Exacerbated Ischemia-reperfusion Injury Research Articles

Bile acids attenuate hepatic inflammation during ischemia/reperfusion injury

Background & AimsPersistent cholestasis has been associated with poor prognosis after orthotopic liver transplantation (OLT). In this study, we aimed to investigate how the accumulation of tauro-beta-muricholic acid (TβMCA), resulting from the reprogramming of bile acid (BA) metabolism during liver ischemia/reperfusion (IR) stress, attenuates liver inflammation. MethodsIngenuity Pathway Analysis (IPA) was performed using transcriptome data from a murine hepatic IR model. Three different models of hepatic IR (liver warm IR, bile duct separation-IR, common bile duct ligation-IR) were employed. We generated AAV-transfected mice and CD11b-DTR mice to assess the role of BAs in regulating the myeloid S1PR2-GSDMD axis. Hepatic BA levels were analyzed using targeted metabolomics. Finally, the correlation between the reprogramming of BA metabolism and hepatic S1PR2 levels was validated through RNA-seq of human OLT biopsies. ResultsWe found that BA metabolism underwent reprogramming in murine hepatocytes under IR stress, leading to increased synthesis of TβMCA, catalyzed by the enzyme CYP2C70. The levels of hepatic TβMCA were negatively correlated with the severity of hepatic inflammation, as indicated by the serum IL-1β levels. Inhibition of hepatic CYP2C70 resulted in reduced TβMCA production, which subsequently increased serum IL-1β levels and exacerbated IR injury. Moreover, our findings suggested that TβMCA could inhibit canonical inflammasome activation in macrophages and attenuate inflammatory responses in a myeloid-specific S1PR2-GSDMD-dependent manner. Additionally, Gly-βMCA, a derivative of TβMCA, could effectively attenuate inflammatory injury in vivo and inhibit human macrophage pyroptosis in vitro. ConclusionsIR stress orchestrates hepatic BA metabolism to generate TβMCA, which attenuates hepatic inflammatory injury by inhibiting the myeloid S1PR2-GSDMD axis. Bile acids have immunomodulatory functions in liver reperfusion injury that may guide therapeutic strategies. Impact and implicationsOur research reveals that liver IR stress triggers reprogramming of BA metabolism. This functions as an adaptive mechanism to mitigate inflammatory injury by regulating the S1PR2-GSDMD axis, thereby controlling the release of IL-1β from macrophages. Our results highlight the crucial role of bile acids in regulating hepatocyte-immune cell crosstalk, which demonstrates an immunomodulatory function in liver reperfusion injury that may guide therapeutic strategies targeting bile acids and their receptors.

Exacerbated ischemia-reperfusion injury in fatty livers is mediated by lipid peroxidation stress and ferroptosis

BackgroundIschemia-reperfusion injury is a common problem in liver surgery and transplantation. Although ischemia-reperfusion injury is known to be more pronounced in fatty livers, the underlying mechanisms for this difference remain poorly understood. We hypothesized that ferroptosis plays a significant role in fatty liver ischemia-reperfusion injury due to increased lipid peroxidation in the presence of stored iron in the fatty liver. To test this hypothesis, the ferroptosis pathway was evaluated in a murine fatty liver ischemia-reperfusion injury model. MethodsC57BL6 mice were fed with a normal diet or a high fat, high sucrose diet for 12 weeks. At 22 weeks of age, liver ischemia-reperfusion injury was induced through partial (70%) hepatic pedicle clamping for 60 minutes, followed by 24 hours of reperfusion before tissue harvest. Acyl–coenzyme A synthetase long-chain family member 4 and 4-hydroxynonenal were quantified in the liver tissues. In separate experiments, liproxstatin-1 or vehicle control was administered for 7 consecutive days before liver ischemia-reperfusion injury. ResultsExacerbated ischemia-reperfusion injury was observed in the livers of high fat, high sucrose diet fed mice. High fat, high sucrose diet + ischemia-reperfusion injury (HDF+IRI) livers had a significantly greater abundance of acyl–coenzyme A synthetase long-chain family member 4 and 4-hydroxynonenal compared with normal diet + ischemia-reperfusion injury (ND+IRI) livers or sham fatty livers, which indicated an increase of ferroptosis. HFD fed animals receiving liproxstatin-1 injections had a significant reduction in serum aspartate transaminase and alanine transaminase after ischemia-reperfusion injury, consistent with attenuation of ischemia-reperfusion injury in the liver. ConclusionFerroptosis plays a significant role in ischemia-reperfusion injury in fatty livers. Inhibiting ferroptotic pathways in the liver may serve as a novel therapeutic strategy to protect the fatty liver in the setting of ischemia-reperfusion injury.

