Alexis Desmoulière

Tissue repair, contraction, and the myofibroblast

After the first description of the myofibroblast in granulation tissue of an open wound by means of electron microscopy, as an intermediate cell between the fibroblast and the smooth muscle cell, the myofibroblast has been identified both in normal tissues, particularly in locations where there is a necessity of mechanical force development, and in pathological tissues, in relation with hypertrophic scarring, fibromatoses and fibrocontractive diseases as well as in the stroma reaction to epithelial tumors. It is now accepted that fibroblast/myofibroblast transition begins with the appearance of the protomyofibroblast, whose stress fibers contain only β‐ and γ‐cytoplasmic actins and evolves, but not necessarily always, into the appearance of the differentiated myofibroblast, the most common variant of this cell, with stress fibers containing α‐smooth muscle actin. Myofibroblast differentiation is a complex process, regulated by at least a cytokine (the transforming growth factor‐β1), an extracellular matrix component (the ED‐A splice variant of cellular fibronectin), as well as the presence of mechanical tension. The myofibroblast is a key cell for the connective tissue remodeling that takes place during wound healing and fibrosis development. On this basis, the myofibroblast may represent a new important target for improving the evolution of such diseases as hypertrophic scars, and liver, kidney or pulmonary fibrosis.

Normal and Pathologic Soft Tissue Remodeling: Role of the Myofibroblast, with Special Emphasis on Liver and Kidney Fibrosis

Normal and Pathologic Soft Tissue Remodeling: Role of the Myofibroblast, with Special Emphasis on Liver and Kidney Fibrosis

Fibroblasts and myofibroblasts in wound healing

(Myo)fibroblasts are key players for maintaining skin homeostasis and for orchestrating physiological tissue repair. (Myo)fibroblasts are embedded in a sophisticated extracellular matrix (ECM) that they secrete, and a complex and interactive dialogue exists between (myo)fibroblasts and their microenvironment. In addition to the secretion of the ECM, (myo)fibroblasts, by secreting matrix metalloproteinases and tissue inhibitors of metalloproteinases, are able to remodel this ECM. (Myo)fibroblasts and their microenvironment form an evolving network during tissue repair, with reciprocal actions leading to cell differentiation, proliferation, quiescence, or apoptosis, and actions on growth factor bioavailability by binding, sequestration, and activation. In addition, the (myo)fibroblast phenotype is regulated by mechanical stresses to which they are subjected and thus by mechanical signaling. In pathological situations (excessive scarring or fibrosis), or during aging, this dialogue between the (myo)fibroblasts and their microenvironment may be altered or disrupted, leading to repair defects or to injuries with damaged and/or cosmetic skin alterations such as wrinkle development. The intimate dialogue between the (myo)fibroblasts and their microenvironment therefore represents a fascinating domain that must be better understood in order not only to characterize new therapeutic targets and drugs able to prevent or treat pathological developments but also to interfere with skin alterations observed during normal aging or premature aging induced by a deleterious environment.

Mechanical forces induce scar remodeling. Study in non-pressure-treated versus pressure-treated hypertrophic scars.

Reparative process of second and third degree burns usually results in hypertrophic scar formation that can be treated by pressure. Although this method is efficient, its mechanisms of action are not known. In this work, we have studied the histological organization of hypertrophic scars submitted to pressure. Skin biopsies were performed 2 to 7 months after the onset of treatment in two adjacent regions of the scar, non-pressure- or pressure-treated and analyzed by immunohistochemistry and transmission electron microscopy for extracellular matrix organization and cellular morphology. In non-pressure-treated regions, fibrillin deposits did not present the classical candelabra-like pattern under epidermis and were reduced in dermis; in pressure-treated regions the amount was increased compared to non-pressure-treated regions but the organization was still disturbed. In non-pressure-treated regions, elastin was present in patch deposits; in pressure-treated regions elastin formed fibers, smaller than in normal dermis. Tenascin was present in the whole dermis in non-pressure-treated regions, whereas in pressure-treated regions it was observed only under epidermis and around vessels, as in normal skin. alpha-Smooth muscle actin-expressing myofibroblasts were absent in normal skin, present in large amounts in non-pressure-treated regions, and almost absent in pressure-treated regions. The disturbed ultrastructural organization of dermal-epidermal junction observed in non-pressure-treated regions disappeared after pressure therapy; typical features of apoptosis in fibroblastic cells and morphological aspects of collagen degradation were observed in pressure-treated regions. Our results show that, in hypertrophic scars, pressure therapy restores in part the extracellular matrix organization observed in normal scar and induces the disappearance of alpha-smooth muscle actin-expressing myofibroblasts, probably by apoptosis. We suggest that the pressure acts by accelerating the remission phase of the postburn reparative process.

