of H2S attenuated oxidative stress and inflammation, reduced mesangial cell prolif- dothelial dysfunction in the forearm vasculature (116). The important role of NO in the macrovasculature is also eration, and inhibited the renin-angiotensin-aldosterone 2 2 showninanimalmodelsofdiabetes.Leprdb/db eNOS / system (110). These data indicate that in DN H2Shas fi double knockout mice showed an aggravated vascular phe- predominantly bene cial effects and is therefore a db/db 2/2 promising target for intervention. Thiosulfate might be notype compared with diabetic Lepr or eNOS single knockouts, as evidenced by an increased aortic wall the perfect H2S donor as it is already in use in the clinic thickness and reduced re-endothelialization after arterial for patients with calcifylaxis with end-stage renal disease 2/2 (111) and has been shown to be beneficial in hypertensive injury (117). In ApoE mice and mice with STZ- induced type 1 diabetes, treatment with bone marrow– renal disease in rats (109). The effects of H2Sonthekidney in DN are schematically shown in Fig. 2C. derived mononuclear cells overexpressing eNOS resulted in reduced plaque progression and improved postische- Summary mic neovascularization, an effect that was completely NG Knowledge on the working mechanisms of H2Siscon- inhibited by NOS inhibitor L- -nitro-L-arginine methyl tinuously increasing. H2S-regulated vasodilatation acts ester (L-NAME) (118). diabetes.diabetesjournals.org van den Born and Associates 339

Protective properties of CO in diabetes have been diabetic Sprague-Dawley rats, treatment with H2S donor mainly investigated in STZ-induced type 1 diabetes in rats sodium hydrosulfide (NaHS) improved vascular relaxation or mice. Exposure of the tail artery to CO ex vivo resulted and NO bioavailability. This indicates that H2S is a poten- in vasodilatation, an effect that was reduced in arteries of tial therapeutic agent in diabetic vascular disease via cross STZ-induced diabetic rats, indicating a reduced sensitivity talk with NO (26). Ex vivo administration of H2S sub- for CO in diabetic animals (58). In a myocardial I/R model strate L-cysteine also resulted in dose-dependent vasore- 2 2 in HO-1 / diabetic mice, infarct size and mortality were laxation in rat middle cerebral arteries, which was reduced dramatically worsened compared with wild-type (HO-1+/+) in diabetic animals (125). The vasorelaxant effects of diabetic mice, without affecting glucose levels (119). In di- NaHS are reduced with the addition of KATP blocker gli- abetic rats, CORM-3 or HO-1 inducer cobalt protoporphy- benclamide, demonstrating that NaHS-induced vasorelax- rin (CoPP) preserved endothelial function and vascular ation takes place via activation of KATP channels (126). Ex relaxation, an effect that was reversed by HO inhibitors vivo overexpression of CSE improved vascular relaxation chromium mesoporphyrin (CrMP) and tin mesoporphyrin in hyperglycemic conditions and reduced ROS production, (SnMP) (120–122). In a model of myocardial I/R injury, while CSE mRNA knockdown with small interfering RNA treatment with CO-releasing compound PEGylated carboxy- resulted in a more pronounced ROS production (127). hemoglobin bovine (PEG-COOH) drastically reduced in- Beneficial properties of H2S have been shown in models farct size and troponin levels in STZ-induced diabetic for myocardial injury as well. The addition of NaHS in mice.InmicereceivingPEG-COOHduringreperfusion diabetic rats resulted in preserved cardiac function (128), only, infarct size was reduced, suggesting CO as a potential reduced infarct size, reduced ROS and inflammatory pa- therapeutic agent for patients after myocardial infarction rameters such as tumor necrosis factor-a and interleu- (123). In STZ-induced diabetes in rats, induction of HO-1 kin-10, and inhibited expression of adhesion molecules with hemin, and treatment with CORM-2 to lesser ex- such as intracellular adhesion molecule 1 (129). In a tent, attenuated vascular damage and oxidative stress model of myocardial I/R injury in diabetic Leprdb/db mice, fi and improved vascular relaxation compared with non- treatment with H2S donor sodium sul de (Na2S), either treated rats (124). before I/R or only during reperfusion, diminished infarct H2S as a therapeutic agent in diabetic vascular disease size, troponin levels, and ROS (130,131). Although studies is evaluated in both mice and rats. In STZ-induced on the role of gasotransmitters in the development of

Table 4—Effect of gasotransmitters in diabetic macrovascular disease Model Intervention ↑ / ↓ Outcome References NO Mouse: Leprdb/db eNOS2/2 ↓ Increased arterial injury 117 Mouse: STZ-induced eNOS overexpression ↑ Reduced atherosclerosis and 118 diabetes of BM-MNCs improved angiogenesis Human: type 1 diabetes NO donor SNP ↑ Induced vasodilatation, but SNP-induced 114 vasodilatation is reduced in patients with diabetes

Human: type 2 diabetes NOS cofactor BH4 ↑ Improved forearm blood flow 115 L-NMMA ↓ Reduced forearm blood flow 115