Formyl peptide receptor 2 activation by mitochondrial formyl peptides stimulates the neutrophil proinflammatory response via the ERK pathway and exacerbates ischemia–reperfusion injury

BackgroundIschemia–reperfusion injury (IRI) is an inevitable process in renal transplantation that significantly increases the risk of delayed graft function, acute rejection, and even graft loss. Formyl peptide receptor 2 (FPR2) is an important receptor in multiple septic and aseptic injuries, but its functions in kidney IRI are still unclear. This study was designed to reveal the pathological role of FPR2 in kidney IRI and its functional mechanisms.MethodsTo explore the mechanism of FPR2 in kidney IRI, the model rats were sacrificed after IRI surgery. Immunofluorescence, enzyme-linked immunosorbent assays, and western blotting were used to detect differences in the expression of FPR2 and its ligands between the IRI and control groups. WRW4 (WRWWWW-NH2), a specific antagonist of FPR2, was administered to kidney IRI rats. Kidney function and pathological damage were detected to assess kidney injury and recovery. Flow cytometry was used to quantitatively compare neutrophil infiltration among the experimental groups. Mitochondrial formyl peptides (mtFPs) were synthesized and administered to primary rat neutrophils together with the specific FPR family antagonist WRW4 to verify our hypothesis in vitro. Western blotting and cell function assays were used to examine the functions and signaling pathways that FPR2 mediates in neutrophils.ResultsFPR2 was activated mainly by mtFPs during the acute phase of IRI, mediating neutrophil migration and reactive oxygen species production in the rat kidney through the ERK1/2 pathway. FPR2 blockade in the early phase protected rat kidneys from IRI.ConclusionsmtFPs activated FPR2 during the acute phase of IRI and mediated rat kidney injury by activating the migration and reactive oxygen species generation of neutrophils through the ERK1/2 pathway.

Serpine1 Regulates Peripheral Neutrophil Recruitment and Acts as Potential Target in Ischemic Stroke.

IntroductionPeripheral neutrophil infiltration can exacerbate ischemia–reperfusion injury. We focused on the relationship between various peripheral immune cells and cerebral ischemia–reperfusion (I/R) injury.MethodsIn this study, we investigated the effects of dauricine on neuronal injury induced by ischemia–reperfusion and peripheral immune cells after ischemic stroke in mouse model, and we explored the undefined mechanisms of regulating peripheral immune cells through RNA sequencing and various biochemical verification in vitro and in vivo.ResultsWe found that dauricine improved the neurological deficits of I/R injury, reduced the infarct volume, and improved the neurological scores. Furthermore, dauricine reduced the infiltration of neutrophils into the brain after MCAO-R and increased peripheral neutrophils but unchanged the permeability of the endotheliocyte Transwell system in an in vitro blood-brain barrier (BBB) model. RNA sequencing showed that chemotaxis factors, such as CXCL3, CXCL11, CCL20, CCL22, IL12a, IL23a, and serpine1, might play a crucial role. Overexpression of serpine1 reversed LPS-induced migration of neutrophils. Dauricine can directly bind with serpine1 in ligand–receptor docking performed with the Autodock and analyzed with PyMOL.ConclusionWe identified chemotaxis factor serpine1 played a crucial role in peripheral neutrophil infiltration, which may contribute to reduce the neuronal injury induced by ischemia–reperfusion. These findings reveal that serpine1 may act as a potential treatment target in the acute stage of ischemic stroke.

Reduced Neutrophil Extracellular Trap Formation During Ischemia Reperfusion Injury in C3 KO Mice: C3 Requirement for NETs Release.

