Differential Response of Fibroblasts from Type I Diabetics to Proliferative and Migratory Signals

Abstract

Type I diabetics experience significant morbidity from failure of lower extremity injuries to heal. This impaired healing response leads to chronic ulceration, infection and potential amputation of the extremity. Numerous hypotheses have been proposed to explain this impaired healing response, but little is known about intrinsic differences in the fibroblasts populating the diabetic extremity prior to wounding.Nontransformed fibroblast cell lines from type I diabetics were obtained from the NIGMS‐NIH cell repository at the Coriell Institute. Cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, penicillin, streptomycin and either 450 mg/dL (high) or 100 mg/dL (low) glucose. Cell migration was measured over 72 hr by movement of cells away from a circular source of cells, deposited within cloning rings, under high or low glucose conditions. The initial (0, 6, 24 and 48 hr) proliferative response of cells was measured by 3H‐thymidine incorporation into confluent cell monolayers following partial scraping of the cell layer. Growth curves were obtained by seeding 50,000 cells into 6‐well plates and counting cells at 24, 48, 72, and 168 hrs.Fibroblasts from diabetic individuals showed impaired migration compared to nondiabetic cells (34% vs. 67% increase in diameter) in high glucose over an initial 72 hrs. This difference was minimized if the cells were cultured and tested in low glucose medium. In contrast, diabetic derived cells proliferated more rapidly at early times (13.5%[24 hr], 97%[48 hr], 209%[72 hr] diabetic vs. 4%, 52.5%, 156% increase in cell number) when cultured under high glucose conditions. This trend continued under low glucose conditions, although the magnitude difference was diminished. These results suggest that the fibroblast population present in the type I diabetic connective tissue possesses a phenotype distinctly different from normal cells, characterized by a reduced capacity to migrate, but a normal or heightened proliferative response. This may indicate that injury to this connective tissue fails to produce the required influx of fibroblasts to deposit new matrix.