Purification and Identification of the Bioactive Subs tance That Mediates Cardiac Response to the Ischemia Reperfusion Stresses and Development of Treatment

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1. Function of the bioactive substance that mediates cardiac response to hypoxia/reoxygenation : We showed that the conditioned medium from cardiac myocytes subjected to hypoxia/reoxygenation activated three MAPK family member kinases within 5 to 10 min and induced apoptosis of cardiac myocytes within 24 to 48 hrs. 2. Purification and identification of the bioactive substance released from cardiac myocytes in response to hypoxia/reoxygenation : (A) We showed that the activity for MAPK activation and apoptosis induction of conditioned medium from cardiac myocytes subjected to hypoxia/reoxygenation exists in the protein fraction with MW higher than 10kD.Then, we further purified the substance by chromatograpy such as isoelectric point, ion exchange, get filtration, and hydrophobic We finally analyzed the bands of SDS-PAGE by an amino acid sequencer or mass-spectrometry. We found that the substance consisted of several proteins and at least one novel protein. We are currently going on the process of affinity-purification with an antibody. 3. Autocrine mechanism of humoral factors involved in the activation of intracellular signaling in cardiac myocytes in response to hypoxia : (A) We found that VEGF plays a protective role in cardiac response to hypoxic stresses not only by suppressing apoptotic process but increases the adhesion between cardiac myocytes and extracellular matrix through activation of p125^<FAK> and paxillin in an autocrine fashion. (B) We found that similar autocrine mechanism mediated by VEGF is also involved in cardiac response to pulsatile mechanical stretch (relative hypoxia) and that there is further autocrine mechanism mediated by TGF-β upstream of that by VEGF.