Complement C3 plays a prominent role in inflammatory processes, and its increase exacerbates ischemia reperfusion injury (IRI)-induced acute kidney injury (AKI). Infiltrated neutrophils can be stimulated to form neutrophil extracellular traps (NETs), leading to renal injury. However, the relationship between the increase of C3 and the release of NETs in AKI was not clear. Here we found that IRI in the mouse kidney leads to increased neutrophils infiltration and NET formation. Furthermore, neutrophils depletion by anti-Ly6G IgG (1A8) did not reduce C3 activation but reduced kidney injury and inflammation, indicating a link between neutrophils infiltration and renal tissue damage. Pretreatment with 1A8 suppressed ischemia-induced NET formation, proving that extracellular traps (ETs) in renal tissue were mainly derived from neutrophils. Renal ischemia injury also leads to increased expression of C3. Moreover, C3 KO mice (C3 KO) with IRI exhibited attenuated kidney damage and decreased neutrophils and NETs. In vitro, C3a primed neutrophils to form NETs, reflected by amorphous extracellular DNA structures that colocalized with CitH3 and MPO. These data reveal that C3 deficiency can ameliorate AKI by reducing the infiltration of neutrophils and the formation of NETs. Targeting C3 activation may be a new therapeutic strategy for alleviating the necroinflammation of NETs in AKI.

SIRT3 protects kidneys from ischemia-reperfusion injury by modulating the DRP1 pathway to induce mitochondrial autophagy

Renal ischemia-reperfusion injury (IRI) is a leading cause of acute kidney injury (AKI) and may influence renal graft survival. In this study, we investigate the involvement of SIRT3 and DRP1 in mitochondrial autophagy and AKI in a mouse model of IRI. Autophagy was detected in the absence of SIRT3, and hypoxic reoxygenation (H/R) experiments using renal tubular epithelial cells NRK52E were performed in vitro to validate these results. We found that autophagosomes increased following IRI and that the expression of autophagy-related genes was up-regulated. The inhibition of autophagy with 3-methyladenine exacerbated IRI, whereas the DRP1 inhibitor Mdivi-1 reversed this inhibition. Mdivi-1 did not reverse the inhibition of autophagy in the absence of SIRT3. During IRI, Mdivi-1 reduced autophagy and DRP1 expression, whereas SIRT3 overexpression attenuated this condition. Rescue experiment showed that autophagy was increased when both SIRT3 or DRP1 were over- or under-expressed or just DRP1 was under-expressed but expression was reduced when just SIRT3 was under-expressed. However, the expression of DRP1-related molecules was reduced when SIRT3 was overexpressed and when DRP1 was under-expressed. Taken together, these findings indicate that SIRT3 protects against kidney damage from IRI by modulating the DRP1 pathway to induce mitochondrial autophagy.

In Situ Pre-Treatment of Vascularized Composite Allografts With a Targeted Complement Inhibitor Protects Against Brain Death and Ischemia Reperfusion Induced Injuries.

IntroductionDonor brain death (BD) is an unavoidable component of vascularized composite allograft (VCA) transplantation and a key contributor to ischemia-reperfusion injury (IRI). Complement is activated and deposited within solid organ grafts as a consequence of BD and has been shown to exacerbate IRI, although the role of BD and complement in VCA and the role it plays in IRI and VCA rejection has not been studied.MethodsBD was induced in Balb/c donors, and the VCA perfused prior to graft procurement with UW solution supplemented with or without CR2-Crry, a C3 convertase complement inhibitor that binds at sites of complement activation, such as that induced on the endothelium by induction of BD. Following perfusion, donor VCAs were cold stored for 6 hours before transplantation into C57BL/6 recipients. Donor VCAs from living donors (LD) were also procured and stored. Analyses included CR2-Crry graft binding, complement activation, toxicity, injury/inflammation, graft gene expression and survival.ResultsCompared to LD VCAs, BD donor VCAs had exacerbated IRI and rejected earlier. Following pretransplant in-situ perfusion of the donor graft, CR2-Crry bound within the graft and was retained post-transplantation. CR2-Crry treatment significantly reduced complement deposition, inflammation and IRI as compared to vehicle-treated BD donors. Treatment of BD donor VCAs with CR2-Crry led to an injury profile not dissimilar to that seen in recipients of LD VCAs.ConclusionPre-coating a VCA with CR2-Crry in a clinically relevant treatment paradigm provides localized, and therefore minimally immunosuppressive, protection from the complement-mediated effects of BD induced exacerbated IRI.

KCNQ1OT1 Exacerbates Ischemia-Reperfusion Injury Through Targeted Inhibition of miR-140-3P.

