ca-074-methyl-ester and Brain-Ischemia

Many studies have demonstrated the key role of lysosomes in ischemic cell death in the brain and have led to the "lysosomocentric" hypothesis. In this hypothesis, the release of cathepsin-B due to a change of lysosomal membrane permeabilization (LMP) or rupture is critical, and this can be prevented by its inhibitors CA074 and CA074-me. However, the role of CA074-me in neuronal death and its effect on the change of lysosomal membrane integrity after global cerebral ischemia/reperfusion (I/R) injury is not clear, so we investigated this here. Rat hippocampal CA1 neuronal death was evaluated after 20-min global cerebral I/R injury. CA074-me (1 μg, 10 μg) were given intracerebroventricularly 1h before ischemia or 1h post reperfusion. The changes of heat shock protein 70 (Hsp70), cathepsin-B, lysosomal-associated membrane protein 1 (LAMP-1), receptor-interacting protein 3 (RIP3), and the change of lysosomal pH were evaluated respectively. Hippocampal CA1 neuronal programmed necrosis induced by global cerebral I/R injury was prevented by CA074-me both pre-treatment and post-treatment. Diffuse cytoplasmic cathepsin-B and LAMP-1 immunostaining synchronized with the pyknotic nuclear changes 2 days post reperfusion, and a rise of lysosomal pH with the leakage of DND-153, a dye of lysosomes, after oxygen-glucose deprivation (OGD) was detected. Both of these changes demonstrated the rupture of lysosomal membrane and the leakage of cathepsin-B, and this was strongly inhibited by CA074-me pre-treatment. The overexpression and nuclear translocation of RIP3 and the reduction of NAD(+) level after I/R injury were also inhibited, while the upregulation of Hsp70 was strengthened by CA074-me pre-treatment. Delayed fulminant leakage of cathepsin-B due to lysosomal rupture is a critical harmful factor in neuronal programmed necrosis induced by 20-min global I/R injury. In addition to being an inhibitor of cathepsin-B, CA074-me may have an indirect neuroprotective effect by maintaining lysosomal membrane integrity and protecting against lysosomal rupture.

Topics: Active Transport, Cell Nucleus; Animals; Brain Ischemia; CA1 Region, Hippocampal; Cathepsin B; Cell Hypoxia; Cells, Cultured; Dipeptides; Disease Models, Animal; Glucose; HSP70 Heat-Shock Proteins; Male; NAD; Necrosis; Neurons; Neuroprotective Agents; Rats, Sprague-Dawley; Receptor-Interacting Protein Serine-Threonine Kinases; Reperfusion Injury

Endostatin (ES) has been recognized as a potent anti-angiogenic factor. We here investigated the expression of ES in ischemic brain and the consequence of cells expressing ES after stroke in adult stroke-prone renovascular hypertensive rats. A single dose of Ca-074ME, a membrane-permeable cathepsin B (CB) specific inhibitor, or vehicle was given by intraperitoneal injection immediately after distal middle cerebral artery occlusion (dMCAO), ES expression was evaluated using fluorescent immunohistochemistry staining, and CB enzyme activity was tested by measuring the free 7-amino-4-methylcoumarin (AMC) released by CB from its' specific substrate, the Z-Arg-Arg-7-amido-4-methylcoumarin. ES immunoreactivity (IR) was significantly up-regulated as early as 6 h and returned to baseline level at 3 days in peri-infarct area following dMCAO. Double-staining experiment revealed that the majority of ischemia-induced ES positive cells were neurons. Furthermore, ES was co-labeled with CB and Cleaved Caspase-3(Asp175) whereas treatment with Ca-074ME reduced up-regulation of ES expression and attenuated apoptosis in peri-infarct neurons. Collectively, our data suggest that peri-infarct neurons express ES during the early stage of cerebral ischemia and treatment with Ca-074ME attenuates ES expression and apoptosis in peri-infarct neurons.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Caspase 3; Cathepsin B; Coumarins; Dipeptides; Disease Models, Animal; Endostatins; Enzyme Inhibitors; Hypertension; Infarction, Middle Cerebral Artery; Male; Neurons; Random Allocation; Rats; Rats, Sprague-Dawley; Stroke; Time Factors