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 InternationalResearch Training Group 1874 Diabetic Microvascular Complications (DIAMICOM)
Gasotransmitters in VascularComplications of DiabetesDiabetes 2016;65:331–345 | DOI: 10.2337/db15-1003
In the past decades three gaseous signaling molecules—so-called gasotransmitters—have been identified: nitricoxide (NO), carbon monoxide (CO), and hydrogen sulfide(H2S). These gasotransmitters are endogenously pro-duced by different enzymes in various cell types andplay an important role in physiology and disease. Despitetheir specific functions, all gasotransmitters share thecapacity to reduce oxidative stress, induce angiogenesis,and promote vasorelaxation. In patients with diabetes, alower bioavailability of the different gasotransmitters isobserved when compared with healthy individuals. Asyet, it is unknown whether this reduction precedes orresults from diabetes. The increased risk for vasculardisease in patients with diabetes, in combination withthe extensive clinical, financial, and societal burden, callsfor action to either prevent or improve the treatment ofvascular complications. In this Perspective, we present aconcise overview of the current data on the bioavailabilityof gasotransmitters in diabetes and their potential role inthe development and progression of diabetes-associatedmicrovascular (retinopathy, neuropathy, and nephropa-thy) and macrovascular (cerebrovascular, coronary ar-tery, and peripheral arterial diseases) complications.Gasotransmitters appear to have both inhibitory andstimulatory effects in the course of vascular disease de-velopment. This Perspective concludes with a discussionon gasotransmitter-based interventions as a therapeuticoption.
DIABETES AND ITS COMPLICATIONS
Diabetes is characterized by hyperglycemia and insulinresistance or deficiency. Diabetes is a top 10 cause ofdeath worldwide; its prevalence is increasing and currentlyestimated to be 9% among adults (1,2). Both type 1 and
type 2 diabetes are important risk factors for vascular dis-eases, with a two- to fourfold increased risk when com-pared with individuals without diabetes (3). These vascularcomplications are divided into microvascular (retinopathy,neuropathy, and nephropathy) and macrovascular (cerebro-vascular, coronary artery, and peripheral arterial diseases)complications with respective clinical symptoms (Fig. 1).Although the pathophysiology of type 1 and type 2 diabetesis different, the proposed underlying mechanism leading tovascular complications seems to be similar and is thought tobe related to endothelial dysfunction (4,5) and the associ-ated formation of reactive oxygen species (ROS). Chronichyperglycemia promotes multiple biochemical pathways tooverproduce ROS, either through mitochondrial overpro-duction or through enzymatic responses to high glucose (6).
Diabetic retinopathy is the main cause of blindness inadults. The worldwide prevalence is approximately 35%in patients with diabetes (7). The essentials of diabeticretinopathy can be best characterized by the combina-tion of increased vessel permeability and progressivevascular occlusion. Although the clinical diagnosis ofretinopathy is still made by the changes in small (early)and larger (later) vessels, it has become clear that almostevery cell type in the retina can be subject to damage bycomplex metabolic changes, induced by chronic hyper-glycemia (8–11).
Polyneuropathy is defined as a diffuse and bilateraldisturbance of functions or pathological changes in multi-ple peripheral nerves. Diabetic peripheral polyneuropathyis very frequent in the course of diabetes and even inprediabetes, affecting up to 50% of all patients withdiabetes (12–14). However, although being frequent andsevere, it is inadequately treated in most patients—77%of those with chronic painful peripheral neuropathy report
1Department of Pathology and Medical Biology, Division of Pathology, UniversityMedical Center Groningen, University of Groningen, Groningen, the Netherlands25th Medical Department, Medical Faculty Mannheim, University of Heidelberg,Mannheim, Germany3Department of Neurophysiology, Centre for Biomedicine and Medical TechnologyMannheim, Medical Faculty Mannheim, University of Heidelberg, Mannheim,Germany
persistent pain over 5 years (15). Experimental studiessuggest the importance of neurovascular vasodilation indiabetic neuropathy (16); however, the mechanisms remainpoorly understood, which may explain the current lack ofadequate treatment in (diabetic) neuropathic pain.
Diabetic nephropathy (DN) is one of the leading causesof end-stage renal disease in the Western world, occurringin ;30% of patients with type 1 and type 2 diabetes andaccounting for about 40% of new cases of end-stage renaldisease based on U.S. data (17). At the structural level, theglomeruli are often affected as evidenced by basementmembrane thickening, mesangial lesions (Kimmelstiel-Wilson lesions), and nodular sclerosis. Clinically, DN isaccompanied by proteinuria and chronic renal failure. Inaddition, arterioles are often affected. The mechanismsleading to renal changes include the metabolic defect,nonenzymatic glycation of proteins, and hemodynamicchanges, such as hypertension leading to glomerular hy-pertrophy (18).
Macrovascular complications are characterized by thedevelopment of atherosclerosis in arteries throughout thebody. Atherosclerosis results from a proinflammatorystate starting with endothelial dysfunction and culminat-ing in the narrowing of the arterial lumen as a result of
atherosclerotic plaque formation. As opposed to stableplaques, vulnerable, nonstable plaques are prone torupture, causing downstream ischemic events such astransient ischemic attack and stroke (Fig. 1).