Potassium voltage-gated channel subfamily Q member 1 opposite strand 1 (KCNQ1OT1), a long non-coding RNA found in the KCNQ1 locus, has been evidenced to play important roles in the aggravation of inflammatory and oxidative stresses under hypoxia, but whether and how KCNQ1OT1 contributes to neuronal damages in the cerebral ischemic stroke remains unknown. In the present study, we found a dominant upregulation of KCNQ1OT1 both in the plasma of cerebral ischemia patients and in an oxygen-glucose deprivation and reperfusion (OGD/R) model in PC12 cells. KCNQ1OT1 knocking-down significantly ameliorated the inflammation, oxidative stress, and cell apoptosis induced by OGD/R. We further demonstrated that KCNQ1OT1 directly bound to and suppressed the expression of miR-140-3p. Overexpressing miR-140-3p significantly alleviated both the inflammation, oxidative stress, and cell apoptosis in OGD/R, while all those cytoprotective effects of miR-140-3p-overexpression were hindered by the co-overexpression of KCNQ1OT1. Furthermore, we found a direct interaction between miR-140-3p and the hypoxia-inducible factor-1α (HIF-1α), which was suppressed by the upregulation of KCNQ1OT1 in OGD/R. Our results indicate that KCNQ1OT1 exacerbates cerebral ischemia-reperfusion injury by targeted binding to miR-140-3p, thus interfering its direct interaction with HIF-1α. These data provide novel therapeutic targets in the cerebral ischemic stroke.

Endothelial Stabilization Reduces the Effects of Brain Death Induced Acute Lung Injury Post-Transplant

Purpose Donor Brain death (BD) provokes activation of inflammatory pathways resulting in acute lung injury (BD-ALI), characterized by the breakdown of endothelial and epithelial barrier function. These injuries are further propagated in the early transplant period by ischemia reperfusion injury (IRI) which together adversely affect post-transplant outcomes. We have developed and characterized a novel small molecule gap/tight junction stabilizer, aCT1, which we have shown to improve cellular communication and integrity. Herein we explored the use of aCT1 as a pretreatment therapeutic to protect donor lungs from the effects of BD-ALI. Methods A mouse model of BD and orthotopic left lung transplant (LTx) were utilized to examine the effects of aCT1 on barrier function stabilization in BD and subsequent impact on LTx outcomes. Following BD induction donors were nebulized with aCT1 or vehicle and ventilated for a further 3 hours. Lungs were then removed to be flushed with and stored in Perfadex solution at 4°C. After 18 hours of cold storage the right lung was processed for histological analysis and the left lung transplanted into CD45.1 B6 mice. Transplanted lungs were then harvested 6 hours post-Tx for analysis of graft injury via histopathology and protein quantification. For controls a standard living donor (LD) model was used for comparison. Results After 3 hrs of BD induction and 18 hours cold ischemia, lungs had BD-ALI features which included significant increases in immune cell infiltration, edema, and endothelial activation. Treatment with aCT1 had no impact on BD-ALI features. 6 hrs post-Tx lungs from BD donors had significantly increased IRI as compared to LD controls, as determined by increased immune cell infiltration and pathological assessment of injury. Treatment of BD donors with aCT1 significantly reduced this BD injury profile, with pathological injury not dissimilar to that seen in LD controls (BD+aCT1 3 vs. BD 8.25, BD+aCT1 3 vs. LD 3, respectively. p Conclusion These data support gap junction stabilization as a viable option for protecting donor lungs against BD-ALI exacerbated ischemia reperfusion injury.

Age-dependent loss of induced regulatory T cell function exacerbates liver ischemia-reperfusion injury

Previous studies demonstrate that the number of induced regulatory T cells (iTregs) increases in aged mice. However, these studies do not characterize iTregs across different ages or how these immune modulators contribute to the dysregulation of immunity in murine disease models. Therefore, this study aimed to examine the relationship between age and iTreg function using a mouse model of hepatic ischemia-reperfusion injury (IRI). In this model, aged-mice suffered more serious injury than Young-mice, with higher serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and higher histological scores from liver biopsies. iTregs isolated from Young-mice exhibited stronger immunosuppressive ability in vitro and had a greater response during IRI in vivo. In addition, aged-mice that were pretreated with iTregs generated in Young-mice (Y-iTregs) had alleviated injury compared with mice pretreated with iTregs from aged-mice (A-iTregs) or no treatment group. Adoptive transfer of iTregs ameliorated liver IRI and promoted liver recovery with decreased levels of interferon-γ (IFN-γ) and interleukin-17 (IL-17). These results demonstrate that the exacerbated IRI observed in aged-mice is a result of decreased iTreg function. Therefore, improving iTreg function is important for disease treatment in elder patients.

