| Coronary heart disease is the major cause of death in many countries and regions. Procedures used to rescure ischecmic myocardium include endovascular thrombolysis in acute myocardial infarction treatment, percutaneous transluminal coronary angioplasty and cardiopulmonary bypass (CPB) in coronary artery bypass grafting. However, these procedures per se may lead to myocardial ischemia-reperfusion injury (IRI), which is receiving more and more attention. Therefore, protecting myocardium against IRI have been an important target for researchers around the world. During myocardial ischemia/reperfusion, reactive oxygen species (ROS) and lipid peroxidation production increased, leading to increased oxidative stress that play a key role in IRI.15-F2t-isoprostane (IsoP), prostaglandin (PG)-like compounds derived from the free radical-catalyzed peroxidation of arachidonic acid.As the end production of the lipid peroxidation generated independently from cyclooxygenase, IsoP is stable in human biological fluid and tissue.For these characteristics, it is considered to be a reliable and specific biomarker of oxidase stress.It also has been found to be a sensitive and independent risk marker of coronary artery disease. It increased during myocardial ischemia-reperfusion, accompanied by increased release of the potent vasoconstrictor endothelin-1(ET-1). Inflammatory factor such as tumour necrosis factor-a (TNF-a) can stimulate the formation of ET-1, and ET-1 blockade with a ET-1 receptor antagonist can protect the myocardium from injury evidenced by reducing infarct size in experimental models of ischemia-reperfusion.We hypothesized that IsoP exerts deleterious effects on ischemic-reperfused hearts at least in a part by stimulating ET-1 formation and release.We postulatd the ET-1 blockade by bosentan,a dual ET-1 receptor A and B antagonist, can attenuate the deleterious effects of IsoP. The hypothesis was tested in an established isolated rat heart ischemia-reperfusion model.Of interest, circulatory pro-apoptotic inflammatory cytokines TNF-a and ROS, which are increased during myocardial IRI, may promote cardiomyocyte apoptosis subsequent to the induction of endothelial cells apoptosis. Thus, inhibition of TNF-a and ROS induced endothelial cell apoptosis may represent an effective therapy for myocardial IRI. It is of note that NO reacts with superoxide anion (O2-) to form peroxynitrite anion (ONOO-). Thus, we hypothesized that in the presence of oxidative stress, supplementing L-arginine, which is the NO precursor, may exacerbate TNF-a induced endothelial cell apoptosis by enhancing peroxynitrite mediated nitrative stress.Part I Bosentan Affects 15-F2t-isoprostane Adverse Effects on Postischemic Rat HeartsBackground and purpose:15-F2t-isoprostane (IsoP), a reliable and specific biomarker of ROS-induced oxidative stress, is increased after myocardial ischemia and reperfusion. It exerts deleterious effects on postischemic myocardium accompanied with increased release of ET-1, a potent vasoconstrictor. We hypothesized that IsoP exacerbates myocardial IRI by stimulating ET-1 production, and that ET-1 blockade can attenuate or prevent these deleterious effects of IsoP.Methods:Adult rat hearts were perfused by the Langendorff technique with Krebs-Henseleit solution (KH) at a constant flow rate of 10 mL/min. Global myocardial ischemia was induced by stopping KH perfusion for 40min followed by 60 min of reperfusion. Hearts were randomized to one of the five groups (n=8 each):untreated control, treated with ET-1 receptor A/B antagonist bosentan (1 mM), or the IsoP (100 nM) alone or in combination 10 min prior to, during 40 min global ischemia and 15 min of reperfusion, or treated with IsoP as above plus delayed administration of bosentan after 15 min of reperfusion.Results:Coronary effluent ET-1 concentrations in the IsoP group were higher than those in the control group during ischemia and reperfusion (P< 0.05), which was associated with increased release of cardiac-specific creatine kinase, reduced cardiac contractility during reperfusion, and increased myocardial infarct size (all P< 0.05 vs. control). Bosentan administration during early reperfusion exacerbated the IsoP deleterious effects, while delayed administration attenuated it.Conclusion:IsoP-induced ET-1 production during later reperfusion is detrimental to functional recovery of damaged myocardium, while ET-1 increase during early reperfusion seems to improve it. Part II Mechanism and Effects of L-arginine on TNF-a induced human endothelial cell apoptosisBackground