GASOTRANSMITTERS IN MICROVASCULARDISEASE
Nitric Oxide and Microvascular Complications ofDiabetesNitric oxide (NO) was first recognized as an endothelium-derived relaxing factor (19). It is endogenously formedfrom its substrate L-arginine by three different nitric ox-ide synthase (NOS) enzymes. Endothelial NOS (eNOS) ispredominantly associated with vascular tone. InducibleNOS (iNOS), although also present in the vascular system,is mainly active in the immune system under conditionsof oxidative stress. It functions as a promoter of inflam-mation. Neuronal NOS (nNOS), present in neurons andskeletal muscle cells, is important for neuronal cell-cellinteractions (20). NO acts as a vasodilator and inhibitsplatelet aggregation and stabilizes atherosclerotic plaques(21). In humans, NO-dependent vasodilatation is im-paired in patients with type 2 diabetes, and lower eNOSexpression and reduced NO production are the suggested
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, transientischemic attack.
332 Gasotransmitters in Diabetic Angiopathy Diabetes Volume 65, February 2016
underlying cause (22,23). Blockade of NOS causes insulinresistance in a rat model, indicating that in this modelloss of NO synthesis precedes type 2 diabetes (24). Indifferent animal models for diabetes, lower bioavailabilityof NO is observed. Reduced NO production was found inspontaneous type 1 diabetic BioBreeding rats (25) as wellas streptozotocin (STZ)-induced type 1 diabetes in maleSprague-Dawley rats (26). In mouse models of diet-in-duced obesity and type 2 diabetes, NO bioavailability isreduced, leading to endothelial dysfunction and impairedNO-mediated vasodilatation (27,28). In contrast to theseprotective effects of NO, iNOS-produced NO seems toplay an important role in inducing nitrosative stressand inflammation, also in the course of diabetes. Thus,NO seems to play a dual role in the development andprogression of diabetes as well as in the development ofvascular dysfunction (29).
Effects of NO depletion and supplementation on thedevelopment of microvascular complications have beenprimarily studied in experimental models as summarizedin Table 1 and discussed below.
RetinopathyIn the retina, iNOS is sensitive to hyperglycemia andresponsible for overproduction of NO (30,31). The result-ing surplus of NO is either quenched by advanced glyca-tion end products (AGEs) or leads, through the reactionwith superoxide, to the formation of peroxynitrite withsubsequent nitrosylation of proteins, lipids, and DNA. NOproduction is important in inflammatory signaling, andinflammation is thought to be important in incipient di-abetic retinopathy (32). Increased reactive nitrogen spe-cies (RNS) has been observed in diabetic rat retinae and invitro, and these changes were corrigible by aminoguani-dine, an inhibitor of NO synthases (33). As an inhibitor of
AGEs, aminoguanidine reduces vascular cell damage inseveral animal models (33,34). Zheng et al. (30) foundthat nitrosative stress was reduced in the retinae ofiNOS2/2 mice, together with an inhibition of vasoregres-sion and retinal thinning. However, the essential role ofiNOS for the development of diabetic retinopathy seemsnot to be the case for other NOS isoforms, as deletion ofeNOS exacerbates diabetic retinopathy (35). In STZ-in-duced type 1 diabetes, eNOS2/2 mice developed moresevere retinopathy compared with wild-type diabetic con-trol mice. The worsened phenotype in these eNOS2/2
mice was accompanied by increased iNOS expression, fur-ther suggesting an important role for iNOS in the devel-opment of diabetic retinopathy. However, eNOS2/2 micesuffer from higher blood pressure, so the worsened retinalphenotype can partly be explained by hypertensive injury.In essence, NO appears to have a dual role (i.e., protectiveand noxious effects) in the diabetic retina, as schemati-cally shown in Fig. 2A.
NeuropathyUntil now, NO was the best-characterized gasotransmittercontributing to nociception and pain. Its downstreamtargets within the peripheral nervous system (PNS)include cyclic guanosine monophosphate (cGMP) pro-duction by activation of soluble guanylyl cyclase (sGC)and phosphorylation of membrane receptors and chan-nels by cGMP-dependent protein kinases (36)—mecha-nisms usually associated with increased nociception.Consistently, different members of the large family oftransient receptor potential channels, several of whichare known as nociceptive sensor molecules such asTRPV1 and TRPA1, are activated by NO via cysteine S-nitrosylation (37). In contrast, several mechanisms wereidentified that may induce antinociception and analgesia
Table 1—Effect of NO in diabetic microvascular disease
diabetes.diabetesjournals.org van den Born and Associates 333
and increase efficacy of analgesic compounds. In the cen-tral nervous system, NO interacts with the descendinginhibitory control mechanisms of nociception (38). In pa-tients with type 2 diabetes suffering from painful diabeticneuropathy, treatment with the NO donors glyceryl trini-trate (39) and isosorbide dinitrate (40) significantly im-proved pain symptoms, indicative of the beneficial actionof NO in diabetic neuropathy. Similar effects were ob-served when locally applying a NO-releasing cutaneouspatch (41). These effects may, however, also be indirectlyexplained by variations in local microcirculation: transientchanges in sciatic nerve microcirculation were observed inresponse to NO in animals with STZ-induced diabetesdeveloping diabetic neuropathy (42). In STZ-induced di-abetic rats, NOS activity is increased in primary sensoryneurons (43). The potent oxidant peroxynitrite, a productof a superoxide anion radical reaction with NO, was
suggested to play a role in the induction of peripheraldiabetic neuropathy and neuropathic pain via inductionof RNS (44), including protein nitrosylation, lipid perox-idation, DNA damage, and cell death (29). Hyperglycemiaactivates iNOS and therefore generally increased nitrosa-tive stress in the PNS (44,45). Absence of iNOS reducednitrosative stress in peripheral nerve fibers displayingnormal nerve conduction velocities; diabetic neuropathywas also less severe in diabetic iNOS2/2 mice than indiabetic wild-type mice. Thus, diabetic neuropathy de-pends on nitrosative stress induced in axons and Schwanncells by NO produced from iNOS. In contrast, nNOS isrequired for maintaining PNS function and nerve fiberdensity and contributes to a lesser extent to the develop-ment of diabetic polyneuropathy (45). In summary, NOmay play pivotal direct and indirect roles in the progres-sion of diabetic neuropathy, presumably by impairing
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, gasotransmittersare 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 oftransmitters, synaptic plasticity).
