Summary
Skin fibroblasts from patients with diabetes mellitus display abnormalities in cell
proliferation. The use of exogenous growth factors on diabetic wounds has been found
to stimulate fibroblast proliferation and facilitate wound healing. However, the results
of application of FGF-2 alone to diabetic wounds in clinical trials have been disappointing.
The objective of this experiment was to study the effects of FGF-2 and media supplements
on in vitro proliferation of skin fibroblasts from patients with type II diabetes and nondiabetic
controls, and to evaluate the association between fibroblast proliferation and cAMP
production. Fibroblast cell lines (n = 5 from diabetic and n = 5 from control individuals)
were cultured in DMEM + 20% FBS for 7 days. Cells were then counted, plated into 24-well
plates at a concentration of 2 × 104 cells/well and incubated for 24 h in DMEM with serum. The next day, medium was changed
to serum-free DMEM alone or DMEM with supplements (albumin, transferrin, insulin and
hydrocortisone). Cells were cultured in the presence or absence of varying doses of
FGF-2 (0, 0.3, 1, 3, 10 and 30 ng/ml) for 72 hrs then counted and medium was collected
for cAMP radioimmunoassay. The doubling time for cell number tended to be greater
(p < 0.2) for diabetic fibroblasts than for control fibroblasts. The addition of supplements
to the medium reduced (p < 0.05) the doubling time for both fibroblast types. FGF-2
stimulated (p < 0.05) proliferation of diabetic fibroblasts only in medium containing
supplements. In contrast, FGF-2 stimulated proliferation of control fibroblasts in
medium with or without supplements. The maximal effects of FGF-2 on fibroblast proliferation
were greater (p < 0.02) in medium with supplements than in medium without supplements.
The KD of FGF-2 for fibroblast proliferation was greater (p < 0.06) for diabetic than for
control fibroblasts, and lower (p < 0.02) for medium with supplements than for medium
without supplements. Fibroblasts from patients with diabetes mellitus produced more
(p < 0.05) cAMP than control fibroblasts. These results demonstrate that FGF-2 requires
the presence of supplements to enhance proliferation of fibroblasts from patients
with type II diabetes mellitus. In addition, fibroblasts from diabetic patients showed
a greater KD for FGF-2 in terms of cell proliferation. These data suggest a defective FGF receptor
or down-regulation of the FGF receptor-mediated cascade that leads to cell proliferation.
Identifying methods of reducing the KD of FGF-2 in stimulating the proliferation of diabetic fibroblasts may improve the
clinical response of diabetic wounds to FGF-2.
Key words:
Diabetes mellitus - Fibroblasts - FGF-2 - cAMP - Wound healing
References
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1 Supported by grants No. 1813-0496-2090 and No. 6371-8183-2661 from University of
North Dakota, Grand Forks, ND to K.M.A. and A.T.G.-B.
Anna.Grazul-Bilska@ndsu.nodak.edu
