Inhibition of Syk activity by R788 in platelets prevents remote lung tissue damage after mesenteric ischemia-reperfusion injury

Tissue injury following ischemia-reperfusion (I/R) occurs as a consequence of actions of soluble factors and immune cells. Growing evidence supports a role for platelets in the manifestation of tissue damage following I/R. Spleen tyrosine kinase has been well documented to be important in lymphocyte activation and more recently in platelet activation. We performed experiments to evaluate whether inhibition of platelet activation through inhibition of spleen tyrosine kinase prevents tissue damage after mesenteric I/R injury. Platelets isolated from C57BL/6J mice fed with R788 for 10 days were transfused into C57BL/6J mice depleted of platelets 2 days before mesenteric I/R injury. Platelet-depleted mice transfused with platelets from R788-treated mice before mesenteric I/R displayed a significant reduction in the degree of remote lung damage, but with little change in the degree of local intestinal damage compared with control I/R mice. Transfusion of R788-treated platelets also decreased platelet sequestration, C3 deposition, and immunoglobulin deposition in lung, but not in the intestine, compared with control groups. These findings demonstrate that platelet activation is a requisite for sequestration in the pulmonary vasculature to mediate remote tissue injury after mesenteric I/R. The use of small-molecule inhibitors may be valuable to prevent tissue damage in remote organs following I/R injury.     

Platelet-associated CD40/CD154 mediates remote tissue damage after mesenteric ischemia/reperfusion injury

Several innate and adaptive immune cell types participate in ischemia/reperfusion induced tissue injury. Amongst them, platelets have received little attention as contributors in the process of tissue damage after ischemia reperfusion (I/R) injury. It is currently unknown whether platelets participate through the immunologically important molecules including, CD40 and when activated, CD154 (CD40L), in the pathogenesis of I/R injury. We hypothesized that constitutive expression of CD40 and activation-induced expression of CD154 on platelets mediate local mesenteric and remote lung tissue damage after I/R injury. Wild type (WT; C57BL/6J), CD40 and CD154 deficient mice underwent mesenteric ischemia for 30 minutes followed by reperfusion for 3 hours. WT mice subjected to mesenteric I/R injury displayed both local intestinal and remote lung damage. In contrast, there was significantly less intestinal damage and no remote lung injury in CD40 and CD154 deficient mice when compared to WT mice. Platelet-depleted WT mice transfused with platelets from CD40 or CD154 deficient mice failed to reconstitute remote lung damage. In contrast, when CD40 or CD154 deficient mice were transfused with WT platelets lung tissue damage was re-established. Together, these findings suggest that multiple mechanisms are involved in local and remote tissue injury and also identify platelet-expressed CD40 and/or CD154 as mediators of remote tissue damage.     

Fas-independent mitochondrial damage triggers cardiomyocyte death after ischemia-reperfusion

The Fas/Fas ligand and mitochondria pathways have been involved in cell death in several cell types. We combined the genetic inactivation of the Fas receptor (lpr mice), on the one hand, to the pharmacological inhibition of the mitochondrial permeability transition pore (mPTP), on the other hand, to investigate which of these pathways is predominantly activated during prolonged ischemia-reperfusion. Anesthetized C57BL/6JICO (control) and C57BL/6-lpr mice were pretreated with either saline or cyclosporin A (CsA; 40 mg/kg, 3 times a day), an inhibitor of the mPTP, and underwent 25 min of ischemia and 24 h of reperfusion. After 24 h of reperfusion, hearts were harvested: infarct size was assessed by 2,3,5-triphenyltetrazolium chloride staining, myocardial apoptosis by caspase 3 activity, and mitochondrial permeability transition by Ca2+-induced mPTP opening using a potentiometric approach. Infarct size was comparable in untreated control and lpr mice, ranging from 77 +/- 5% to 83 +/- 3% of the area at risk. CsA significantly reduced infarct size in control and lpr hearts. Control and lpr hearts exhibited comparable increase in caspase 3 activity that averaged 57 +/- 18 and 49 +/- 5 pmol x min(-1) x mg(-1), respectively. CsA treatment significantly reduced caspase 3 activity in control and lpr hearts. The Ca2+ overload required to open the mPTP was decreased to a similar extent in lpr and controls. CsA significantly attenuated Ca2+-induced mPTP opening in both groups. Our results suggest that the Fas pathway likely plays a minor role, whereas mitochondria are preferentially involved in mice cardiomyocyte death after a lethal ischemia-reperfusion injury.     

