The applicant's group has identified a 5 kb mRNA that is differentially expressed at high levels in isolated human endothelial cells. They have cloned and sequenced this mRNA which has revealed its identity with a cDNA which was named """"""""testican"""""""" because it was cloned from testes, although it is transcribed in most human organs. It remains functionally uncharacterized. The encoded amino acid sequence and other data indicates that the predicted protein is an extracellular proteoglycan with partial homologies to certain matrix proteins and contains a distinctive region of negative charge density. Based on these properties and its differential expression by endothelial cells, it is their hypothesis that testican is a basal lamina protein of the vessel wall and functions as a molecular barrier to anionic plasma proteins. They will test this hypothesis by determining where and when testican mRNA is expressed in vivo, localizing the protein product in tissues, and documenting its functional correlates in culture. They have used nucleic acid techniques to establish the range of cell types that express testican mRNA in culture and they will use in situ hybridization to establish what cells express this gene in vivo and how its expression is influenced by remodeling processes. Immunological reagents have been developed to determine whether the testican protein occurs most abundantly in the predicted endothelial cell basal lamina. Antibodies to specific portions of the encoded sequence will be used to determine what parts of the encoded sequence are retained in the mature protein. They will use homologous recombination to produce testican-deficient endothelial cells to evaluate the effects of this gene product on endothelial cell functions including the ability of monolayers to retain anionic plasma proteins.
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