HUMAN OMENTAL TISSUE MICROVASCULAR ENDOTHELIAL CELLS (HOTMEC): ISOLATION AND NEW ASPECTS OF CHARACTERIZATION
To study the function of microvascular endothelial cells in comparison to large vessel endothelial cells, HOTMEC were enzymatically isolated from human omental tissue and plated on petri dishes precoated with an extracellular matrix prepared from isolated fibroblasts of the same tissue or precoated with fibronectin. The culture medium was supplemented with 10% fetal calf serum; growth factors were not needed. HOTMEC were subcultured in a split ratio of 1:3 and maintained in culture for up to 3 month. Cultured HOTMEC were identified and discriminated from other non-endothelial cells by different characteristics and functions. 1. The cells demonstrated the typical polygonal shape as known for endothelial cells isolated from umbilical veins. In comparison to human umbilical vein endothelial cells (HUVEC), however, HOTMEC showed prominant nuclei with several nucleoli and presented a pronounced granulation of the perinuclear cytoplasm. 2. A monoclonal antibody specific for endothelial cells was bound to cultured HOTMEC. 3. Von Willebrand Factor (vWF) antigen was demonstrated within the cells by immunofluorescence staining; measurable amounts of vWF were only found in HUVEC in contrast to HOTMEC using an ELISA. 4. The addition of purified human protein C to HOTMEC preincubated with thrombin led to the activation of the zymogen as demonstrated by a chromogenic assay system. The kinetics of protein C activation were identical for HOTMEC and HUVEC. Tissue plasminogen activator (tPA) as well as plasminogen activator inhibitor (PAI) activity were detected in the culture supernatant of HOTMEC. After incubation period of 12 h in serum-free medium, the conditioned medium of confluent HOTMEC contained 100-fold higher levels of tPA than that of HUVEC. The data demonstrate that the cells isolated from human omental tissue have morphological as well as functional characteristics typical for endothelial cells. Furthermore, the study indicates that HOTMEC and HUVEC present quantitative differences in coagulant and fibrinolytic activities.