Effect of interferon-alpha2b on guinea pig wound closure and the expression of cytoskeletal proteins in vivo.

Scar contraction following the healing of deep partial-thickness or full-thickness dermal injury is a leading cause of functional and cosmetic morbidity. The therapeutic use of interferon for the treatment of fibroproliferative disorders associated with scar contraction, including hypertrophic scar, has been suggested because of its antifibrotic properties. Treatment of fibroblasts with interferon has been shown to reduce the rate and extent of contraction using the in vitro fibroblast-populated collagen lattice model. In order to establish the effect of interferon-alpha2b on full-thickness wound contraction in vivo, osmotic pumps loaded with interferon or sterile saline were implanted intraperitoneally in guinea pigs. Seven days following implantation, six full-thickness punch biopsy wounds were created and were monitored by daily assessment of the wound. There was a significant reduction in the rate of wound contraction in the interferon-treated animals after day 3 (p < 0.01). Western blot analysis was used to quantitate selected cytoskeletal proteins in the normal skin and tissue biopsied from the wound at days 7, 14, and 21 postinjury. The amount of vimentin in the contracted wound increased following injury as compared with the amount present in normal skin (p < 0.0001); however, the relative amounts of the myofibroblast-associated cytoskeletal proteins alpha-smooth muscle actin and smooth muscle myosin were less than those found in normal, uninjured skin. By using vimentin to adjust the levels of cytoskeletal proteins for the increase in cellularity in the wounds, both alpha-smooth muscle actin and smooth muscle myosin significantly increased after closure of the wounds on day 14, as compared with the open-wound stage (day 7), before further reductions occurred with remodeling on day 21. Measurement of apoptotic cells using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay revealed an increase in apoptosis in the interferon-alpha2b-treated animals at 21 days following wounding (p < 0.001), which did not colocalize with alpha-smooth muscle actin staining. Taken together, these findings suggest that interferon-alpha2b inhibits wound contraction in vivo, not through an appreciable alteration in myofibroblast number or cytoskeletal protein expression, but possibly through a reduction in fibroblast cellularity by the induction of apoptosis.

Human dermal fibroblasts produce nitric oxide and express both constitutive and inducible nitric oxide synthase isoforms.

Nitric oxide (NO) is produced by a variety of human and animal cells and is involved in a broad array of physiological and pathophysiological processes. It can cause vasodilation, serve as a neurotransmitter, and have anti-neoplastic, anti-microbial, and anti-proliferative effects. In this study, we have demonstrated that fibroblasts derived from human skin spontaneously produce NO and that this production can be enhanced by stimulating the cells with interferon-gamma and lipopolysaccharide. The production of NO by human dermal fibroblasts can be blocked by NG-monomethyl-L-arginine (L-NMMA). The inhibitory effect of L-NMMA on NO production was restored by addition of L-arginine but not D-arginine. By measuring the rate of conversion of [14C]L-arginine to [14C]L-citrulline, we show that unstimulated cells expressed only Ca2+-dependent NO synthase (NOS) activity (1.36 +/- 0.57 pmol/mg/min; n = 4) whereas stimulated cells expressed both Ca2+-dependent (2.60 +/- 0.54 pmol/mg/min; n = 4) and -independent (1.59 +/- 0.14 pmol/mg/min; n = 4) NOS activities. With reverse transcription polymerase chain reaction (RT-PCR), the 422-bp RT-PCR product for human endothelial constitutive NOS and the 462-bp RT-PCR product for human hepatocyte inducible NOS were detected in proportion to the amount of mRNA-related RT-cDNA added to the reaction mixture. Further evidence by immunocytochemistry demonstrated that human dermal fibroblasts express both constitutive and inducible NOS proteins. These data collectively suggest that in addition to macrophages and other inflammatory cells, nitric oxide production by dermal fibroblasts could be important during the inflammatory stages of wound healing and possibly also in the later stages of proliferation and tissue remodeling after skin injury in humans.