Fibroblasts cultured from distal lower extremities in patients with venousreflux display cellular characteristics of senescence

Purpose: Venous reflux precedes the development of venous ulcers. Our earli er work showed that the fibroblasts that are cultured from these wounds dis play more characteristics of senescence. We evaluated fibroblast senescence in patients with venous reflux but without ulcers to further investigate t he role of venous reflux in the predisposition to venous ulcers. Methods: Fibroblasts that were isolated from skin biopsy specimens of the " gaiter" area (distal) and of the ipsilateral thigh of the same patient (pro ximal) were compared. Twelve patients with venous reflux (9 patients in cli nical, etiologic, anatomic, and pathologic classification 4; 3 patients in classification 5) with an average venous filling index of 5.45 mL/s and 4 p atients without venous reflux were enrolled in the study. The growth rates, the response to basic fibroblast growth factor (b-FGF), and the senescence markers (beta-galactosidase activity at a pH level of 6, unstimulated fibr oblasts fibronectin protein, and messenger RNA levels) were determined for each cell population. Results: The number of senescence-associated beta-galactosidase positive ce lls (8.3%, +/- 1.9% vs 2.2% +/- 0.8%; P = .008) and the level of cellular f ibronectin protein (455.7 +/- 80 vs 210 +/- 51; P = .04) and messenger RNA (16.8 +/- 6.8 vs 13.5 +/- 5.7; P = .042) were significantly higher in the d istal fibroblasts as compared with the proximal; fibroblast cultures. The g rowth rates of the distal fibroblasts were lower when compared with the pro ximal fibroblasts (15,746 +/- 4287 cells/day vs 29,550 +/- 5035 cells/day; P < .002) but were not different in the presence of b-FGF (41,717 +/- 9542 cells/day vs 47,030 +/- 6133 cells/day; P = .53). In the patients without v enous reflux, no site differences were noted in the growth rates or the sen escence markers between the proximal and distal fibroblasts. Conclusion: Distal fibroblasts that are isolated from patients with venous reflux display more senescence characteristics than do proximal fibroblasts and have significantly lower growth rates. Despite senescence, b-FGF resto red the distal-fibroblasts growth rate to that of the stimulated proximal f ibroblasts, which proposes a therapeutic role for b-FGF. These changes prec ede ulcer formation and suggest a mechanism that is focal and intrinsically related to venous reflux.