Cellular Retinol-Binding Protein-1 Expression and Modulation during In Vivo and In Vitro Myofibroblastic Differentiation of Rat Hepatic Stellate Cells and Portal Fibroblasts

Cellular retinol-binding protein-1 (CRBP-1) is involved in vitamin A metabolism because it mediates both retinol esterification to retinyl esters and retinol oxidation to retinal and retinoic acid. CRBP-1 is highly expressed in the liver, particularly in hepatic stellate cells (HSC). In this study, we investigated the liver expression of CRBP-1 during experimental fibrogenesis. We also studied the regulation of CRBP-1 expression in cultured HSC and portal fibroblasts, two fibroblastic cell types involved in liver fibrogenesis. Fibrosis was induced in rats by carbon tetrachloride (CCl4) or bile duct ligation. Immunohistochemical staining was performed for CRBP-1 and α-smooth muscle (SM) actin, an activation marker of fibrogenic cells. CRBP-1 and α-SM actin expression was studied by Western blotting and/or Northern blot in primary cultures of HSC isolated by conventional methods and in portal fibroblasts that were obtained by outgrowth from the biliary tree after enzymatic digestion. In normal liver, contrary to HSC, portal fibroblasts did not express CRBP-1. After CCl4 injury, CRBP-1 expression was maintained in myofibroblastic α-SM actin-positive HSC. After bile duct ligation, portal fibroblasts (which proliferated around ductular structures) acquired expression of both CRBP-1 and α-SM actin. During HSC activation in culture, CRBP-1 expression gradually increased until Day 5 when α-SM actin expression was obvious. Cultured portal fibroblasts developed both CRBP-1 and α-SM actin expression. In both cell populations, transforming growth factor-β1 treatment increased CRBP-1 expression. Thus, in normal liver, CRBP-1 expression was different among fibroblastic cells, a finding that adds to the concept of heterogeneity of liver fibrogenic cells. Furthermore, during myofibroblastic differentiation, HSC that lost their stores of retinol maintained a high level of CRBP-1 expression, whereas portal fibroblasts acquired CRBP1 expression. Together, these data suggest a correlation between CRBP-1 expression and myofibroblastic differentiation.

Effects of bile acids on biliary epithelial cells: Proliferation, cytotoxicity, and cytokine secretion

Hydrophobic bile acids, which are known to be cytotoxic for hepatocytes, are retained in high amount in the liver during cholestasis. Thus, we have investigated the effects of bile acids with various hydrophobicities on biliary epithelial cells. Biliary epithelial cells were cultured in the presence of tauroursodeoxycholate (TUDC), taurocholate (TC), taurodeoxycholate (TDC), taurochenodeoxycholate (TCDC), or taurolithocholate (TLC). Cell proliferation, viability, apoptosis and secretion of monocyte chemotactic protein-1 (MCP-1) and of interleukin-6 (IL-6) were studied. Cell proliferation was increased by TDC, and markedly decreased by TLC in a dose dependent manner (50-500 microM). Cell viability was significantly decreased by TLC and TCDC at 500 microM. TLC, TDC and TCDC induced apoptosis at high concentrations. The secretion of MCP-1 and IL-6 was markedly stimulated by TC. TUDC had no significant effect on any parameter. These findings demonstrate that hydrophobic bile acids were cytotoxic and induced apoptosis of biliary epithelial cells. Furthermore, TC, a major biliary acid in human bile, stimulated secretion of cytokines involved in the inflammatory and fibrotic processes occurring during cholestatic liver diseases.

Vascularization pattern in hypertrophic scars and keloids: a stereological analysis.

Wound healing is a complex process that does not always occur harmoniously and may lead to pathological scar development, such as hypertrophic scars and keloids. Considering that vascularization can play a role in the development of these scars, and that the literature is controversial, we performed a stereological analysis of dermal for vessels of normal skin, normal scars, hypertrophic scars, and keloids. The parameters studied concerned vessels: surface density, length density; for vessels and myofibroblasts: volume density, in papillary and reticular dermis. The pattern of dermal vascularization in normal skin and normal scar showed no differences. In papillary demis, the number of vessels was higher in hypertrophic scars and keloids than in normal skin (p < 0.05). Vessels of hypertrophic scars were more dilated than those of normal skin (p < 0.01). In reticular dermis, vessels were present in higher amount in hypertrophic scars and keloids than in normal skin (p < 0.025; p < 0.001, respectively). The pattern of vascularization did not show any differences between hypertrophic scars and keloids. Our results show that hypertrophic scars and keloids have a distinct pattern of vascularization compared to normal skin and normal scars. This indicates that abnormal vascularization can be involved in the development of hypertrophic scars and keloids.

