Deletion of CD39 on natural killer cells attenuates hepatic ischemia/reperfusion injury in mice

Natural killer (NK) cells play crucial roles in innate immunity and express CD39 (Ecto-nucleoside triphosphate diphosphohydrolase 1 [E-NTPD1]), a rate-limiting ectonucleotidase in the phosphohydrolysis of extracellular nucleotides to adenosine. We have studied the effects of CD39 gene deletion on NK cells in dictating outcomes after partial hepatic ischemia/reperfusion injury (IRI). We show in mice that gene deletion of CD39 is associated with marked decreases in phosphohydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate on NK cells, thereby modulating the type-2 purinergic (P2) receptors demonstrated on these cells. We note that CD39-null mice are protected from acute vascular injury after single-lobe warm IRI, and, relative to control wild-type mice, display significantly less elevation of aminotransferases with less pronounced histopathological changes associated with IRI. Selective adoptive transfers of immune cells into Rag2/common gamma null mice (deficient in T cells, B cells, and NK/NKT cells) suggest that it is CD39 deletion on NK cells that provides end-organ protection, which is comparable to that seen in the absence of interferon gamma. Indeed, NK effector mechanisms such as interferon gamma secretion are inhibited by P2 receptor activation in vitro. Specifically, ATPgammaS (a nonhydrolyzable ATP analog) inhibits secretion of interferon gamma by NK cells in response to interleukin-12 and interleukin-18, providing a mechanistic link between CD39 deletion and altered cytokine secretion. CONCLUSION: We propose that CD39 deficiency and changes in P2 receptor activation abrogate secretion of interferon gamma by NK cells in response to inflammatory mediators, thereby limiting tissue damage mediated by these innate immune cells during IRI.

Level of G protein-coupled receptor kinase-2 determines myocardial ischemia/reperfusion injury via pro- and anti-apoptotic mechanisms

Activation of prosurvival kinases and subsequent nitric oxide (NO) production by certain G protein-coupled receptors (GPCRs) protects myocardium in ischemia/reperfusion injury (I/R) models. GPCR signaling pathways are regulated by GPCR kinases (GRKs), and GRK2 has been shown to be a critical molecule in normal and pathological cardiac function.

Ablation of the tumor suppressor histidine triad nucleotide binding protein 1 is protective against hepatic ischemia/reperfusion injury

The identification of cellular pathways capable of limiting ischemia/reperfusion (I/R) injury remains a frontier in medicine, and its clinical relevance is urgent. Histidine triad nucleotide binding protein 1 (HINT1) is a tumor suppressor that influences apoptosis. Because apoptotic pathways are a feature of I/R injury, we asked whether Hint1 influences hepatic I/R injury. Hint1(-/-) and C57BL/6 mice were subjected to 70% liver ischemia followed by reperfusion for 3 or 24 hours or to a sham operation. The serum aminotransferase levels, histological lesions, apoptosis, reactive oxygen species, and expression of B cell lymphoma 2-associated X protein (Bax), heme oxygenase 1 (HO-1), interleukin-6 (IL-6), IL-10, tumor necrosis factor-a, Src, nuclear factor kappa B (p65/RelA), and c-Jun were quantified. The responses to toll-like receptor ligands and nicotinamide adenine dinucleotide phosphate oxidase activity in Kupffer cells were compared in Hint1(-/-) mice and C57BL/6 mice. After I/R, the levels of serum aminotransferases, parenchymal necrosis, and hepatocellular apoptosis were significantly lower in Hint1(-/-) mice versus control mice. Furthermore, Bax expression decreased more than 2-fold in Hint1(-/-) mice, and the increases in reactive oxygen species and HO-1 expression that were evident in wild-type mice after I/R were absent in Hint1(-/-) mice. The phosphorylation of Src and the nuclear translocation of p65 were increased in Hint1(-/-) mice, whereas the nuclear expression of phosphorylated c-Jun was decreased. The levels of the protective cytokines IL-6 and IL-10 were increased in Hint1(-/-) mice. These effects increased survival after I/R in mice lacking Hint1. Hint1(-/-) Kupffer cells were less activated than control cells after stimulation with lipopolysaccharides. CONCLUSION: The Hint1 protein influences the course of I/R injury, and its ablation in Kupffer cells may limit the extent of the injury.

