Hydrogen Sulfide Donor GYY4137 Acts Through Endothelial Nitric Oxide to Protect Intestine in Murine Models of Necrotizing Enterocolitis and Intestinal Ischemia

BACKGROUND: Necrotizing enterocolitis (NEC) in premature infants is often a devastating surgical condition with poor outcomes. GYY4137 is a long-acting donor of hydrogen sulfide, a gasotransmitter that is protective against intestinal injury in experimental NEC, likely through protection against injury secondary to ischemia. We hypothesized that administration of GYY4137 would improve mesenteric perfusion, reduce intestinal injury, and reduce inflammatory responses in experimental NEC and ischemia-reperfusion injury, and that these benefits would be mediated through endothelial nitric oxide synthase-dependent pathways. METHODS: NEC was induced in C57BL/6 wild-type (WT) and endothelial nitric oxide synthase (eNOS) knockout (eNOSKO) pups via maternal separation, formula feeding, enteral lipopolysaccharide, and intermittent hypoxic and hypothermic stress. Pups received daily intraperitoneal injections of 50 mg/kg GYY4137 or phosphate buffered saline vehicle. In separate groups, adult male WT and eNOSKO mice underwent superior mesenteric artery occlusion for 60 min. Before abdominal closure, 50 mg/kg GYY4137 or phosphate buffered saline vehicle was administered into the peritoneal cavity. Laser doppler imaging was used to assess mesenteric perfusion of pups at baseline and on postnatal day 9, and the adult mice at baseline and 24 h after ischemic insult. After euthanasia, the terminal ileum of each animal was fixed, paraffin embedded, sectioned, and stained with hematoxylin and eosin. Sections were blindly graded using published injury scores. Intestinal tissue was homogenized and cytokines measured by ELISA. Data were compared using Mann-Whitney U test, and P-values <0.05 were significant. RESULTS: After NEC and ischemia reperfusion (I/R) injury, GYY4137 improved perfusion in WT mice compared to vehicle, but this effect was lost in the eNOSKO animals. Histologic injury followed a similar pattern with reduced intestinal injury in WT mice treated with GYY4137, and no significant improvement in the eNOSKO group. Cytokine expression after GYY4137 administration was altered by the ablation of eNOS in both NEC and I/R injury groups, with significant differences noted in Interleukin 6 and vascular endothelial growth factor. CONCLUSIONS: GYY4137, a long-acting donor of hydrogen sulfide, has potential as a therapeutic compound for NEC. It improves mesenteric perfusion and intestinal injury in experimental NEC and intestinal I/R injury, and these benefits appear to be mediated through eNOS-dependent pathways

The route and timing of hydrogen sulfide therapy critically impacts intestinal recovery following ischemia and reperfusion injury

PURPOSE: Hydrogen sulfide (H2S) has many beneficial properties and may serve as a novel treatment in patients suffering from intestinal ischemia-reperfusion injury (I/R). The purpose of this study was to examine the method of delivery and timing of administration of H2S for intestinal therapy during ischemic injury. We hypothesized that 1) route of administration of hydrogen sulfide would impact intestinal recovery following acute mesenteric ischemia and 2) preischemic H2S conditioning using the optimal mode of administration as determined above would provide superior protection compared to postischemic application. METHODS: Male C57BL/6J mice underwent intestinal ischemia by temporary occlusion of the superior mesenteric artery. Following ischemia, animals were treated according to one of the following (N=6 per group): intraperitoneal or intravenous injection of GYY4137 (H2S-releasing donor, 50mg/kg in PBS), vehicle, inhalation of oxygen only, inhalation of 80ppm hydrogen sulfide gas. Following 24-h recovery, perfusion was assessed via laser Doppler imaging, and animals were euthanized. Perfusion and histology data were assessed, and terminal ileum samples were analyzed for cytokine production following ischemia. Once the optimal route of administration was determined, preischemic conditioning with H2S was undertaken using that route of administration. All data were analyzed using Mann-Whitney. P-values <0.05 were significant. RESULTS: Mesenteric perfusion following intestinal I/R was superior in mice treated with intraperitoneal (IP) GYY4137 (IP vehicle: 25.6±6.0 vs. IP GYY4137: 79.7±15.1; p=0.02) or intravenous (IV) GYY4137 (IV vehicle: 36.3±5.9 vs. IV GYY4137: 100.7±34.0; p=0.03). This benefit was not observed with inhaled H2S gas (O2 vehicle: 66.6±11.4 vs. H2S gas: 81.8±6.0; p=0.31). However, histological architecture was only preserved with intraperitoneal administration of GYY4127 (IP vehicle: 3.4±0.4 vs. IP GYY4137: 2±0.3; p=0.02). Additionally, IP GYY4137 allowed for significant attenuation of inflammatory chemokine production of IL-6, IP-10 and MIP-2. We then analyzed whether there was a difference between pre- and postischemic administration of IP GYY4137. We found that preconditioning of animals with intraperitoneal GYY4137 only added minor improvements in outcomes compared to postischemic application. CONCLUSION: Therapeutic benefits of H2S are superior with intraperitoneal application of an H2S donor compared to other administration routes. Additionally, while intraperitoneal treatment in both the pre- and postischemic period is beneficial, preischemic application of an H2S donor was found to be slightly better. Further studies are needed to examine long term outcomes and further mechanisms of action prior to widespread clinical application. TYPE OF STUDY: Basic science. LEVEL OF EVIDENCE: N/A

