Role of reactive oxygen species in regulating collagen metabolism in fibroblasts

Abstract

Listen

Translate article

Introduction. Reactive oxygen species (ROS) are traditionally viewed as detrimental to cells by means of causing oxidative damage. However, recent studies have shown that micromolar concentrations of oxidants actually help facilitate wound repair by inducing vascular endothelial growth factor (VEGF) in wound-related cells such as macrophages and keratinocytes. It is hypothesized that aged fibroblasts will have a lower basal cell proliferation, VEGF expression, and production of other cytokines that will be tolerant of exogenous oxidant treatment due to elevated levels of the endogenous antioxidant system as compared to young fibroblasts. Methods. Primary cultures of neonatal, adult, and aged human fibroblasts derived from tissue explants were utilized at low passage number. Triplicate cultures were grown to confluence in enriched media and changed to serum-free media for 48 h before exposure to H 2O 2 (0.05–5 μM; stabilized; Sigma) or xanthine (500 μM; Sigma) plus xanthine oxidase (0.001–0.1 mU/ml; bovine milk; Boehringer--Mannheim) (XXO). Control cells were treated identically in serum-free media. Cell proliferation was assayed using a commercially available kit. VEGF was quantified by ELISA. Results. Cell proliferation in neonatal fibroblasts was greatest in 0.5 μM H 2O 2-treated group as compared to control (0.05 μM = 88% of control, 0.5 μM = 114%, 5.0 μM = 51%). Cell proliferation in adult fibroblasts was greatest in 0.5 μM H 2O 2-treated group (0.05 μM = 111% of control, 0.5 μM = 152%, 5.0 μM = 106%). Cell proliferation in aged cells was greatest in 5.0 μM H 2O 2-treated group (0.05 μM = 89% of control, 0.5 μM = 108%, 5.0 μM = 186%). VEGF in neonatal fibroblasts was greatest in 0.5 μM H 2O 2-treated group (0.05 μM = 105% of control, 0.5 μM = 171%, 5.0 μM = 50%). VEGF in adult cells was greatest in 0.05 μM H 2O 2-treated group (0.05 μM = 84% of control, 0.5 μM = 66%, 5.0 μM = 7%). Production of collagen, degradative enzymes, PDGF, TGF-β 1, and VEGF assays are in progress. Conclusion. In neonatal cells, micromolar concentrations of H 2O 2 induced VEGF at the expense of cell proliferation, while adult fibroblast proliferation was increased without a corresponding increase in VEGF. The proliferation of adult cells resembles the neonates, indicating that tolerance to oxidative stress increases with age.