Repercussões pulmonares após isquemia hepática parcial e reperfusão: modelo experimental

OBJETIVO: Descrever um modelo experimental de lesão de isquemia/reperfusão hepática com manifestações sistêmicas, representadas pelo envolvimento pulmonar, que possa ser utilizado por aqueles que pretendem compreender esse fenômeno. MÉTODOS: Ratos Wistar machos (200-250g) foram usados. Quatorze foram alocados em dois grupos, sendo G1 com oito submetidos somente à laparotomia e G2, seis à isquemia e reperfusão hepática. As funções hepática (aminotransferases séricas, respiração mitocondrial, histologia) e pulmonar (teste do azul de Evans) foram analisadas. RESULTADOS: houve diferença estatística significativa entre G1 e G2 ao se comparar valores de AST (24,3 ± 108 e 5406 ± 2263), ALT (88,5 ± 28,5 e 5169 ± 2690), razão de controle respiratório (3,41 ± 0,17 e 1,91 ± 0,55) e relação ADP/O (1,93 ± 0,03 e 1,45 ± 0,27), lesões histológicas (necrose, células inflamatórias, hemorragia, microesteatose) e teste do azul de Evans (194,31 ± 53 e 491,8 ± 141). CONCLUSÃO: O modelo mostrou-se útil para o estudo de lesão de isquemia/reperfusão hepática.

Cytoprotective role of heme oxygenase-1 and heme degradation derived end products in liver injury

The activation of heme oxygenase-1 (HO-1) appears to be an endogenous defensive mechanism used by cells to reduce inflammation and tissue damage in a number of injury models. HO-1, a stress-responsive enzyme that catabolizes heme into carbon monoxide (CO), biliverdin and iron, has previously been shown to protect grafts from ischemia/reperfusion and rejection. In addition, the products of the HO-catalyzed reaction, particularly CO and biliverdin/bilirubin, have been shown to exert protective effects in the liver against a number of stimuli, as in chronic hepatitis C and in transplanted liver grafts. Furthermore, the induction of HO-1 expression can protect the liver against damage caused by a number of chemical compounds. More specifically, the CO derived from HO-1-mediated heme catabolism has been shown to be involved in the regulation of inflammation; furthermore, administration of low concentrations of exogenous CO has a protective effect against inflammation. Both murine and human HO-1 deficiencies have systemic manifestations associated with iron metabolism, such as hepatic overload (with signs of a chronic hepatitis) and iron deficiency anemia (with paradoxical increased levels of ferritin). Hypoxia induces HO-1 expression in multiple rodent, bovine and monkey cell lines, but interestingly, hypoxia represses expression of the human HO-1 gene in a variety of human cell types (endothelial cells, epithelial cells, T cells). These data suggest that HO-1 and CO are promising novel therapeutic molecules for patients with inflammatory diseases. In this review, we present what is currently known regarding the role of HO-1 in liver injuries and in particular, we focus on the implications of targeted induction of HO-1 as a potential therapeutic strategy to protect the liver against chemically induced injury.

Effect of hypoxia and hyperglycemia on cell bioenergetics

Mitochondria have a central role in energy supply in cells, ROS production and apoptosis and have been implicated in several human disease and mitochondrial dysfunctions in hypoxia have been related with disorders like Type II Diabetes, Alzheimer Disease, inflammation, cancer and ischemia/reperfusion in heart. When oxygen availability becomes limiting in cells, mitochondrial functions are modulated to allow biologic adaptation. Cells exposed to a reduced oxygen concentration readily respond by adaptive mechanisms to maintain the physiological ATP/ADP ratio, essential for their functions and survival. In the beginning, the AMP-activated protein kinase (AMPK) pathway is activated, but the responsiveness to prolonged hypoxia requires the stimulation of hypoxia-inducible factors (HIFs). In this work we report a study of the mitochondrial bioenergetics of primary cells exposed to a prolonged hypoxic period . To shine light on this issue we examined the bioenergetics of fibroblast mitochondria cultured in hypoxic atmospheres (1% O2) for 72 hours. Here we report on the mitochondrial organization in cells and on their contribution to the cellular energy state. Our results indicate that prolonged hypoxia cause a significant reduction of mitochondrial mass and of the quantity of the oxidative phosphorylation complexes. Hypoxia is also responsible to damage mitochondrial complexes as shown after normalization versus citrate synthase activity. HIF-1α plays a pivotal role in wound healing, and its expression in the multistage process of normal wound healing has been well characterized, it is necessary for cell motility, expression of angiogenic growth factor and recruitment of endothelial progenitor cells. We studied hypoxia in the pathological status of diabetes and complications of diabetes and we evaluated the combined effect of hyperglycemia and hypoxia on human dermal fibroblasts (HDFs) and human dermal micro-vascular endothelial cells (HDMECs) that were grown in high glucose, low glucose concentrations and mannitol as control for the osmotic challenge.

