Appearance and regression of rat pouch tissue.

Fibrosis, a consequence of tissue repair, can become a final common pathway to organ failure, if progressive. Prevention and regression of organ fibrosis represent targets of considerable interest. The natural fate of fibrosis differs among various tissues being either persistent, progressive or regressive. Cellular and molecular responses involving myofibroblasts (myoFb), a phenotypically transformed fibroblast-like cell of considerable functional diversity, is involved in collagen turnover at sites of repair, where they govern the fate of fibrosis. Insights gained from the natural regression of established fibrous tissue may offer strategies to remove unwanted fibrosis in failing organs. In the present study, we addressed the temporal sequence to various components of collagen synthesis and degradation involved in the appearance and subsequent regression of pouch tissue induced in the rat by subcutaneous injection of air followed by instillation of the phorbol ester croton oil. Pouch tissue was collected on day 2, 4, 10, 14, 21, 28 and 35 (n=6 at each time point). Activities of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of MMP-1 (TIMP-1) were determined by zymography and reverse zymography, respectively; collagen accumulation by hydroxyproline concentration; gene expression of TIMP-1 or tissue inhibitor of MMP-1, type I collagen and transforming growth factor-beta1 (TGF-beta1) by in situ hybridization; TGF-beta1 concentration by sandwich enzyme-linked immunosorbant assay (ELISA); and myoFb and its phenotypes by immunohistochemistry using antibodies to alpha-smooth muscle actin (alpha-SMA), vimentin or desmin. During pouch tissue formation, we found: (1) pouch weight increased progressively from day 2 to day 14 and then declined progressively thereafter; (2) type I collagen mRNA expression, barely detectable at day 2, increased at day 4, together with tissue hydroxyproline concentration (P<0.05) reaching a peak on day 10, and gradually decreased thereafter in association with declining tissue hydroxyproline concentration; (3) mRNA expression and concentration of TGF-beta1, detectable at day 2, significantly (P<0.05) increased at day 4, reached a peak at day 10, and gradually declined thereafter; (4) MMP-1 activity, low at day 2, increased continually over the course of 35 days; (5) TIMP-1 mRNA, detectable at day 2 and significantly (P<0.05) increased at day 4, gradually decreased thereafter; (6) activity of TIMP-1 increased continuously from day 2 to day 14 and then was markedly reduced thereafter; and (7) myoFb were first observed in pouch tissue at day 4 and became more extensive thereafter with their phenotype changing over time. Early appearing myoFb (day 4, 10, 14, and 21) expressed alpha -SMA and vimentin (VA phenotype), while later appearing cells (day 28 and 35) additionally expressed desmin (VAD phenotype). Thus, in croton oil-induced rat pouch model, the subcutaneous accumulation of pouch tissue hydroxyproline over the course of 10 days is initially associated with a VA-positive myoFb phenotype and its transcription of TGF-beta1, type I collagen and TIMP-1. Beyond day 10, a regression of pouch tissue collagen begins in association with the appearance of a VAD-positive myoFb phenotype and progressive increase in MMP-1 activity as the expression of TIMP-1 and TGF-beta1 are withdrawn. Regression of established fibrosis in failing organs may, therefore, be attainable through manipulation of myoFb phenotype and/or enhanced collagen degradation relative to collagen synthesis.