Cell-type-specific Hox regulatory strategies orchestrate tissue identity

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The role of platelet factor 4 in local and remote tissue damage in a mouse model of mesenteric ischemia/reperfusion injury

The robust inflammatory response that occurs during ischemia reperfusion (IR) injury recruits factors from both the innate and adaptive immune systems. However the contribution of platelets and their products such as Platelet Factor 4 (PF4; CXCL4), during the pathogenesis of IR injury has not been thoroughly investigated. We show that a deficiency in PF4 protects mice from local and remote tissue damage after 30 minutes of mesenteric ischemia and 3 hours of reperfusion in PF4-/- mice compared to control B6 mice. This protection was independent from Ig or complement deposition in the tissues. However, neutrophil and monocyte infiltration were decreased in the lungs of PF4-/- mice compared with B6 control mice. Platelet-depleted B6 mice transfused with platelets from PF4-/- mice displayed reduced tissue damage compared with controls. In contrast, transfusion of B6 platelets into platelet depleted PF4-/- mice reconstituted damage in both intestine and lung tissues. We also show that PF4 may modulate the release of IgA. Interestingly, we show that PF4 expression on intestinal epithelial cells is increased after IR at both the mRNA and protein levels. In conclusion, these findings demonstrate that may PF4 represent an important mediator of local and remote tissue damage.     

Alpha-melanocyte-stimulating hormone protects against mesenteric ischemia-reperfusion injury

Mesenteric ischemia-reperfusion (I/R) injury to the intestine is a common and often devastating clinical occurrence for which there are few therapeutic options. alpha-Melanocyte-stimulating hormone (alpha-MSH) is a tridecapeptide released by the pituitary gland and immunocompetent cells that exerts anti-inflammatory actions and abrogates postischemic injury to the kidneys and brainstem of rodents. To test the hypothesis that alpha-MSH would afford similar protection in the postischemic small intestine, we analyzed the effects of this peptide on intestinal transit, histology, myeloperoxidase activity, and nuclear factor-kappaB (NF-kappaB) activation after 45 min of superior mesenteric artery occlusion and 相似文献   

Orexigenic hormone ghrelin attenuates local and remote organ injury after intestinal ischemia-reperfusion

Background

Gut ischemia/reperfusion (I/R) injury is a serious condition in intensive care patients. Activation of immune cells adjacent to the huge endothelial cell surface area of the intestinal microvasculature produces initially local and then systemic inflammatory responses. Stimulation of the vagus nerve can rapidly attenuate systemic inflammatory responses through inhibiting the activation of macrophages and endothelial cells. Ghrelin, a novel orexigenic hormone, is produced predominately in the gastrointestinal system. Ghrelin receptors are expressed at a high density in the dorsal vagal complex of the brain stem. In this study, we investigated the regulation of the cholinergic anti-inflammatory pathway by the novel gastrointestinal hormone, ghrelin, after gut I/R.

Methods and Findings

Gut ischemia was induced by placing a microvascular clip across the superior mesenteric artery for 90 min in male adult rats. Our results showed that ghrelin levels were significantly reduced after gut I/R and that ghrelin administration inhibited pro-inflammatory cytokine release, reduced neutrophil infiltration, ameliorated intestinal barrier dysfunction, attenuated organ injury, and improved survival after gut I/R. Administration of a specific ghrelin receptor antagonist worsened gut I/R-induced organ injury and mortality. To determine whether ghrelin''s beneficial effects after gut I/R require the intact vagus nerve, vagotomy was performed in sham and gut I/R animals immediately prior to the induction of gut ischemia. Our result showed that vagotomy completely eliminated ghrelin''s beneficial effect after gut I/R. To further confirm that ghrelin''s beneficial effects after gut I/R are mediated through the central nervous system, intracerebroventricular administration of ghrelin was performed at the beginning of reperfusion after 90-min gut ischemia. Our result showed that intracerebroventricular injection of ghrelin also protected the rats from gut I/R injury.

Conclusions

These findings suggest that ghrelin attenuates excessive inflammation and reduces organ injury after gut I/R through activation of the cholinergic anti-inflammatory pathway.     

Platelets mediate cytotoxic T lymphocyte-induced liver damage

We found that platelet depletion reduces intrahepatic accumulation of virus-specific cytotoxic T lymphocytes (CTLs) and organ damage in mouse models of acute viral hepatitis. Transfusion of normal but not activation-blocked platelets in platelet-depleted mice restored accumulation of CTLs and severity of disease. In contrast, anticoagulant treatment that prevented intrahepatic fibrin deposition without reducing platelet counts did not avert liver injury. Thus, activated platelets contribute to CTL-mediated liver immunopathology independently of procoagulant function.     

