Relationship between the distribution of myofibroblasts, and stellar and circular scar formation due to the contraction of square and circular wound healing.

Square skin wounds can heal to form a stellar scar with four protrusions at the four angles, whereas circular wounds can heal to form an ellipsoid scar. It is not clear why these differences occur and the aim of the present study was to clarify this phenomenon. Two square or circular full-thickness skin wounds were made on the dorsum of mice, and covered with hydrocolloid dressing. They were observed from day 0 to 15 after wounding, and used to prepare paraffin sections stained with anti-alpha-smooth muscle actin antibody to detect myofibroblasts. The square wound was transiently enlarged by edema and skin tension on day 3, at which time the angles became round, and thus the square form became more circular. Thereafter, the wound contracted rapidly and the circular form was maintained until day 11. On day 11 distinct angles appeared where the scar formation had progressed further, and there were fewer myofibroblasts than in any other section. A stellar scar with protrusions from the four angles was formed on day 15, when myofibroblasts almost disappeared in the protrusions. This indicates that due to the earlier disappearance of myofibroblasts and earlier scarring in the angles of the square wound, the scar angle cannot be pulled into the center of the wound but residual myofibroblasts on the side can pull the side into the center due to myofibroblastic contraction and consequently a stellar scar is formed. Thus, the earlier disappearance of myofibroblasts in the angles is very important for the formation of stellar scars.

Subepithelial myofibroblasts are novel nonprofessional APCs in the human colonic mucosa.

The human gastrointestinal mucosa is exposed to a diverse normal microflora and dietary Ags and is a common site of entry for pathogens. The mucosal immune system must respond to these diverse signals with either the initiation of immunity or tolerance. APCs are important accessory cells that modulate T cell responses which initiate and maintain adaptive immunity. The ability of APCs to communicate with CD4+ T cells is largely dependent on the expression of class II MHC molecules by the APCs. Using immunohistochemistry, confocal microscopy, and flow cytometry, we demonstrate that alpha-smooth muscle actin(+), CD90+ subepithelial myofibroblasts (stromal cells) constitutively express class II MHC molecules in normal colonic mucosa and that they are distinct from professional APCs such as macrophages and dendritic cells. Primary isolates of human colonic myofibroblasts (CMFs) cultured in vitro were able to stimulate allogeneic CD4+ T cell proliferation. This process was dependent on class II MHC and CD80/86 costimulatory molecule expression by the myofibroblasts. We also demonstrate that CMFs, engineered to express a specific DR4 allele, can process and present human serum albumin to a human serum albumin-specific and DR4 allele-restricted T cell hybridoma. These studies characterize a novel cell phenotype which, due to its strategic location and class II MHC expression, may be involved in capture of Ags that cross the epithelial barrier and present them to lamina propria CD4+ T cells. Thus, human CMFs may be important in regulating local immunity in the colon.

Isoform-specific regulation of the actin-organizing protein palladin during TGF-beta1-induced myofibroblast differentiation.

Contractile myofibroblasts are responsible for remodeling of extracellular matrix during wound healing; however, their continued activity results in various fibrocontractive diseases. Conversion of fibroblasts into myofibroblasts is induced by transforming growth factor-beta1 (TGF-beta1) and is hallmarked by the neo-expression of alpha-smooth muscle actin (alpha-SMA), a commonly used myofibroblast marker. Moreover, myofibroblast differentiation and acquisition of the contractile phenotype involves functionally important alterations in the expression of actin-organizing proteins. We investigated whether myofibroblast differentiation is accompanied by changes in the expression of palladin, a cytoskeletal protein that controls stress fiber integrity. Palladin is expressed as several isoforms, including major 3Ig (90 kDa) and 4Ig (140 kDa) forms that differ in their N-terminal sequence. Expression of the 4Ig isoform is strongly induced in fibroblast stress fibers upon TGF-beta1 treatment preceding alpha-SMA upregulation. TGF-beta1 induced upregulation of palladin is mediated both by Smad and mitogen-activated protein kinase pathways. Furthermore, palladin 4Ig-isoform is co-expressed with alpha-SMA in vivo in experimental rat wounds and in human myofibroblast-containing lesions. Taken together these results identify palladin 4Ig as a novel marker of myofibroblast conversion in vitro and in vivo. They also provide for the first time information about the signaling cascades involved in the regulation of palladin expression.

