Angiogenesis, the process of forming new blood vessels, is central to a number of essential physiologic and pathologic prosesses, such as wound healing, placental development and tumor metastasis. Endothelial cells, which form the lining of all blood vessels, appear to play a central role in this process. By altering the conditions under which they are grown, human umbilical vein endothelial cells (HUVECs) can be induced to form capillarylike networks in vitro. Using a subtraction/amplification method developed in this lab, mRNA differentially expressed in differentiating HUVECs compared to HUVECs growing as a monolayer will be isolated, amplified by PCR, and used to differentially screen cDNA libraries constructed from PMA-treated HUVECs and differentiated HUVECS. Unique cDNAs will be sequenced and predictions made concerning their structure/function. Northern blots performed with RNA from various cell lines and tissues will determine the tissue distribution of the clones. The functions of the differentially expressed proteins will be elucidated by the addiiion of antisense oligomers or antibodies added to cell cultures and transfection of the clones into a spontaneously transformed human endothelial cell line. Hybridization of fluorescein-labeled antibodies and radiolabed RNA to normal tissue sections and pathologic specimens (tumors, experimentally-induced chronic arthritis in rats) will help elucidate the role of the novel proteins in health and disease.
