Glucose Modulates the Growth and Specific Growth Factors of Cultured Human Retinal Pericytes

Abstract

Abstract: : Purpose: Human retinal pericytes (HRP) are cells that support and regulate the growth and differentiation of their underlying capillary endothelial cells. Under the hyperglycemic conditions associated with diabetes, HRP begin to weaken and eventually die, compromising the stability and integrity of the capillary and its component endothelial cells, which begin the process of angiogenesis and neovascularization associated with diabetic retinopathy (DR). This study focuses on the effect of high glucose on the growth of HRP and on their secretion of TGFß2 and VEGF, cytokines known to be associated with the development and progression of DR. Methods: HRP (gift of Dr XJ Ma, MUSC) at P8-9 were seeded in 24-well plates at a density of 1.8 x 10₃ cells/ml, 72% viability, and grown to confluence in DMEM F-12 with euglycemic glucose (5.5mM), 15% FBS at 37°C, 5% CO₂, 100% humidity. At confluence, cells were switched to serum-free media containing either 5.5mM or hyperglycemic (17mM) glucose. At 48 hours, cells were counted by hemacytometer using Trypan blue to assess viability, total protein was determined using the Bradford assay, and TGFß2 and VEGF in the conditioned media (CM) were quantified using ELISA. Results: At the end of 48 hours, HRP grown in 17mM media had the same cell number per well, 1.1 x 10₄, as their 5.5mM counterparts, but the number of viable cells differed. In the 5.5mM condition there were 7.0 x 10₃ viable cells/well vs 4.4 x 10₃ viable cells/well in the hyperglycemic condition. These numbers were used to adjust total protein to the amount that could be attributed to viable cells. TGFß2 in CM of pericytes grown in 5.5mM media was 21.36 pg/µg protein from viable cells. For cells grown in 17mM media, TGFß2 was 40.35 pg/µg protein from viable cells. VEGF in the CM of pericytes grown in 5.5mM media was 5.53 pg/µg protein from viable cells; cells grown in 17mM media had 9.41 pg/µg protein from viable cells. Conclusion: HRP, after 48 hours of exposure to 17mM media, have 25% fewer viable cells as well as an 85% increase of TGFß2, and a 70% increase of VEGF in the CM as compared with cells grown in 5.5mM media. These factors could play a role in the angiogenesis and neovasculaization of the apposite endothelial cells, and on the subsequent development of DR.

Keywords: 388 diabetic retinopathy • 423 growth factors/growth factor receptors