benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Myocardial-Ischemia

In order to improve medical treatment of ischemic injury such as myocardial infarction, it is important to elucidate hypoxia-induced changes to endothelial cells. An in vitro blood vessel model, in which HUVECs are stimulated to form a network of capillary-like tubes, was used to analyze hypoxia-induced morphological and biochemical changes. When exposed to hypoxia, the network of capillary tubes broke down into small clusters. This tube breakdown was accompanied by chromatin condensation and cell nuclear fragmentation, morphological markers of apoptosis, and activation of two apoptotic signals, caspase-3 and p38. We investigated what roles caspase cascade and p38 play in hypoxia-induced apoptosis and tube breakdown by using zVAD-fmk and SB203580, specific inhibitors of these two apoptotic signals, respectively. Chromatin condensation and cell nuclear fragmentation and tube breakdown were effectively inhibited by SB203580, but not by zVAD-fmk. SB203580 caused dephosphorylation of p38, which indicates that p38 was autophosphorylated. Inhibition by zVAD-fmk caused slight MW increase in p17 and emergence of p19, which indicates that the inhibitor caused partial processing of caspase-3. Inhibition of p38 suppressed activation of caspase-3 but not vice versa. In addition, these two inhibitors were shown to differentially inhibit cleavage of so-called caspase substrates. SB203580 inhibited cleavage of PARP and lamin A/C, while zVAD-fmk inhibited cleavage of lamin A/C but not that of PARP. Taken together, these results show that p38 is located upstream of caspase cascade and that, although caspase-3 is activated, a p38-regulated caspase-independent pathway is crucial for the execution of hypoxia-induced apoptosis and tube breakdown.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Capillaries; Caspase 3; Caspase Inhibitors; Cell Nucleus; Cells, Cultured; Cysteine Proteinase Inhibitors; Endothelium, Vascular; Enzyme Inhibitors; Humans; Hypoxia; Imidazoles; In Vitro Techniques; Myocardial Ischemia; p38 Mitogen-Activated Protein Kinases; Pyridines; Signal Transduction; Umbilical Veins

The efficacy of a novel, nonpeptidic, caspase 3/7-selective inhibitor, (S)-(+)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (MMPSI) for reducing ischemic injury in isolated rabbit hearts or cardiomyocytes was evaluated. MMPSI (0.1-10 microM) evoked a concentration-dependent reduction in infarct size (up to 56% vs. control; IC(50)=0.2 microM). Furthermore, apoptosis (DNA laddering, soluble nucleosomes) was reduced in the ischemic area-at-risk. MMPSI inhibited recombinant human caspase-3 with an IC(50)=1.7 microM. Apoptosis in H9c2 cells after 16-h simulated ischemia and 2-h simulated reperfusion was significantly reduced by MMPSI in a concentration-dependent manner (IC(50)=0.5 microM); similar effects were observed in isolated adult rabbit cardiomyocytes (IC(50)=1.5 microM). These data support an important role for caspase-3/7 in mediating myocardial ischemic injury. Furthermore, these data indicate that cardioprotection via caspase-3/7 inhibition is attainable via a small molecule (nonpeptidic) inhibitor, a necessary step in making this approach therapeutically viable.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspase 7; Caspase Inhibitors; Caspases; Cell Line; Cells, Cultured; Coronary Circulation; Cysteine Proteinase Inhibitors; Dipeptides; Dose-Response Relationship, Drug; Heart Rate; In Situ Nick-End Labeling; Isatin; Ketones; Male; Microscopy, Electron; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; Pyrrolidines; Rabbits

Some infarcted myocytes undergo caspase-dependent DNA fragmentation, but serine protease-dependent DNA fragmentation may also be involved. There is controversy regarding whether caspase inhibitors can reduce infarct size, so the present study investigated whether serine protease inhibitor can reduce the DNA fragmentation of infarcted myocytes and whether serine protease or caspase inhibitors attenuates myocardial infarct size in Japanese white rabbits without collateral circulation. Rabbits were subjected to 30-min coronary occlusion followed by 48-h reperfusion. A vehicle (dimethylsulfoxide, control group, n=8) or Z-Val-Ala-Asp(Ome)-CH2F (ZVAD-fmk, a caspase inhibitor, ZVAD group, 0.8 mg/kg iv at 20 min before coronary occlusion and 0.8 mg/kg at 90 min after reperfusion, n=8) or 3,4-dichloroisocoumarin (DCI, a serine protease inhibitor, 2 mg/kg iv at 20 min before coronary occlusion, DCI group, n=8) was administered. Animals were killed at 48h after reperfusion for the detection of myocardial infarct size and at 4h after reperfusion for the detection of dUTP nick end-labeling (TUNEL)-positive myocytes, the electrophoretic pattern of DNA fragmentation and ultrastructural analysis. The left ventricle (LV) was excised and sliced. The myocardial infarct size as a percentage of the area at risk was assessed by triphenyltetrazolium chloride staining. DNA fragmentation was assessed by in situ TUNEL at the light microscopic level. ZVAD and DCI significantly reduced the mean blood pressure during reperfusion without affecting heart rate. There was no significant difference in the % area at risk (AAR) of LV among the 3 groups (control: 26.3+/-3.0%; ZVAD: 25.6+/-2.6%; DCI: 25.6+/-2.0%). The % infarct size as a percentage of the AAR in the ZVAD group (41.3+/-4.5%) and the DCI group (50.4+/-3.8%) was not significantly different from the control group (43.5+/-4.5%). However, the percent DNA fragmentation in the infarcted area in the ZVAD (3.5+/-0.8%) and DCI groups (4.2+/-0.9%) was significantly reduced compared with the control group (10.7+/-1.9%). The DNA ladder pattern observed in the control group was attenuated in both the ZVAD and DCI groups. There was no difference in electron microscopic changes among the 3 groups. Serine protease-dependent DNA fragmentation is present in infarcted myocytes, in addition to caspase-dependent DNA fragmentation, but an infarct-size reducing effect was not observed with either of these inhibitors.