The lectin-like domain of tumor necrosis factor improves lung function after rat lung transplantation--potential role for a reduction in reactive oxygen species generation

To test the hypothesis that the lectin-like domain of tumor necrosis factor, mimicked by the TIP peptide, can improve lung function after unilateral orthotopic lung isotransplantation. Because of a lack of a specific treatment for ischemia reperfusion-mediated lung injury, accompanied by a disrupted barrier integrity and a dysfunctional alveolar liquid clearance, alternative therapies restoring these parameters after lung transplantation are required.

A role for GRK2 in myocardial ischemic injury: indicators of a potential future therapy and diagnostic

Morbidity and mortality of myocardial infarction remains significant with resulting left ventricular function presenting as a major determinant of clinical outcome. Protecting the myocardium against ischemia reperfusion injury has become a major therapeutic goal and the identification of key signaling pathways has paved the way for various interventions, but until now with disappointing results. This article describes the recently discovered new role of G-protein-coupled receptor kinase-2 (GRK2), which is known to critically influence the development and progression of heart failure, in acute myocardial injury. This article focuses on potential applications of the GRK2 peptide inhibitor βARKct in ischemic myocardial injury, the use of GRK2 as a biomarker in acute myocardial infarction and discusses the challenges of translating GRK2 inhibition as a cardioprotective strategy to a possible future clinical application.

Evaluation of multimeric tyrosine-O-sulfate as a cytoprotectant in an in vivo model of acute myocardial infarction in pigs

Intracoronary administration of glycosaminoglycan analogs, including the complement inhibitor dextran sulfate, attenuates myocardial ischemia/reperfusion injury (I/R injury). However, dextran sulfate has a distinct anticoagulatory effect, possibly limiting its use in specific situations in vivo. We therefore developed multimeric tyrosine sulfate (sTyr-PAA), a novel, minimally anticoagulatory, fully synthetic non-carbohydrate-containing polyacrylamide conjugate, for in vivo testing in an acute closed-chest porcine model of acute myocardial infarction.

The Emerging Role of TLR and Innate Immunity in Cardiovascular Disease

Cardiovascular disease is a complex disorder involving multiple pathophysiological processes, several of which involve activation of toll-like receptors (TLRs) of the innate immune system. As sentinels of innate immunity TLRs are nonclonally germline-encoded molecular pattern recognition receptors that recognize exogenous as well as tissue-derived molecular dangers signals promoting inflammation. In addition to their expression in immune cells, TLRs are found in other tissues and cell types including cardiomyocytes, endothelial and vascular smooth muscle cells. TLRs are differentially regulated in various cell types by several cardiovascular risk factors such as hypercholesterolemia, hyperlipidemia, and hyperglycemia and may represent a key mechanism linking chronic inflammation, cardiovascular disease progression, and activation of the immune system. Modulation of TLR signaling by specific TLR agonists or antagonists, alone or in combination, may be a useful therapeutic approach to treat various cardiovascular inflammatory conditions such as atherosclerosis, peripheral arterial disease, secondary microvascular complications of diabetes, autoimmune disease, and ischemia reperfusion injury. In this paper we discuss recent developments and current evidence for the role of TLR in cardiovascular disease as well as the therapeutic potential of various compounds on inhibition of TLR-mediated inflammatory responses.