Ischemia/reperfusion injury of porcine limbs after extracorporeal perfusion

Revascularization of amputated extremities after prolonged ischemia is complicated by reperfusion injury. We assessed ischemia/reperfusion (I/R) injury of porcine extremities after prolonged preservation using extracorporeal circulation (ECC).

Exogenous surfactant application in a rat lung ischemia reperfusion injury model: effects on edema formation and alveolar type II cells

BACKGROUND: Prophylactic exogenous surfactant therapy is a promising way to attenuate the ischemia and reperfusion (I/R) injury associated with lung transplantation and thereby to decrease the clinical occurrence of acute lung injury and acute respiratory distress syndrome. However, there is little information on the mode by which exogenous surfactant attenuates I/R injury of the lung. We hypothesized that exogenous surfactant may act by limiting pulmonary edema formation and by enhancing alveolar type II cell and lamellar body preservation. Therefore, we investigated the effect of exogenous surfactant therapy on the formation of pulmonary edema in different lung compartments and on the ultrastructure of the surfactant producing alveolar epithelial type II cells. METHODS: Rats were randomly assigned to a control, Celsior (CE) or Celsior + surfactant (CE+S) group (n = 5 each). In both Celsior groups, the lungs were flush-perfused with Celsior and subsequently exposed to 4 h of extracorporeal ischemia at 4 degrees C and 50 min of reperfusion at 37 degrees C. The CE+S group received an intratracheal bolus of a modified natural bovine surfactant at a dosage of 50 mg/kg body weight before flush perfusion. After reperfusion (Celsior groups) or immediately after sacrifice (Control), the lungs were fixed by vascular perfusion and processed for light and electron microscopy. Stereology was used to quantify edematous changes as well as alterations of the alveolar epithelial type II cells. RESULTS: Surfactant treatment decreased the intraalveolar edema formation (mean (coefficient of variation): CE: 160 mm3 (0.61) vs. CE+S: 4 mm3 (0.75); p < 0.05) and the development of atelectases (CE: 342 mm3 (0.90) vs. CE+S: 0 mm3; p < 0.05) but led to a higher degree of peribronchovascular edema (CE: 89 mm3 (0.39) vs. CE+S: 268 mm3 (0.43); p < 0.05). Alveolar type II cells were similarly swollen in CE (423 microm3(0.10)) and CE+S (481 microm3(0.10)) compared with controls (323 microm3(0.07); p < 0.05 vs. CE and CE+S). The number of lamellar bodies was increased and the mean lamellar body volume was decreased in both CE groups compared with the control group (p < 0.05). CONCLUSION: Intratracheal surfactant application before I/R significantly reduces the intraalveolar edema formation and development of atelectases but leads to an increased development of peribronchovascular edema. Morphological changes of alveolar type II cells due to I/R are not affected by surfactant treatment. The beneficial effects of exogenous surfactant therapy are related to the intraalveolar activity of the exogenous surfactant.

Temporary hypoxic stress protects the liver from Fas-mediated apoptosis and ischemia reperfusion injury

