No abstract

Transforming growth factor-beta (TGF-beta) is a potent stimulus of connective tissue accumulation, and is implicated in the pathogenesis of scleroderma and other fibrotic disorders. Smad3 functions as a key intracellular signal transducer for profibrotic TGF-beta responses in normal skin fibroblasts. The potential role of Smad3 in the pathogenesis of scleroderma was investigated in Smad3-null (Smad3(-/-)) mice using a model of skin fibrosis induced by subcutaneous injections of bleomycin. At early time points, bleomycin-induced macrophage infiltration in the dermis and local TGF-beta production were similar in Smad3(-/-) and wild-type mice. In contrast, at day 28, lesional skin from Smad3(-/-) mice showed attenuated fibrosis, lower synthesis and accumulation of collagen, and reduced collagen gene transcription in situ, compared to wild-type mice. Connective tissue growth factor and alpha-smooth muscle actin expression in lesional skin were also significantly attenuated. Electron microscopy revealed an absence of small diameter collagen fibrils in the dermis from bleomycin-treated Smad3(-/-) mice. Compared to fibroblasts derived from wild-type mice, Smad3(-/-) fibroblasts showed reduced in vitro proliferative and profibrotic responses elicited by TGF-beta. Together, these results indicate that ablation of Smad3 is associated with markedly altered fibroblast regulation in vivo and in vitro, and confers partial protection from bleomycin-induced scleroderma in mice. Reduced fibrosis is due to deregulated fibroblast function, as the inflammatory response induced by bleomycin was similar in wild-type and Smad3(-/-) mice.

Wound healing in the oral mucosa is clinically distinguished by rapid healing and lack of scar formation compared with dermal wounds. Mechanisms of favorable mucosal healing are yet to be elucidated. Utilizing a murine model of equivalent-size mucosal and skin wounds, we verified the rapid reepithelializaton and reduction in scarring of oral wounds reported in humans. Collagen fibrillar structure in oral wounds rapidly approached the size of normal collagen fibrils, while the collagen ultrastructure in skin remained immature through the later phases of healing. To determine whether the transforming growth factor-beta (TGF-beta) contributes to the lack of scar formation in oral mucosa, we compared the expression and production in oral and skin wounds. The RNase protection assay demonstrated significantly lower levels of TGF-beta1 expression in oral wounds compared with dermal wounds, and no changes were observed in the expression levels of TGF-beta2 or TGF-beta 3. ELISA analysis confirmed that oral wounds contained lower levels of TGF-beta1 levels compared with dermal wounds, along with a significant increase in the ratio of TGF-beta 3 to -beta1. These findings showed reduced scarring in oral wounds at the ultrastructural level, and provide evidence that site-specific differences in TGF-beta production contributes to the superior healing of oral wounds.

Upon stimulation, mast cells release a heterogeneous group of factors that promote inflammation and influence cell proliferation. Mast cells accumulate at sites of injury, further suggesting a critical role in wound healing. To assess the importance of mast cells in tissue repair, we compared wound healing in mast cell-deficient WBB6F1/J-KitW/KitW-v (KitW/KitW-v) and wild type WBB6F1/++ (WT) mice. During the inflammatory phase, neutrophil infiltration into wounds of the KitW/KitW-v mice was significantly less than that of WT mice (84.6 +/- 10.3 vs. 218 +/- 26.0 cells/10 high-power fields at day 3, p< 0.001), while wound macrophage and T cell infiltration were similar in both strains. The decrease in neutrophils could not be explained by changes in tumor necrosis factor-alpha or macrophage inflammatory protein-2 levels, because the amounts of these two neutrophil chemoattractants were similar in both KitW/KitW-v and WT mice. Surprisingly, the absence of mast cells had no effect on the proliferative aspects of wound healing, including reepithelialization, collagen synthesis, and angiogenesis. Although mast cells are known to release proangiogenic mediators, vascular endothelial growth factor levels were similar in WT and KitW/KitW-v mice. Moreover, levels of fibroblast growth factor-2 were increased in KitW/KitW-v mice (4206 +/- 107 vs. 1865 +/- 249 pg/ml, p < 0.01). These results suggest that mast cells modulate the recruitment of neutrophils into sites of injury, yet indicate that mast cells are unlikely to exert a major influence on the proliferative response within healing wounds.

Scar and fibrosis are often the end result of mechanical injury and inflammatory diseases. One chemokine that is repeatedly linked to fibrotic responses is monocyte chemoattractant protein-1 (MCP-1). We utilized a murine fibrosis model that produces dermal lesions similar to scleroderma to evaluate collagen fibrillogenesis in the absence of MCP-1. Dermal fibrosis was induced by subcutaneous injection of bleomycin into the dorsal skin of MCP-1-/- and wild-type C57BL/6 mice. After 4 weeks of daily injections, bleomycin treatment led to thickened collagen bundles with robust inflammation in the lesional dermis of wild-type mice. In contrast, the lesional skin of MCP-1-/- mice exhibited a dermal architecture similar to phosphate-buffered saline (PBS)-injected control and normal skin, with few inflammatory cells. Ultrastructural analysis of the lesional dermis from bleomycin-injected wild-type mice revealed markedly abnormal arrangement of collagen fibrils, with normal large diameter collagen fibrils replaced by small collagen fibrils of 41.5 nm. In comparison, the dermis of bleomycin-injected MCP-1-/- mice displayed a uniform pattern of fibril diameters that was similar to normal skin (average diameter 76.7 nm). The findings implicate MCP-1 as a key determinant in the development of skin fibrosis induced by bleomycin, and suggest that MCP-1 may influence collagen fiber formation in vivo.