334 Gasotransmitters in Diabetic Angiopathy Diabetes Volume 65, February 2016
microcirculation in PNS at pathophysiological levels andcontributing to oxidative stress and inflammation andtissue injury (29), as schematically shown in Fig. 2B.
NephropathyThere is still controversy regarding whether the genera-tion of NO is enhanced or decreased in DN. In the earlystages of DN, Chiarelli et al. (46) found significantlyhigher concentrations of NO end products (nitrite/nitrate)in the serum of DN patients with microalbuminuriacompared with the serum of healthy individuals. How-ever, an association as such does not imply causality perse. This excess of NO can indicate an upregulated inflam-matory response by iNOS or a (protective) compensatoryresponse against renal injury, mediated by eNOS. In ex-perimental STZ-induced type 1 diabetes, renal NO pro-duction is decreased in the early phase of the disease (47).Deficiency of eNOS results in accelerated nephropathy indiabetic mice (48,49), also supporting a protective role forNO in DN (50). Supplementation of tetrahydrobiopterin(BH4), a cofactor of NOS, reduced proteinuria and renaldamage in type 2 diabetic rats (51). Taken together, NOproduction is clearly modulated in DN, and decrements inits expression point to a contributing role for this gaso-transmitter in DN. Scavenging of ROS positively influ-ences the redox status and may mechanistically underliethese findings. The modes of action of NO in the devel-opment of DN are schematically shown in Fig. 2C.
SummaryAs discussed above, NO demonstrates both protective anddamaging properties in the development of microvasculardisease. The producing enzyme seems to play a major rolein the contrasting actions of NO: eNOS- and nNOS-derivedNO exerts the vast majority of their positive effects viaupregulation of the production of cGMP by activation ofsGC. However, iNOS-produced NO is involved in inflam-matory signaling and is an important contributor to thedevelopment of diabetic angiopathy. In addition, the pres-ence of ROS is important in the actions of NO. An excess ofNO in the presence of abundant ROS (superoxide) pro-duction leads to the formation of peroxynitrite withsubsequent nitrosylation of proteins, lipids, and DNA.There is also increasing evidence for harmful effects ofNO in protein tyrosine nitration (52). Protein nitration is aposttranslational modification that takes place in the com-bined presence of oxidative stress and NO, which is the casein disease conditions such as diabetes (53). Given the dataavailable, we conclude that NO plays a dual role in theprogression and maintenance of diabetic microvascularcomplications, which is mostly driven by the expressionof its producing enzymes (NOS) and the presence of ROS.
Carbon Monoxide and Microvascular Complications ofDiabetesCarbon monoxide (CO), the second gasotransmitter, isproduced by the different heme oxygenase (HO) enzymesas a product of heme metabolism (54). Heme is converted
to biliverdin, iron, and CO by HO. Three different iso-forms of HO exist; the inducible form, HO-1, and theconstitutive isoforms, HO-2 and HO-3. HO-1 and HO-2are physiologically active, whereas the role of HO-3 in hu-man physiology remains unclear (55). CO has numerousphysiological functions, including vasodilation and inhibi-tion of platelet aggregation. In skeletal muscle biopsies andcirculating leukocytes from patients with type 2 diabetes,mRNA expression of HO-1 was dramatically decreasedcompared with age-matched control subjects without dia-betes (56,57). In STZ-induced type 1 diabetic rats, a de-creased vasorelaxant function of CO was demonstrated,despite higher HO-1 expression levels (58). In Zucker di-abetic fatty (ZDF) rats, CO production was decreased inaortic tissue compared with that in nondiabetic controls.Increasing HO-1 activity with cobalt protoporphyrinresulted in higher levels of CO, lower glucose levels, andincreased insulin sensitivity (59). These data are in favor ofreduced vascular risk in the presence of higher CO levels,which might be mediated via effects on insulin sensitivity(60). Taken together, reduced bioavailability of CO in thediabetic state is accompanied by insulin resistance and areduction of endothelial health, indicating a potential rolefor the HO-1/CO pathway in the development of diabetesand its associated complications. The effects of CO deple-tion and supplementation in diabetic mice and rats on thedevelopment of microvascular complications are summa-rized in Table 2 and will be discussed below.
RetinopathyOxidative stress in the diabetic retina promotes theactivation of HOs (61). In the diabetic retina, HO-1 ispredominantly found in glial cells, in particular in Müllercells, and to some extent in the microvasculature (62). Invitro, HO-1 overexpression protects retinal endothelialcells from high glucose and oxidative/nitrosative stress(63). In a STZ-induced type 1 diabetes model in rats, HO-1upregulation by hemin resulted in protection against thedevelopment of diabetic retinopathy (64). This protectionis reflected by the downregulation of p53, vascular endothe-lial growth factor (VEGF), and HIF-1a and a reduction ofdiabetes-induced apoptosis in retinal ganglion cells (RGCs).On the contrary, HO-1–derived CO is proangiogenic (65),and angiogenesis, causing increased retinal blood flow, is akey factor in the development of diabetic retinopathy (66).This implies that the proangiogenic effects of CO may actu-ally aggravate diabetic retinopathy. The effects of CO in thediabetic retina are schematically shown in Fig. 2A.
