During the previous grant cycle we undertook the determination of whether microvascular cells from skin possessed unique characteristics, capabilities, and functional properties that would provide insights into the role of endothelial cells in cutaneous homeostasis and pathology. We reasoned that since pathologic and indeed most physiologic events take place at the level of the microvasculature, cells derived from this source would be the most appropriate to study regarding organ specific responsiveness. Inherent in this approach was our hypothesis that not all endothelial cells are alike and that the information derived from studies of large vessel endothelial cells would not be necessarily applicable to microvascular endothelial cells. At that time, and even now, almost all laboratories engaged in the study of endothelial cells used large vessel endothelial cells derived from human umbilical veins, because of the relative ease of isolating and culturing these cells. Conversely, microvascular cells are relatively difficult to isolate and propagate, and their culture requirements are very expensive. We have clearly determined that microvascular endothelial cells from skin differ profoundly from HUVEC in a variety of ways, including growth requirements, state of differentiation, expression of cell adhesion molecules, regulation of cell adhesion molecules at the biochemical and molecular levels, and function of cell adhesion molecules as they relate to cell-cell and cell-matrix interactions. In this next grant cycle, we have chosen to pursue in greater detail three important observations that we have made that are biologically significant and apparently unique to microvascular endothelial cells. These observations involve the microvascular-specific regulated expression of vascular cell adhesion molecule-1 (VCAM1), a cell adhesion molecule fundamentally involved in leukocyte trafficking and localization of immune and inflammatory responses; CD36, a cell surface glycoprotein critically involved in pathologic vaso-occlusive processes within the microvasculature; and TNF-alpha and IL-1, two essential pro- inflammatory cytokines implicated in the initiation and localization of cutaneous inflammation and hypoxic damage. Our studies indicate that the induction and expression profiles of these important proteins are uniquely different in HDMEC, and that these differences are critically important in the pathogenesis of localized inflammatory processes and vaso-occlusive events which occur within the microvascular bed. Therefore, the Specific Aims of this project are: l) To characterize at the molecular, biochemical, and functional levels the regulation of VCAM-1 on HDMEC; 2) To delineate the biochemical, binding, and molecular regulatory characteristics of the HDMEC CD36, and compare these characteristics to those of CD36 described in other tissues; and 3) To determine the conditions and mechanisms by which TNF-alpha, IL-1alpha, and IL-1beta are synthesized and released by HDMEC.
