Small molecule anti-angiogenic probes of the ubiquitin proteasome pathway: potential application to choroidal neovascularization.
OBJECTIVE
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 IkappaB-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 IkappaB-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.
Bargagna-Mohan
Paola
P
Department of Ophthalmology and Visual Sciences, University of Kentucky, Lexington, KY 40536, USA.
Ravindranath
Padma Priya
PP
Mohan
Royce
R
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D015153:Blotting, Western; D002478:Cells, Cultured; D002829:Choroid / Q000098:blood supply; D020256:Choroidal Neovascularization / Q000517:prevention & control*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004875:Ergosterol / Q000031:analogs & derivatives* / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology; D051547:Heme Oxygenase-1 / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010936:Plant Extracts / Q000494:pharmacology; D010946:Plants, Medicinal; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology; D054358:Withanolides
D020533:Angiogenesis Inhibitors; D006133:Growth Substances; D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; D010936:Plant Extracts; D025801:Ubiquitin; D054358:Withanolides; D000072000:NF-KappaB Inhibitor alpha; C010376:withanolide D; D051547:Heme Oxygenase-1; D046988:Proteasome Endopeptidase Complex; C009684:withaferin A; D004875:Ergosterol