Small molecule anti-angiogenic probes of the ubiquitin proteasome pathway: Potential application to choroidal neovascularization

PURPOSE. To characterize the angiogenic and inflammatory responses of human choroidal endothelial cells (HCECs) to stimulators and inhibitors of the ubiquitin proteasome pathway (UPP). METHODS. The regulation of the UPP by the inhibitor withaferin A and its congener, withanolide D, two natural products derived from the medicinal plant Withania somnifera was assessed in the three-dimensional endothelial cell sprouting assay (3D-ECSA), by using HCEC- and human umbilical vein endothelial cell (HUVEC)-derived spheroids embedded in a collagen I matrix. Western blot analysis was used to investigate the effect of withanolides on I kappa B-alpha, polyubiquitination, and heme oxygenase (HO)-1 regulation in HCEC and HUVEC cultures. RESULTS. HCECs, like HUVECs, responded to fibroblast growth factor-2, vascular endothelial growth factor, and tumor necrosis factor (TNF)-alpha stimulation and sprouted vessel-like structures in collagen I matrix. However, HCECs were slower to generate these sprouting vessels, when compared with HUVECs. The extent of inhibition of endothelial cell sprouting in 3D matrix, the blockade of TNF-alpha-induced I kappa B-alpha degradation, levels of global polyubiquitinated proteins, and induced production of HO-1 in response to treatment by the withanolides in cultured endothelial cells was similarly regulated between HCECs and HUVECs. CONCLUSIONS. HCECs share with HUVECs a similar response to UPP inhibitors, suggesting that this well-conserved pathway that regulates angioinflammatory mechanisms could be exploited for drug-targeting in the development of novel agents for CNV treatment.