Topics: Amino Acid Chloromethyl Ketones; Animals; Caspase Inhibitors; Caspases; Disease Models, Animal; DNA Fragmentation; Enzyme Inhibitors; In Situ Nick-End Labeling; Male; Microscopy, Electron; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Rabbits; Serine Proteinase Inhibitors

The death of cardiac cells during ischemia and reperfusion is partially mediated by apoptosis, as seen, eg, in autopsy material of patients after acute myocardial infarction.. To study the role of CD95/Fas/Apo1 for induction of postischemic cell death, we used an ischemia/reperfusion model of isolated rat and mouse hearts in Langendorff perfusion. In this model, caspase-dependent apoptosis occurred during postischemic reperfusion. Moreover, soluble CD95 ligand/Fas ligand was released by the postischemic hearts early after the onset of reperfusion. In addition, this ligand was synthesized de novo under these circumstances. Similar findings were observed for other "death-inducing" ligands, such as tumor necrosis factor (TNF)-alpha and TNF-related apoptosis-inducing ligand. In primary adult rat myocyte culture, hypoxia and reoxygenation caused a marked increase in sensitivity to the apoptotic effects of CD95 ligand. Isolated hearts from mice lacking functional CD95 (lpr) display marked reduction in cell death after ischemia and reperfusion compared with wild-type controls.. These data suggest that CD95/Apo1/Fas is directly involved in cell death after myocardial ischemia. The CD95 system might thus represent a novel target for therapeutic prevention of postischemic cell death in the heart.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Apoptosis Regulatory Proteins; Caspase Inhibitors; Caspases; Cell Death; Cells, Cultured; Cysteine Proteinase Inhibitors; Fas Ligand Protein; fas Receptor; Heart; In Vitro Techniques; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Rats; Rats, Wistar; Solubility; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Many cell types undergo apoptosis under conditions of ischemia. Little is known, however, about the molecular pathways that mediate this response. A cellular and biochemical approach to elucidate such signaling pathways was undertaken in primary cultures of cardiac myocytes, a cell type that is especially sensitive to ischemia-induced apoptosis. Deprivation of serum and glucose, components of ischemia in vivo, resulted in myocyte apoptosis, as determined by nuclear fragmentation, internucleosomal cleavage of DNA, and processing of caspase substrates. These manifestations of apoptosis were blocked by zVAD-fmk, a peptide caspase inhibitor, indicating that caspase activity is necessary for the progression of apoptosis in this model. In contrast to control cells, apoptotic myocytes exhibited cytoplasmic accumulation of cytochrome c, indicating release from the mitochondria. Furthermore, both caspase-9 and caspase-3 were processed to their active forms in serum-/glucose-deprived myocytes. Caspase processing, but not cytochrome c release, was inhibited by zVAD-fmk, placing the latter event upstream of caspase activation. This evidence demonstrates that components of ischemia activate the mitochondrial death pathway in cardiac myocytes.

Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Apoptosis; Biomarkers; Blood Physiological Phenomena; Caspase 3; Caspase 9; Caspase Inhibitors; Caspases; Cells, Cultured; Culture Media; Cysteine Proteinase Inhibitors; Cytochrome c Group; Glucose; Membrane Potentials; Mitochondria, Heart; Muscle Proteins; Myocardial Ischemia; Myocardium; Oxidoreductases; Rats; Signal Transduction

Z-Val-Ala-Asp(OMe)-CH2F (ZVAD-fmk), a tripeptide inhibitor of the caspase interleukin-1beta-converting enzyme family of cysteine proteases, may reduce myocardial reperfusion injury in vivo by attenuating cardiomyocyte apoptosis within the ischemic area at risk.. Sprague-Dawley rats were subjected to a 30-minute coronary occlusion followed by a 24-hour reperfusion. An inert vehicle (dimethylsulfoxide; group 1, n=8) or ZVAD-fmk, at a total dose of 3.3 mg/kg (group 2, n=8), was administered intravenously every 6 hours starting at 30 minutes before coronary occlusion until 24 hours of reperfusion. At this 24-hour point, hemodynamics were assessed by means of cardiac catheterization; then, the rats were killed, and the left ventricle was excised and sliced. The myocardial infarct size/ischemic area at risk and the count of presumed apoptotic cardiomyocytes (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling [TUNEL]-positive cells) within the ischemic area at risk were assessed through triphenyltetrazolium chloride staining and TUNEL methods, respectively. Peak positive left ventricular dP/dt was higher (P=.02) and left ventricular end-diastolic pressure was lower (P=.04) in group 2 than in group 1. The infarct size/ischemic area at risk of group 2 (52.4+/-4.0%) was smaller (P=.02) than that of group 1 (66.6+/-3.7%), and TUNEL-positive cells were fewer (P=.0002) (group 2, 3.1+/-0.9%; group 1, 11.1+/-1.0%). Agarose gel electrophoresis revealed DNA laddering in the border zone myocardium of group 1, but DNA ladder formation was attenuated in group 2.. ZVAD-fmk was effective in reducing myocardial reperfusion injury, which could at least be partially attributed to the attenuation of cardiomyocyte apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Animals; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Disease Models, Animal; Electrophoresis, Agar Gel; Genetic Techniques; Heart; Hemodynamics; Leukocyte Count; Male; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Rats; Rats, Sprague-Dawley