L-arginine, BH4 ↑ Reduced endothelial dysfunction 116 CO Mouse: STZ-induced HO-12/2 ↓ Induced oxidative stress and increased infarct 119 diabetes size in myocardial I/R model CO donor PEG-COOH ↑ Reduced myocardial injury and oxidative 123 stress in myocardial I/R model Rat: STZ-induced diabetes CO gas ↑ Induced vasodilatation, but CO-dependent– 58 vasodilatation is reduced in diabetic rats CORM-2, CORM-3, biliverdin ↑ Improved vascular function and 120–122,124 reduced endothelial damage HO inducers hemin, CoPP ↑ Improved vascular function and 120,122,124 reduced oxidative stress HO inhibitor SnMP ↓ Diminished protective effects of CORM-3 121 db/db H2S Mouse: Lepr Na2S ↑ Reduced myocardial injury in 130,131 myocardial I/R model Rat: STZ-induced diabetes NaHS, L-cysteine ↑ Improved vascular function and reduced 26,125–129 myocardial injury CSE overexpression ↑ Improved vascular function ex vivo 127 CSE inhibitor PPG ↓ Increased myocardial injury and 125,129 inhibited vasorelaxation ex vivo ↑ indicates increased gasotransmitter availability; ↓ indicates decreased gasotransmitter availability. BM-MNCs, bone marrow–derived mononuclear cells. 340 Gasotransmitters in Diabetic Angiopathy Diabetes Volume 65, February 2016 macrovascular disease are limited to endothelial dysfunction functional similarities, it is likely that gasotransmitters rather than atherosclerosis, we propose that gasotransmit- have mutual interactions. Such a relationship between CO ters may also modulate atherogenesis via different mecha- and NO has been investigated intensively and is mainly nisms as schematically depicted in Fig. 3. Considering the mediated via the sGC/cGMP pathway. These effects include data described above and summarized in Table 4, gasotrans- blood pressure regulation and inflammation (138). Cyto- mitters seem to be promising targets for intervention in the protective effects of NO donors in endothelial cells were course of diabetic macrovascular disease. abolished in the presence of the HO inhibitor tin pro- toporphyrin (139). It has already been shown that H2S Protective Mechanisms and Mutual Gasotransmitter exertsitseffectsviaNOproduction,asHSpromotes Interactions 2 eNOS production and activity (140). Additionally, vasore- Although NO, CO, and H S have different molecular struc- 2 laxant effects of H S were diminished when aortic rings tures and routes of endogenous production, they do share 2 were pretreated with NOS inhibitor L-NAME (141). Recip- various physiological properties such as the ability to bind rocally, NO donor SNP stimulates endogenous H Sproduc- to heme groups (132) and to promote vasorelaxation by 2 tion via upregulation of CBS (142). Although CO and H S stimulation the sGC/cGMP pathway. NO and CO stimu- 2 share a lot of functional characteristics, the mutual rela- late cGMP production by targeting sGC, and H S by inhib- 2 tionship between these two gasotransmitters has barely iting phosphodiesterase type 5 activity (133). NO as well been studied. In one study of a myocardial I/R injury mouse as CO and H S are direct ROS scavengers, partly by direct 2 model, HO-1 expression was upregulated 24 h after intra- interaction with the mitochondrial respiratory chain. venous H S treatment, which was accompanied by a pro- They all engage on the K channels to achieve this an- 2 ATP tection against I/R-induced damage (143). tioxidant effect, both in the vasculature and nervous sys- tem (134–136). In addition, they share several common Methods to Measure Gasotransmitters intracellular pathways, such as nuclear factor-kB, nuclear In order to study the association of gasotransmitters and factor-like 2, and mitogen-activated protein kinases, the development of diabetes-associated vascular compli- therebyexertingantiapoptotic, antioxidant, and anti- cations, reliable and sensitive assays to measure NO, CO, inflammatory effects (133). NO, CO, and H2S inhibit the and H2S are indispensable. Various methodologies are used expression of intracellular adhesion molecule 1, vascular to measure the different gasotransmitters and these will cell adhesion molecule 1, and E-selectin, thereby pro- be briefly described. First, measuring NO is quite a chal- moting endothelial health and integrity. Finally, all lenge because of its instability. There are different methods three gasotransmitters act as proangiogenic substances of measuring NO. Most commonly used is the relatively via the VEGF pathway (113,137). On the basis of these simple Griess method, which does not measure NO directly

Figure 3—Beneficial and deleterious effects of gasotransmitters in the development of atherosclerosis in diabetes-associated macro- vascular complications. In the panel, the development of an atherosclerotic plaque (yellow layer) is schematically depicted. Gasotrans- mitters are depicted in green when having beneficial effects (numbers 1–4) and depicted in red when having deleterious effects (numbers 5 and 6) on the development of atherosclerosis as indicated in the panel and explanatory text. Ox, oxidized low-density lipoprotein. diabetes.diabetesjournals.org van den Born and Associates 341 but rather its oxidated products nitrite and nitrate. However, other organic nitrates, which are well established for their nitrite and nitrate can be detected more precise by high- vasodilatory effects during angina. Organic nitrates act performance liquid chromatography (144). NO can also as NO donors by enzymatic or nonenzymatic breakdown be directly measured using gas phase chemiluminescence, of nitrates into nitrite and NO (149). Molsidomine and which involves the reaction of NO with ozone (O3)toform Linsidomine are registered in several European countries as excited nitrogen dioxide. During relaxation to (unexcited) antianginal drugs and act as vasodilators by the nonenzy- nitrogen dioxide, a photon is released that is then detected matic release of NO. Finally, dietary products with high by chemiluminescence. Using this method, NO release from nitrate content can act as NO donors. The intake of beet- different body fluids and tissues can be measured. In addition root juice lowered blood pressure significantly, an effect to the aforementioned methods, different fluorescent probes that was accompanied by higher levels of total urinary and electrodes are currently available, with the possibility to nitrite/nitrate (150). CO administration or CORMs are measure NO in fluids and tissues and intracellularly in cells in not in clinical use yet, albeit that some of