scholarly journals Cerebral Endothelial Cell Apoptosis after Ischemia—Reperfusion: Role of PARP Activation and AIF Translocation

2005 ◽  
Vol 25 (7) ◽  
pp. 868-877 ◽  
Author(s):  
Yunhong Zhang ◽  
Xiaochun Zhang ◽  
Tae S Park ◽  
Jeffrey M Gidday
Keyword(s):  
Endothelial Cell ◽  
Cell Viability ◽  
Nuclear Translocation ◽  
Ischemia Reperfusion ◽  
Total Duration ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Microvascular Endothelial Cell ◽  
Parp Activation ◽  
Cerebral Endothelial Cell ◽  
Parp 1

Cerebral ischemia-reperfusion leads to vascular dysfunction characterized by endothelial cell injury or death. In the present study, we used an in vitro model to elucidate mechanisms of human brain microvascular endothelial cell (HBMEC) injury after episodic ischemia-reperfusion. Near-confluent HBMEC cultures were exposed to intermittent hypoxia-reoxygenation (HX/RO) and, at different recovery time points, cell viability was assessed by the MTT assay, apoptotic death by fluorescence microscopy of terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL)-positive cells, and nuclear translocation of apoptosis-inducing factor (AIF) and cleavage of poly(ADP-ribose) polymerase-1 (PARP-1) by immunoblotting of subcellular fractions. Reductions in HBMEC viability were proportional to the number of HX/RO cycles, and not the total duration of hypoxia. Using four cycles of 1-h HX with 1 h of intervening normoxic RO, cell viability was reduced 30% to 40% between 12 and 48 h. Treatment with the PARP-1 inhibitors 3-aminobenzamide or 4-amino-1,8-naphthalimide during the insult improved HBMEC viability at 24 h after insult, and resulted in dose-dependent reductions in TUNEL-positivity at 16 h after insult, but not if these treatments were delayed by 4 h. HX/RO-induced increases in nuclear AIF translocation, as well as PARP-1 cleavage, were also reduced dose-dependently at 4 h after insult by the inhibitors. The caspase inhibitor z-VAD-fmk blocked PARP-1 cleavage, but did not affect AIF translocation and was only modestly cytoprotective. These findings indicate that PARP-1 activation and a PARP-1-dependent, caspase-independent, nuclear translocation of AIF contribute to apoptotic cerebral endothelial cell death after ischemia-reperfusion, underscoring the potential for ischemic microvascular protection by inhibiting PARP activation or preventing AIF translocation.

scholarly journals Cyclophilin D Regulates the Nuclear Translocation of AIF, Cardiac Endothelial Cell Necroptosis and Murine Cardiac Transplant Injury

2021 ◽  
Vol 22 (20) ◽  
pp. 11038
Author(s):  
Adnan Qamar ◽  
Jianqi Zhao ◽  
Laura Xu ◽  
Patrick McLeod ◽  
Xuyan Huang ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Nuclear Translocation ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Transplant Rejection ◽  
Microvascular Endothelial Cell ◽  
Cardiac Transplant ◽  
Cyclophilin D ◽  
Iri Model ◽  
Organ Rejection

Ischemia-reperfusion injury (IRI) is an inevitable consequence of organ transplant procedure and associated with acute and chronic organ rejection in transplantation. IRI leads to various forms of programmed cell death, which worsens tissue damage and accelerates transplant rejection. We recently demonstrated that necroptosis participates in murine cardiac microvascular endothelial cell (MVEC) death and murine cardiac transplant rejection. However, MVEC death under a more complex IRI model has not been studied. In this study, we found that simulating IRI conditions in vitro by hypoxia, reoxygenation and treatment with inflammatory cytokines induced necroptosis in MVECs. Interestingly, the apoptosis-inducing factor (AIF) translocated to the nucleus during MVEC necroptosis, which is regulated by the mitochondrial permeability molecule cyclophilin D (CypD). Furthermore, CypD deficiency in donor cardiac grafts inhibited AIF translocation and mitigated graft IRI and rejection (n = 7; p = 0.002). Our studies indicate that CypD and AIF play significant roles in MVEC necroptosis and cardiac transplant rejection following IRI. Targeting CypD and its downstream AIF may be a plausible approach to inhibit IRI-caused cardiac damage and improve transplant survival.

