Up-regulation by human recombinant transforming growth factor β-1 of collagen production in cultured dermal fibroblasts is mediated by the inhibition of nitric oxide signaling

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Abstract
Hypertrophic scarring remains the most disabling sequela for burn survivors. Little is known about its pathogenesis. Collagen accumulation, however, has been consistently observed in burn hypertrophic scars (HS). We have studied collagen production in the dermal fibroblasts derived from HS, which has developed for 9 months to 2 years. Reconstructive surgery was performed to remove HS from which the fibroblasts were cultured. Similarly, the normal cells were grown from the patient's donor site (DS), which provided autografting to the HS site. Collagen production in HS and DS fibroblasts was compared and analyzed in minimal essential amino acid medium containing 5% fetal bovine serum with inclusion of L-ascorbic acid (100 microg/mL) and beta-aminopropoinitrile (100 microg/mL) by monitoring a 20-h [3H]proline incorporation into bacterial collagenase III-digestible protein in the conditioned media. We failed to detect any significant difference in collagen production in vitro between HS and DS. Irrespective of the fibroblasts from HS or DS, collagen production was substantially stimulated by human recombinant transforming growth factor beta-1 (TGF-beta1) (20 ng/mL) by approximately 250% after a 3-day pretreatment. In contrast, sodium nitroprusside (SNP) at 100 microM exhibited significant suppression (68%), which was rescued by hemoglobin (10 microM). TGF-beta1 significantly decreased nitric oxide (NO) production by 55%. In contrast, NO level drastically increased by 350% following SNP treatment. Epidermal growth factor showed no effect on either collagen production or NO level. The linear regression analysis shows a significant inverse correlation (r = 0.72; p < 0.05) of NO level with collagen production, suggesting the involvement of NO signaling in the modulation of collagen production. Consistent with the notion, we further showed that N-nitro-L-arginine methyl ester (100 microM) caused a synergistic stimulation and an arrested inhibition of collagen production in the presence of TGFbeta-1 and SNP, respectively. 8-BrcGMP (300 microM) mimicked the NO inhibitory action, while methylene blue (50 microM) restored the collagen production which was inhibited by SNP. Moreover, 8-BrcGMP offset the stimulation of collagen production. The dermal fibroblasts derived from HS were not different from normals with respect to collagen production and their responses to regulations. The inhibition of collagen production was achieved by a cGMP-dependent NO action. TGFbeta-1 inhibited NO/cGMP signaling to ensure its stimulatory effect on collagen production in the dermal fibroblasts.