Molecular responses to hypoxia restore oxygen homeostasis and promote cell survival, and are mainly regulated through the activation of the hypoxia-inducible transcription factor (HIF)-1 and its target genes. In this study we questioned whether surgically depleting the liver s arterial blood supply, by clamping the hepatic artery (HA), would be sufficient to mount a hypoxia-driven molecular response, the up-regulation of hepatoprotective genes and thereby protect the liver from subsequent damaging insults.;;The HA of normal male Balb/c mice was clamped with a micro vascular clip for 2 hours. The liver s saturated oxygen concentration (SO2) was measured using an O2C surface probe (LEA-Medizintechnik) and interstitial fluid was collected with microdialysis membranes to monitor tissue damage. Mice without clamping served as sham operated controls. Interstitial fluid was assessed for lactate pyruvate (L/P) and glycerol content and the mRNA of hepatoprotective genes was analyzed by real time PCR. Subsequently, mice received either a tail vein injection of anti-Fas antibody (Jo2, 0.2 mg/kg) or the liver was made ischemic (60min) followed by 6 hours reperfusion. Caspase 3-activity and cleaved lamin A were used to assess apoptosis. In separate groups, animal were monitored for survival.;;After 30min of clamping the HA the SO2 of the liver decreased and remained at a reduced level for up to 2 hours, without an increase in L/P ratio or glycerol release. We demonstrate the activation of a hypoxia-inducible signaling pathway by the stabilization of HIF-1 protein (Western blot) and by an increase of its target gene, Epo, mRNA. There was an up-regulation of the hepatoprotective genes IL-6, IGFBP-1, HO-1 and A20 mRNA. When subsequently injected with Jo2, animals preconditioned with HA clamping, had a significantly decreased caspase-3 activity (avg21044 vs. avg3637; p=0.001, T-test) and there were fewer positive cells for cleaved Lamin A. The survival probability (10.5 hours, n=12) of mice with HA clamping was significantly higher (3.2 hours, n=13; p=0.014, Logrank test). Likewise, survival after 60 minutes of partial hepatic ischemia and 6 hours of reperfusion was reduced from 86% in mice with pretreatment by HA clamping to 56% in sham treated controls.;;This study demonstrates that a localized hypoxic stress can be achieved by surgically removing the livers arterial blood supply. Furthermore it can stimulate a hepatoprotective response that protects the liver against Fas-mediated apoptosis and ischemia-reperfusion injury. Our findings offer an innovative approach to induce hepatoprotective genes to defend the liver against subsequent insults.

Helium breathing provides modest antiinflammatory, but no endothelial protection against ischemia-reperfusion injury in humans in vivo

BACKGROUND: The noble gas helium is devoid of anesthetic effects, and it elicits cardiac preconditioning. We hypothesized that inhalation of helium provides protection against postocclusive endothelial dysfunction after ischemia-reperfusion of the forearm in humans. METHODS: Eight healthy male subjects were enrolled in this study with a crossover design. Each volunteer was randomly exposed to 15 min of forearm ischemia in the presence or absence of helium inhalation. Helium was inhaled at an end-tidal concentration of 50 vol% from 15 min before ischemia until 5 min after the onset of reperfusion ("helium conditioning"). Hyperemic reaction, a marker of nitric oxide bioavailability and endothelial function, was determined at 15 and 30 min of reperfusion on the forearm using venous occlusion plethysmography. Expression of the proinflammatory markers CD11b, ICAM-1, PSGL-1, and L-selectin (CD62L) on leukocytes and P-selectin (CD62P), PSGL-1, and CD42b on platelets were measured by flow cytometry during reperfusion. RESULTS: Ischemia-reperfusion consistently reduced the postocclusive endothelium-dependent hyperemic reaction at 15 and 30 min of reperfusion. Periischemic inhalation of helium at 50 vol% did not improve postocclusive hyperemic reaction. Helium decreased expression of the proinflammatory marker CD11b and ICAM-1 on leukocytes and attenuated the expression of the procoagulant markers CD42b and PSGL-1 on platelets. CONCLUSIONS: Although inhalation of helium diminished the postischemic inflammatory reaction, our data indicate that human endothelium, which is a component of all vital organs, is not amenable to protection by helium at 50 vol% in vivo. This is in contrast to sevoflurane, which protects human endothelium at low subanesthetic concentrations.

Myocardial Ablation of G Protein-Coupled Receptor Kinase 2 (GRK2) Decreases Ischemia/Reperfusion Injury through an Anti-Intrinsic Apoptotic Pathway