Endothelial cell protection in xenotransplantation: looking after a key player in rejection

The endothelium, as an organ at the interface between the intra- and extravascular space, actively participates in maintaining an anti-inflammatory and anti-coagulant environment under physiological conditions. Severe humoral as well as cellular rejection responses, which accompany cross-species transplantation of vascularized organs as well as ischemia/reperfusion injury, primarily target the endothelium and disrupt this delicate balance. Activation of pro-inflammatory and pro-coagulant pathways often lead to irreversible injury not only of the endothelial layer but also of the entire graft, with ensuing rejection. This review focuses on strategies targeted at protecting the endothelium from such damaging effects, ranging from genetic manipulation of the donor organ to soluble, as well as membrane-targeted, protective strategies.

Fructose-1,6-bisphosphate preserves intracellular glutathione and protects cortical neurons against oxidative stress

Fructose-1,6-bisphosphate (FBP), an endogenous intermediate of glycolysis, protects the brain against ischemia-reperfusion injury. The mechanisms of FBP protection after cerebral ischemia are not well understood. The current study was undertaken to determine whether FBP protects primary neurons against hypoxia and oxidative stress by preserving reduced glutathione (GSH). Cultures of pure cortical neurons were subjected to oxygen deprivation, a donor of nitric oxide and superoxide radicals (3-morpholinosydnonimine), an inhibitor of glutathione synthesis (L-buthionine-sulfoximine) or glutathione reductase (1,3-bis(2-chloroethyl)-1-nitrosourea) in the presence or absence of FBP (3.5 mM). Neuronal viability was determined using an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. FBP protected neurons against hypoxia-reoxygenation and oxidative stress under conditions of compromised GSH metabolism. The efficacy of FBP depended on duration of hypoxia and was associated with higher intracellular GSH concentration, an effect partly mediated via increased glutathione reductase activity.

The role of purinergic signaling in the liver and in transplantation: effects of extracellular nucleotides on hepatic graft vascular injury, rejection and metabolism

Extracellular nucleotides (e.g. ATP, UTP, ADP) are released by activated endothelium, leukocytes and platelets within the injured vasculature and bind specific cell-surface type-2 purinergic (P2) receptors. This process drives vascular inflammation and thrombosis within grafted organs. Importantly, there are also vascular ectonucleotidases i.e. ectoenzymes that hydrolyze extracellular nucleotides in the blood to generate nucleosides (viz. adenosine). Endothelial cell NTPDase1/CD39 has been shown to critically modulate levels of circulating nucleotides. This process tends to limit the activation of platelet and leukocyte expressed P2 receptors and also generates adenosine to reverse inflammatory events. This vascular protective CD39 activity is rapidly inhibited by oxidative reactions, such as is observed with liver ischemia reperfusion injury. In this review, we chiefly address the impact of these signaling cascades following liver transplantation. Interestingly, the hepatic vasculature, hepatocytes and all non-parenchymal cell types express several components co-ordinating the purinergic signaling response. With hepatic and vascular dysfunction, we note heightened P2- expression and alterations in ectonucleotidase expression and function that may predispose to progression of disease. In addition to documented impacts upon the vasculature during engraftment, extracellular nucleotides also have direct influences upon liver function and bile flow (both under physiological and pathological states). We have recently shown that alterations in purinergic signaling mediated by altered CD39 expression have major impacts upon hepatic metabolism, repair mechanisms, regeneration and associated immune responses. Future clinical applications in transplantation might involve new therapeutic modalities using soluble recombinant forms of CD39, altering expression of this ectonucleotidase by drugs and/or using small molecules to inhibit deleterious P2-mediated signaling while augmenting beneficial adenosine-mediated effects within the transplanted liver.

Low molecular weight dextran sulfate as complement inhibitor and cytoprotectant in solid organ and islet transplantation

Complement is an essential part of the innate immune system and plays a crucial role in organ and islet transplantation. Its activation, triggered for example by ischemia/reperfusion (I/R), significantly influences graft survival, and blocking of complement by inhibitors has been shown to attenuate I/R injury. Another player of innate immunity are the dendritic cells (DC), which form an important link between innate and adaptive immunity. DC are relevant in the induction of an immune response as well as in the maintenance of tolerance. Modulation or inhibition of both components, complement and DC, may be crucial to improve the clinical outcome of solid organ as well as islet transplantation. Low molecular weight dextran sulfate (DXS), a well-known complement inhibitor, has been shown to prevent complement-mediated damage of the donor graft endothelium and is thus acting as an endothelial protectant. In this review we will discuss the evidence for this cytoprotective effect of DXS and also highlight recent data which show that DXS inhibits the maturation of human DC. Taken together the available data suggest that DXS may be a useful reagent to prevent the activation of innate immunity, both in solid organ and islet transplantation.