the vascular vitro (145). protective effects of acetylsalicylic acid and statins are at- CO levels also can be measured using different tributed to induction of HO-1. In human endothelial cells methods. The most commonly used and relatively simple in vitro, a dose-dependent increase of HO-1 expression was way to measure CO is in the air using gas chromatog- seen after statin (151,152) or acetylsalicylic acid (153) raphy. In vivo, CO is generally measured in red blood treatment. However, this effect was not reproduced in hu- cells by determining the percentage of carboxyhemoglo- man subjects as no differences in HO-1 expression were bin relative to total hemoglobin concentration. Finally, observed between patients treated with acetylsalicylic some studies use [14C]Hemeinvitrotomeasureendog- acid, statin, or placebo (154). The antioxidative actions of enous 14CO production (146). polyphenol resveratrol are also partly attributed to HO-1 When considering H2S as therapeutic target, reliable upregulation as shown by increased HO-1 expression levels methods to determine H2Slevelsinbodyfluids and tissues in STZ-induced type 1 diabetes in Sprague-Dawley rats are needed. However, measurements of H2S are difficult (122). Although the HO-1–inducing effects of resveratrol because of its volatility. For that reason, stable end prod- have not yet been described in humans, this dietary sup- ucts like sulfate or thiosulfate can be measured in serum or plement is readily available for human use. Similar to CO, urine (147), although a few methodologies have been de- H2S is also not clinically used in humans yet, although in- scribed to measure H2S itself. The methylene blue assay is travenous Na2S administration has been performed in a the most commonly used technique. It is based on the phase 1 safety study (155). This study revealed increased oxidative coupling of H2S with two N,N-dimethyl-p- H2S and thiosulfate levels after Na2S administration, indi- phenylenediamine molecules, forming the methylene blue cating that circulating H2S levels can be achieved following dye that can be detected spectrophotometrically. However, parenteral administration. The H2S metabolite thiosulfate this technique is extremely pH dependent and not very can also act as a H2S donor via enzymatic conversion by sensitive and reliable. A more sensitive method is based rhodanese (also known as thiosulfate sulfurtransferase) on monobromobimane in which two monobromobimane (156). Thiosulfate is used as a treatment for calcifylaxis molecules form the stable sulfide dibimane in the presence in patients with end-stage renal disease (111) and has of H2S. Sulfide dibimane can be separated by reverse-phase been described as a protective agent in hypertensive heart chromatography and detected by a fluorescence detector. and renal disease in rats (109,157). Sulfhydrylated ACE Now, fluorescent probes and sulfide selective electrodes are inhibitors, such as zofenopril and captopril, show additional extensively used however, with different sensitivity and beneficial effects in different trials (158). Recently, it was reliability (148). As yet, H2S measurements are difficult, demonstrated that the beneficial effects of sulfhydrylated with variable reliability, thereby complicating studies on ACE inhibitors are explained by H2S release (159). Finally, the role of H2S and its use as therapeutic target in various H2S is also generated by various species of sulfate-reducing diseases, including diabetes-associated vascular disease. bacteria in the gut. Germ-free mice showed significantly lower levels of H2S (160), indicating that the addition of Future Perspectives and Treatment Options dietary sulfate or sulfur-containing amino acids can act as Patients with diabetes have a two- to fourfold increased natural H2Sdonors. risk for cardiovascular disease, and adequate treatment and preventive strategies are still lacking. As discussed in CONCLUSION this Perspective, the different gasotransmitters appear to Various gasotransmitter-based strategies are currently be- be important mediators in the development of diabetic ing studied as potential strategy to treat vascular dysfunc- angiopathy and therefore are potential targets for in- tion.Sofar,thesestrategieshavenotbeenexploredinthe tervention. As aforementioned, NO-based interventions context of diabetes-associated vascular disease. Because of are already applied in humans and readily available. SNP the toxicity of high concentrations of gasotransmitters, as is clinically used and acts as a direct NO donor by releasing well as their potential deleterious effects on the develop- NO from the ferrous ion center without the need for ment of vascular disease (as discussed in this Perspec- enzymatic action. The same is true for nitroglycerin and tive), prudence is called for when considering exogenous 342 Gasotransmitters in Diabetic Angiopathy Diabetes Volume 65, February 2016 administration of gasotransmitters. However, gasotransmitter- 20. Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthases: structure, based interventions are relatively safe, mainly because function and inhibition. Biochem J 2001;357:593–615 these gases are also produced endogenously and there- 21. Bauer V, Sotníková R. Nitric oxide–the endothelium-derived relaxing – fore are highly promising candidate therapeutics. factor and its role in endothelial functions. Gen Physiol Biophys 2010;29:319 340 22. Elliott TG, Cockcroft JR, Groop PH, Viberti GC, Ritter JM. Inhibition of nitric Acknowledgments. The authors would like to thank Amanda Gautier oxide synthesis in forearm vasculature of insulin-dependent diabetic patients: from Gautier Scientific Illustration for preparing the artwork. blunted vasoconstriction in patients with microalbuminuria. Clin Sci (Lond) 1993; – Funding. This work was supported by grants from the Deutsche For- 85:687 693 schungsgemeinschaft (IRTG 1874-1 DIAMICOM) (to H.