Breaking free from the NETs

Neutrophil extracellular traps exacerbate ischemia reperfusion injury associated with neonatal midgut volvulus and serve as a therapeutic target.

The Effect of Diazepam on the Function of Hypertrophied Rats’ Hearts in Ischemia-Reperfusion Conditions

: Background: Hypertrophied hearts are susceptible to ischemic injury. Besides, cardiac vulnerability could be changed in the presence of diazepam. Objectives: The current study aimed to investigate the effect of diazepam on hypertrophied rats’ hearts in ischemia-reperfusion conditions. Materials and Methods: Male Wistar rats (body weight 210 - 270 gr) were administered with isoproterenol (4 mg/kg body weight, intraperitoneally for 7 days) alone or along with diazepam (1 and 5 mg/kg body weight, intraperitoneally for 5 days). The control rats received normal saline intraperitoneally. The animal s’ hearts were isolated according to langendorff setup and were passed through baseline, ischemia, and reperfusion stages. Then, cardiac mass index (ratio of heart weight to body weight) was measured. Cardiac functional parameters, including left ventricular developed pressure and rate pressure product, were also assessed at baseline and following ischemia. The data were analyzed using ANOVA and P < 0.05 was considered to be statistically significant. Results: Isoproterenol-induced cardiac hypertrophy was significantly reduced by both doses of diazepam compared to the group only treated with isoproterenol (P < 0.05) although it did not reach the control level. However, diazepam administration (1 and 5 mg/kg) did not change isoproterenol-induced exacerbated ischemia-reperfusion injury compared to the control group (P = 0.001 and P = 0.013, respectively). Conclusions: Diazepam relatively prevented the isoproterenol–induced cardiac hypertrophy in the animal model. This effect could be probably explained by the modification of oxidative stress and preservation of intracellular calcium concentration. Considering the common clinical usage of diazepam, as a peripheral benzodiazepine ligand, antihypertrophic effects of diazepam are recommended to be investigated in clinical trials.

Treatment With Tetrahydrobiopterin Overcomes Brain Death–Associated Injury in a Murine Model of Pancreas Transplantation

Brain death (BD) has been associated with an immunological priming of donor organs and is thought to exacerbate ischemia reperfusion injury (IRI). Recently, we showed that the essential nitric oxide synthase co‐factor tetrahydrobiopterin (BH4) abrogates IRI following experimental pancreas transplantation. We therefore studied the effects of BD in a murine model of syngeneic pancreas transplantation and tested the therapeutic potential of BH4 treatment. Compared with sham‐operated controls, donor BD resulted in intragraft inflammation reflected by induced IL‐1ß, IL‐6, VCAM‐1, and P‐selectin mRNA expression levels and impaired microcirculation after reperfusion (p < 0.05), whereas pretreatment of the BD donor with BH4 significantly improved microcirculation after reperfusion (p < 0.05). Moreover, BD had a devastating impact on cell viability, whereas BH4‐treated grafts showed a significantly higher percentage of viable cells (p < 0.001). Early parenchymal damage in pancreatic grafts was significantly more pronounced in organs from BD donors than from sham or non‐BD donors (p < 0.05), but BH4 pretreatment significantly ameliorated necrotic lesions in BD organs (p < 0.05). Pretreatment of the BD donor with BH4 resulted in significant recipient survival (p < 0.05). Our data provide novel insights into the impact of BD on pancreatic isografts, further demonstrating the potential of donor pretreatment strategies including BH4 for preventing BD‐associated injury after transplantation.

Hypoxia-Inducible Factor-2α Limits Natural Killer T Cell Cytotoxicity in Renal Ischemia/Reperfusion Injury.