Dextran sulfate modulates MAP kinase signaling and reduces endothelial injury in a rat aortic clamping model

OBJECTIVE: Mitogen-activated protein kinases (MAPKs), including JNK, p38, and ERK1/2, noticeably influence ischemia/reperfusion injury (IRI). The complement inhibitor dextran sulfate (DXS) associates with damaged endothelium denudated of its heparan sulfate proteoglycan (HSPG) layer. Other glycosaminoglycan analogs are known to influence MAPK signaling. Hypothetically therefore, targeted intravascular cytoprotection by DXS may function in part through influencing MAPK activation to reduce IRI-induced damage of the vasculature. METHODS: IRI of the infrarenal aorta of male Wistar rats was induced by 90 minutes clamping followed by 120 minutes reperfusion. DXS (5 mg/mL) or physiologic saline (NaCl controls) was infused locally into the ischemic aortic segment immediately prior to reperfusion. Ninety minutes ischemia-only and heparinase infusion (maximal damage) experiments, as well as native rat aorta, served as controls. Aortas were excised following termination of the experiments for further analysis. RESULTS: DXS significantly inhibited IRI-induced JNK and ERK1/2 activation (P = .043; P =.005) without influencing the p38 pathway (P =.110). Reduced aortic injury, with significant inhibition of apoptosis (P = .032 for DXS vs NaCl), correlated with decreased nuclear factor kappaB translocation within the aortic wall. DXS treatment clearly reduced C1q, C4b/c, C3b/c, and C9 complement deposition, whilst preserving endothelial cell integrity and reducing reperfusion-induced HSPG shedding. Protection was associated with binding of fluorescein labeled DXS to ischemically damaged tissue. CONCLUSIONS: Local application of DXS into ischemic vasculature immediately prior to reperfusion reduces complement deposition and preserves endothelial integrity, partially through modulating activation of MAPKs and may offer a new approach to tackle IRI in vascular surgical procedures. CLINICAL RELEVANCE: The purpose of the present study was to determine the role of dextran sulfate (DXS), a glycosaminoglycan analog and complement inhibitor, in modulating intracellular MAPK signaling pathways, reducing complement activation and ultimately attenuating ischemia/reperfusion injury (IRI) in a rat aortic-clamping model, in part a surrogate model to study the microvasculature. The study shows a role for DXS in ameliorating endothelial injury by reducing IRI-mediated damage and intravascular, local inflammation in the affected aortic segment. DXS may be envisaged as an endothelial protectant in vascular injury, such as occurs during vascular surgical procedures.

Activation of the lectin pathway of complement in pig-to-human xenotransplantation models

BACKGROUND Natural IgM containing anti-Gal antibodies initiates classic pathway complement activation in xenotransplantation. However, in ischemia-reperfusion injury, IgM also induces lectin pathway activation. The present study was therefore focused on lectin pathway as well as interaction of IgM and mannose-binding lectin (MBL) in pig-to-human xenotransplantation models. METHODS Activation of the different complement pathways was assessed by cell enzyme-linked immunosorbent assay using human serum on wild-type (WT) and α-galactosyl transferase knockout (GalTKO)/hCD46-transgenic porcine aortic endothelial cells (PAEC). Colocalization of MBL/MASP2 with IgM, C3b/c, C4b/c, and C6 was investigated by immunofluorescence in vitro on PAEC and ex vivo in pig leg xenoperfusion with human blood. Influence of IgM on MBL binding to PAEC was tested using IgM depleted/repleted and anti-Gal immunoabsorbed serum. RESULTS Activation of all the three complement pathways was observed in vitro as indicated by IgM, C1q, MBL, and factor Bb deposition on WT PAEC. MBL deposition colocalized with MASP2 (Manders' coefficient [3D] r=0.93), C3b/c (r=0.84), C4b/c (r=0.86), and C6 (r=0.80). IgM colocalized with MBL (r=0.87) and MASP2 (r=0.83). Human IgM led to dose-dependently increased deposition of MBL, C3b/c, and C6 on WT PAEC. Colocalization of MBL with IgM (Pearson's coefficient [2D] rp=0.88), C3b/c (rp=0.82), C4b/c (rp=0.63), and C6 (rp=0.81) was also seen in ex vivo xenoperfusion. Significantly reduced MBL deposition and complement activation was observed on GalTKO/hCD46-PAEC. CONCLUSION Colocalization of MBL/MASP2 with IgM and complement suggests that the lectin pathway is activated by human anti-Gal IgM and may play a pathophysiologic role in pig-to-human xenotransplantation.

