Posters
Academic Level (Author 1)
Staff
Discipline/Specialty (Author 1)
Neuroscience
Discipline Track
Biomedical Science
Abstract
Background: Human retinal pericytes (HRP) are contractile cells providing support for endothelial cells of capillaries, essential in the regulation of retinal vasculature. Early stages of DR are characterized by the loss of HRP, leading to angiogenesis. Our preliminary studies identified monocyte-derived macrophages secrete TGF-β1, inducing the expression and secretion of a TGFβ1-Induced, pro-apoptotic BIGH3 protein leading to apoptosis of HRP. Based on a preliminary study in renal cells (unpublished data), CTP with an RGD domain is released from BIGH3 by proteolysis leading to renal cell apoptosis. In the present study, we employed Western Blots to determine if a similar molecular event also takes place in the BIGH3 protein to induce HRP apoptosis.
Methods: HRP cells were cultured in complete media with 10% FBS, 1% penicillin/streptomycin, in a 5% CO2 incubator at 37°C. HRP were starved for 24hrs with media composed of DMEM and 1% penicillin/streptomycin prior to getting treated 24 hours with and without 15ug/mL of TGF-β, 50ug/mL of Leupeptin, and with both TGF-β and Leupeptin. Conditioned media was collected and stored in -80C until protein concentration assay and probed with an anti-BIGH3 polyclonal antibody. Western blot analyses of the cleaved BIGH3 protein band were quantified using ImageJ.
Results/Conclusion: Consistent with our previous observation, culture HPR secret BIGH3 protein. Western blots show two BIGH3 protein bands: un-cleaved protein and cleaved. Image analyses of band intensity of the cleaved proteins was significantly reduced in the presence of Leupeptin. Similarly, a reduction of the cleaved protein was also observed when Leupeptin was added to the cell media in with TGFβ. Thus, our results are consistent with prior observations in renal cells that CTP release from BIGH3 is a molecular event in the HRP apoptosis.
Presentation Type
Poster
Recommended Citation
Valdez, Laura, "Molecular event in HRP Apoptosis" (2023). Research Colloquium. 27.
https://scholarworks.utrgv.edu/colloquium/2022/posters/27
Molecular event in HRP Apoptosis
Background: Human retinal pericytes (HRP) are contractile cells providing support for endothelial cells of capillaries, essential in the regulation of retinal vasculature. Early stages of DR are characterized by the loss of HRP, leading to angiogenesis. Our preliminary studies identified monocyte-derived macrophages secrete TGF-β1, inducing the expression and secretion of a TGFβ1-Induced, pro-apoptotic BIGH3 protein leading to apoptosis of HRP. Based on a preliminary study in renal cells (unpublished data), CTP with an RGD domain is released from BIGH3 by proteolysis leading to renal cell apoptosis. In the present study, we employed Western Blots to determine if a similar molecular event also takes place in the BIGH3 protein to induce HRP apoptosis.
Methods: HRP cells were cultured in complete media with 10% FBS, 1% penicillin/streptomycin, in a 5% CO2 incubator at 37°C. HRP were starved for 24hrs with media composed of DMEM and 1% penicillin/streptomycin prior to getting treated 24 hours with and without 15ug/mL of TGF-β, 50ug/mL of Leupeptin, and with both TGF-β and Leupeptin. Conditioned media was collected and stored in -80C until protein concentration assay and probed with an anti-BIGH3 polyclonal antibody. Western blot analyses of the cleaved BIGH3 protein band were quantified using ImageJ.
Results/Conclusion: Consistent with our previous observation, culture HPR secret BIGH3 protein. Western blots show two BIGH3 protein bands: un-cleaved protein and cleaved. Image analyses of band intensity of the cleaved proteins was significantly reduced in the presence of Leupeptin. Similarly, a reduction of the cleaved protein was also observed when Leupeptin was added to the cell media in with TGFβ. Thus, our results are consistent with prior observations in renal cells that CTP release from BIGH3 is a molecular event in the HRP apoptosis.