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The effect of halofuginone, a plant alkaloid known to inhibit collagen type I synthesis, on skin collagen content and skin morphology was evaluated in two in vivo models of scleroderma: the murine chronic graft-versus-host disease (cGvHD) and the tight skin mouse. Skin collagen was assessed by hydroxyproline levels in skin biopsies and by immunohistochemistry using anti-collagen type I antibodies. Daily intraperitoneal injections of halofuginone (1 microgram/mouse) for 52 d starting 3 d before spleen cell transplantation, abrogated the increase in skin collagen and prevented the thickening of the dermis and the loss of the subdermal fat, all of which are characteristic of the cGvHD mice. Halofuginone had a minimal effect on collagen content of the control mice. The halofuginone-dependent decrease in skin collagen content was concentration-dependent and was not accompanied by changes in body weight in either the cGvHD or the control mice. Injections of halofuginone (1 microgram/mouse) for 45 d caused a decrease in the collagen content and dermis width in tight skin mice, but did not affect the dermis width of control mice. Collagen content determination from skin biopsies confirmed the immunohistochemical results in the same mice. The low concentration of halofuginone needed to prevent collagen deposition in fibrotic skin without affecting body weight suggests that halofuginone may serve as a novel and promising anti-fibrotic therapy.

Age-dependent gene expression and protein synthesis associated with chondrocyte differentiation were evaluated in the epiphyseal growth plates of normal and tibial dyschondroplasia (TD)-afflicted chickens. In the normal growth plate, collagen type II gene is expressed mainly by chondrocytes at the upper zone of the growth plate and by the chondrocytes in the articular cartilage. Collagen type X and osteopontin (OPN) genes are expressed in the lower zone of the growth plate and in the zone of cartilage-to-bone transition. No age-dependent changes in the pattern of OPN and collagen type II or X gene expression were observed up to 20 days of age. In the TD-afflicted growth plates, the lesion is enlarged with age, and chondrocytes expressing the collagen type II gene were observed in the hypertrophic zone as early as 8 days posthatching. Abnormal expression of OPN and collagen type X genes was also observed starting at 13 days of age. At day 20, the entire TD lesion-which was significantly enlarged-was surrounded by collagen type II, collagen type X, and OPN expressing cells. The level of OPN in TD was reduced with increasing age, and at 20 days almost no OPN could be detected in either the upper or the lower hypertrophic zones. The level of bone sialoprotein (BSP) also diminished with increasing age in the TD growth plates. In contrast to OPN, the age-dependent reduction in BSP levels was mainly in the lower hypertrophic zone (LHZ), and at 20 days of age, BSP was barely detected in the LHZ, whereas in the upper hypertrophic zone, the levels of BSP were similar to those in normal growth plate. In summary, our results suggest that the primary event of the TD lesion occurs in cells of proliferative phenotype within the hypertrophic zone. These cells divide and form the TD lesion, which consists of cells that do not express the genes associated with hypertrophy.

MBA-15, a marrow stromal-derived cell line, was shown to express an estrogen receptor. This finding was confirmed by in situ hybridization and receptor binding assay. An exposure to estrogen (10(-12)-10(-6) M) in a dose response manner resulted in a decrease of cell proliferation as measured by MTT assay. Cell function was measured by enzymatic activities of two osteoblastic markers, CD10/NEP and alkaline phosphatase. These enzymatic activities were elevated following the estrogen treatment. This model enabled direct evaluation of the estrogen effect on stromal osteoblast cells.