Studies from our lab have shown that decreasing myocardial G protein-coupled receptor kinase 2 (GRK2) activity and expression can prevent heart failure progression after myocardial infarction. Since GRK2 appears to also act as a pro-death kinase in myocytes, we investigated the effect of cardiomyocyte-specific GRK2 ablation on the acute response to cardiac ischemia/reperfusion (I/R) injury. To do this we utilized two independent lines of GRK2 knockout (KO) mice where the GRK2 gene was deleted in only cardiomyocytes either constitutively at birth or in an inducible manner that occurred in adult mice prior to I/R. These GRK2 KO mice and appropriate control mice were subjected to a sham procedure or 30 min of myocardial ischemia via coronary artery ligation followed by 24 hrs reperfusion. Echocardiography and hemodynamic measurements showed significantly improved post-I/R cardiac function in both GRK2 KO lines, which correlated with smaller infarct sizes in GRK2 KO mice compared to controls. Moreover, there was significantly less TUNEL positive myocytes, less caspase-3, and -9 but not caspase-8 activities in GRK2 KO mice compared to control mice after I/R injury. Of note, we found that lowering cardiac GRK2 expression was associated with significantly lower cytosolic cytochrome C levels in both lines of GRK2 KO mice after I/R compared to corresponding control animals. Mechanistically, the anti-apoptotic effects of lowering GRK2 expression were accompanied by increased levels of Bcl-2, Bcl-xl, and increased activation of Akt after I/R injury. These findings were reproduced in vitro in cultured cardiomyocytes and GRK2 mRNA silencing. Therefore, lowering GRK2 expression in cardiomyocytes limits I/R-induced injury and improves post-ischemia recovery by decreasing myocyte apoptosis at least partially via Akt/Bcl-2 mediated mitochondrial protection and implicates mitochondrial-dependent actions, solidifying GRK2 as a pro-death kinase in the heart.

Inhibition of Fas-associated death domain-containing protein (FADD) protects against myocardial ischemia/reperfusion injury in a heart failure mouse model

AIM As technological interventions treating acute myocardial infarction (MI) improve, post-ischemic heart failure increasingly threatens patient health. The aim of the current study was to test whether FADD could be a potential target of gene therapy in the treatment of heart failure. METHODS Cardiomyocyte-specific FADD knockout mice along with non-transgenic littermates (NLC) were subjected to 30 minutes myocardial ischemia followed by 7 days of reperfusion or 6 weeks of permanent myocardial ischemia via the ligation of left main descending coronary artery. Cardiac function were evaluated by echocardiography and left ventricular (LV) catheterization and cardiomyocyte death was measured by Evans blue-TTC staining, TUNEL staining, and caspase-3, -8, and -9 activities. In vitro, H9C2 cells transfected with ether scramble siRNA or FADD siRNA were stressed with chelerythrin for 30 min and cleaved caspase-3 was assessed. RESULTS FADD expression was significantly decreased in FADD knockout mice compared to NLC. Ischemia/reperfusion (I/R) upregulated FADD expression in NLC mice, but not in FADD knockout mice at the early time. FADD deletion significantly attenuated I/R-induced cardiac dysfunction, decreased myocardial necrosis, and inhibited cardiomyocyte apoptosis. Furthermore, in 6 weeks long term permanent ischemia model, FADD deletion significantly reduced the infarct size (from 41.20 ± 3.90% in NLC to 26.83 ± 4.17% in FADD deletion), attenuated myocardial remodeling, improved cardiac function and improved survival. In vitro, FADD knockdown significantly reduced chelerythrin-induced the level of cleaved caspase-3. CONCLUSION Taken together, our results suggest FADD plays a critical role in post-ischemic heart failure. Inhibition of FADD retards heart failure progression. Our data supports the further investigation of FADD as a potential target for genetic manipulation in the treatment of heart failure.

Slit2 prevents neutrophil recruitment and renal ischemia-reperfusion injury.

Neutrophils recruited to the postischemic kidney contribute to the pathogenesis of ischemia-reperfusion injury (IRI), which is the most common cause of renal failure among hospitalized patients. The Slit family of secreted proteins inhibits chemotaxis of leukocytes by preventing activation of Rho-family GTPases, suggesting that members of this family might modulate the recruitment of neutrophils and the resulting IRI. Here, in static and microfluidic shear assays, Slit2 inhibited multiple steps required for the infiltration of neutrophils into tissue. Specifically, Slit2 blocked the capture and firm adhesion of human neutrophils to inflamed vascular endothelial barriers as well as their subsequent transmigration. To examine whether these observations were relevant to renal IRI, we administered Slit2 to mice before bilateral clamping of the renal pedicles. Assessed at 18 hours after reperfusion, Slit2 significantly inhibited renal tubular necrosis, neutrophil and macrophage infiltration, and rise in plasma creatinine. In vitro, Slit2 did not impair the protective functions of neutrophils, including phagocytosis and superoxide production, and did not inhibit neutrophils from killing the extracellular pathogen Staphylococcus aureus. In vivo, administration of Slit2 did not attenuate neutrophil recruitment or bacterial clearance in mice with ascending Escherichia coli urinary tract infections and did not increase the bacterial load in the livers of mice infected with the intracellular pathogen Listeria monocytogenes. Collectively, these results suggest that Slit2 may hold promise as a strategy to combat renal IRI without compromising the protective innate immune response.