-P.H., W.G., and J.-L.H.), 23. Sobrevia L, Mann GE. Dysfunction of the endothelial nitric oxide signalling – the Dutch Kidney Foundation (NSN C08-2254 and IP13-114) (to H.v.G.), and the pathway in diabetes and hyperglycaemia. Exp Physiol 1997;82:423 452 Graduate School of Medical Sciences of the University of Groningen (to J.C.v.d.B.) 24. Sadri P, Lautt WW. Blockade of hepatic nitric oxide synthase causes in- – Duality of Interest. No potential conflicts of interest relevant to this article sulin resistance. Am J Physiol 1999;277:G101 G108 were reported. 25. Wu G, Meininger CJ. Impaired arginine metabolism and NO synthesis in coronary endothelial cells of the spontaneously diabetic BB rat. Am J Physiol References 1995;269:H1312–H1318 1. World Health Organization. Global Status Report on Noncommunicable 26. Zheng YF, Dai DZ, Dai Y. NaHS ameliorates diabetic vascular injury by Diseases 2014. Geneva, Switzerland, World Health Organization, 2014 correcting depressed connexin 43 and 40 in the vasculature in streptozotocin- 2. Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: injected rats. J Pharm Pharmacol 2010;62:615–621 estimates for the year 2000 and projections for 2030. Diabetes Care 2004;27: 27. Bender SB, Herrick EK, Lott ND, Klabunde RE. Diet-induced obesity and 1047–1053 diabetes reduce coronary responses to nitric oxide due to reduced bioavailability 3. Fox CS, Coady S, Sorlie PD, et al. Trends in cardiovascular complications in isolated mouse hearts. Diabetes Obes Metab 2007;9:688–696 of diabetes. JAMA 2004;292:2495–2499 28. Kim F, Pham M, Maloney E, et al. Vascular inflammation, insulin re- 4. Sena CM, Pereira AM, Seiça R. Endothelial dysfunction - a major mediator sistance, and reduced nitric oxide production precede the onset of peripheral of diabetic vascular disease. Biochim Biophys Acta 2013;1832:2216–2231 insulin resistance. Arterioscler Thromb Vasc Biol 2008;28:1982–1988 5. Schalkwijk CG, Stehouwer CD. Vascular complications in diabetes 29. Yamagishi S, Matsui T. Nitric oxide, a janus-faced therapeutic target for mellitus: the role of endothelial dysfunction. Clin Sci (Lond) 2005;109:143– diabetic -friend or foe? Pharmacol Res 2011;64:187–194 159 30. Zheng L, Du Y, Miller C, et al. Critical role of inducible nitric oxide synthase 6. Giacco F, Brownlee M. Oxidative stress and diabetic complications. Circ in degeneration of retinal capillaries in mice with streptozotocin-induced di- Res 2010;107:1058–1070 abetes. Diabetologia 2007;50:1987–1996 7. Yau JW, Rogers SL, Kawasaki R, et al.; Meta-Analysis for Eye Disease 31. Leal EC, Manivannan A, Hosoya K, et al. Inducible nitric oxide synthase (META-EYE) Study Group. Global prevalence and major risk factors of diabetic isoform is a key mediator of leukostasis and blood-retinal barrier breakdown in retinopathy. Diabetes Care 2012;35:556–564 diabetic retinopathy. Invest Ophthalmol Vis Sci 2007;48:5257–5265 8. Antonetti DA, Klein R, Gardner TW. Diabetic retinopathy. N Engl J Med 32. Joussen AM, Poulaki V, Le ML, et al. A central role for inflammation in the 2012;366:1227–1239 pathogenesis of diabetic retinopathy. FASEB J 2004;18:1450–1452 9. Kern TS, Tang J, Berkowitz BA. Validation of structural and functional 33. Du Y, Smith MA, Miller CM, Kern TS. Diabetes-induced nitrative stress in lesions of diabetic retinopathy in mice. Mol Vis 2010;16:2121–2131 the retina, and correction by aminoguanidine. J Neurochem 2002;80:771–779 10. Hammes HP, Feng Y, Pfister F, Brownlee M. Diabetic retinopathy: targeting 34. Hammes HP, Martin S, Federlin K, Geisen K, Brownlee M. Aminoguanidine vasoregression. Diabetes 2011;60:9–16 treatment inhibits the development of experimental diabetic retinopathy. Proc Natl 11. Calcutt NA, Cooper ME, Kern TS, Schmidt AM. Therapies for hyper- Acad Sci U S A 1991;88:11555–11558 glycaemia-induced diabetic complications: from animal models to clinical trials. 35. Li Q, Verma A, Han PY, et al. Diabetic eNOS-knockout mice develop Nat Rev Drug Discov 2009;8:417–429 accelerated retinopathy. Invest Ophthalmol Vis Sci 2010;51:5240–5246 12. Singh R, Kishore L, Kaur N. Diabetic peripheral neuropathy: current per- 36. Cury Y, Picolo G, Gutierrez VP, Ferreira SH. Pain and analgesia: The dual spective and future directions. Pharmacol Res 2014;80:21–35 effect of nitric oxide in the nociceptive system. Nitric Oxide 2011;25:243–254 13. Papanas N, Vinik AI, Ziegler D. Neuropathy in prediabetes: does the clock 37. Yoshida T, Inoue R, Morii T, et al. Nitric oxide activates TRP channels by start ticking early? Nat Rev Endocrinol 2011;7:682–690 cysteine S-nitrosylation. Nat Chem Biol 2006;2:596–607 14. Ziegler D, Papanas N, Vinik AI, Shaw JE. Epidemiology of polyneuropathy 38. Millan MJ. Descending control of pain. Prog Neurobiol 2002;66:355–474 in diabetes and prediabetes. Handb Clin Neurol 2014;126:3–22 39. Agrawal RP, Choudhary R, Sharma P, et al. Glyceryl trinitrate spray in the 15. Daousi C, Benbow SJ, Woodward A, MacFarlane IA. The natural history of management of painful diabetic neuropathy: a randomized double blind placebo chronic painful peripheral neuropathy in a community diabetes population. Diabet controlled cross-over study. Diabetes Res Clin Pract 2007;77:161–167 Med 2006;23:1021–1024 40. Yuen KC, Baker NR, Rayman G. Treatment of chronic painful diabetic 16. Hamdy O, Abou-Elenin K, LoGerfo FW, Horton ES, Veves A. Contribution of neuropathy with isosorbide dinitrate spray: a double-blind placebo-controlled nerve-axon reflex-related vasodilation to the total skin vasodilation in diabetic cross-over study. Diabetes Care 2002;25:1699–1703 patients with and without neuropathy. Diabetes Care 2001;24:344–349 41. Agrawal RP, Jain S, Goyal S, Singhal S, Lindgren L, Sthengel E. A clinical 17. Molitch ME, DeFronzo RA, Franz MJ, et al.; American Diabetes Association. trial of nitrosense patch for the treatment of patients with painful diabetic neu- Nephropathy in diabetes. Diabetes Care 2004;27(Suppl. 