Hydrogen Sulfide: A Potential Novel Therapy for the Treatment of Ischemia

Hydrogen sulfide (H2S) is a novel signaling molecule most recently found to be of fundamental importance in cellular function as a regulator of apoptosis, inflammation, and perfusion. Mechanisms of endogenous H2S signaling are poorly understood; however, signal transmission is thought to occur via persulfidation at reactive cysteine residues on proteins. Although much has been discovered about how H2S is synthesized in the body, less is known about how it is metabolized. Recent studies have discovered a multitude of different targets for H2S therapy, including those related to protein modification, intracellular signaling, and ion channel depolarization. The most difficult part of studying hydrogen sulfide has been finding a way to accurately and reproducibly measure it. The purpose of this review is to: elaborate on the biosynthesis and catabolism of H2S in the human body, review current knowledge of the mechanisms of action of this gas in relation to ischemic injury, define strategies for physiological measurement of H2S in biological systems, and review potential novel therapies that use H2S for treatment

Direct Peritoneal Resuscitation Improves Mesenteric Perfusion by Nitric Oxide Dependent Pathways

Background Direct peritoneal resuscitation (DPR) has been shown to increase survival after intestinal ischemia and reperfusion injury (I/R). We have previously appreciated that minimum essential medium (MEM), a synthetic cell culture medium with bovine serum, glutamine, and antibiotics, contributes to these benefits. We hypothesized that (1) DPR using MEM as a dialysate would increase mesenteric perfusion, improve intestinal mucosal injury, and limit intestinal and hepatic inflammation after intestinal I/R and (2) these improvements would be dependent on endothelial nitric oxide pathways. Methods Eight-week-old C57Bl6J wild-type (WT) and eNOS Knock Out (eNOS KO) male mice were anesthetized and intestinal ischemia was induced for 60 min. After ischemia, 1 mL of phosphate buffered saline vehicle or MEM was injected into the abdominal cavity. Intestinal perfusion was reassessed after 48 h. Animals were then euthanized, and intestines and livers explanted for histologic and molecular analyses. Results DPR with MEM significantly improved mesenteric perfusion compared with vehicle (phosphate buffered saline) as measured by Laser Doppler Imaging (WT + MEM 91.58 ± 13.74%, WT + Vehicle 44.27 ± 11.93%, P 0.05). Intestinal levels of interleukin (IL)-1β were increased in WT animals treated with MEM compared with eNOS KOs, whereas concentrations of intestinal IL-6 were similar between groups. Hepatic levels of both IL-1β and IL-6 were significantly elevated in eNOS KOs compared with WT treated with MEM. Conclusions DPR with MEM has significant therapeutic potential for improving mesenteric perfusion, intestinal injury, and the local inflammatory response after intestinal I/R. These benefits appear to be dependent on nitric oxide signaling within the endothelium

Mesenchymal Stromal Cell Therapy for the Treatment of Intestinal Ischemia: Defining the Optimal Cell Isolate for Maximum Therapeutic Benefit

Intestinal ischemia is a devastating intraabdominal emergency that often necessitates surgical intervention. Mortality rates can be high, and patients who survive often have significant long-term morbidity. The implementation of traditional medical therapies to prevent or treat intestinal ischemia have been sparse over the last decade, and therefore, the use of novel therapies are becoming more prevalent. Cellular therapy using mesenchymal stromal cells is one such treatment modality that is attracting noteworthy attention in the scientific community. Several groups have seen benefit with cellular therapy, but the optimal cell line has not been identified. The purpose of this review is to: 1) Review the mechanism of intestinal ischemia and reperfusion injury, 2) Identify the mechanisms of how cellular therapy may be therapeutic for this disease, and 3) Compare various MSC tissue sources to maximize potential therapeutic efficacy in the treatment of intestinal I/R diseases

