ISOLATION AND CHARACTERIZATION OF CEREBRAL RESISTANCE VESSEL ENDOTHELIUM IN CULTURE

Organ-derived endothelia have been shown to exhibit distinct patterns of morphology and growth responsiveness in vitro. This report describe s the development, cloning and establishment of long-term serial cultu res of rat vascular endothelial cells derived from cerebrocortical res istance vessels (small arteries and arterioles). Modification of our p revious published technique for establishing resistance vessel-derived smooth muscle cells (RV-SMC) resulted in enhanced levels of endotheli al outgrowth from collagenase-treated microvessel fragments. Although primary culture growth consisted predominantly of SMC, subsequent subc ultivation of these cultures revealed the presence of distinct endothe lial cell clusters within the SMC monolayer. Serial cloning of these i solates resulted in a homogeneous population of cells with the charact eristic endothelial cobblestone growth pattern and positive immuno-flu orescence for factor VIII-related antigen. Previously established RV-S MC frozen stocks provided an additional source for obtaining resistanc e vessel endothelial cells. This was made possible by the slow prolife ration rate of early-passage RV-SMC and their inability to withstand f reezing procedures. Endothelial cells from both preparations were iden tical and designated resistance vessel derived endothelial cells RV-EC . Upon long-term cultivation (>P-15), confluent RV-EC cultures express ed spontaneous multicellular cord development that stained positive fo r factor VIII-related antigen. Cell growth studies demonstrated that R V-EC were capable of significant growth when maintained in serum-free conditions. Growth kinetics using serum-free conditioned medium demons trated mitogenic activity indicating the presence of an autocrine grow th factor. Increase growth responsiveness was also noted in RV-EC when treated with a variety of peptide growth factors. These results indic ate that resistance vessel endothelium can be successfully isolated an d maintained in long-term serial cultures. Furthermore, the availabili ty of cultured EC and SMC from this unique microvascular site will ena ble examination of cerebrovascular endothelial-smooth muscle cell inte ractions in vitro and may help to elucidate the mechanisms of altered vascular function in disease states.