The common bile duct ligation in rat: A relevant in vivo model to study the role of mechanical stress on cell and matrix behaviour.

Common bile duct ligation leads to bile accumulation and liver fibrosis. In this model, little attention has been dedicated to the modification of the common bile duct. We have studied by histochemistry and immunohistochemistry, 3 and 5 days after ligation, the connective tissue modifications of the common bile duct wall. After bile duct ligation, compared with normal bile duct, a strong increase of the bile duct diameter, due to bile stasis, and a thickness of the bile duct wall were observed; numerous myofibroblasts expressing alpha-smooth muscle actin appeared in parallel with the detection of many proliferating connective tissue cells. These myofibroblasts secreted very early high amount of elastic fibre components, elastin and fibrillin-1. Elastic fibre increase was also observed close to the epithelial cell layer. Procollagen type III deposition was also induced 3 days after ligation but decreased thereafter, underlining that myofibroblasts modify their synthesis of extracellular matrix components to comply with the request. We show here that common bile duct ligation represents an invaluable model to study myofibroblastic differentiation and extracellular matrix adaptation produced by an acute mechanical stress.

Immunohistochemical study of NG2 chondroitin sulfate proteoglycan expression in the small and large intestines.

The intestinal subepithelial myofibroblasts (ISEMFs) are located in the lamina propria under the epithelial cells. ISEMFs are thought to have an important role in protecting and maintaining the integrity of the epithelial cell layer and also in the process of wound healing. In this study, we report that the membrane-bound proteoglycan NG2 is abundantly distributed in the ISEMF layer of the mouse and human intestines. NG2 immunostaining in this layer is distributed with similar intensity from the crypt to villi. NG2 is also immunolocalized along the membranes of smooth muscle cells in the intestinal muscle layer. However, skeletal and cardiac muscles are not immunostained for NG2, demonstrating selective expression of the proteoglycan by smooth muscle cells. Using electron microscopy, NG2 immunoreactivity was strongly observed along the cell membranes of ISEMF, with weak diffusion into the neighboring matrix, indicative of the presence of some "shed" NG2. This first report of NG2 proteoglycan expression by ISEMF provides insights into the nature of the interaction of these cells with extracellular matrix and/or intestinal epithelial cells.

The effect of myofibroblast on contracture of hypertrophic scar.

Wound contraction in humans has both positive and negative effects. It is beneficial to wound healing by narrowing the wound margins, but the formation of undesirable scar contracture brings cosmetic and even functional problems. The entire mechanism of wound healing and scar contracture is not clear yet, but it is at least considered that both the fibroblasts and the myofibroblasts are responsible for contraction in healing wounds. The myofibroblast is a cell that possesses all the morphologic and biochemical characteristics of both a fibroblast and a smooth muscle cell. Normally, the myofibroblasts appear in the initial wound healing processes and generate contractile forces to pull both edges of an open wound until it disappears by apoptosis. But as an altered regulation of myofibroblast disappearance, they remain in the dermis and continuously contract the scar, eventually causing scar contracture. In this research, to compare and directly evaluate the influence on scar contracture of the myofibroblast versus the fibroblast, dermal tissues were taken from 10 patients who had highly contracted hypertrophic scars. The myofibroblasts were isolated and concentrated from the fibroblasts using the magnetic activating cell-sorting column to obtain the myofibroblast group, which contained about 28 to 41 percent of the myofibroblasts, and the fibroblast group, which contained less than 0.9 percent of the myofibroblasts. Each group was cultured in the fibroblast-populated collagen lattice for 13 days, and the contraction of the collagen gel was measured every other day. In addition, they were selectively treated with tranilast [N-(3',4'-dimethoxycinnamoyl) anthranilic acid] to evaluate the influence on the contraction of the collagen gel lattice. During the culture, the myofibroblast group, compared with the fibroblast group, showed statistically significant contraction of the collagen gel lattice day by day, except on the first day, and only the myofibroblast group was affected by tranilast treatment, showing significant inhibition of gel contraction. By utilizing an in vitro model, the authors have demonstrated that myofibroblasts play a more important role in the contracture of the hypertrophic scar.