Abstract
Abstract: :
Purpose: To employ the three–dimensional endothelial cell sprouting assay as a model to investigate the role of the ubiquitin–proteasome pathway during endothelial cell differentiation and migration, and as a tool to identify novel stimulators and inhibitors of angiogenesis. Methods: We have essentially followed Kroff and Augustin’s procedure (Journal of Cell Biology 1998;143(5):1341–52) for the development of the endothelial cell sprouting assay, a surrogate model of angiogenesis in which endothelial cell spheroids are cultured in a three–dimensional collagen I matrix and these cells initiate differentiation and invasive processes when stimulated by angiogenic growth factors. In our studies, we use cultures of human umbilical vein endothelial cells (HUVECs) and human choroidal endothelial cells (HCECs) to prepare the spheroids. Approximately, 5 spheroids are plated into each well of a 96–well cluster dish in a total volume of 80 micro liters of collagen I solution which contains culture medium with 10% fetal calf serum. To stimulate growth of vessel sprouting, we have employed fibroblast growth factor–2 (FGF–2; 10 ng/ml) and vascular endothelial growth factor–2 (VEGF; 10 ng/ml). Small molecule drugs and natural products are diluted in 100 microliter of medium containing 0.5% dimethylsulfoxide and added to the spheroids and incubated for 18 to 24 hours. The growth of endothelial cell sprouts is monitored under an inverted microscope. Results: We have successfully demonstrated that cultured HCECs form sprouts in collagen I matrix like HUVECs, and respond to the stimulatory effects of VEGF and FGF–2. Employing small molecule inhibitors of the UPP (e.g. the 20S proteasome inhibitor epoxomicin), we show that proteasome function is critical to vessel growth as we observe potent inhibition of vessel sprouting with 5 µM epoxomicin. In continuing analysis of natural product inhibitors and stimulators of angiogenesis, we observe that HCEC spheroids are highly sensitive to the natural product UK–102. In addition, we have also discovered another natural product (UK 201) which demonstrates inhibitory activity at high (1–12 µM) and stimulatory activity at low (100–500 nM) concentrations, while UK 201 is non–toxic at these tested concentrations to proliferating endothelial cells plated on plastic. Conclusions: The growth of HCECs in three–dimension has allowed us to investigate novel small molecule agents which stimulate and inhibit angiogenic differentiation of endothelial cells. We are pursuing the mechanism of these natural products with complimentary signal transduction assays and confocal microscopy.
Keywords: choroid: neovascularization • vascular cells • drug toxicity/drug effects