scholarly journals Upregulation of FoxM1 protects against ischemia/reperfusion-induced myocardial injury

2021 ◽  
Author(s):  
Gang Zhang ◽  
Kun Yu ◽  
Zhi Bao ◽  
Xiaofeng Sun ◽  
Dongying Zhang
Keyword(s):  
Infarct Size ◽  
Cell Viability ◽  
Myocardial Injury ◽  
Tissue Injury ◽  
Ischemia Reperfusion ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tetrazolium Chloride ◽  
Creatine Kinase Mb ◽  
Hypoxia Reoxygenation

Background. Ischemia/reperfusion (I/R) induced lethal tissue injury in myocardium. FoxM1 (Forkhead Box M1), expressed in proliferating cardiac progenitor cells, could regulate myocardial development. However, the role of FoxM1 in I/R-induced myocardial injury has not been reported yet. Methods. Rats were conducted with regional ischemia followed by reperfusion in myocardium through ligation of the left anterior descending coronary artery. Triphenyl-tetrazolium chloride staining was utilized to assess the infarct size. ELISA was performed to detect activities of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH). Protein expression of FoxM1 in heart tissues and H9c2 were determined by western blot. H9c2 cells were used to establish a hypoxia/reoxygenation cell model, and the cell viability, proliferation and apoptosis were evaluated by MTT, EdU (5-ethynyl-2’-deoxyuridine) staining and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining, respectively. Adenovirus (Ad)-mediated over-expression of FoxM1 was injected into the anterior wall of the left ventricle of rats to evaluate the role of FoxM1 on in vivo I/R-induced myocardial injury. Results. FoxM1 was reduced in heart tissues isolated from rats post myocardial I/R injury. Forced FoxM1 expression increased cell viability and proliferation of hypoxia/reoxygenation-induced H9c2, while repressed the cell apoptosis with increased Bcl-2 and decreased Bax and cleaved caspase-3. Injection of Ad-FoxM1 suppressed infarct size of the heart and decreased activities of CK-MB and LDH. Conclusion. FoxM1 attenuated I/R-induced myocardial injury, providing potential therapeutic target for the disease.

HIF-1 activation attenuates postischemic myocardial injury: role for heme oxygenase-1 in modulating microvascular chemokine generation

2005 ◽  
Vol 289 (2) ◽  
pp. H542-H548 ◽  
Author(s):  
Ramzi Ockaili ◽  
Ramesh Natarajan ◽  
Fadi Salloum ◽  
Bernard J. Fisher ◽  
Drew Jones ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Cell Line ◽  
Heme Oxygenase ◽  
Ischemia Reperfusion ◽  
Microvascular Endothelial Cell ◽  
Endothelial Cell Line ◽  
Human Microvascular Endothelial Cell ◽  
Microvascular Endothelial

