A new in vitro model of venous hypertension: the effect of pressure on dermal fibroblasts.
OBJECTIVE
Venous hypertension leads to venous stasis ulcers. White cell activation, protein leakage from pressurized capillaries, and cytokine imbalances have all been implicated as indirect effects of venous hypertension that contribute to dermal changes seen in chronic venous insufficiency. The direct effect of increased tissue pressures on dermal elements has not been investigated. Prior studies have shown that fibroblasts isolated from venous ulcers have altered growth rates, morphologies, and protein production similar to senescent or aged fibroblasts. We hypothesize that neonatal fibroblasts (NNFs) cultured in conditions of increased atmospheric pressure will demonstrate altered cell function when compared with those grown at normal atmospheric pressure (ATM).


METHODS
A pressure incubator was used to culture populations of NNFs at ATM, 60 mm Hg over ATM (ATM + 60 mm Hg), and 120 mm Hg over ATM (ATM + 120 mm Hg). NNF population growth rates were determined by periodic flow cytometry analysis over a 2-week period. Light microscopy and digital imaging were used to evaluate cell morphology. Senescence-associated B-galactosidase (SA-beta-Gal) activity was determined using the X-Gal stain. Fibronectin production was assessed by exposing cells sequentially to anti-fibronectin antibodies and Oregon Green-conjugated goat anti-mouse secondary antibodies. Flow cytometry then was used to determine relative proportions of cells staining positively for fibronectin. Statistical analysis was accomplished with analysis of variance.


RESULTS
Populations of cells grown under increased pressures (both ATM + 60 and ATM + 120) showed reduced growth rates (P <.001). Similarly, morphologies of cells grown under pressure had increased cytoplasm to nuclear ratios with abnormal nuclear shapes. Populations of cells grown under pressure had higher percentages of cells staining positive for fibronectin (ATM = 45%, ATM + 60 = 59%, ATM + 120 = 79%). After 14 days of growth under pressure, fibroblast populations did not demonstrate augmented productions of the senescence marker SA-beta-Gal (ATM =.5%, ATM + 60 =.25%, ATM + 120 =.75%).


CONCLUSIONS
This study demonstrated that NNFs grown in culture under increased pressures undergo a transformation not seen in cells grown at atmospheric pressure. Cells grown under pressure demonstrated reduced growth rates, increased fibronectin production, and abnormal morphologies similar to fibroblasts isolated from venous ulcers. This study suggests that pressure elevations (like venous hypertension) can directly result in altered cell function and morphology that may contribute to the delayed wound healing seen in patients with venous ulcers. This model uses a pressurized incubator that may prove to be a valuable adjunct in studying the effects of venous hypertension.
Healey
Chris
C
Department of Surgery, University of Vermont, College of Medicine, Burlington, 05405, USA.
Forgione
Patrick
P
Lounsbury
Karen M
KM
Corrow
Kim
K
Osler
Turner
T
Ricci
Michael A
MA
Stanley
Andrew
A
D001274:Atmospheric Pressure; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division / Q000502:physiology*; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology; D020405:Dermis; D005347:Fibroblasts / Q000737:chemistry / Q000166:cytology* / Q000502:physiology*; D005353:Fibronectins / Q000032:analysis; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D008954:Models, Biological; D014690:Venous Pressure / Q000502:physiology*; D001616:beta-Galactosidase / Q000032:analysis
D005353:Fibronectins; D001616:beta-Galactosidase