1):S79–S83 ropathy. J Assoc Physicians India 2014;62:385–390 18. Fagerudd JA, Tarnow L, Jacobsen P, et al. Predisposition to essential 42. Thomsen K, Rubin I, Lauritzen M. NO- and non-NO-, non-prostanoid-de- hypertension and development of diabetic nephropathy in IDDM patients. Di- pendent vasodilatation in rat sciatic nerve during maturation and developing abetes 1998;47:439–444 experimental diabetic neuropathy. J Physiol 2002;543:977–993 19. Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium- 43. Zochodne DW, Verge VM, Cheng C, et al. Nitric oxide synthase activity and derived relaxing factor produced and released from artery and vein is nitric oxide. expression in experimental diabetic neuropathy. J Neuropathol Exp Neurol 2000; Proc Natl Acad Sci U S A 1987;84:9265–9269 59:798–807 diabetes.diabetesjournals.org van den Born and Associates 343

44. Vareniuk I, Pacher P, Pavlov IA, Drel VR, Obrosova IG. Peripheral neu- 66. Crawford TN, Alfaro DV 3rd, Kerrison JB, Jablon EP. Diabetic retinopathy ropathy in mice with neuronal nitric oxide synthase gene deficiency. Int J Mol and angiogenesis. Curr Diabetes Rev 2009;5:8–13 Med 2009;23:571–580 67. Fan W, Huang F, Wu Z, Zhu X, Li D, He H. Carbon monoxide: a gas that 45. Vareniuk I, Pavlov IA, Obrosova IG. Inducible nitric oxide synthase gene modulates nociception. J Neurosci Res 2011;89:802–807 deficiency counteracts multiple manifestations of peripheral neuropathy in a 68. Bijjem KR, Padi SS, lal Sharma P. Pharmacological activation of heme streptozotocin-induced mouse model of diabetes. Diabetologia 2008;51:2126–2133 oxygenase (HO)-1/carbon monoxide pathway prevents the development of pe- 46. Chiarelli F, Cipollone F, Romano F, et al. Increased circulating nitric oxide ripheral neuropathic pain in Wistar rats. Naunyn Schmiedebergs Arch Pharmacol in young patients with type 1 diabetes and persistent microalbuminuria: relation 2013;386:79–90 to glomerular hyperfiltration. Diabetes 2000;49:1258–1263 69. Hervera A, Gou G, Leánez S, Pol O. Effects of treatment with a carbon 47. Keynan S, Hirshberg B, Levin-Iaina N, et al. Renal nitric oxide production during monoxide-releasing molecule and a heme oxygenase 1 inducer in the anti- the early phase of experimental diabetes mellitus. Kidney Int 2000;58:740–747 nociceptive effects of morphine in different models of acute and chronic pain in 48. Zhao HJ, Wang S, Cheng H, et al. Endothelial nitric oxide synthase de- mice. Psychopharmacology (Berl) 2013;228:463–477 ficiency produces accelerated nephropathy in diabetic mice. J Am Soc Nephrol 70. Hervera A, Leánez S, Negrete R, Motterlini R, Pol O. Carbon monoxide 2006;17:2664–2669 reduces neuropathic pain and spinal microglial activation by inhibiting nitric oxide 49. Nakagawa T, Sato W, Glushakova O, et al. Diabetic endothelial nitric oxide synthesis in mice. PLoS One 2012;7:e43693 synthase knockout mice develop advanced diabetic nephropathy. J Am Soc 71. Ndisang JF, Jadhav A, Mishra M. The heme oxygenase system suppresses Nephrol 2007;18:539–550 perirenal visceral adiposity, abates renal inflammation and ameliorates diabetic 50. Kamijo H, Higuchi M, Hora K. Chronic inhibition of nitric oxide production nephropathy in Zucker diabetic fatty rats. PLoS One 2014;9:e87936 aggravates diabetic nephropathy in Otsuka Long-Evans Tokushima fatty rats. 72. Ndisang JF, Jadhav A. Hemin therapy improves kidney function in male Nephron, Physiol 2006;104:12–22 streptozotocin-induced diabetic rats: role of the heme oxygenase/atrial natriuretic 51. Okumura M, Masada M, Yoshida Y, et al. Decrease in tetrahydrobiopterin as peptide/adiponectin axis. Endocrinology 2014;155:215–229 a possible cause of nephropathy in type II diabetic rats. Kidney Int 2006;70:471–476 73. Elmarakby AA, Faulkner J, Baban B, Saleh MA, Sullivan JC. Induction of 52. Radi R. Nitric oxide, oxidants, and protein tyrosine nitration. Proc Natl Acad hemeoxygenase-1 reduces glomerular injury and apoptosis in diabetic sponta- Sci U S A 2004;101:4003–4008 neously hypertensive rats. Am J Physiol Renal Physiol 2012;302:F791–F800 53. Turko IV, Li L, Aulak KS, Stuehr DJ, Chang JY, Murad F. Protein tyrosine 74. Ptilovanciv EO, Fernandes GS, Teixeira LC, et al. Heme oxygenase 1 im- nitration in the mitochondria from diabetic mouse heart. Implications to dys- proves glucoses metabolism and kidney histological alterations in diabetic rats. functional mitochondria in diabetes. J Biol Chem 2003;278:33972–33977 Diabetol Metab Syndr 2013;5:3 54. Wu L, Wang R. Carbon monoxide: endogenous production, physiological 75. Goodman AI, Chander PN, Rezzani R, et al. Heme oxygenase-2 deficiency functions, and pharmacological applications. Pharmacol Rev 2005;57:585–630 contributes to diabetes-mediated increase in superoxide anion and renal dys- 55. Kikuchi G, Yoshida T, Noguchi M. Heme oxygenase and heme degradation. function. J Am Soc Nephrol 2006;17:1073–1081 Biochem Biophys Res Commun 2005;338:558–567 76. Abe K, Kimura H. The possible role of hydrogen sulfide as an endogenous 56. Bruce CR, Carey AL, Hawley JA, Febbraio MA. Intramuscular heat shock neuromodulator. J Neurosci 1996;16:1066–1071 protein 72 and heme oxygenase-1 mRNA are reduced in patients with type 2 77. Wang R. Two’s company, three’s a crowd: can H2S be the third endog- diabetes: evidence that insulin resistance is associated with a disturbed anti- enous gaseous transmitter? FASEB J 