Effect of Adenosine Agonists on the Proliferation and Differentiation of Chick Embryo Fibroblasts in Three Dimensional Reconstituted Tissue Constructs

Previous studies indicate that organ fibroblasts play an important role in wound healing, collagen production, remodeling processes and pathogenesis of progressive heart, lung, renal and hepatic fibrotic diseases. Several studies suggest a possible inhibitory role for adenosine in the regulation of fibroblast proliferation. The effect of adenosine A2 agonists on proliferation and differentiation of chick embryo skin/muscle fibroblasts was studied in collagen-based 3-dimensional tissue constructs and also in plated monolayer cells. Chick embryo primary fibroblasts were plated in separate groups and were synchronized by growth arrest before stimulation by different doses of adenosine, and A2 receptor agonists, CV1808, NECA and an A2 receptor antagonist, CGS15943, and control, in the presence of serum or serum free medium. The cell counts for each treatment of monolayer fibroblasts were compared to determine fibroblast proliferation. Western blot analysis, immunostaining and myofibroblast size measurements were conducted to measure the effect of adenosine on the fibroblast differentiation into myofibroblasts. Cell proliferation was also gauged with DNA assays in the 3-D constructs. Adenosine agonists at low doses significantly reduced fibroblast proliferation in monolayer and 3-D cell culture in the presence of 5% Fetal Calf Serum (FCS) demonstrating a potential antifibrotic activity possibly by activation of the A2B receptor. Western blot analysis and immunostaining of cells revealed no significant inhibition of the expression of ?- smooth muscle actin on a per cell basis by adenosine agonists. Cell size measurements indicated increased numbers of smaller fibroblasts suggesting that adenosine may inhibit the conversion of fibroblasts to myofibroblasts. This study suggests that agents that increase tissue cAMP levels may be of beneficial therapeutic value in organ tissue fibrosis.