Myocardial ischemia-reperfusion damage after pacing-induced tachycardia in patients with cardiac syndrome X

The presence of myocardial ischemia in syndrome X (chest pain, "ischemia-like" electrocardiogram changes, and normal coronary angiograms) is uncertain possibly because, when focally distributed, it may not cause contractile dysfunction or lactate production. We measured lipid hydroperoxides (ROOHs) and conjugated dienes (CDs), two sensitive, independent markers of ischemia-reperfusion oxidative stress, in paired aortic and great cardiac vein blood samples before and after pacing-induced tachycardia in nine patients with syndrome X. Diagnostic ischemic S-T segment changes during pacing were followed by a consistent increase in ROOH and CD levels in the great cardiac vein (from 4.83 +/- 1.18 micromol/l at baseline to 7.88 +/- 1.12 micromol/l and from 0.038 +/- 0.002 to 0.051 +/- 0.003 arbitrary units, respectively, P < 0.01). In controls, ROOH and CD levels did not change after pacing. The large postpacing cardiac release of lipid peroxidation products, consistently observed in all patients and similar to that previously observed after ischemia caused by percutaneous transluminal coronary angioplasty, is consistent with an ischemic origin of syndrome X.     

Galectins and TRIMs directly interact and orchestrate autophagic response to endomembrane damage

Macroautophagy/autophagy is a homeostatic process delivering cytoplasmic targets, including damaged organelles, to lysosomes for degradation; however, it is not completely understood how compromised endomembranes are recognized by the autophagic apparatus. We have described previously that the TRIM family of proteins act as receptors for selective autophagy. In this study we uncovered the property of TRIMs to directly interact with members of the family of cytosolic lectins termed galectins. Galectins patrol the cytoplasm and recognize compromised membranes. We show that TRIM16 uses LGALS3 (galectin 3) to detect damaged lysosomes and phagosomes. TRIM16 assembles the core autophagic machinery and is found in protein complexes with MTOR and TFEB, thus regulating their activity to set in motion endomembrane quality control. The TRIM16-LGALS3 system plays a key role in autophagic homeostasis of lysosomes and in the control of Mycobacterium tuberculosis in vivo.     

Effects of NF-kappa B inhibition on mesenteric ischemia-reperfusion injury

Mesenteric ischemia-reperfusion injury is a serious complication of shock. Because activation of nuclear factor-kappaB (NF-kappaB) has been implicated in this process, we treated rats with vehicle or the IkappaB-alpha inhibitor BAY 11-7085 (25 mg/kg ip) 1 h before mesenteric ischemia-reperfusion (45 min of ischemia followed by reperfusion at 30 min or 6 h) and examined the ileal injury response. Vehicle-treated rats subjected to ischemia-reperfusion exhibited severe mucosal injury, increased myeloperoxidase (MPO) activity, increased expression of interleukin-6 and intercellular adhesion molecule 1 protein, and a biphasic peak of NF-kappaB DNA-binding activity during the 30-min and 6-h reperfusion courses. In contrast, BAY 11-7085-pretreated rats subjected to ischemia-reperfusion exhibited less histological injury and less interleukin-6 and intercellular adhesion molecule 1 protein expression at 30 min of reperfusion but more histological injury at 6 h of reperfusion than vehicle-treated rats subjected to ischemia-reperfusion. Studies with phosphorylation site-specific antibodies demonstrated that IkappaB-alpha phosphorylation at Ser(32),Ser(36) was induced at 30 min of reperfusion, whereas tyrosine phosphorylation of IkappaB-alpha was induced at 6 h of reperfusion. BAY 11-7085 inhibited the former, but not the latter, phosphorylation pathway, whereas alpha-melanocyte-stimulating hormone, which is effective in limiting late ischemia-reperfusion injury to the intestine, inhibited tyrosine phosphorylation of IkappaB-alpha. Thus NF-kappaB appears to play an important role in the generation and resolution of intestinal ischemia-reperfusion injury through different activation pathways.     