NeuropathyIn the case of diabetic neuropathy, CO acts as a pain-modulating second messenger within the nervous system(67). The activation of HO/CO signaling reduced symp-toms of neuropathic pain, presumably by the activation ofanti-inflammatory and antioxidant mechanisms (68). COexerts antinociceptive effects and increases the anti-allodynic and antihyperalgesic efficacy of morphine in
diabetes.diabetesjournals.org van den Born and Associates 335
chronic inflammation and neuropathic pain (69)—the lat-ter strictly dependant on NO produced by nNOS and iNOS.Furthermore, CO relieves neuropathic pain symptoms byreducing the expression of iNOS and nNOS as well as byreducing the activation of spinal microglia (70). Interestingly,the constitutive isoform HO-2 is coexpressed with NOS inthe PNS and central nervous system (67), and CO, similar toNO, is also capable of activating the proalgesic cGMP proteinkinase pathway (70). In fact, there is a close interaction be-tween the CO and NO systems in the course of neuropathicpain, suggesting that they might act as cotransmitters inneuronal signaling transmission (67). In nociception, themore stable CO may set basal activity by tonic backgroundstimulation and NO may transiently amplify nociceptivesignaling. Substances increasing endogenous CO (e.g., CO-releasing molecules [CORMs] or HO inducers alone or incombination with analgesics) may be useful for the treat-ment of (diabetic) neuropathic pain. The effects of CO indiabetic neuropathy are depicted in Fig. 2B.
NephropathyIn the kidney, HO-1 and HO-2 are important in cytopro-tection and serve as physiologic regulators of heme-dependent protein synthesis during which CO is produced.Inducers of the HO pathway (like hemin) are protectiveagainst renal inflammation and ameliorate DN in type 2diabetic ZDF rats and STZ-induced type 1 diabetic rats (71–73). The antioxidant effect of HO-1 is believed to play a rolein renal protection in diabetic rats (74). The opposite, de-ficiency of HO-2, results in higher superoxide anion levelsand increased renal dysfunction after STZ-induced diabetes(75). Enhanced production of CO seems to be beneficial forthe kidney in DN, suggesting possibilities for therapeuticintervention. The effects of CO in DN are shown in Fig. 2C.
SummaryBesides the beneficial effects of CO, high CO concentra-tions are toxic because of the high affinity of CO to bind
heme proteins such as hemoglobin. Due to the high levelsof CO bound to hemoglobin (forming carboxyhemoglobin),oxygen is not able to bind to that particular hemoglobinmolecule, and disrupted oxygen transport develops. However,endogenous production of CO by HO enzymes obviouslydoes not result in toxic levels. In contrast to NO, the exactworking mechanism and molecular targets of CO are mostlyunknown. Nevertheless, one of the known pathways is thatCO is able to increase cGMP production by activation of sGC,albeit with lower affinity than NO. Moreover, CO is able tobind to complex IV (cytochrome c oxidase) of the mitochon-drial electron transport chain and thereby regulate ROSproduction. In summary, CO is mainly protective in diabeticvascular disease via inhibition of ROS formation, via in-teraction with NO, and via the sGC/cGMP pathway.
Hydrogen Sulfide and Microvascular Complicationsof DiabetesHydrogen sulfide (H2S) is the third gasotransmitter and wasrecognized as such in the 1990s (76,77). It is endogenouslyproduced by three different enzymes. The pyridoxal-59-phosphate (PLP)–dependent enzymes cystathionine b-synthase(CBS) and cystathionine g-lyase (CSE) are the two majorH2S-producing enzymes. The third H2S-producing enzymeis 3-mercaptopyruvate sulfurtransferase (3MST). The mainsubstrates for H2S production are homocysteine and cyste-ine. 3MST produces H2S from 3-mercaptopyruvate, which isproduced by the enzymes cysteine aminotransferase andD-amino acid oxidase from L-cysteine and D-cysteine, respec-tively (78). H2S is a physiologically active compound and iscalled endothelium-derived hyperpolarizing factor (79,80); itcauses vasodilatation but also acts as scavenger of ROS andstimulating angiogenesis (81). Renal CSE and CBS expressionand H2S production are markedly lowered in spontaneousdiabetic Ins2Akita mice (82). In nonobese diabetic mice, an-other mouse model of type 1 diabetes, it was also shownthat diabetic mice had lower H2S levels compared with
Table 2—Effect of CO in diabetic microvascular disease
Model Intervention ↑ / ↓ Outcome References
Retinopathy Rat: STZ-induceddiabetes
HO inhibitor SnPP ↓ Prevented diabetes-induced vascular injury 61Hemin ↑ Maintained RGCs and reduced ROS in retina 64
Neuropathy Mouse CORM-2, CORM-3,HO-inducer CoPP
↑ Reduced neuropathic pain 70
Rat Hemin, CORM-2 ↑ Reduced neuropathic pain, inflammation,and ROS/RNS
68
Nephropathy Mouse: STZ-induceddiabetes
HO-22/2 ↓ Enhanced renal injury and loss of renal function 75HO inducer CoPP ↑ Reduced glomerular injury and renal
insufficiency75
Rat: STZ-induceddiabetes
HO inducers hemin, CoPP ↑ Improved renal injury, inflammation, ROS, andrenal function
72–74
HO inhibitors SnMP, CrMP ↓ Enhanced renal injury and renal function andcounteracted the protective effects of hemin
72,73
Rat: ZDF Hemin ↑ Improved renal injury, inflammation, andrenal function
71
HO inhibitor SnMP ↓ Enhanced renal injury and renal insufficiency 71
↑ indicates increased CO; ↓ indicates reduced CO.