2002;16:1792–1798 oxidant defense mechanism. Diabetes 2003;52:2338–2345 78. Kimura H. The physiological role of hydrogen sulfide and beyond. Nitric 57. Adaikalakoteswari A, Balasubramanyam M, Rema M, Mohan V. Differential Oxide 2014;41:4–10 gene expression of NADPH oxidase (p22phox) and hemoxygenase-1 in patients 79. Mustafa AK, Sikka G, Gazi SK, et al. Hydrogen sulfide as endothelium- with type 2 diabetes and microangiopathy. Diabet Med 2006;23:666–674 derived hyperpolarizing factor sulfhydrates potassium channels. Circ Res 2011; 58. Wang R, Wang Z, Wu L, Hanna ST, Peterson-Wakeman R. Reduced 109:1259–1268 vasorelaxant effect of carbon monoxide in diabetes and the underlying mecha- 80. Tang G, Yang G, Jiang B, Ju Y, Wu L, Wang R. H₂S is an endothelium- nisms. Diabetes 2001;50:166–174 derived hyperpolarizing factor. Antioxid Redox Signal 2013;19:1634–1646 59. Nicolai A, Li M, Kim DH, et al. Heme oxygenase-1 induction remodels 81. Polhemus DJ, Lefer DJ. Emergence of hydrogen sulfide as an endogenous adipose tissue and improves insulin sensitivity in obesity-induced diabetic rats. gaseous signaling molecule in cardiovascular disease. Circ Res 2014;114:730– Hypertension 2009;53:508–515 737 60. Li M, Kim DH, Tsenovoy PL, et al. Treatment of obese diabetic mice with a 82. Kundu S, Pushpakumar SB, Tyagi A, Coley D, Sen U. Hydrogen sulfide heme oxygenase inducer reduces visceral and subcutaneous adiposity, increases deficiency and diabetic renal remodeling: role of matrix metalloproteinase-9. Am adiponectin levels, and improves insulin sensitivity and glucose tolerance. Di- J Physiol Endocrinol Metab 2013;304:E1365–E1378 abetes 2008;57:1526–1535 83. Brancaleone V, Roviezzo F, Vellecco V, De Gruttola L, Bucci M, Cirino G. 61. Cukiernik M, Mukherjee S, Downey D, Chakabarti S. Heme oxygenase in Biosynthesis of H2S is impaired in non-obese diabetic (NOD) mice. Br J Phar- the retina in diabetes. Curr Eye Res 2003;27:301–308 macol 2008;155:673–680 62. Hammes HP, Bartmann A, Engel L, Wülfroth P. Antioxidant treatment of 84. Jain SK, Bull R, Rains JL, et al. Low levels of hydrogen sulfide in the blood experimental diabetic retinopathy in rats with nicanartine. Diabetologia 1997;40: of diabetes patients and streptozotocin-treated rats causes vascular in- 629–634 flammation? Antioxid Redox Signal 2010;12:1333–1337 63. Castilho Á, Aveleira CA, Leal EC, et al. Heme oxygenase-1 protects retinal 85. Yang G, Tang G, Zhang L, Wu L, Wang R. The pathogenic role of cys- endothelial cells against high glucose- and oxidative/nitrosative stress-induced tathionine g-lyase/hydrogen sulfide in streptozotocin-induced diabetes in mice. toxicity. PLoS One 2012;7:e42428 Am J Pathol 2011;179:869–879 64. Fan J, Xu G, Jiang T, Qin Y. Pharmacologic induction of heme oxygenase-1 86. Geng B, Cai B, Liao F, et al. Increase or decrease hydrogen sulfide exert plays a protective role in diabetic retinopathy in rats. Invest Ophthalmol Vis Sci opposite lipolysis, but reduce global insulin resistance in high fatty diet induced 2012;53:6541–6556 obese mice. PLoS One 2013;8:e73892 65. Li Volti G, Sacerdoti D, Sangras B, et al. Carbon monoxide signaling in 87. Oosterhuis NR, Frenay AR, Wesseling S, et al. DL-propargylglycine reduces promoting angiogenesis in human microvessel endothelial cells. Antioxid Redox blood pressure and renal injury but increases kidney weight in angiotensin-II Signal 2005;7:704–710 infused rats. Nitric Oxide 2015;49:56–66 344 Gasotransmitters in Diabetic Angiopathy Diabetes Volume 65, February 2016

88. Elfarra AA, Lash LH, Anders MW. Metabolic activation and detoxication of 109. Snijder PM, Frenay AR, Koning AM, et al. Sodium thiosulfate attenuates nephrotoxic cysteine and homocysteine S-conjugates. Proc Natl Acad Sci U S A angiotensin II-induced hypertension, proteinuria and renal damage. Nitric Oxide 1986;83:2667–2671 2014;42:87–98 89. Whiteman M, Gooding KM, Whatmore JL, et al. Adiposity is a major de- 110. Zhou X, Feng Y, Zhan Z, Chen J. Hydrogen sulfide alleviates diabetic terminant of plasma levels of the novel vasodilator hydrogen sulphide. Dia- nephropathy in a streptozotocin-induced diabetic rat model. J Biol Chem 2014; betologia 2010;53:1722–1726 289:28827–28834 90. Persa C, Osmotherly K, Chao-Wei Chen K, Moon S, Lou MF. The distri- 111. Nigwekar SU, Brunelli SM, Meade D, Wang W, Hymes J, Lacson E Jr. bution of cystathionine beta-synthase (CBS) in the eye: implication of the pres- Sodium thiosulfate therapy for calcific uremic arteriolopathy. Clin J Am Soc ence of a trans-sulfuration pathway for oxidative stress defense. Exp Eye Res Nephrol 2013;8:1162–1170 2006;83:817–823 112. di Villa Bianca Rd, Cirino G, Sorrentino R. Hydrogen sulfide and urogenital 91. Ganapathy PS, Moister B, Roon P, et al. Endogenous elevation of homo- tract. Handbook Exp Pharmacol 2015;230:111–136 cysteine induces retinal neuron death in the cystathionine-beta-synthase mutant 113. Tao BB, Liu SY, Zhang CC, et al. VEGFR2 functions as an H2S-targeting mouse. Invest Ophthalmol Vis Sci 2009;50:4460–4470 receptor protein kinase with its novel Cys1045-Cys1024 disulfide bond serving 92. Biermann J, Lagrèze WA, Schallner N, Schwer CI, Goebel U. Inhalative as a specific molecular switch for hydrogen sulfide actions in vascular endothelial preconditioning with hydrogen sulfide attenuated apoptosis after retinal ischemia/ cells. Antioxid Redox Signal 2013;19:448–464 reperfusion injury. Mol Vis 2011;17:1275–1286 114. Calver A, Collier J, Vallance P. Inhibition and stimulation of nitric oxide 93. Si YF, Wang J, Guan J, Zhou L, Sheng Y, Zhao J. Treatment with hydrogen synthesis in the human forearm arterial bed of patients with insulin-dependent sulfide alleviates streptozotocin-induced diabetic retinopathy in rats. Br J Phar- diabetes. J Clin Invest 1992;90:2548–2554 macol 2013;169:619–631 115. Heitzer T, Krohn K, Albers S, Meinertz T. Tetrahydrobiopterin improves 94. Ran R, Du L, Zhang X, et al. Elevated hydrogen sulfide levels in vitreous endothelium-dependent vasodilation by increasing nitric oxide activity in patients body and plasma in patients with proliferative diabetic retinopathy. Retina 2014; with type II diabetes mellitus. Diabetologia 2000;43:1435–1438 34:2003–2009 116. Settergren M, Böhm F, Malmström RE, Channon KM, Pernow J. L-arginine 95. Kawabata A, Ishiki T, Nagasawa K, et al. Hydrogen sulfide as a novel and tetrahydrobiopterin protects against ischemia/reperfusion-induced endothelial nociceptive messenger. Pain 2007;132:74–81 dysfunction in patients with type 2 diabetes mellitus and coronary artery disease. 