Platelets activated by transient coronary occlusion exacerbate ischemia-reperfusion injury in rat hearts

Platelets (Plt) accumulate in reperfused myocardium but their effect on myocardial necrosis has not been established. We tested the hypothesis that the effect of Plt depends on their activation status. Pig Plt were obtained before 48 min of coronary occlusion (pre-CO-Plt), 10 min after reperfusion (R-Plt), or after a 60-min sham operation (sham-Plt). Plt were infused into isolated rat hearts (n = 124) and subsequently submitted to 60 min of ischemia and 60 min of reperfusion. P-selectin expression was higher (P = 0.02) in R-Plt than in pre-CO-Plt or sham-Plt. Lactate dehydrogenase (LDH) release during reperfusion was similar in hearts receiving pre-CO-Plt, sham-Plt, or no Plt, but R-Plt increased LDH release by 60% (P = 0.004). Activation of pre-CO-Plt with thrombin increased P-selectin expression and LDH release (P < 0.001), and these results were unaffected by tirofiban. There was a close correlation between P-selectin expression and LDH release (r = 0.84; P < 0.001), and myocardial Plt accumulation (r = 0.85; P < 0.001). We conclude that the deleterious effect of Plt on reperfused myocardium depends on their activation status as represented by P-selectin expression, which is enhanced by ischemia-reperfusion.     

Domain V peptides inhibit beta2-glycoprotein I-mediated mesenteric ischemia/reperfusion-induced tissue damage and inflammation

Reperfusion of ischemic tissue induces significant tissue damage in multiple conditions, including myocardial infarctions, stroke, and transplantation. Although not as common, the mortality rate of mesenteric ischemia/reperfusion (IR) remains >70%. Although complement and naturally occurring Abs are known to mediate significant damage during IR, the target Ags are intracellular molecules. We investigated the role of the serum protein, β2-glycoprotein I as an initiating Ag for Ab recognition and β2-glycoprotein I (β2-GPI) peptides as a therapeutic for mesenteric IR. The time course of β2-GPI binding to the tissue indicated binding and complement activation within 15 min postreperfusion. Treatment of wild-type mice with peptides corresponding to the lipid binding domain V of β2-GPI blocked intestinal injury and inflammation, including cellular influx and cytokine and eicosanoid production. The optimal therapeutic peptide (peptide 296) contained the lysine-rich region of domain V. In addition, damage and most inflammation were also blocked by peptide 305, which overlaps with peptide 296 but does not contain the lysine-rich, phospholipid-binding region. Importantly, peptide 296 retained efficacy after replacement of cysteine residues with serine. In addition, infusion of wild-type serum containing reduced levels of anti-β2-GPI Abs into Rag-1(-/-) mice prevented IR-induced intestinal damage and inflammation. Taken together, these data suggest that the serum protein β2-GPI initiates the IR-induced intestinal damage and inflammatory response and as such is a critical therapeutic target for IR-induced damage and inflammation.     

Alteration of Mrp2 and P-gp expression, including expression in remote organs, after intestinal ischemia-reperfusion

The present study was carried out in order to identify the changes in expression of multidrug resistance-associated protein (Mrp) 2 and P-glycoprotein (P-gp) in the intestine and remote organs after intestinal ischemia-reperfusion (I/R). Mrp2 expression in the jejunum and liver was decreased at 6 h after I/R. This decrease in Mrp2 expression was associated with an increase in the serum level of IL-6. These results suggest that the decreased Mrp2 expression after intestinal I/R was regulated by IL-6. The expression level of mdr1a in the ileum, which encodes P-gp, was decreased at 6 and 24 h after I/R, and the expression level of mdr1b, also encodes P-gp, was not altered at any time. P-gp protein expression in the ileum was decreased at 6 h after I/R. In the liver, mdr1a expression was decreased at 6 h after I/R, but mdr1b expression was increased at 6 h after I/R. P-gp protein was not altered at any time. In the kidney, mdr1a expression was decreased at 24 h after I/R, but mdr1b expression was not altered at any time. P-gp protein expression in the kidney was decreased at 24 h after I/R, as was mdr1a expression. These results suggest that P-gp expression after intestinal I/R differs in each organ. This is the first report to provide evidence that expression levels of transporters in remote organs are altered intestinal after I/R.     