C1 esterase inhibitor reduces lower extremity ischemia/reperfusion injury and associated lung damage

BACKGROUND Ischemia/reperfusion injury of lower extremities and associated lung damage may result from thrombotic occlusion, embolism, trauma, or surgical intervention with prolonged ischemia and subsequent restoration of blood flow. This clinical entity is characterized by high morbidity and mortality. Deprivation of blood supply leads to molecular and structural changes in the affected tissue. Upon reperfusion inflammatory cascades are activated causing tissue injury. We therefore tested preoperative treatment for prevention of reperfusion injury by using C1 esterase inhibitor (C1 INH). METHODS AND FINDINGS Wistar rats systemically pretreated with C1 INH (n = 6), APT070 (a membrane-targeted myristoylated peptidyl construct derived from human complement receptor 1, n = 4), vehicle (n = 7), or NaCl (n = 8) were subjected to 3h hind limb ischemia and 24h reperfusion. The femoral artery was clamped and a tourniquet placed under maintenance of a venous return. C1 INH treated rats showed significantly less edema in muscle (P<0.001) and lung and improved muscle viability (P<0.001) compared to controls and APT070. C1 INH prevented up-regulation of bradykinin receptor b1 (P<0.05) and VE-cadherin (P<0.01), reduced apoptosis (P<0.001) and fibrin deposition (P<0.01) and decreased plasma levels of pro-inflammatory cytokines, whereas deposition of complement components was not significantly reduced in the reperfused muscle. CONCLUSIONS C1 INH reduced edema formation locally in reperfused muscle as well as in lung, and improved muscle viability. C1 INH did not primarily act via inhibition of the complement system, but via the kinin and coagulation cascade. APT070 did not show beneficial effects in this model, despite potent inhibition of complement activation. Taken together, C1 INH might be a promising therapy to reduce peripheral ischemia/reperfusion injury and distant lung damage in complex and prolonged surgical interventions requiring tourniquet application.

Refinement of tourniquet-induced peripheral ischemia/reperfusion injury in rats: comparison of 2 h vs 24 h reperfusion.

Prolonged ischemia of skeletal muscle tissue, followed by reperfusion, leads to ischemia/reperfusion injury (IRI), which is a feared local and systemic inflammatory reaction. With respect to the 3Rs, we wanted to determine which parameters for assessment of IRI require a reperfusion time of 24 h and for which 2 h of reperfusion are sufficient. Rats were subjected to 3 h of hind limb ischemia and 2 h or 24 h of reperfusion. Human plasma derived C1 inhibitor was used as a drug to prevent reperfusion injury. For 2 h of reperfusion the rats stayed under anesthesia throughout (severity grade 1), whereas for 24 h they were awake under analgesia during reperfusion (grade 2). The femoral artery was clamped and a tourniquet was placed, under maintenance of venous return. C1 esterase inhibitor was systemically administered 5 min before the induction of ischemia. No differences in local muscle edema formation and depositions of immunoglobulin G and immunoglobulin M were observed between 2 h and 24 h (P > 0.05), whereas lung edema was only observed after 24 h. Muscle viability was significantly lower after 24 h vs 2 h reperfusion (P < 0.05). Increased plasma creatine kinase (CK)-MM and platelet-derived growth factor (PDGF)-bb could be detected after 2 h, but not after 24 h of reperfusion. By contrast, depositions of C3b/c and fibrin in muscle were only detected after 24 h (P < 0.001). In conclusion, for a first screening of drug candidates to reduce IRI, 2 h reperfusions are sufficient, and these reduce the severity of the animal experiment. Twenty-four-hour reperfusions are only needed for in-depth analysis of the mechanisms of IRI, including lung damage.