The CXC chemokine IL-8, which promotes adhesion, activation, and transmigration of polymorphonuclear neutrophils (PMN), has been associated with production of tissue injury in reperfused myocardium. Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric peptide that is a key regulator of genes such as heme oxygenase (HO)-1 expressed under hypoxic conditions. We hypothesized that HO-1 plays an important role in regulating proinflammatory mediator production under conditions of ischemia-reperfusion. HIF-1 was activated in the human microvascular endothelial cell line (HMEC-1) with the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG). DMOG significantly attenuated cytokine-induced IL-8 promoter activity and protein secretion and cytokine-induced PMN migration across human microvascular endothelial cell line HMEC-1 monolayers. In vivo studies in a rabbit model of myocardial ischemia-reperfusion showed that rabbits pretreated with a 20 mg/kg DMOG infusion ( n = 6) 24 h before study exhibited a 21.58 ± 1.76% infarct size compared with 35.25 ± 2.06% in saline-treated ischemia-reperfusion animals ( n = 6, change in reduction = 39%; P < 0.001). In DMOG-pretreated (20 mg/kg) animals, plasma IL-8 levels at 3 h after onset of reperfusion were 405 ± 40 pg/ml vs. 790 ± 40 pg/ml in saline-treated ischemia-reperfusion animals ( P < 0.001). DMOG pretreatment reduced myocardial myeloperoxidase activity, expressed as number of PMN per gram of myocardium, to 1.43 ± 0.59 vs. 4.86 ± 1.1 ( P = 0.012) in saline-treated ischemia-reperfused hearts. Both in vitro and in vivo DMOG-attenuated IL-8 production was associated with robust HO-1 expression. Thus our data show that HIF-1 activation induces substantial HO-1 expression that is associated with attenuated proinflammatory chemokine production by microvascular endothelium in vitro and in vivo.

scholarly journals Overexpression of IκBα in cardiomyocytes alleviates hydrogen peroxide-induced apoptosis and autophagy through inhibiting NF-κB translocation

2020 ◽  
Author(s):  
Min Han ◽  
Xiao-Cui Chen ◽  
Ming-Hui Sun ◽  
Min-Tao Gai ◽  
Yi-Ning Yang ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Cell Viability ◽  
Nuclear Translocation ◽  
Ischemia Reperfusion ◽  
Annexin V ◽  
Neonatal Rat ◽  
Cell Counting ◽  
Pyrrolidine Dithiocarbamate ◽  
Dependent Manner ◽  
Induced Apoptosis

Abstract Background: Inflammation and oxidative stress play a predominant role in the initiation and progression of ischemia/reperfusion (I/R) injury, of which nuclear factor kappa B (NF-κB) is considered to be a crucial mediator. Inactivation of NF-κB could benefit cardiomyocytes through inhibiting apoptosis. IκBα, an inhibitor of NF-κB, is hypothesized to protects cardiomyocytes from H2O2-induced apoptosis and autophagy through inhibiting the NF-κB pathway.Methods: We designed an AAV9-delivered mutated IκBαS32A, S36A and investigated its effect on neonatal rat ventricular cardiomyocytes (NRVMs) in response to hydrogen peroxide (H2O2). NRVMs were divided into Normal (blank), Control (H2O2), GFP +H2O2, IκBα+H2O2, and Pyrrolidine dithiocarbamate (PDTC)+H2O2 groups. NF-κB p65 nuclear translocation was evaluated by immunofluorescence and western blot. Cell viability was assessed by a cell counting kit-8 kit. Supernatant lactate dehydrogenase (LDH) and intracellular malondialdehyde (MDA) were measured to identify H2O2-stimulated cytotoxicity. Apoptosis was determined by Annexin V-PE/7-AAD, and the mitochondrial membrane potential (△Ψm) was detected by JC-1. Western bolt was used to detect apoptosis and autophagy related proteins.Results: Consequently, H2O2-treated NRVMs showed reductions in cell viability but increased IκBα degradation and NF-κB p65 nuclear translocation in a time-dependent manner. Furthermore, LDH and MDA content, LC3-Ⅱ/LC3-Ⅰ ratio, Bax and Beclin-1 expressions, and apoptotic cells were upregulated in NRVMs exposed to H2O2, whereas △Ψm and Bcl-2 expression were downregulated. Additionally, IκBα transduction or PDTC pretreatment both attenuated the nuclear translocation of the p65 subunit and reversed the H2O2-stimulated effects in NRVMs.Conclusion: These findings suggest that IκBα could ameliorate H2O2-induced apoptosis and autophagy through targeted inhibition of NF-κB activation, which may guide strategies to prevent cardiac I/R jury.

Sign in / Sign up

Export Citation Format

Share Document