The effect of passage number on fibroblast cellular senescence in patients with chronic venous insufficiency with and without ulcer

Background: Fibroblasts (fb) cultured from venous ulcer patients and patients with venous reflux disease without ulcer demonstrate characteristics of cellular senescence, such as increased fibronectin level and senescence-associated β-galactosidase (SA β-gal) positive cells. Cellular senescence is an in vitro event characterized by the progressive loss of proliferative capacity with increased passage number, and has been associated with impaired healing in vivo. This report examines progressive stages of cellular senescence in fb from the distal area (du-fb) and proximal fb (pu-fb) of patients with venous ulcer, as well as in distal fb (dr-fb) and proximal fb (pr-fb) from patients with venous reflux without ulcer, by comparing the population doubling time (T) and percent SA β-gal expression. Results: The mean value of T over 6 passages for fb in the ulcer group was 132.5 ± 29.0 hours for pu-fb and 492.9 ± 146.2 hours for du-fb (P = 0.0009). For fb in the reflux group the mean value of T over 5 passages was 79.3 ± 12.8 hours for pr-fb and 94.2 ± 16.8 hours for dr-fb (P = 0.8). Comparing ulcer and reflux fb, no difference in T was observed between pu-fb and pr-fb (P = 0.6), but a difference was noted between du-fb and dr-fb (P = 0.0004). The mean percent SA β-gal activity for fb in the ulcer group was 11.2% ± 3.1% for pu-fb and 63.8% ± 8.9% for du-fb (P = 0.0001). Individual passages demonstrated significant difference (P <0.05) in SA β-gal activity between pu-fb and du-fb at early and late passages. No difference was noted in SA β-gal activity for fb in the reflux group or between pu-fb and pr-fb, but comparison between du-fb and dr-fb was significant (63.8% ± 8.9% versus 7.8% ± 2.9%; P = 0.0001). Conclusions: The in vitro passage of du-fb and pu-fb in chronic venous ulcer patients has an effect on T and cellular senescence as measured by SA β-gal activity. Our data further suggest that du-fb are at a more progressive stage of cellular senescence when compared with pu-fb, and more importantly with fb cultured from patients with venous reflux without ulcer. These findings are consistent with impaired wound healing of venous stasis ulcer. The accumulation of senescent fb and a more advanced stage of cellular senescence of du-fb may explain why repeated episodes of venous ulceration are resistant to conservative treatment and require more aggressive measures of therapy.