Effect of Nitric Oxide Donor on IL-6 and PDGF-BB: Comparison of Diabetic Versus Normal Tissue Under Hypoxic and Normoxic Conditions

Abstract

Statement of the Problem: CDC estimates that 20.8 million people in the US have diabetes. Vascular changes in diabetic tissues result in decreases in oxygenation, platelet derived growth factor (PDGF), and nitric oxide (NO). Fibroblasts and collagen production are significantly reduced in cells from diabetic patients1. Decreased NO production and increased matrix metalloproteinase 8 and 9 occur in wounds of diabetic patients2. Diabetic wounds demonstrate increased inflammatory cytokine secretion, including Interleukin-6. This lab has also demonstrated that the addition of an NO donor can reduce the MMP-8 and MMP-9 expression by fibroblastic cells3. Materials and Methods: Human skin fibroblasts from a diabetic subject were compared to fibroblasts from a non-diabetic patient grown under normoxic and hypoxic conditions. IL-6 and PDGF production were measured. Fibroblasts from a 31yo diabetic and 32yo non-diabetic patients were obtained from the Coriell Institute. Fibroblasts were cultured to confluence and treated with 1nM, 10nM, or 100nM of long-acting SNitrosoglutathione(SNOG) or short-acting FK409 (NOR3) NO donor for 1, 3, or 7 days. Cells were cultured under normoxic (20% oxygen) or hypoxic (2% oxygen) conditions. Appropriate untreated controls were included in all experiments. At harvest, conditioned media from the cultures were assayed for production of IL-6 or PDGF-BB. IL-6 was measured using a ChemiKine® Human Interleukin-6 Sandwich ELISA kit (Chemicon International). PDGF-BB was measured using a Quantikine® Human Immunoassay (RD where significant differences were found, a two-tailed t-test was performed. P-values less than 0.05 were considered significant. Results: After 1, 3, and 7 days in culture, normal fibroblasts produced 23% as much IL-6 as diabetic cells under normoxic conditions. Under hypoxic conditions, normal cells produced an even smaller relative amount of IL-6 (14%). The production of IL-6 by diabetic, but not normal, fibroblasts increased with time in culture. Under normoxic conditions, IL-6 production by diabetic fibroblasts increased from 4.34ng/mg protein to 6.94ng/mg protein from day 1 to day 7, under hypoxic conditions, IL-6 increased from 11.23ng/mg protein to 17.98ng/mg protein. NOR3 had no effect on IL-6 production by any cells under either condition. Treatment with SNOG, decreased IL-6 production by both normal and diabetic fibroblasts, with greater effect on diabetic fibroblasts. Under normoxic conditions, IL-6 production by diabetic cells was dosedependently decreased by up to 81% at the 100nM dose. A smaller response was observed with normal fibroblasts. Only the 100nM dose resulted in a significant decrease in IL-6 production at all three time points. Cells cultured under hypoxic conditions had similar results, but the magnitude was greater in diabetic cells. PDGF-BB was produced by both normal and diabetic fibroblasts. Normal fibroblasts under normoxic conditions synthesized twice as much PDGF-BB as diabetic cells. Under hypoxic conditions, normal cells produced five times the amount of PDGF synthesized by diabetic cells. There were no significant changes in PDGF-BB production with NO donor treatment. Conclusions: Production of PDGF-BB, is significantly reduced under hypoxic conditions in both normal and diabetic cells, but the effect is much greater in the diabetic cells. Synthesis of PDGF-BB by diabetic cells is more influenced by hypoxia than normal cells. Treatment with the NO donor compounds had no effect on PDGF-BB production. Production of IL-6, an inflammatory mediator, is increased in diabetic cells under both normoxic and hypoxic conditions. The addition of SNOG, significantly decreased the amount of IL-6 produced by normal and diabetic cells, but the effect was greater in the diabetic cells.