Pressure-induced cellular senescence: a mechanism linking venous hypertension to venous ulcers.
BACKGROUND
Slow healing of ulcers in chronic venous insufficiency (CVI) has long been thought secondary to venous hypertension. Dermal fibroblasts isolated from venous ulcers have morphologies and protein production suggestive of premature aging. In this study, we hypothesized that neonatal fibroblasts (NNF) cultured under elevated pressure will demonstrate premature aging and that this effect will be augmented by an inflammatory mediator, transforming growth factor beta (TGF-beta).


METHODS
A unique pressure incubator was used to culture NNF at atmospheric pressure (ATM), ATM + 30 mmHg, ATM + 60 mmHg, and ATM +120 mmHg. Some pressure-exposed NNF were also cultured with TGF- beta (1 ng/ml). Growth rates were determined by flow cytometry. Senescent cells were identified by staining with a marker for cellular senescence, beta-galactosidase (SA-beta-Gal). Light microscopy and digital imaging were used to evaluate cell morphology. Paired linear models and comparison of the slopes were used for statistical analysis of growth. chi2 analysis was used to compare senescence rates.


RESULTS
NNF cultured at ATM + 60 mmHg and ATM + 120 mmHg showed increased SA-beta-Gal activity (P <0.05), and reduced growth rates (P <0.05) at 11 days. These effects were not seen at ATM + 30 mmHg. NNF grown with TGF-beta did not show augmented SA-beta-Gal staining.


CONCLUSIONS
Pressure-exposed NNF demonstrated an accelerated aging phenomenon similar to fibroblasts isolated from venous ulcers. This aging effect was directly related to the level of pressure. TGF-beta did not augment the aging effect. This study suggests that pressure elevations result in altered cell function and accelerated aging that may contribute to the slowed healing seen in patients with venous insufficiency.
Stanley
Andrew C
AC
The Division of Vascular Surgery, University of Vermont College of Medicine, Burlington, Vermont 05405-0680, USA. andrew.stanley@uvm.edu
Fernandez
Nathanial N
NN
Lounsbury
Karen M
KM
Corrow
Kim
K
Osler
Turner
T
Healey
Christopher
C
Forgione
Patrick
P
Shackford
Steven R
SR
Ricci
Michael A
MA
D018929:Cell Culture Techniques; D049109:Cell Proliferation; D016922:Cellular Senescence / Q000502:physiology*; D005347:Fibroblasts / Q000276:immunology / Q000502:physiology*; D006801:Humans; D007231:Infant, Newborn; D011312:Pressure / Q000009:adverse effects*; D012867:Skin; D016212:Transforming Growth Factor beta / Q000009:adverse effects / Q000276:immunology; D014647:Varicose Ulcer / Q000209:etiology / Q000276:immunology / Q000503:physiopathology*; D014690:Venous Pressure / Q000502:physiology*; D001616:beta-Galactosidase / Q000032:analysis
D016212:Transforming Growth Factor beta; D001616:beta-Galactosidase