Altered proliferative responses of dermal fibroblasts to TGF-beta1 may contribute to chronic venous stasis ulcer.
OBJECTIVE
Venous ulcer fibroblasts demonstrate decreased proliferative responses to growth factor stimulation, suggesting cellular senescence. However, the role of chronic venous insufficiency (CVI) disease progression and extracellular matrix (ECM) proteins in agonist-induced cellular proliferation is ill-defined. We hypothesize that CVI-induced fibroblast proliferative resistance to growth factors worsens with disease progression and is regulated by the composition of ECM.


METHODS
Fibroblast explants were isolated from biopsy specimens from two patients without CVI and 16 patients with CVI of the lower calf (LC) and lower thigh (LT) and stratified according to CEAP disease severity: non-CVI (NC; n = 2), class 2-3 (n = 5), class 4 (n = 5), class 5 (n = 3), and class 6 (n = 3). Proliferation experiments were standardized with a neonatal foreskin fibroblast cell line (HS68). A 10-day course and dose response experiment with 0, 0.5, 1.0, 2.5, 5, 10, and 20 ng/mL of transforming growth factor-beta(1) (TGF-beta(1)) demonstrated maximal cell proliferation at 5 ng/mL of TGF-beta(1) on day 4. Under these conditions, CVI dermal fibroblasts were challenged with and without TGF-beta(1) and evaluated for proliferative responses on plates coated with polystyrene, collagen, and fibronectin.


RESULTS
No differences in unstimulated proliferation were observed in LT and LC fibroblasts from patients with class 2-3 disease and LT fibroblasts from patients with class 4 and 5 disease, compared with NC and HS68 cells. LC fibroblasts from patients with class 4 disease (P <.05) and class 5 disease (P <.001), and LC (P <.001), and LT fibroblasts from patients with class 6 disease (P <.001) proliferated to a lesser degree than did NC and HS68 cells. The diminished proliferation observed in class 4 LC cells was reversible with TGF-beta(1) stimulation (P <.004); however, class 5 and class 6 LC and LT fibroblasts did not respond to stimulation with TGF-beta(1). Collagen increased proliferation of HS68 cells with (P <.05) and without (P <.01) TGF-beta(1), compared with cells grown on polystyrene, but did not increase proliferative responses in NC or CVI fibroblasts with and without TGF-beta(1). Similarly, fibronectin increased proliferation of HS68 cells (P <.05) compared with cells grown on polystyrene, but did not alter proliferation in CVI fibroblasts. Fibronectin did seem to inhibit TGF-beta(1)-induced proliferation observed in class 4 LC cells.


CONCLUSIONS
These data indicate that clinical disease progression correlates with cellular dysfunction. Fibroblasts from patients with class 2-3 disease retain their unstimulated and agonist- induced proliferative capacity, compared with NC and HS68 cells. The onset of inflammatory skin changes (class 4 and class 5 disease) diminishes agonist-induced proliferation, and ulcer formation (class 6 disease) severely inhibits it. In addition, the composition of ECM does not affect TGF-beta(1)-induced proliferation of fibroblasts in CVI.
Lal
Brajesh K
BK
Division of Vascular Surgery, Department of Surgery, H 578, MSB, UMDNJ-New Jersey Medical School, 185 S Orange Ave, , Newark, NJ 07013, USA. lalbk@umdnj.edu
Saito
Satoshi
S
Pappas
Peter J
PJ
Padberg
Frank T
FT
Cerveira
Joaquim J
JJ
Hobson
Robert W
RW
Durán
Walter N
WN
D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D002468:Cell Physiological Phenomena / Q000187:drug effects*; D002908:Chronic Disease; D020405:Dermis / Q000187:drug effects* / Q000503:physiopathology*; D016326:Extracellular Matrix Proteins / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects* / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D012307:Risk Factors; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D014647:Varicose Ulcer / Q000209:etiology / Q000503:physiopathology*
D016326:Extracellular Matrix Proteins; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1