Biological Function of Type VIII Collagen

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In order to know which cells or organs produce type VIII collagen, we extracted RNA from various cells and tissues and performed Northern blot analysis using specific cDNAs. Cultured human aortic endothelial cells were found to keep synthesizing alpha1 (VIII) and alpha2 (VIII) chains until tenth passage. These cells synthesize collagen type I,III,and V at the same time. In separate experiment rat aortic smooth muscle cells were tested whether they produce collagens. What we detected was that the cells synthesized 75 kDa bacterial collagenase sensitive materials. Of interest was that production of this materials was enhanced 20 to 30 times by addition of heparin into the culture medium. Currently we are testing whether this bacterial collagenase sensitive material is the alpha-chain of type VIII by Northern blotting analysis. We also demonstrated that mouse keratinocytes produce collagen VIII at the mRNA level using Northern-blotting and in situ hybridization.Several lines of transgenic mice did show strange expression pattern of type VIII collagen gene during development. It seemed that its pattern was strict and developmentally regulated. However, we did not know whether the pattern was artifact or real, since we did not predict the expression pattern ; transient in certain tissues. Direct in situ hybridization was performed using the specific probes for mouse collagen type VIII at the developmental stage and proved that the expression pattern was real.