Involvement of matrix metalloproteinase type-3 in hepatocyte growth factor-induced invasion of human hepatocellular carcinoma cells

Intra‐hepatic invasion is a key feature of hepatocellular carcinoma (HCC) progression. We have shown that human liver myofibroblasts induce invasion of HCC cells through Matrigel, via the secretion of hepatocyte growth factor (HGF). In our study, we investigated the role of matrix metalloproteinases (MMP) in HGF‐induced HCC cells invasion. Marimastat, a synthetic MMP inhibitor, dose‐dependently decreased HGF‐induced invasion of HepG2 cells with a maximum of 82.7 ± 13.3% at 20 μM. TIMP‐2, a natural inhibitor, decreased invasion up to 51.2 ± 11.2% at 200 ng/ml. To determine the target for these inhibitors, we examined MMP expression using RT‐PCR. MMPs 1, 7–9 and 10 were not expressed in HepG2 cells either in the absence or in the presence of HGF. MMP‐2 and MMP‐13 transcripts were detected in unstimulated cells but their expression was unchanged after exposition to HGF. MMP‐3 transcripts were undetectable in unstimulated HepG2 cells. They became clearly expressed in HGF‐stimulated cells, however, and this was confirmed by Northern blot. By Western blot, HGF dose‐dependently stimulated the secretion of pro‐MMP‐3 in the culture medium. The role of MMP‐3 in HGF‐induced invasion was directly confirmed by using an antibody to MMP‐3, that blocked invasion. Finally, RT‐PCR demonstrated MMP‐3 expression in 10/16 human HCCs tested, but not in normal liver. In conclusion, our data demonstrate that MMPs, most likely MMP‐3, mediate HGF‐induced invasion of HCC cells. The in vivo expression of MMP‐3 in HCC suggests a role for this protease in HCC progression.

Direct evidence that hepatocyte growth factor-induced invasion of hepatocellular carcinoma cells is mediated by urokinase

BACKGROUND/AIMS We have shown that hepatocyte growth factor secreted by human hepatic myofibroblasts increased the in vitro invasion of the hepatocarcinoma cell line HepG2 through Matrigel. Our aim in this study was to evaluate the role of urokinase in this process. METHODS Expression of urokinase in HepG2 cells was measured by Northern blot and zymography, and plasminogen activation was shown by a chromogenic substrate assay. Cell invasion was assayed on Matrigel-coated filters. Urokinase and urokinase receptor transcripts in hepatocarcinoma were detected by reverse transcription-polymerase chain reaction. Activated hepatocyte growth factor was detected by Western blot with a hepatocyte growth factor-beta chain-specific antibody. RESULTS HepG2 cells expressed urokinase mRNA and secreted active urokinase. Urokinase expression was enhanced by hepatocyte growth factor at the protein and mRNA level. Notably, cell-surface-associated urokinase was increased 22-fold by hepatocyte growth factor. Hepatocyte growth factor also increased urokinase receptor mRNA expression. B428, a urokinase inhibitor, decreased by up to 70% HepG2 invasion induced by myofibroblasts and by 90% that induced by recombinant hepatocyte growth factor. This was not due to a decrease in the generation of activated hepatocyte growth factor by myofibroblasts. Finally, all 17 hepatocarcinoma samples tested expressed urokinase and urokinase receptor transcripts. CONCLUSION Hepatocyte growth factor-dependent, myofibroblasts-induced invasion of HepG2 cells is secondary to the induction of urokinase expression on tumor cells.

Down-regulation of connective tissue growth factor and type I collagen mRNA expression by connective tissue growth factor antisense oligonucleotide during experimental liver fibrosis

Transforming growth factor (TGF)‐β1 is a major mediator of liver fibrosis. Connective tissue growth factor (CTGF) mediates TGF‐β1 pro‐fibrogenic effects in vitro, but its in vivo role is unknown. Both TGF‐β1 and CTGF are overexpressed in hepatic stellate cells during liver fibrosis. We have used antisense oligonucleotides to examine the role of CTGF in carbon tetrachloride‐induced liver fibrosis in mice. Mice received carbon tetrachloride together with CTGF or TGF‐β1 antisense oligonucleotides for 2 weeks (preventive model), or carbon tetrachloride for 2 weeks followed by carbon tetrachloride and oligonucleotides for 2 more weeks (curative model). In both models, CTGF and TGF‐β1 oligonucleotides decreased by more than 50 percent the mRNA expression of their targets. Type I collagen mRNA was also decreased by about 40 percent in the preventive experiment. Tissue inhibitor of matrix metalloproteinase‐1 mRNA expression and fibrotic deposition evaluated by Sirius red staining were not modified in any group. In summary, our results suggest that hepatic stellate cells can be targeted in vivo with oligonucleotides, and that reducing CTGF levels can lead to a decrease in fibrogenesis as shown by the reduction in type I collagen expression. The lack of effect on fibrosis may be due to the persistence of high tissue inhibitor of matrix metalloproteinase‐1 expression.