Small molecule anti-angiogenic probes of the ubiquitin proteasome pathway: potential application to choroidal
Paola Bargagna-Mohan1, Padma Priya Ravindranath, Royce Mohan
1Department of Ophthalmology and Visual Sciences, University of Kentucky, Lexington, KY 40536, USA.
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.
Insights
Human choroidal endothelial cells (HCECs) and HUVECs respond similarly to ubiquitin proteasome pathway (UPP) inhibitors. This suggests UPP targeting could be a novel therapeutic strategy for conditions like choroidal neovascularization (CNV).
Area of Science:
- Endothelial cell biology
- Molecular pathways
- Ocular disease mechanisms
Background:
- The ubiquitin proteasome pathway (UPP) regulates key cellular processes, including angiogenesis and inflammation.
- Human choroidal endothelial cells (HCECs) are crucial for retinal health, and their dysfunction is implicated in diseases like choroidal neovascularization (CNV).
- Understanding how HCECs respond to UPP modulators is vital for developing targeted therapies.
Purpose of the Study:
- To investigate the angiogenic and inflammatory responses of HCECs to UPP stimulators and inhibitors.
- To compare the cellular responses of HCECs with human umbilical vein endothelial cells (HUVECs) under UPP modulation.
- To assess the potential of UPP inhibitors as therapeutic agents for CNV.
Main Methods:
- Utilized a three-dimensional endothelial cell sprouting assay (3D-ECSA) with HCEC and HUVEC spheroids in a collagen I matrix.
- Assessed the effects of withaferin A and withanolide D (UPP inhibitors) on endothelial cell sprouting.
- Employed Western blot analysis to examine the impact of withanolides on key proteins like IkappaB-alpha, polyubiquitinated proteins, and heme oxygenase (HO)-1.
Main Results:
- Both HCECs and HUVECs exhibited vessel-like structure formation in response to growth factors and TNF-alpha.
- HCECs demonstrated a slower sprouting response compared to HUVECs.
- UPP inhibitors similarly affected endothelial cell sprouting, IkappaB-alpha degradation, polyubiquitination, and HO-1 induction in both cell types.
Conclusions:
- HCECs share conserved responses to UPP inhibitors with HUVECs.
- The UPP plays a significant role in angioinflammatory mechanisms within endothelial cells.
- Targeting the UPP presents a promising avenue for developing novel therapeutic agents for CNV.
