The proliferative capacity of neonatal skin fibroblasts is reduced after exposure to venous ulcer wound fluid: A potential mechanism for senescence in venous ulcers

Purpose: We have previously shown that fibroblasts cultured from venous ulc ers display characteristics of senescence and have reduced growth rates. Su sceptibility of young fibroblasts to the microcirculatory changes associate d with venous ulcers, such as macrophage trapping and activation, could exp lain the prevalence of senescent fibroblasts in these wounds. Methods: We tested the in vitro effect of venous ulcer wound fluid (VUWF), as well as pro-inflammatory cytokines known to be present in VUWP (TNF-alph a, IL-1 beta, and TGF-beta 1), on neonatal foreskin fibroblasts (NFFs). NPP growth rates, cellular morphology, and senescence-associated beta-galactos idase (SA-beta-Gal) activity were determined in the presence or absence of VUWP and the above cytokines. VUWF TNF-alpha concentration and the effect o f anti-TNF-alpha antibody on VUWF inhibitory activity were determined in sa mples obtained from four patients with venous ulcers. Results: NFF growth rates were significantly reduced by VUWF (42,727 +/- 63 01 vs 3902 +/- 2191 P = .006). TNF-alpha r also significantly reduced NPP g rowth rates in a dose-dependent manner (P = .01). No significant growth-inh ibitory activity was seen for IL-1 alpha or TGF-beta. Incubation with VUWF significantly increased the percentage of SA-beta-Gal-positive fibroblasts in vitro on culture day 12 (P = .02). TNF-alpha and TGF-beta 1 had similar effects. TNP-alpha was detected in all VUWF tested, with a mean of 254 +/- 19 pg/mL. Conclusion: These data suggest that the venous ulcer microenvironment adver sely affects young, rapidly proliferating fibroblasts such as NFFs and indu ces fibroblast senescence. Pro-inflammatory cytokines such as TNP-alpha and TGP-beta 1 might be involved in this process. The role of other unknown in hibitory mediators, as well as pro-inflammatory cytokines, in venous ulcer development and impaired healing must be considered.