Stimulation of autophagy by rapamycin protects neurons from remote degeneration after acute focal brain damage

Autophagy is the evolutionarily conserved degradation and recycling of cellular constituents. In mammals, autophagy is implicated in the pathogenesis of many neurodegenerative diseases. However, its involvement in acute brain damage is unknown. This study addresses the function of autophagy in neurodegeneration that has been induced by acute focal cerebellar lesions. We provide morphological, ultrastructural, and biochemical evidence that lesions in a cerebellar hemisphere activate autophagy in axotomized precerebellar neurons. Through time course analyses of the apoptotic cascade, we determined mitochondrial dysfunction to be the early trigger of degeneration. Further, the stimulation of autophagy by rapamycin and the employment of mice with impaired autophagic responses allowed us to demonstrate that autophagy protects from damage promoting functional recovery. These findings have therapeutic significance, demonstrating the potential of pro-autophagy treatments for acute brain pathologies, such as stroke and brain trauma.     

Stimulation of autophagy by rapamycin protects neurons from remote degeneration after acute focal brain damage

Autophagy is the evolutionarily conserved degradation and recycling of cellular constituents. In mammals, autophagy is implicated in the pathogenesis of many neurodegenerative diseases. However, its involvement in acute brain damage is unknown. This study addresses the function of autophagy in neurodegeneration that has been induced by acute focal cerebellar lesions. We provide morphological, ultrastructural, and biochemical evidence that lesions in a cerebellar hemisphere activate autophagy in axotomized precerebellar neurons. Through time course analyses of the apoptotic cascade, we determined mitochondrial dysfunction to be the early trigger of degeneration. Further, the stimulation of autophagy by rapamycin and the employment of mice with impaired autophagic responses allowed us to demonstrate that autophagy protects from damage promoting functional recovery. These findings have therapeutic significance, demonstrating the potential of pro-autophagy treatments for acute brain pathologies, such as stroke and brain trauma.     

Haemosiderin and tissue damage

High levels of haemosiderin occur in iron overload syndromes such as idiopathic haemochromatosis or secondary iron overload in thalassaemic patients; haemosiderin is the predominant iron-storage compound in such cases. It consists of a large aggregate of FeOOH cores, many of which have an incomplete shell of protein, and is probably derived from ferritin by lysosomal proteolysis. In addition, some chemical degradation of the ferritin cores appears to occur on conversion to haemosiderin. Other biochemical components are phosphate and magnesium, which may be adsorbed to the core surface, and perhaps certain lipids. Haemosiderin may have a central role, either directly or indirectly, in iron cytotoxicity and therefore the chemistry and biochemistry of this material warrants further study.     

Effect of leptin on renal ischemia-reperfusion damage in rats

Tumor necrosis factor-alpha (TNF-alpha) has been established as an important mediator in renal ischemia-reperfusion (I/R) injury. Leptin, a product of the ob gene, has been known to exhibit cytoprotective effects on renal tissue, but its effect on renal tissue TNF-alpha level after renal I/R injury in rats remains unknown. The purpose of the study was to evaluate the effects of leptin on renal tissue TNF-alpha, malondialdehyde (MDA), protein carbonyls (PCs) and total sulfydryl group (SH) levels, and plasma nitrite levels after renal I/R injury in rats. The animals were divided into three groups: control, I/R and I/R+leptin. Rats were subjected to renal ischemia by clamping the left pedicle for 45 min, and then reperfused for 1 h. The I/R+leptin group was pretreated intraperitoneally with leptin (10 microg/kg) 30 min before the induction of ischemia. Our results indicate that MDA, TNF-alpha levels, and PCs were significantly higher in the I/R group than those in the control group (p < 0.05). The administration of leptin decreased these parameters (p < 0.05) significantly. The SH level was observed to significantly decrease after I/R injury when compared to the control group (p < 0.05). Leptin treatment significantly increased tissue SH and plasma nitrite levels when compared to the I/R group (p < 0.05). Plasma nitrite levels did not change significantly in I/R when compared to the control. These results suggest that leptin could exert a protective effect on I/R induced renal damage by decreasing TNF-alpha levels and increasing nitrite level.     

Thematic review series: adipocyte biology. Adipose tissue function and plasticity orchestrate nutritional adaptation

This review focuses on adipose tissue biology and introduces the concept of adipose tissue plasticity and expandability as key determinants of obesity-associated metabolic dysregulation. This concept is fundamental to our understanding of adipose tissue as a dynamic organ at the center of nutritional adaptation. Here, we summarize the current knowledge of the mechanisms by which adipose tissue can affect peripheral energy homeostasis, particularly in the context of overnutrition. Two mechanisms emerge that provide a molecular understanding for obesity-associated insulin resistance. These are a) the dysregulation of adipose tissue expandability and b) the abnormal production of adipokines. This knowledge has the potential to pave the way for novel therapeutic concepts and strategies for managing and/or correcting complications associated with obesity and the metabolic syndrome.