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

Invest Ophthalmol Vis Sci. 2006 Sep;47(9):4138-45. doi: 10.1167/iovs.05-1452.


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 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.

MeSH terms

  • Angiogenesis Inhibitors / pharmacology*
  • Blotting, Western
  • Cells, Cultured
  • Choroid / blood supply
  • Choroidal Neovascularization / prevention & control*
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / drug effects*
  • Endothelium, Vascular / metabolism
  • Ergosterol / analogs & derivatives*
  • Ergosterol / pharmacology
  • Growth Substances / pharmacology
  • Heme Oxygenase-1 / metabolism
  • Humans
  • I-kappa B Proteins / metabolism
  • NF-KappaB Inhibitor alpha
  • Neovascularization, Physiologic / drug effects
  • Plant Extracts / pharmacology
  • Plants, Medicinal
  • Proteasome Endopeptidase Complex / metabolism*
  • Ubiquitin / metabolism
  • Umbilical Veins / cytology
  • Withanolides


  • Angiogenesis Inhibitors
  • Growth Substances
  • I-kappa B Proteins
  • NFKBIA protein, human
  • Plant Extracts
  • Ubiquitin
  • Withanolides
  • NF-KappaB Inhibitor alpha
  • withanolide D
  • Heme Oxygenase-1
  • Proteasome Endopeptidase Complex
  • withaferin A
  • Ergosterol