Use of mesenchymal stem cells for cutaneous repair and skin substitute elaboration.

Human mesenchymal stem cells (MSCs) are a heterogeneous population of fibroblast-like cells, which are present in different locations, including bone marrow, adipose tissue, extra-foetal tissues, gingiva and dermis. MSCs, which present multipotency capacities, important expansive potential and immunotolerance properties, remain an attractive tool for tissue repair and regenerative medicine. Currently, several studies and clinical trials highlight the use of MSCs in cutaneous repair underlining that their effects are essentially due to the numerous factors that they release. MSCs are also used in skin substitute development. In this study, we will first discuss the different sources of MSCs actually available. We will then present results showing that bone marrow-derived MSCs prepared according to Good Manufacturing Practices and included in a dermal equivalent are able to promote appropriate epidermis growth and differentiation. These data demonstrate that bone marrow-derived MSCs represent a satisfactory alternative to dermal fibroblasts in order to develop skin substitute. In addition, MSCs could provide a useful alternative to sustain epidermis development and to promote wound healing.

The complex dialogue between (myo)fibroblasts and the extracellular matrix during skin repair processes and ageing.

The fibroblasts and the myofibroblasts are key players for maintaining skin homeostasis and for orchestrating physiological tissue repair. The (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. The composition of the ECM around (myo)fibroblasts is variable depending on the situation and, in addition to the secretion of the ECM, the (myo)fibroblasts, by secreting matrix metalloproteinases and tissue inhibitors of metalloproteinases can remodel this ECM. The (myo)fibroblasts and their microenvironment form a changing network with reciprocal actions leading to cell differentiation, proliferation, quiescence or apoptosis, and also acting on growth factor biodisponibility. In pathological situations (such as chronic wounds or excessive scarring), or during ageing, especially due to ultraviolet exposition, this dialogue between the (myo)fibroblasts and their microenvironment is disrupted, leading to repair defects or to skin injuries with unaesthetic alterations such as wrinkles. Knowing the intimate exchanges between the (myo)fibroblasts and their microenvironment represents a fascinating domain important not only for characterizing new targets and drugs able to prevent pathological developments but also for interfering with skin alterations observed during ageing.

Investigation of liver fibrosis in clinical practice.

The liver is composed of different hepatic fibrogenic cells: hepatic stellate cells, portal fibroblasts, fibroblasts of the Glisson capsule surrounding the liver and vascular smooth muscle cells and the second layer cells present around centrolobular veins. During liver disease, one or several populations of these cells are activated, transformed into myofibroblasts and secrete the extra-cellular matrix. There are markers to identify hepatic stellate cells either quiescent (CRBP-1) or activated (alpha-smooth muscle actin). Liver biopsy, the current "gold-standard" to estimate liver fibrosis cannot be used anymore as a "gold standard". Furthermore, it is a costly procedure with adverse effects feared by patients and clinicians. Alternative to liver biopsy using non-invasive-tests or technics include FibroTest-ActiTest, transient-elastography, hepatic vein transit time using contrast ultrasonography, magnetic resonance imaging. As a routine test, the FibroTest-ActiTest is a validated one for patients with chronic hepatitis C. The advantage of the non-invasive tests or technics is that they provide a rapid and quantitative estimation of fibrosis. With these new methods, it is possible to follow the progression of the disease and its regression either spontaneously or under treatment. In conclusion, clinicians have in their hands several painless tools to explore liver fibrosis that can be easily repeated.

A role for thrombin in liver fibrosis.