336 Gasotransmitters in Diabetic Angiopathy Diabetes Volume 65, February 2016
nondiabetic mice (83). In STZ-induced type 1 diabetic rats,H2S levels were lower compared with age-matched nondia-betic rats (84). However, CSE deficiency delayed the onset ofSTZ-induced type 1 diabetes, and diabetes was accompaniedby increased pancreatic H2S production without changes inpancreatic CSE of CBS protein expression (85). Anotherplayer in the development of vascular complications is in-sulin resistance. Insulin resistance is affected by H2S in amouse model with high-fat diet–induced obesity. Interest-ingly, both the inhibition of H2S production by DL-propar-gylglycine (PPG) and the treatment with slow-release H2Sdonor GYY4137 improved insulin resistance in these mice(86). This unexpected beneficial effect of PPG could beexplained by an upregulation of HO-1 resulting in higherCO levels, an effect of PPG that was recently described(87). In addition, this contradiction could be explained bythe fact that PPG is an unspecific CSE inhibitor (based onits cofactor PLP), thereby potentially inhibiting other PLP-dependent enzymes as well (88). In humans, diabetes isassociated with lower levels of H2S. In a small group ofpatients with type 2 diabetes, plasma H2S levels were re-duced by 73% compared with those in healthy (lean) indi-viduals (89). Interestingly, obesity is correlated with lowerlevels of H2S compared with those in lean volunteers. Col-lectively, human and experimental diabetes are associatedwith reduced H2S bioavailability, which might be related toincreased cardiovascular risk as observed in subjects withdiabetes. The effects of H2S depletion and supplementationin diabetic mice and rats on the development of microvas-cular complications are summarized in Table 3 and will bediscussed below.
RetinopathyH2S has recently received attention in research on diabeticretinopathy as some H2S-related changes are compatiblewith a significant role of H2S in the development and prop-agation of diabetic retinopathy. Reduced H2S-mediated cellprotection supposedly plays a role in retinal diseases as CBSexpression is found in various eye compartments, includingthe retina, suggesting that the trans-sulfuration pathway ispresent in the eye (90). Many CBS deficiency–related eyedisorders are associated with increased homocysteine
levels, and the retinae of CBS+/2 mice are characterizedby RGC loss, which is mediated by mitochondrial dysfunc-tion (91). It is thus conceivable that H2S is neuroprotectiveand there is indeed experimental proof for protective prop-erties of H2S in the retina, as evidenced by a decreased RGCloss in H2S-pretreated animals after retinal ischemia/reper-fusion (I/R) (92). Si et al. (93) investigated the effect of H2Sin experimental retinopathy of STZ-induced type 1 diabeticrats. They reported beneficial effects on neuronal dysfunction(based on electroretinography) and retinal structure (i.e., in-hibition of diabetes-induced retinal thickening and extracel-lular matrix proteins), while others clearly showed a link toimproved endothelial function, such as tightened blood-retinal barrier and reduced vasoregression. H2S is a knownproangiogenic signaling molecule and can thereby also con-tribute to enhanced angiogenesis in the diabetic retina. Inline with this, increased levels of H2S were observed in vit-reous body of patients with proliferative diabetic retinopathycompared with patients with rhegmatogenous retinal de-tachment (94). The effects of H2S in the diabetic retinaare schematically shown in Fig. 2A.
NeuropathyH2S has mainly been reported to increase pain sensitivityvia several proposed modes of action (95). These includesensitization of voltage-gated sodium and calcium chan-nels (95–97) and/or suppression of potassium channels.Furthermore, the pronociceptive transient receptor po-tential channels TRPV1 and TRPA1 (98,99), as well asNMDA receptors, were suggested to be sensitized byH2S. H2S displayed pronociceptive actions in inflammatorypain, both in STZ-induced type 1 diabetes and nondia-betic control animals. Interestingly, when treated withantagonists of the H2S-producing enzymes, pain reduc-tion was much more pronounced in diabetic animalsthan it was in nondiabetic animals, indicative of an in-creased H2S sensitivity of the nociceptive system in ratssuffering from diabetes (100). Conversely, reduction of H2Sreduced the tactile allodynia developed in course of diabe-tes. The T-type voltage-gated calcium channel CaV3.2 issensitized by H2S, leading to increased pain sensitivity(101). An increased H2S tissue content and hyperactivity
Table 3—Effect of H2S in diabetic microvascular disease
diabetes.diabetesjournals.org van den Born and Associates 337
of CaV3.2 were observed in chemotherapy-induced neuro-pathic hyperalgesia and pain that could be reversed byblocking H2S production (96). Painful peripheral diabeticneuropathy is accompanied by an enhancement of CaV3.2T-type calcium channels and neuronal excitability. Thus,CaV3.2 T-channels are thought to represent signal ampli-fiers in peripheral sensory neurons, contributing to hyper-excitability that ultimately leads to the development ofpain in diabetic neuropathy (102). Conversely, antinocicep-tive effects also have been reported for H2S. These effectswere antagonized by the ATP-sensitive potassium (KATP)channel blocker glibenclamide and by NOS inhibition(103). Inhalation of H2S reduced the development of neu-ropathic pain by reducing the resulting increase in inter-leukin-6 and chemokines, which was attributed to aninhibition of microglia activation in course of neuropathy(104). Furthermore, H2S functions as a neuroprotectiveagent by enhancing the production of glutathione, a majorintracellular antioxidant that scavenges mitochondrial ROS(105). Collectively, these data indicate that H2S displays bothpro- and antinociceptive actions in diabetic neuropathy. Theeffects of H2S in diabetic neuropathy are depicted in Fig. 2B.