Natural killer T (NKT) cells are the major early-acting immune cell type and fundamental immune modulators in ischemia-reperfusion injury (IRI). Because lymphocytes are exposed to various oxygen tensions under pathophysiologic conditions, we hypothesize that hypoxia-inducible factors (HIFs) have roles in NKT cell activation, and thus determine the final outcome of renal IRI. In this study, we used Lck-Cre transgenic mice to specifically disrupt HIF-2α in T/NKT cells and found that HIF-2α knockout led to upregulated Fas ligand expression on peripheral NKT cells, but not on conventional T cells. HIF-2α knockout promoted infiltration of NKT cells into ischemic kidneys and exacerbated IRI, which could be mitigated by in vivo NK1.1(+) cell depletion or Fas ligand blockade. Compared with wild-type NKT cells, HIF-2α(-/-) NKT cells adoptively transferred to Rag1-knockout mice elicited more severe renal injury, and these mice were not protected by CGS21680, an adenosine A2A receptor agonist. Mechanistically, hypoxia-induced expression of adenosine A2A receptor in NKT cells and CGS21680-induced cAMP production in thymocytes were HIF-2α-dependent. Hydrogen peroxide-induced Fas ligand expression on thymic wild-type NKT cells was significantly attenuated by CGS21680 treatment, but this effect was lost in HIF-2α(-/-) NKT cells. Finally, CGS21680 and LPS, an inducer of HIF-2α in endothelium, synergistically reduced renal IRI substantially, but this effect was absent in Mx1-Cre-induced global HIF-2α-knockout mice. Taken together, our results reveal a hypoxia/HIF-2α/adenosine A2A receptor axis that restricts NKT cell activation when confronted with oxidative stress and thus protects against renal IRI.

Hydrogen sulphide as a novel therapy to ameliorate cyclosporine nephrotoxicity

BackgroundCalcineurin inhibitors have significant nephrotoxic side effects, which can exacerbate ischemia–reperfusion injury in renal transplantation. Novel therapeutic agents such as hydrogen sulphide (H2S) may reduce these harmful effects. This study investigated the effects of H2S on cyclosporine (CsA) induced nephrotoxicity. Materials and methodsPorcine kidneys were subjected to 15 min of warm ischemia and 2 h of static cold storage. They were reperfused for 3 h with oxygenated normothermic autologous whole blood on an isolated organ reperfusion apparatus. Kidneys were treated with CsA during reperfusion (n = 6) or cyclosporine and 0.25 mmol/L of H2S infused 10 min before and 20 min after reperfusion (n = 6). These were compared with untreated controls (n = 7). ResultsCsA caused a significant reduction in renal blood flow during reperfusion, which was reversed by H2S (area under the curve renal blood flow CsA 257 ± 93 versus control 477 ± 206 versus CsA + H2S 478 ± 271 mL/min/100 g.h; P = 0.024). Urine output was higher after 2 h of reperfusion in the CsA + H2S group (CsA + H2S 305 ± 218 versus CsA 78 ± 180 versus control 210 ± 45 mL; P = 0.034). CsA treatment was associated with an increase in tubular injury, which was not reversed by H2S (area under the curve fractional excretion of sodium, control 77 ± 53 versus CsA 100 ± 61 versus CsA + H2S 111 ± 57%.h; P = 0.003). Histologic evaluation showed significant vacuolation and glomerular shrinkage in the CsA group. These were significantly reduced by H2S (P = 0.005, 0.002). ConclusionsH2S reversed the vasoconstriction changes associated with CsA treatment during reperfusion.

Tetrahydrobiopterin Saves Pancreatic Isografts From Brain Death Exacerbated Ischemia Reperfusion Injury.

Background: Brain death (BD) has been shown to immunologically prime grafts in part by aggravating ischemia reperfusion injury (IRI). Herein we assessed the effects of BD on IRI in an experimental setting furthermore the therapeutic potential of tetrahydrobiopterin (BH4), an essential NOS-cofactor was tested. Methods and Materials: Pancreas transplantation was performed using C57BL/6-mice. Animals underwent BD induction and were followed for 3h. Experimental groups included: non-treated BD-donors, BD-donors treated with 50mg/kg BH4, ventilated non-treated donors and living donors. Following 2 hours of reperfusion, microcirculation (functional capillary density, FCD; capillary diameter, CD) and cell viability was assessed by intravital fluorescence microscopy. Parenchymal graft damage was assessed by histology, ROS were quantified by immunohistochemistry against nitrotyrosin and mRNA expression of inflammatory candidate markers was measured by real-time RT-PCR. Results: Compared with controls, BD exacerbated IRI reflected by significantly reduced FCD and CD values (p<0.05). Moreover BD induced IL-1ß, TNFa, IL-6 and ICAM-1 mRNA expression (p<0.05). In contrast treated grafts displayed significantly higher FCD and CD values (p<0.05). BD had devastating impact on cell viability whereas treatment resulted in significantly higher numbers of viable cells after reperfusion (p<0.01). Parenchymal damage in grafts from BD-donors was significantly more pronounced when compared to controls (p<0.05). Treatment resulted in significantly better histology (p<0.05). Nitrotyrosin immunostaining showed significantly higher score values in grafts from BD donors when compared to BH4 treated pancreata (p<0.05). Conclusion: Our data gain new insights into the impact of BD on pancreatic grafts. Donor pre-treatment with BH4 offers a novel option for preventing BD exacerbated IRI.