96. Okubo K, Takahashi T, Sekiguchi F, et al. Inhibition of T-type calcium Atherosclerosis 2009;204:73–78 channels and hydrogen sulfide-forming enzyme reverses paclitaxel-evoked 117. Mohan S, Reddick RL, Musi N, et al. Diabetic eNOS knockout mice neuropathic hyperalgesia in rats. Neuroscience 2011;188:148–156 develop distinct macro- and microvascular complications. Lab Invest 2008; 97. Qu R, Tao J, Wang Y, et al. Neonatal colonic inflammation sensitizes 88:515–528 voltage-gated Na(+) channels via upregulation of cystathionine b-synthetase 118. Mees B, Récalde A, Loinard C, et al. Endothelial nitric oxide synthase expression in rat primary sensory neurons. Am J Physiol Gastrointest Liver overexpression restores the efficiency of bone marrow mononuclear cell-based Physiol 2013;304:G763–G772 therapy. Am J Pathol 2011;178:55–60 98. Macpherson LJ, Dubin AE, Evans MJ, et al. Noxious compounds activate 119. Liu X, Wei J, Peng DH, Layne MD, Yet SF. Absence of heme oxygenase-1 TRPA1 ion channels through covalent modification of cysteines. Nature 2007; exacerbates myocardial ischemia/reperfusion injury in diabetic mice. Diabetes 445:541–545 2005;54:778–784 99. Trevisani M, Patacchini R, Nicoletti P, et al. Hydrogen sulfide causes va- 120. Di Pascoli M, Rodella L, Sacerdoti D, Bolognesi M, Turkseven S, Abraham nilloid receptor 1-mediated neurogenic inflammation in the airways. Br J Phar- NG. Chronic CO levels have [corrected] a beneficial effect on vascular relaxation macol 2005;145:1123–1131 in diabetes. Biochem Biophys Res Commun 2006;340:935–943 100. Velasco-Xolalpa ME, Barragán-Iglesias P, Roa-Coria JE, et al. Role of 121. Rodella L, Lamon BD, Rezzani R, et al. Carbon monoxide and biliverdin hydrogen sulfide in the pain processing of non-diabetic and diabetic rats. Neu- prevent endothelial cell sloughing in rats with type I diabetes. Free Radic Biol Med roscience 2013;250:786–797 2006;40:2198–2205 101. Takahashi T, Aoki Y, Okubo K, et al. Upregulation of Ca(v)3.2 T-type 122. Rodella LF, Vanella L, Peterson SJ, et al. Heme oxygenase-derived carbon calcium channels targeted by endogenous hydrogen sulfide contributes to monoxide restores vascular function in type 1 diabetes. Drug Metab Lett 2008;2: maintenance of neuropathic pain. Pain 2010;150:183–191 290–300 102. Messinger RB, Naik AK, Jagodic MM, et al. In vivo silencing of the Ca(V)3.2 123. Ananthakrishnan R, Li Q, O’Shea KM, et al. Carbon monoxide form of T-type calcium channels in sensory neurons alleviates hyperalgesia in rats with PEGylated hemoglobin protects myocardium against ischemia/reperfusion streptozocin-induced diabetic neuropathy. Pain 2009;145:184–195 injury in diabetic and normal mice. Artif Cells Nanomed Biotechnol 2013;41: 103. Distrutti E, Sediari L, Mencarelli A, et al. Evidence that hydrogen sulfide 428–436 exerts antinociceptive effects in the gastrointestinal tract by activating KATP 124. Wang Y, Ying L, Chen YY, et al. Induction of heme oxygenase-1 amelio- channels. J Pharmacol Exp Ther 2006;316:325–335 rates vascular dysfunction in streptozotocin-induced type 2 diabetic rats. Vascul 104. Kida K, Marutani E, Nguyen RK, Ichinose F. Inhaled hydrogen sulfide prevents Pharmacol 2014;61:16–24 neuropathic pain after peripheral nerve injury in mice. Nitric Oxide 2015;46:87–92 125. Streeter EY, Badoer E, Woodman OL, Hart JL. Effect of type 1 diabetes on 105. Kimura H. Physiological role of hydrogen sulfide and polysulfide in the the production and vasoactivity of hydrogen sulfide in rat middle cerebral arteries. central nervous system. Neurochem Int 2013;63:492–497 Physiol Rep 2013;1:e00111 106. Li H, Feng SJ, Zhang GZ, Wang SX. Correlation of lower concentrations of 126. Denizalti M, Bozkurt TE, Akpulat U, Sahin-Erdemli I, Abacıoglu N. The hydrogen sulfide with atherosclerosis in chronic hemodialysis patients with di- vasorelaxant effect of hydrogen sulfide is enhanced in streptozotocin-induced abetic nephropathy. Blood Purif 2014;38:188–194 diabetic rats. Naunyn Schmiedebergs Arch Pharmacol 2011;383:509–517 107. Andrésdóttir G, Bakker SJ, Hansen HP, Parving HH, Rossing P. Urinary 127. Suzuki K, Olah G, Modis K, et al. Hydrogen sulfide replacement therapy sulphate excretion and progression of diabetic nephropathy in type 1 diabetes. protects the vascular endothelium in hyperglycemia by preserving mitochondrial Diabet Med 2013;30:563–566 function. Proc Natl Acad Sci U S A 2011;108:13829–13834 108. Ahmad FU, Sattar MA, Rathore HA, et al. Exogenous hydrogen sulfide 128. Zhong X, Wang L, Wang Y, et al. Exogenous hydrogen sulfide attenuates (H2S) reduces blood pressure and prevents the progression of diabetic ne- diabetic myocardial injury through cardiac mitochondrial protection. Mol Cell phropathy in spontaneously hypertensive rats. Ren Fail 2012;34:203–210 Biochem 2012;371:187–198 diabetes.diabetesjournals.org van den Born and Associates 345