NephropathyIn DN patients with atherosclerosis who are on dialysis,lower plasma levels of H2S were measured, which couldindicate a loss of the supposed protective effects of H2Sin these patients (106). This might be caused by endo-thelial damage or downregulation via other pathways ofthe enzymes producing H2S. Also, high urinary sulfate,as a proxy for H2S, is significantly associated with aslower decline in glomerular filtration rate in patientswith type 1 diabetes and DN (107). In the experimentalsetting, exogenous H2S reduces blood pressure and pre-vents the progression of DN in spontaneously hyper-tensive rats (108). Renal protection via blood pressurereduction is also shown in angiotensin II–induced hy-pertension and proteinuria in rats (109). Other studiesalso suggest that H2S is a key modulator in renalremodeling and that its actions can be affected by thematrix metalloproteinase 9, which is shown to modu-late CBS and CSE (82). In STZ-induced type 1 diabeticmice, the administration of H2S attenuated oxidativestress and inflammation, reduced mesangial cell prolif-eration, and inhibited the renin-angiotensin-aldosteronesystem (110). These data indicate that in DN H2S haspredominantly beneficial effects and is therefore apromising target for intervention. Thiosulfate might bethe perfect H2S donor as it is already in use in the clinicfor patients with calcifylaxis with end-stage renal disease(111) and has been shown to be beneficial in hypertensiverenal disease in rats (109). The effects of H2S on the kidneyin DN are schematically shown in Fig. 2C.
SummaryKnowledge on the working mechanisms of H2S is con-tinuously increasing. H2S-regulated vasodilatation acts
partly via activation of KATP channels, and a rathernew hypothesis is the interference of H2S with thecGMP pathway by inhibition of phosphodiesterase type5 activity, a mode of action comparable with “naturalsildenafil” (112). ROS production is decreased by H2Sthrough direct interference with the mitochondrial res-piration chain. It binds to cytochrome c oxidase, therebydirectly inhibiting the formation of ROS. Another impor-tant effect of H2S in terms of diabetic angiopathy isangiogenesis. The VEGF receptor 2 is the natural targetfor H2S to achieve its proangiogenic effect (113). In di-abetic retinopathy, the development of new vessels re-flects the severity. However, increased angiogenesismight also have some protective effects, e.g., angiogen-esis of vaso nervorum in diabetic neuropathy. The effectsof H2S on different ion channels are mainly important indiabetic neuropathy. Its interference with, for instance,TRPV1, TRPA1, and CaV3.2 channels contributes to in-creased nociception. Taken together, H2S exerts dual ef-fects in diabetic angiopathy, positive effects via itsvasodilatory actions, and unwanted detrimental effectsvia different ion channels and angiogenesis.
GASOTRANSMITTERS IN MACROVASCULARDISEASE
Gasotransmitters have been studied in diabetes-associatedmacrovascular disease and therapeutically used in clinicalpractice (NO only, CO and H2S have not yet been used). Theeffects of gasotransmitters depletion and supplementationin human and experimental diabetes on the developmentof endothelial function and macrovascular disease are sum-marized in Table 4 and briefly discussed below. In 1992,NO donor sodium nitroprusside (SNP) was used to mea-sure NO-dependent vasorelaxation in patients with type 1diabetes. SNP-stimulated vasodilatation was decreased inpatients without diabetes compared with subjects with di-abetes, indicating a lower NO sensitivity (114). In patientswith type 2 diabetes, the addition of NOS cofactor BH4
resulted in improved forearm blood flow, an effect thatwas nullified by NOS-inhibitor NG-monomethyl-L-arginine(L-NMMA) (115). In patients with type 2 diabetes andcoronary artery disease, treatment with NO substrateL-arginine and NOS cofactor BH4 protected against I/R en-dothelial dysfunction in the forearm vasculature (116). Theimportant role of NO in the macrovasculature is alsoshown in animal models of diabetes. Leprdb/db eNOS2/2
double knockout mice showed an aggravated vascular phe-notype compared with diabetic Leprdb/db or eNOS2/2singleknockouts, as evidenced by an increased aortic wallthickness and reduced re-endothelialization after arterialinjury (117). In ApoE2/2 mice and mice with STZ-induced type 1 diabetes, treatment with bone marrow–derived mononuclear cells overexpressing eNOS resultedin reduced plaque progression and improved postische-mic neovascularization, an effect that was completelyinhibited by NOS inhibitor L-NG-nitro-L-arginine methylester (L-NAME) (118).
338 Gasotransmitters in Diabetic Angiopathy Diabetes Volume 65, February 2016
Protective properties of CO in diabetes have beenmainly investigated in STZ-induced type 1 diabetes in ratsor mice. Exposure of the tail artery to CO ex vivo resultedin vasodilatation, an effect that was reduced in arteries ofSTZ-induced diabetic rats, indicating a reduced sensitivityfor CO in diabetic animals (58). In a myocardial I/R modelin HO-12/2 diabetic mice, infarct size and mortality weredramatically worsened compared with wild-type (HO-1+/+)diabetic mice, without affecting glucose levels (119). In di-abetic rats, CORM-3 or HO-1 inducer cobalt protoporphy-rin (CoPP) preserved endothelial function and vascularrelaxation, an effect that was reversed by HO inhibitorschromium mesoporphyrin (CrMP) and tin mesoporphyrin(SnMP) (120–122). In a model of myocardial I/R injury,treatment with CO-releasing compound PEGylated carboxy-hemoglobin bovine (PEG-COOH) drastically reduced in-farct size and troponin levels in STZ-induced diabeticmice. In mice receiving PEG-COOH during reperfusiononly, infarct size was reduced, suggesting CO as a potentialtherapeutic agent for patients after myocardial infarction(123). In STZ-induced diabetes in rats, induction of HO-1with hemin, and treatment with CORM-2 to lesser ex-tent, attenuated vascular damage and oxidative stressand improved vascular relaxation compared with non-treated rats (124).