Hydrogen sulfide provides intestinal protection during a murine model of experimental necrotizing enterocolitis

Background Necrotizing enterocolitis (NEC) continues to be a morbid surgical condition among preterm infants. Novel therapies for this condition are desperately needed. Hydrogen sulfide (H2S) is an endogenous gasotransmitter that has been found to have beneficial properties. We therefore hypothesized that intraperitoneal injection of various H2S donors would improve clinical outcomes, increase intestinal perfusion, and reduce intestinal injury in an experimental mouse model of necrotizing enterocolitis. Methods NEC was induced in five-day-old mouse C57BL/6 mouse pups through maternal separation, formula feeding, and intermittent hypoxic and hypothermic stress. The control group (n = 10) remained with their mother and breastfed ad lib. Experimental groups (n = 10/group) received intraperitoneal injections of phosphate buffered saline (PBS) vehicle or one of the following H2S donors: (1) GYY4137, 50 mg/kg daily; (2) Sodium sulfide (Na2S), 20 mg/kg three times daily; (3) AP39, 0.16 mg/kg daily. Pups were monitored for weight gain, clinical status, and intestinal perfusion via transcutaneous Laser Doppler Imaging (LDI). After sacrifice on day nine, intestinal appearance and histology were scored and cytokines were measured in tissue homogenates of intestine, liver, and lung. Data were compared with Mann–Whitney and p < 0.05 was considered significant. Results Clinical score and weight gain were significantly improved in all three H2S-treated groups as compared to vehicle (p < 0.05 for all groups). Intestinal perfusion of the vehicle group was 22% of baseline while the GYY4137 group was 38.7% (p = 0.0103), Na2S was 47.0% (p = 0.0040), and AP39 was 43.0% (p = 0.0018). The vehicle group had a median histology score of 2.5, while the GYY4137 group's was 1 (p = 0.0013), Na2S was 0.5 (p = 0.0004), and AP39 was 0.5 (p = 0.0001). Cytokine analysis of the intestine of the H2S-treated groups revealed levels closer to breastfed pups as compared to vehicle (p < 0.05 for all groups). Conclusion Intraperitoneal administration of H2S protects against development of NEC by improving mesenteric perfusion, and by limiting mucosal injury and altering the tissue inflammatory response. Further experimentation is necessary to elucidate downstream mechanisms prior to clinical implementation

Umbilical mesenchymal stromal cells provide intestinal protection through nitric oxide dependent pathways

Background Umbilical-derived mesenchymal stromal cells (USCs) have shown promise in the protection of ischemic organs. We hypothesized that USCs would improve mesenteric perfusion, preserve intestinal histological architecture, and limit inflammation by nitric oxide–dependent mechanisms following intestinal ischemia/reperfusion (IR) injury. Methods Adult wild-type C57BL/6J (WT) and endothelial nitric oxide synthase knock out (eNOS KO) mice were used: (1) WT IR + vehicle, (2) WT IR + USC, (3) eNOS KO IR + vehicle, and (4) eNOS KO IR + USC. Mice were anesthetized, and a midline laparotomy was performed. The superior mesenteric artery was clamped with a nonoccluding clamp for 60-min. Following IR, mice were treated with an injection of 250 μL phosphate buffered saline or 2 × 106 USCs suspended in 250-μL phosphate buffered saline solution. Mesenteric perfusion images were acquired using laser Doppler imaging. Perfusion was analyzed as a percentage of baseline. At 24 h, mice were euthanized, and intestines were harvested. Intestines were evaluated for injury, and data were analyzed using the Mann–Whitney or Kruskal–Wallis tests. Results Intestinal mesenteric perfusion was significantly improved in WT mice treated with USC therapy compared with eNOS KOs. Intestinal histological architecture was preserved with USC therapy in WT mice. However, in eNOS KO mice, this benefit was abolished. Finally, the presence of several cytokines and growth factors were significantly improved in WT mice compared with eNOS KO mice treated with USCs. Conclusions The benefits of USC-mediated therapy following intestinal IR injury likely occur via nitric oxide–dependent pathways. Further studies are required to define the molecular mechanisms by which USCs activate endothelial nitric oxide synthase to bring about their protective effects

Harvest Tissue Source Does Not Alter the Protective Power of Stromal Cell Therapy Following Intestinal Ischemia and Reperfusion Injury