and purpose:The morbidity and mortality attendant with myocardial ischemic injury/infarction remains significant despite advancement in surgical techniques and pharmacological therapies, in part due to the increased disease severity in the elderly or in patients with diabetes. Of interest, TNF-a and ROS, which are increased during myocardial IRI, may promote cardiomyocyte apoptosis subsequent to the induction of vascular endothelial cells apoptosis. L-arginine, the exclusive precursor for the biosynthesis of nitric oxide, has beneficial effect in prevention of endothelium dysfunction. However, supplementing L-arginine may be associated with higher postinfarction mortality in patients following acute myocardial infarction(AMI), a situation that is related with increased TNF-a level. Preliminary study has shown that TNF-a increases NO formation and ROS at the same time, leading to increased peroxynitrite production. Thus, we hypothesized that in the presence of oxidative stress, L-arginine may exacerbate TNF-a induced endothelial cell apoptosis by enhancing peroxynitrite mediated nitrative stress.Methods:ECV304 cell lines, a spontaneously-transformed and immortalized cell line derived from human umbilical vein endothelial cell, although there is difference on epithelial phenotype between the primary cells and ECV304 cell lines, it can still be used as a model in some other studies.Cultured cells were divided into 8 groups. Cells were not treated (group C), or treated with TNF-a alone for 24 h(group T), or 24h TNF-a treatment were followed by 30min pretreatment with either NO precursor L-arginine(100μM) (group LA+T), or the intravenous anesthetics propofol(50μM) (group P+T),or the non-selective nitric oxide synthase inhibitor N (omega)-nitro-L-arginine (L-NNA)(100μM) (group LN+T), the selective inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine (250μM)(group AG+T), or propofol plus L-arginine(group P+LA+T),or propofol plus L-NNA(group P+LN+T).Cell viability and apoptosis were quantified by MTT coloration and flow cytometry. Cytotoxicity was detected by measuring LDH release in the medium exposed to different treatment. 1. Activity of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were measured to estimate the antioxidant capacity of endothelial cells. Malondialdehyde (MDA), an oxidant-mediated lipid peroxidation production which is a marker of oxidative stress, its concentration can be detectd by assay kit.2. Cells were incubated with dihydroethidium (DHE) and 2’,7’-dichlorofluorescin diacetate (DCFH-DA). DHE reacts with ROS forming the fluorescent product ethidium which partitions into the nucleus upon binding DNA. DCFH-DA is deacetylated by cellular esterases yielding 2’,7’-dichlorofluorescin (DCFH), which reacts with ROS forming the fluorescent product 2’,7’-dichlorofluorescein (DCF).3. The interaction of NO in the system is measured by the determination of the total nitrite and nitrate concentrations in the medium exposed to differente reagents. The endothelial NO synthase (eNOS) and the inducible NO synthase (iNOS) protein expression in vascular endothelial cells were determined by western blot analysis.4. The levels of nitrotyrosine, a footprint of peroxynitrite production, were detected using immunohistochemical assay kit.Results:TNF-a increased intracellular ROS such as superoxide and hydrogen peroxide production accompanied by increases of iNOS protein expression and NO production. These were accompanied by increased protein expression of nitrotyrosine, a fingerprint of peroxynitrite and an index of nitrative stress, and increased endothelial cell apoptosis (all P< 0.05 vs. C). L-arginine did not enhance TNF-a induced increases of superoxide and peroxinitrite production, but further increased TNF-a induced increase of nitrotyrosine production and exacerbated TNF-a mediated cell apoptosis. Both L-NNA and aminoguanidine reduced TNF-a induced nitrative stress and attenuated TNF-a cellular toxicity. Propofol decreased L-arginine mediated enhancement of TNF-a induced increase of nitrotyrosine production and nitrative stress. Propofol plus L-NNA abolished almost all the changes induced by TNF-a except eNOS protein expression (all P< 0.05 vs. C).Conclusion:It is concluded that under pathological conditions associated with increased TNF-a production, supplementing L-arginine may further exacerbate TNF-a cellular toxicity by enhancing nitrative stress, and propofol may exert beneficial effect counteract the deleterious effect of L-arginine. |
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