PACAP-Mediated Neuromodulation Ameliorates Liver Ischemia-Reperfusion Injury Via Autophagy Signaling Pathway.

Background: Consistent with the novel concept of immune - nervous system communication, we have recently shown that treatment of WT mice with neuropeptide PACAP (Pituitary Adenylate Cyclase-Activating Polypeptides) ameliorated hepatic ischemia/reperfusion injury (IRI), whereas PACAP KO mice suffered exacerbated IRI, compared with WT counterparts. Aim: To study putative mechanisms by which PACAP-neuromodulation promotes cytoprotective phenotype and mitigates hepatocellular damage in IR-stressed livers. Methods&Results: In a mouse model of liver warm ischemia (90min) followed by reperfusion (0-24h), both macrophage and autophagy activation peaked at 6h of reperfusion, coinciding with the maximal IR-hepatocellular damage. Preconditioning of mouse recipients with neuropeptide PACAP: 1/ ameliorated liver IRI, evidenced by reduced sALT levels and well-preserved hepatic architecture; 2/ enhanced hepatic autophagy, consistent with increased local LC-3a/Beclin-1 expression, and accompanied by up-regulation of liver regeneration HGF (Hepatocyte Growth Factor) and c-Met signaling; 3/ diminished intrahepatic CD68+ macrophage sequestration; 4/ decreased proinflammatory cytokine/chemokine program (TNF-α, IL-1β, IFN-β and CXCL10) yet enhanced IL-10 expression (8-folds vs. controls) in IR-stressed livers. Strikingly, PACAP-facilitated cytoprotection was abolished and IR-hepatocellular injury fully restored after adjunctive treatment with a chemical autophagy inhibitor (3-MA). In parallel in vitro studies, neuropeptide PACAP-induced autophagy ameliorated hydrogen peroxide (H2O2)-triggered necrosis in primary hepatocyte cultures. The regulatory function of PACAP on hepatocytes was confirmed by decreased LDH release, increased conversion of LC3-I/LC3-II, and abundant visualized endogenous LC3 punctae. In addition, PACAP supplement in vitro suppressed LPS-stimulated macrophage activation, evidenced by depressed TNF-α/IL-6/IL-12 yet increased IL-10 elaboration. Conclusion: Treatment with neuropeptide PACAP prevented tissue damage and promoted cellular homeostasis in IR-stressed mouse livers by: 1) enhancing hepatocellular autophagy; and 2) diminishing macrophage sequestration/activation. Because the enhancement of neural modulation differentially regulates local innate inflammation and hepatocyte survival, these results provide the rationale for novel neuropeptide-based approaches to manage liver IRI in transplant patients.

Human heat shock protein 27 exacerbates ischemia reperfusion injury in rats by reducing the number of T regulatory cells

Ischemia reperfusion injury (IRI) occurs in almost every liver surgery and is associated with the reduction of the liver blood flow. Ischemia impairs liver function and can even cause liver failure following surgery. The present study aimed to identify a new molecular target allowing the reduction of IRI and explore the related cellular mechanism. Adenovirus (~2.5x10(12) viral particles) bearing the human heat shock protein 27 (HSP27) gene was injected into rat liver through the ileocecal vein. Five days following the injection, ischemia was induced by clamping the median and left portal veins, hepatic arteries and bile ducts. The levels of alanine transaminase (ALT), aspartate aminotransferase (AST), glutathione (GSH) and superoxide dismutase (SOD) were measured. The infiltration of inflammatory cells and the expression of pro-inflammatory factors were investigated. The number of regulatory T cells (Tregs) was measured by flow cytometry. At 2 h following reperfusion, the group injected with HSP27 had the highest level of ALT and AST, followed by the group injected with HSP27 and treated with gadolinium trichloride (GdCl3), the empty vector-injected and the vector+GdCl3 groups. The HSP27 group also had the lowest levels of the oxidative stress-protective factors SOD and GSH, and the highest levels of pro-inflammatory factors. The number of Tregs was reduced in the groups injected with HSP27. In conclusion, the human HSP27 protein can effectively accelerate liver damage at the early stages of IRI in rats. Tregs might play a critical role in HSP27‑induced liver injury.

