Abstract
Abstract: :
Purpose: Previous studies have shown that human retinal pigmented epithelium (RPE) cells produce transforming growth factor-beta2 (TGF-ß2; Pfeffer, et al 94) and that the TGF-ß protein production is affected by glucose (Pascal et al 99). In this study, we investigated the production of bone morphogenetic protein-4 (BMP-4, a member of the TGF-ß superfamily) in adult RPE cells grown under hyperglycemic conditions and exposed to exogenous insulin. Methods: ARPE19 (from ATCC) were revived at passage 24 in 5.5mM glucose with 10% Fetal Bovine Serum. They were grown to confluence and transferred to 24 well plates. At confluence, cells were exposed to serum free media with respective treatments: 5.5mM glucose (control), 25mM glucose, 5.5mM glucose + 19.5mM mannitol (osmotic control), and 25mM glucose + 100nM insulin. At the end of the four-day treatment period, conditioned media was refreshed and the levels of BMP-4 protein in the conditioned media collected after 24 hrs were determined by immunabsorbant assays (ELISA). Total cellular proteins were measured by the Bradford assay. Results: Treatment with high glucose did not affect total cellular protein level in ARPE19 cells (253 µg/well in 5.5 mM glucose control vs. 247 and 286 µg/well in 25 mM glucose and 5.5mM glucose + 19.5 mM mannitol, respectively). However, the addition of insulin resulted in a significant increase in total cellular proteins (to 389 µg /well). BMP-4 protein in the conditioned media increased by 189% upon treatment with high glucose (from 2.67 µg BMP-4/mg cell protein to 7.98). The BMP-4 protein level in the osmotic control was 3.42. Furthermore, the addition of exogenous insulin (at high glucose concentration) significantly reduced the level of BMP-4 (to 5.80) but not to the same level as those of the control groups. These results are reproducible with n=3. Conclusions: High glucose significantly increased BMP-4 protein secretion by ARPE19 cells. This increase was negatively modulated by exogenous insulin. Since BMP is associated with differentiation and proliferation of different tissues, our current results suggest further studies on glucose and insulin actions on BMP-4 levels in the eye are warranted.
Keywords: retinal pigment epithelium • growth factors/growth factor receptors • diabetic retinopathy