129. Gao Y, Yao X, Zhang Y, et al. The protective role of hydrogen sulfide in 146. Marks GS, Vreman HJ, McLaughlin BE, Brien JF, Nakatsu K. Measurement myocardial ischemia-reperfusion-induced injury in diabetic rats. Int J Cardiol of endogenous carbon monoxide formation in biological systems. Antioxid Redox 2011;152:177–183 Signal 2002;4:271–277 130. Peake BF, Nicholson CK, Lambert JP, et al. Hydrogen sulfide preconditions 147. van den Berg E, Pasch A, Westendorp WH, et al. Urinary sulfur metabolites the db/db diabetic mouse heart against ischemia-reperfusion injury by activating associate with a favorable cardiovascular risk profile and survival benefit in renal Nrf2 signaling in an Erk-dependent manner. Am J Physiol Heart Circ Physiol transplant recipients. J Am Soc Nephrol 2014;25:1303–1312 2013;304:H1215–H1224 148. Nagy P, Pálinkás Z, Nagy A, Budai B, Tóth I, Vasas A. Chemical aspects of 131. Lambert JP, Nicholson CK, Amin H, Amin S, Calvert JW. Hydrogen sulfide hydrogen sulfide measurements in physiological samples. Biochim Biophys Acta provides cardioprotection against myocardial/ischemia reperfusion injury in the 2014;1840:876–891 diabetic state through the activation of the RISK pathway. Med Gas Res 2014;4:20 149. Feelisch M, Noack E, Schröder H. Explanation of the discrepancy between 132. Ríos-González BB, Román-Morales EM, Pietri R, López-Garriga J. Hydro- the degree of organic nitrate decomposition, nitrite formation and guanylate gen sulfide activation in hemeproteins: the sulfheme scenario. J Inorg Biochem cyclase stimulation. Eur Heart J 1988;9(Suppl. A):57–62 2014;133:78–86 150. Hobbs DA, Kaffa N, George TW, Methven L, Lovegrove JA. Blood pressure- 133. Mustafa AK, Gadalla MM, Snyder SH. Signaling by gasotransmitters. Sci lowering effects of beetroot juice and novel beetroot-enriched bread products in Signal 2009;2:re2 normotensive male subjects. Br J Nutr 2012;108:2066–2074 134. Murphy ME, Brayden JE. Nitric oxide hyperpolarizes rabbit mesenteric 151. Grosser N, Erdmann K, Hemmerle A, et al. Rosuvastatin upregulates the arteries via ATP-sensitive potassium channels. J Physiol 1995;486:47–58 antioxidant defense protein heme oxygenase-1. Biochem Biophys Res Commun 135. Pereira de Ávila MA, Giusti-Paiva A, Giovani de Oliveira Nascimento C. The 2004;325:871–876 peripheral antinociceptive effect induced by the heme oxygenase/carbon mon- 152. Grosser N, Hemmerle A, Berndt G, et al. The antioxidant defense protein oxide pathway is associated with ATP-sensitive K+ channels. Eur J Pharmacol heme oxygenase 1 is a novel target for statins in endothelial cells. Free Radic Biol 2014;726:41–48 Med 2004;37:2064–2071 136. Zhao W, Zhang J, Lu Y, Wang R. The vasorelaxant effect of H(2)S as a 153. Grosser N, Abate A, Oberle S, et al. Heme oxygenase-1 induction may novel endogenous gaseous K(ATP) channel opener. EMBO J 2001;20:6008– explain the antioxidant profile of aspirin. Biochem Biophys Res Commun 2003; 6016 308:956–960 137. Dulak J, Józkowicz A. Regulation of vascular endothelial growth factor syn- 154. Bharucha AE, Choi KM, Saw JJ, et al. Effects of aspirin & simvastatin and thesis by nitric oxide: facts and controversies. Antioxid Redox Signal 2003;5:123–132 aspirin, simvastatin, & lipoic acid on heme oxygenase-1 in healthy human 138. Abraham NG, Kappas A. Heme oxygenase and the cardiovascular-renal subjects. Neurogastroenterol Motil 2014;26:1437–1442 system. Free Radic Biol Med 2005;39:1–25 155. Toombs CF, Insko MA, Wintner EA, et al. Detection of exhaled hydrogen 139. Polte T, Abate A, Dennery PA, Schröder H. Heme oxygenase-1 is a cGMP- sulphide gas in healthy human volunteers during intravenous administration of inducible endothelial protein and mediates the cytoprotective action of nitric sodium sulphide. Br J Clin Pharmacol 2010;69:626–636 oxide. Arterioscler Thromb Vasc Biol 2000;20:1209–1215 156. Mishanina TV, Libiad M, Banerjee R. Biogenesis of reactive sulfur species for 140. Kondo K, Bhushan S, King AL, et al. H₂S protects against pressure signaling by hydrogen sulfide oxidation pathways. Nat Chem Biol 2015;11:457–464 overload-induced heart failure via upregulation of endothelial nitric oxide syn- 157. Snijder PM, Frenay AR, de Boer RA, et al. Exogenous administration of thase. Circulation 2013;127:1116–1127 thiosulfate, a donor of hydrogen sulfide, attenuates angiotensin II-induced hy- 141. Zhao W, Wang R. H(2)S-induced vasorelaxation and underlying cellular and pertensive heart disease in rats. Br J Pharmacol 2015;172:1494–1504 molecular mechanisms. Am J Physiol Heart Circ Physiol 2002;283:H474–H480 158. Buikema H, Monnink SH, Tio RA, Crijns HJ, de Zeeuw D, van Gilst WH. 142. Eto K, Kimura H. A novel enhancing mechanism for hydrogen sulfide-pro- Comparison of zofenopril and lisinopril to study the role of the sulfhydryl-group in ducing activity of cystathionine beta-synthase. J Biol Chem 2002;277:42680–42685 improvement of endothelial dysfunction with ACE-inhibitors in experimental heart 143. Calvert JW, Jha S, Gundewar S, et al. Hydrogen sulfide mediates car- failure. Br J Pharmacol 2000;130:1999–2007 dioprotection through Nrf2 signaling. Circ Res 2009;105:365–374 159. Bucci M, Vellecco V, Cantalupo A, et al. Hydrogen sulfide accounts for the 144. Feelisch M, Rassaf T, Mnaimneh S, et al. Concomitant S-, N-, and heme- peripheral vascular effects of zofenopril independently of ACE inhibition. nitros(yl)ation in biological tissues and fluids: implications for the fate of NO in Cardiovasc Res 2014;102:138–147 vivo. FASEB J 2002;16:1775–1785 160. Shen X, Carlström M, Borniquel S, Jädert C, Kevil CG, Lundberg JO. Mi- 145. Hetrick EM, Schoenfisch MH. Analytical chemistry of nitric oxide. Annu Rev crobial regulation of host hydrogen sulfide bioavailability and metabolism. Free Anal Chem (Palo Alto, Calif) 2009;2:409–433 Radic Biol Med 2013;60:195–200