Abstract
Purpose :
Diabetic Retinopathy (DR) is one of the most prevalently diagnosed complication within patients with diabetes. Additionally, DR is identified as the most readily preventable type of blindness. Previous studies have identified damage within the retina frequently occurs at the initial diagnosis of diabetes. In the early stages and diagnosis of DR, there is a noticeable and significant loss of pericytes, ultimately leading to a disruption of retinal vasculature. Various mechanisms of pericyte apoptosis under hyperglycemic conditions have been studied including angiopoietin2-induced pericyte apoptosis by integrins α3 and β1. This present study aims to demonstrate the expression of integrins a3 and b1 receptors and BIGH3 (TGF beta-Induced Ig Human Clone 3) protein in cultured human retinal pericytes (HRP).
Methods :
HRP cells were obtained from Cell Systems and cultured in complete media with 10% FBS, 1% penicillin/streptomycin, in a humidified 5% CO2 incubator at a temperature of 37oC. Cells were seeded at passage 6 to 7 in a 24 well-plate with coverslips or P10 dishes. Cells were seeded at passage 6 to 7 in a 24 well-plate. Immunostaining/Immunohistochemistry (IHC) was done on HRP cells utilizing antihuman integrin a3 and b1. Gene and protein expressions of α3 β1 and BIGH3 pwere detected by Real-Time PCR and ELISA was used to measure integrin receptor protein in cell lysate.
Results :
IHC results show positive staining from a red conjugated (AF647) to the primary antibody for integrin receptor α3 β1 in HRP. RT-PCR results show both β1 and BIGH3 being expressed in HRP cell cultures. Additionally, the fold changes indicate a significant upregulation of expression the expression of β1 and BIGH3 compared to 18s-rRNA (11 and 22 folds respectively), Additional trials will done in order to confirm the expression of a3. ELISA confirmed both a3 and b1 integrins expression in HRP cell lysates. (900 and 4900 pg/ml respectively)
Conclusions :
Our results show HRP in culture express integrin receptor α3 and β1 and BIGH3 proteins. Further experiments will be carried out to elucidate the molecular pathway of protein interactions and the role of integrin in BIG-induced HRP apoptosis.
This is a 2020 ARVO Annual Meeting abstract.