KEAP1-NRF2 complex in ischemia-induced hepatocellular damage of mouse liver transplants

The Keap1-Nrf2 signaling pathway regulates host cell defense responses against oxidative stress and maintains the cellular redox balance. We investigated the function/molecular mechanisms by which Keap1-Nrf2 complex may influence liver ischemia/reperfusion injury (IRI) in a mouse model of hepatic cold storage (20h at 4°C) followed by orthotopic liver transplantation (OLT). The Keap1 hepatocyte-specific knockout (HKO) in the donor liver ameliorated post-transplant IRI, evidenced by improved hepatocellular function and OLT outcomes (Keap1 HKO→Keap1 HKO; 100% survival), as compared with controls (WT→WT; 50% survival; p<0.01). By contrast, donor liver Nrf2 deficiency exacerbated IRI in transplant recipients (Nrf2 KO→Nrf2 KO; 40% survival). Ablation of Keap1 signaling reduced macrophage/neutrophil trafficking, pro-inflammatory cytokine programs, and hepatocellular necrosis/apoptosis, while simultaneously promoting anti-apoptotic functions in OLTs. At the molecular level, Keap1 HKO increased Nrf2 levels, stimulated Akt phosphorylation, and enhanced expression of anti-oxidant Trx1, HIF-1α, and HO-1. Pretreatment of liver donors with PI3K inhibitor (LY294002) disrupted Akt/HIF-1A signaling and recreated hepatocellular damage in otherwise IR-resistant Keap1 HKO transplants. In parallel in vitro studies, hydrogen peroxide-stressed Keap1-deficient hepatocytes were characterized by enhanced expression of Nrf2, Trx1, and Akt phosphorylation, in association with decreased release of lactate dehydrogenase (LDH) in cell culture supernatants. Keap1-Nrf2 complex prevents oxidative injury in IR-stressed OLTs through Keap1 signaling, which negatively regulates Nrf2 pathway. Activation of Nrf2 induces Trx1 and promotes PI3K/Akt, crucial for HIF-1α activity. HIF-1α-mediated overexpression of HO-1/Cyclin D1 facilitates cytoprotection by limiting hepatic inflammatory responses, and hepatocellular necrosis/apoptosis in a PI3K-dependent manner.

Dendritic Cell Sphingosine 1-Phosphate Receptor-3 Regulates Th1–Th2 Polarity in Kidney Ischemia–Reperfusion Injury

Dendritic cells (DCs) are central to innate and adaptive immunity of early kidney ischemia-reperfusion injury (IRI), and strategies to alter DC function may provide new therapeutic opportunities. Sphingosine 1-phosphate (S1P) modulates immunity through binding to its receptors (S1P1-5), and protection from kidney IRI occurs in S1P3-deficient mice. Through a series of experiments we determined that this protective effect was owing in part to differences between S1P3-sufficient and -deficient DCs. Mice lacking S1P3 on bone marrow cells were protected from IRI, and S1P3-deficient DCs displayed an immature phenotype. Wild-type (WT) but not S1P3-deficient DCs injected into mice depleted of DCs prior to kidney IR reconstituted injury. Adoptive transfer (i.e., i.v. injection) of glycolipid (Ag)-loaded WT but not S1P3-deficient DCs into WT mice exacerbated IRI, suggesting that WT but not S1P3-deficient DCs activated NKT cells. Whereas WT DC transfers activated the Th1/IFN-γ pathway, S1P3-deficient DCs activated the Th2/IL-4 pathway, and an IL-4-blocking Ab reversed protection from IRI, supporting the concept that IL-4 mediates the protective effect of S1P3-deficient DCs. Administration of S1P3-deficient DCs 7 d prior to or 3 h after IRI protected mice from IRI and suggests their potential use in cell-based therapy. We conclude that absence of DC S1P3 prevents DC maturation and promotes a Th2/IL-4 response. These findings highlight the importance of DC S1P3 in modulating NKT cell function and IRI and support development of selective S1P3 antagonists for tolerizing DCs for cell-based therapy or for systemic administration for the prevention and treatment of IRI and autoimmune diseases.