Abstract
Abstract: :
Purpose: In our previous studies, we have shown that adult retinal pigment epithelium –19 (ARPE–19) cells in high glucose increases secretion of BMP–4 into the conditioned media and the addition of exogenous insulin reduces the level of BMP–4 secretion. In the present study, we have added exogenous BMP–4 to the ARPE–19 cells to observe the amount of VEGF secretion into the conditioned media. Methods: ARPE–19 cells (from ATCC) were revived at passage 24 in Dulbecco’s Modified Eagles Media (DMEM) in 5 mM glucose with 10% FBS at pH 7.2. They were grown to confluence and transferred to 24 well plates. At confluence cells were exposed to 5 mM glucose and 25 mM glucose respectively for five days in serum–free media. On the 5th day cells were treated with 10 ng/ml BMP–4. Conditioned media was collected 1, 3, and 5 days after treatment. VEGF levels were measured using ELISA kit from R&D Systems. Total cellular protein levels were measured using the Bradford assay. Results: The addition of BMP–4 resulted in a significant increase of VEGF secretion by ARPE–19 cells in 25 mM and 5 mM glucose. VEGF secretion increased from 312.9 ug/ul to 479.5 ug/ul on day 1, 680 ug/ul to 1031 ug/ul on day 3, and 634.5 ug/ul to 1018 ug/ul on day 5, in 5 mM glucose. In 25 mM glucose VEGF secretion increased from 261.9 ug/ul to 477.5 ug/ul on day 1, 633 ug/ul to 889 ug/ul on day 3, and 713.4 ug/ul to 924.1 ug/ul on day 5. There was no significant difference in total cellular protein levels. Conclusions: The addition of exogenous BMP–4 to ARPE–19 cells in 5 mM and 25mM glucose resulted in a significant increase in overall VEGF production. Since VEGF is related to the proliferation of new blood vessels (or angiogenesis), our current results suggest a possible pathway of glucose mediated BMP–4 in diabetic retinopathy or choroidal neovascularization via VEGF.
Keywords: retinal pigment epithelium • growth factors/growth factor receptors • choroid: neovascularization