Macroreplantations of the upper extremity: a series of 11 patients

INTRODUCTION Micro- or macroreplantation is classified depending on the level of amputation, distal or proximal to the wrist. This study was performed to review our experience in macroreplantation of the upper extremity with special attention to technical considerations and outcomes. MATERIALS AND METHODS Between January 1990 and December 2010, 11 patients with a complete amputation of the upper extremity proximal to the wrist were referred for replantations to our department. The patients, one woman and ten men, had a mean age of 43.4 ± 18.2 years (range 19-76 years). There were two elbow, two proximal forearm, four mid-forearm, and three distal forearm amputations. The mechanism of injury was crush in four, crush-avulsion in five and guillotine amputation in two patients. The Chen classification was used to assess the postoperative outcomes. The mean follow-up after macroreplantation was 7.5 ± 6.3 years (range 2-21 years). RESULTS All but one were successfully replanted and regained limb function: Chen I in four cases (36 %), Chen II in three cases (27 %), Chen III in two cases (18 %), and Chen IV in one patient (9 %). We discuss the steps of the macroreplantation technique, the need to minimize ischemic time and the risk of ischemia reperfusion injuries. CONCLUSION Thanks to improvements in technique, the indications for limb preservation after amputation can be expanded. However, because of their rarity, replantations should be performed at specialist replantation centers. LEVEL OF EVIDENCE Level IV.

Effects of exogenous surfactant on the non-heart-beating donor lung graft in experimental lung transplantation - a stereological study.

The use of non-heart-beating donor (NHBD) lungs may help to overcome the shortage of lung grafts in clinical lung transplantation, but warm ischaemia and ischaemia/reperfusion injury (I/R injury) resulting in primary graft dysfunction represent a considerable threat. Thus, better strategies for optimized preservation of lung grafts are urgently needed. Surfactant dysfunction has been shown to contribute to I/R injury, and surfactant replacement therapy is effective in enhancing lung function and structural integrity in related rat models. In the present study we hypothesize that surfactant replacement therapy reduces oedema formation in a pig model of NHBD lung transplantation. Oedema formation was quantified with (SF) and without (non-SF) surfactant replacement therapy in interstitial and alveolar compartments by means of design-based stereology in NHBD lungs 7 h after cardiac arrest, reperfusion and transplantation. A sham-operated group served as control. In both NHBD groups, nearly all animals died within the first hours after transplantation due to right heart failure. Both SF and non-SF developed an interstitial oedema of similar degree, as shown by an increase in septal wall volume and arithmetic mean thickness as well as an increase in the volume of peribron-chovascular connective tissue. Regarding intra-alveolar oedema, no statistically significant difference could be found between SF and non-SF. In conclusion, surfactant replacement therapy cannot prevent poor outcome after prolonged warm ischaemia of 7 h in this model. While the beneficial effects of surfactant replacement therapy have been observed in several experimental and clinical studies related to heart-beating donor lungs and cold ischaemia, it is unlikely that surfactant replacement therapy will overcome the shortage of organs in the context of prolonged warm ischaemia, for example, 7 h. Moreover, our data demonstrate that right heart function and dysfunctions of the pulmonary vascular bed are limiting factors that need to be addressed in NHBD.