Selective inhibition of adhesion molecule expression by edelfosine (ET-18-OCH3) on human umbilical vein or microvascular endothelium.

An abundance of data is accumulating that suggest that if one can block endothelial cell-leukocyte binding or inhibit cell adhesion molecules (CAM), inflammatory events can be greatly diminished. In this report, we demonstrate that an alkyl-lysophospho-lipid compound (ET-18-OCH3) can decrease adhesion molecule expression on cultured human micro- and macrovascular endothelial cell lines. ET-18 selectively decreased CAM expression; CD31 was decreased, however. Vascular CAM-1 tumor necrosis factor-alpha-induced expression was not altered. Intercellular adhesion molecule 1 expression was decreased, but endoglin expression was not affected. Thus, we have demonstrated nontoxic downmodulation of vascular CAM expression in vitro. Whether this compound will have anti-inflammatory properties needs to be clarified in animal models.

Inhibition of induced angiogenesis in a human microvascular endothelial cell line by ET-18-OCH3.

Alkyl-lysophospholipids are a group of anti-cancer compounds that have previously been shown to have the unique feature of being selectively toxic to neoplastic tissues. One of these compounds, ET-18-OCH3, has been used for purging bone marrow of cancer cells in phase I clinical trials. Tumor-induced angiogenesis has been directly correlated with tumor growth and metastasis. In this study, we examined the effect ET-18-OCH3 has on a human microvascular endothelial cell line (HMEC-1), including the following functions: angiogenesis, cell-adhesion molecule expression, and cell-junction integrity. We found that ET-18-OCH3 (in vitro) reversibly inhibited induced angiogenesis at levels that did not affect viability. At lower concentrations, ET-18-OCH3 down-regulated the expression of cell-adhesion molecules and affected the integrity of cell-to-cell junctions. This observation demonstrates this versatile family of compounds to have additional targets of action.

HMEC-1: establishment of an immortalized human microvascular endothelial cell line.

The study of human microvascular endothelial cells has been limited, because these cells are difficult to isolate in pure culture, are fastidious in their in vitro growth requirements, and have a very limited lifespan. In order to overcome these difficulties, we have transfected human dermal microvascular endothelial cells (HMEC) with a PBR-322-based plasmid containing the coding region for the simian virus 40 A gene product, large T antigen, and succeeded in immortalizing them. These cells, termed CDC/EU.HMEC-1 (HMEC-1), have been passaged 95 times to date and show no signs of senescence, whereas normal microvascular endothelial cells undergo senescence at passages 8-10. HMEC-1 exhibit typical cobblestone morphology when grown in monolayer culture, express and secrete von Willebrand's Factor, take up acteylated low-density lipoprotein, and rapidly form tubes when cultured on matrigel. HMEC-1 grow to densities three to seven times higher than microvascular endothelial cells and require much less stringent growth medium. HMEC-1 will grow in the absence of human serum, whereas microvascular endothelial cells require culture medium supplemented with 30% human serum. These cells express other cell-surface molecules typically associated with endothelial cells, including CD31 and CD36 and epitopes identified by monoclonal antibodies EN4 and PAL-E. They also express the cell adhesion molecules ICAM-1 and CD44 and following stimulation with interferon-gamma express major histocompatibility complex class II antigens. HMEC-1 specifically bind lymphocytes in cell adhesion assays. Thus HMEC-1 is the first immortalized human microvascular endothelial cell line that retains the morphologic, phenotypic, and functional characteristics of normal human microvascular endothelial cells.