H2S as a therapeutic agent in diabetic vascular diseaseis evaluated in both mice and rats. In STZ-induced
diabetic Sprague-Dawley rats, treatment with H2S donorsodium hydrosulfide (NaHS) improved vascular relaxationand NO bioavailability. This indicates that H2S is a poten-tial therapeutic agent in diabetic vascular disease via crosstalk with NO (26). Ex vivo administration of H2S sub-strate L-cysteine also resulted in dose-dependent vasore-laxation in rat middle cerebral arteries, which was reducedin diabetic animals (125). The vasorelaxant effects ofNaHS are reduced with the addition of KATP blocker gli-benclamide, demonstrating that NaHS-induced vasorelax-ation takes place via activation of KATP channels (126). Exvivo overexpression of CSE improved vascular relaxationin hyperglycemic conditions and reduced ROS production,while CSE mRNA knockdown with small interfering RNAresulted in a more pronounced ROS production (127).Beneficial properties of H2S have been shown in modelsfor myocardial injury as well. The addition of NaHS indiabetic rats resulted in preserved cardiac function (128),reduced infarct size, reduced ROS and inflammatory pa-rameters such as tumor necrosis factor-a and interleu-kin-10, and inhibited expression of adhesion moleculessuch as intracellular adhesion molecule 1 (129). In amodel of myocardial I/R injury in diabetic Leprdb/db mice,treatment with H2S donor sodium sulfide (Na2S), eitherbefore I/R or only during reperfusion, diminished infarctsize, troponin levels, and ROS (130,131). Although studieson 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 117Mouse: STZ-induced
diabeteseNOS overexpression
of BM-MNCs↑ Reduced atherosclerosis and
improved angiogenesis118
Human: type 1 diabetes NO donor SNP ↑ Induced vasodilatation, but SNP-inducedvasodilatation is reduced in patientswith diabetes
diabetes.diabetesjournals.org van den Born and Associates 339
macrovascular disease are limited to endothelial dysfunctionrather than atherosclerosis, we propose that gasotransmit-ters may also modulate atherogenesis via different mecha-nisms as schematically depicted in Fig. 3. Considering thedata described above and summarized in Table 4, gasotrans-mitters seem to be promising targets for intervention in thecourse of diabetic macrovascular disease.
Protective Mechanisms and Mutual GasotransmitterInteractionsAlthough NO, CO, and H2S have different molecular struc-tures and routes of endogenous production, they do sharevarious physiological properties such as the ability to bindto heme groups (132) and to promote vasorelaxation bystimulation the sGC/cGMP pathway. NO and CO stimu-late cGMP production by targeting sGC, and H2S by inhib-iting phosphodiesterase type 5 activity (133). NO as wellas CO and H2S are direct ROS scavengers, partly by directinteraction with the mitochondrial respiratory chain.They all engage on the KATP channels to achieve this an-tioxidant effect, both in the vasculature and nervous sys-tem (134–136). In addition, they share several commonintracellular pathways, such as nuclear factor-kB, nuclearfactor-like 2, and mitogen-activated protein kinases,thereby exerting antiapoptotic, antioxidant, and anti-inflammatory effects (133). NO, CO, and H2S inhibit theexpression of intracellular adhesion molecule 1, vascularcell adhesion molecule 1, and E-selectin, thereby pro-moting endothelial health and integrity. Finally, allthree gasotransmitters act as proangiogenic substancesvia the VEGF pathway (113,137). On the basis of these
functional similarities, it is likely that gasotransmittershave mutual interactions. Such a relationship between COand NO has been investigated intensively and is mainlymediated via the sGC/cGMP pathway. These effects includeblood pressure regulation and inflammation (138). Cyto-protective effects of NO donors in endothelial cells wereabolished in the presence of the HO inhibitor tin pro-toporphyrin (139). It has already been shown that H2Sexerts its effects via NO production, as H2S promoteseNOS production and activity (140). Additionally, vasore-laxant effects of H2S were diminished when aortic ringswere pretreated with NOS inhibitor L-NAME (141). Recip-rocally, NO donor SNP stimulates endogenous H2S produc-tion via upregulation of CBS (142). Although CO and H2Sshare a lot of functional characteristics, the mutual rela-tionship between these two gasotransmitters has barelybeen studied. In one study of a myocardial I/R injury mousemodel, HO-1 expression was upregulated 24 h after intra-venous H2S treatment, which was accompanied by a pro-tection against I/R-induced damage (143).
Methods to Measure GasotransmittersIn order to study the association of gasotransmitters andthe development of diabetes-associated vascular compli-cations, reliable and sensitive assays to measure NO, CO,and H2S are indispensable. Various methodologies are usedto measure the different gasotransmitters and these willbe briefly described. First, measuring NO is quite a chal-lenge because of its instability. There are different methodsof measuring NO. Most commonly used is the relativelysimple 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 5and 6) on the development of atherosclerosis as indicated in the panel and explanatory text. Ox, oxidized low-density lipoprotein.
340 Gasotransmitters in Diabetic Angiopathy Diabetes Volume 65, February 2016
but rather its oxidated products nitrite and nitrate. However,nitrite and nitrate can be detected more precise by high-performance liquid chromatography (144). NO can alsobe directly measured using gas phase chemiluminescence,which involves the reaction of NO with ozone (O3) to formexcited nitrogen dioxide. During relaxation to (unexcited)nitrogen dioxide, a photon is released that is then detectedby chemiluminescence. Using this method, NO release fromdifferent body fluids and tissues can be measured. In additionto the aforementioned methods, different fluorescent probesand electrodes are currently available, with the possibility tomeasure NO in fluids and tissues and intracellularly in cells invitro (145).