Background Transplantation of mesenchymal stromal cells (MSCs) may be a novel treatment for intestinal ischemia. The optimal stromal cell source that could yield maximal protection following injury, however, has not been identified. We hypothesized that: 1) MSCs would increase survival and mesenteric perfusion, preserve intestinal histological architecture, and limit inflammation following intestinal ischemia and reperfusion injury (I/R), and 2) MSCs harvested from different sources of tissue would have equivalent protective properties to the intestine following I/R. Methods Adult male mice were anesthetized and a midline laparotomy performed. The intestines were eviscerated, the small bowel mesenteric root identified, and baseline intestinal perfusion was determined using Laser Doppler Imaging (LDI). Intestinal ischemia was established by temporarily occluding the superior mesenteric artery for 60 minutes with a non-crushing clamp. Following ischemia, the clamp was removed and the intestines were allowed to recover. Prior to abdominal closure, 2 × 106 human umbilical (USCs), bone-marrow (BMSCs) derived MSCs, or keratinocytes in 250μl of phosphate-buffered saline (PBS) vehicle were injected into the peritoneum. Animals were allowed to recover for 12 or 24 hours (perfusion, histology, inflammatory studies), or 7 days (survival studies). Survival data was analyzed using log rank test. Perfusion was expressed as percentage of baseline and 12 and 24 hour data was analyzed using one way ANOVA and student’s t-test. Non parametric data was compared using Mann-Whitney-U test. A p-value of less than 0.05 was significant. Results All MSCs increased seven day survival following I/R and were superior to vehicle or keratinocytes (P<0.05). All MSCs increased mesenteric perfusion above vehicle at 12 and 24 hours following injury (P<0.05). All MSCs provided superior perfusion compared to keratinocytes at 24 hours post-injury (P<0.05). Administration of each MSC line improved intestinal histology after I/R (P<0.05). Multiple pro-inflammatory chemokines were down-regulated following application of MSCs suggesting a decreased inflammatory response following MSC therapy. Conclusion Transplantation of MSCs following intestinal I/R, irrespective of source tissue, significantly increases survival and mesenteric perfusion while limiting intestinal damage and inflammation. Further studies are needed to identify the mechanism that these cells utilize to promote improved outcomes following injury

Hydrogen sulfide improves intestinal recovery following ischemia by endothelial nitric oxide-dependent mechanisms

Hydrogen sulfide (H2S) is an endogenous gasotransmitter that has vasodilatory properties. It may be a novel therapy for intestinal ischemia-reperfusion (I/R) injury. We hypothesized that 1) H2S would improve postischemic survival, mesenteric perfusion, mucosal injury, and inflammation compared with vehicle and 2) the benefits of H2S would be mediated through endothelial nitric oxide. C57BL/6J wild-type and endothelial nitric oxide synthase knockout (eNOS KO) mice were anesthetized, and a midline laparotomy was performed. Intestines were eviscerated, the small bowel mesenteric root identified, and baseline intestinal perfusion was determined using laser Doppler. Intestinal ischemia was established by temporarily occluding the superior mesenteric artery. Following ischemia, the clamp was removed, and the intestines were allowed to recover. Either sodium hydrosulfide (2 nmol/kg or 2 µmol/kg NaHS) in PBS vehicle or vehicle only was injected into the peritoneum. Animals were allowed to recover and were assessed for mesenteric perfusion, mucosal injury, and intestinal cytokines. P values < 0.05 were significant. H2S improved mesenteric perfusion and mucosal injury scores following I/R injury. However, in the setting of eNOS ablation, there was no improvement in these parameters with H2S therapy. Application of H2S also resulted in lower levels of intestinal cytokine production following I/R. Intraperitoneal H2S therapy can improve mesenteric perfusion, intestinal mucosal injury, and intestinal inflammation following I/R. The benefits of H2S appear to be mediated through endothelial nitric oxide-dependent pathways.NEW & NOTEWORTHY H2S is a gaseous mediator that acts as an anti-inflammatory agent contributing to gastrointestinal mucosal defense. It promotes vascular dilation, mucosal repair, and resolution of inflammation following intestinal ischemia and may be exploited as a novel therapeutic agent. It is unclear whether H2S works through nitric oxide-dependent pathways in the intestine. We appreciate that H2S was able to improve postischemic recovery of mesenteric perfusion, mucosal integrity, and inflammation. The beneficial effects of H2S appear to be mediated through endothelial nitric oxide-dependent pathways