Inhibition of hyaluronan synthesis with 4‐methylumbelliferone (4‐MU) prevents in vitro endothelial network formation following stimulation with phorbol 12‐myristate 13‐acetate (PMA)

To determine the possible role of hyaluronan in angiogenesis, bovine adrenal microvascular endothelial cells were grown on top of a type I collagen gel. After reaching confluence, the cells were stimulated to form endothelial networks within the gel using 40 ng/ml PMA. Hyaluronan content of the gel was measured using radiolabeled hyaluronan binding proteins (HABP). One day following stimulation, when the cells had separated but remained on the gel surface, the hyaluronan content was two times control. Four days after stimulation, the cells had formed networks resembling capillaries within the collagen gel but the hyaluronan content had decreased to control values. 4‐MU given with PMA produced a dose dependent decrease in total length of networks. A dose of 500 μM completely prevented network formation and cell migration beneath the gel surface. Adding 4‐MU after the first day, instead of with PMA, had no effect on cell migration or network formation, consistent with changes in hyaluronan synthesis occurring initially. Measurements of intercellular area following one day of stimulation showed that 4‐MU produced a significant decrease in cell separation. The data suggest that hyaluronan is important in the initial cell separation following endothelial stimulation and that 4‐MU may be an effective inhibitor of angiogenesis. (AMC Research Bridge Award)