CO levels also can be measured using differentmethods. The most commonly used and relatively simpleway to measure CO is in the air using gas chromatog-raphy. In vivo, CO is generally measured in red bloodcells by determining the percentage of carboxyhemoglo-bin relative to total hemoglobin concentration. Finally,some studies use [14C]Heme in vitro to measure endog-enous 14CO production (146).
When considering H2S as therapeutic target, reliablemethods to determine H2S levels in body fluids and tissuesare needed. However, measurements of H2S are difficultbecause of its volatility. For that reason, stable end prod-ucts like sulfate or thiosulfate can be measured in serum orurine (147), although a few methodologies have been de-scribed to measure H2S itself. The methylene blue assay isthe most commonly used technique. It is based on theoxidative coupling of H2S with two N,N-dimethyl-p-phenylenediamine molecules, forming the methylene bluedye that can be detected spectrophotometrically. However,this technique is extremely pH dependent and not verysensitive and reliable. A more sensitive method is basedon monobromobimane in which two monobromobimanemolecules form the stable sulfide dibimane in the presenceof H2S. Sulfide dibimane can be separated by reverse-phasechromatography and detected by a fluorescence detector.Now, fluorescent probes and sulfide selective electrodes areextensively used however, with different sensitivity andreliability (148). As yet, H2S measurements are difficult,with variable reliability, thereby complicating studies onthe role of H2S and its use as therapeutic target in variousdiseases, including diabetes-associated vascular disease.
Future Perspectives and Treatment OptionsPatients with diabetes have a two- to fourfold increasedrisk for cardiovascular disease, and adequate treatmentand preventive strategies are still lacking. As discussed inthis Perspective, the different gasotransmitters appear tobe important mediators in the development of diabeticangiopathy and therefore are potential targets for in-tervention. As aforementioned, NO-based interventionsare already applied in humans and readily available. SNPis clinically used and acts as a direct NO donor by releasingNO from the ferrous ion center without the need forenzymatic action. The same is true for nitroglycerin and
other organic nitrates, which are well established for theirvasodilatory effects during angina. Organic nitrates actas NO donors by enzymatic or nonenzymatic breakdownof nitrates into nitrite and NO (149). Molsidomine andLinsidomine are registered in several European countries asantianginal drugs and act as vasodilators by the nonenzy-matic release of NO. Finally, dietary products with highnitrate content can act as NO donors. The intake of beet-root juice lowered blood pressure significantly, an effectthat was accompanied by higher levels of total urinarynitrite/nitrate (150). CO administration or CORMs arenot in clinical use yet, albeit that some of the vascularprotective effects of acetylsalicylic acid and statins are at-tributed to induction of HO-1. In human endothelial cellsin vitro, a dose-dependent increase of HO-1 expression wasseen after statin (151,152) or acetylsalicylic acid (153)treatment. However, this effect was not reproduced in hu-man subjects as no differences in HO-1 expression wereobserved between patients treated with acetylsalicylicacid, statin, or placebo (154). The antioxidative actions ofpolyphenol resveratrol are also partly attributed to HO-1upregulation as shown by increased HO-1 expression levelsin STZ-induced type 1 diabetes in Sprague-Dawley rats(122). Although the HO-1–inducing effects of resveratrolhave not yet been described in humans, this dietary sup-plement is readily available for human use. Similar to CO,H2S is also not clinically used in humans yet, although in-travenous Na2S administration has been performed in aphase 1 safety study (155). This study revealed increasedH2S and thiosulfate levels after Na2S administration, indi-cating that circulating H2S levels can be achieved followingparenteral administration. The H2S metabolite thiosulfatecan also act as a H2S donor via enzymatic conversion byrhodanese (also known as thiosulfate sulfurtransferase)(156). Thiosulfate is used as a treatment for calcifylaxisin patients with end-stage renal disease (111) and hasbeen described as a protective agent in hypertensive heartand renal disease in rats (109,157). Sulfhydrylated ACEinhibitors, such as zofenopril and captopril, show additionalbeneficial effects in different trials (158). Recently, it wasdemonstrated that the beneficial effects of sulfhydrylatedACE inhibitors are explained by H2S release (159). Finally,H2S is also generated by various species of sulfate-reducingbacteria in the gut. Germ-free mice showed significantlylower levels of H2S (160), indicating that the addition ofdietary sulfate or sulfur-containing amino acids can act asnatural H2S donors.
CONCLUSION
Various gasotransmitter-based strategies are currently be-ing studied as potential strategy to treat vascular dysfunc-tion. So far, these strategies have not been explored in thecontext of diabetes-associated vascular disease. Because ofthe toxicity of high concentrations of gasotransmitters, aswell as their potential deleterious effects on the develop-ment of vascular disease (as discussed in this Perspec-tive), prudence is called for when considering exogenous
diabetes.diabetesjournals.org van den Born and Associates 341
administration of gasotransmitters. However, gasotransmitter-based interventions are relatively safe, mainly becausethese gases are also produced endogenously and there-fore are highly promising candidate therapeutics.
Acknowledgments. The authors would like to thank Amanda Gautierfrom Gautier Scientific Illustration for preparing the artwork.Funding. This work was supported by grants from the Deutsche For-schungsgemeinschaft (IRTG 1874-1 DIAMICOM) (to H.-P.H., W.G., and J.-L.H.),the Dutch Kidney Foundation (NSN C08-2254 and IP13-114) (to H.v.G.), and theGraduate School of Medical Sciences of the University of Groningen (to J.C.v.d.B.)Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.
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