BACKGROUND/AIM: Several lines of evidence incriminate the serine proteinase thrombin in liver fibrogenesis either through its procoagulant function or its signaling via cell-surface receptors. The aim of this study was therefore to evaluate the effect of thrombin inhibition on experimental liver fibrosis. METHODS: Fibrosis was induced in rats by administration of CCl4 for either three or seven weeks. Oral administration of the thrombin antagonist SSR182289 started one week after the start of CCl4 intoxication. Fibrosis and the area occupied by alpha smooth muscle actin (ASMA) positive cells were quantified with histomorphometry. Expression of fibrosis related genes was measured by real time RT-PCR. RESULTS: After three weeks of CCl4, treatment with SSR182289 did not significantly decrease the area of fibrosis but significantly decreased the area of ASMA positive cells by 22% (p = 0.03) and the expression of TIMP-1 mRNA by 52% (p = 0.02). There was no effect on gene expression of collagen I, MMP-2, or TIMP-2. After seven weeks of CCl4, treatment with SSR182289 resulted in a significant decrease in fibrosis (-30%, p = 0.04) and ASMA positive areas (-35%, p = 0.05). SSR182289 alone had no effect on the measured parameters. Additionally, it did not alleviate the acute toxicity of CCl4 as shown by measuring levels of serum aminotransferases and the area of necrosis. CONCLUSIONS: These data provide evidence that thrombin antagonism can reduce liver fibrogenesis. The early effect of SSR182289 on ASMA and TIMP-1 expression suggests that it is beneficial in reducing fibrogenic cell activation.

Validation of a morphometric method for evaluating fibroblast numbers in normal and pathologic tissues.

The aim of this work was to validate an image analysis method, based on cell nuclei form factor determination, for counting fibroblasts within human dermis. We first used reconstructed dermal equivalents in which fibroblasts can also be counted directly after lysis of the collagen matrix. We found a good correlation between the results of direct counting and those of image analysis from day 10 to day 28 of culture. When applied to young normal donors' skin biopsies fixed in Bouin's solution and embedded in paraffin, the image analysis method yielded mid-dermis fibroblast counts of between 2100 and 4100 per mm3 of fresh tissue. A nuclear form factor (FF) comprised between 0.35 and 0.84 was found to be a biologic marker of fibroblasts. This was confirmed after fibroblast discrimination from other cell types, which had rounder nuclei (FF >/= 0.85) and were identified either by their location (e.g. endothelial cells) or by labeling with specific antibodies (e.g. lymphocytes and monocytes/macrophages). Similar results were obtained with seven healthy donors' skin biopsies that had been frozen in nitrogen liquid and cryostat-sectioned, showing that this counting method is independent of the histologic procedure. Finally, analysis of samples of hypertrophic scars from two patients revealed that fibroblast density in some parts of the dermis was more than twice the value found in other parts presenting a fibroblast density almost normal, showing that this cell counting method can also be used to assess fibroblast heterogeneity within a given tissue.

[Mechanisms of hepatic fibrogenesis]. / Mécanismes de la fibrogénèse hépatique.

Hepatic fibrosis is a scaring process leading to cirrhosis, a major complication of numerous chronic liver diseases. Hepatic stellate cells play a central role in the fibrotic process. After parenchymal or biliary injury, cytokines and growth factors allow the recruitment, proliferation, and activation, of stellate cells toward myofibroblasts, which secrete the extracellular matrix. Fibrosis, resulting from the failure of the balance between synthesis and degradation of extracellular matrix, is an evolutive and potentially reversible process. Histological examination is the main investigation to quantify fibrosis. Serological tests are warranted to allow a non invasive follow up of patients. Development of antifibrotic therapies should soon permit to slow down the evolution toward cirrhosis, limiting the needs for hepatic transplantation.

Myofibroblast: a prognostic marker and target cell in progressive renal disease.

Myofibroblasts play an important role in many tissue injuries, and particularly in renal disease. The myofibroblast differentiation is an early event in the development of fibrosis. Myofibroblast-like cells express smooth muscle (SM) cytoskeletal markers (alpha-SM actin in particular) and participate actively in the production of extracellular matrix. Identification of early risk factors, particularly histological criteria, could be useful to identify patients at risk of progressive renal failure and needing a treatment. The evaluation of myofibroblast differentiation in renal tissue may reflect the intensity of tissue injury, predict long term outcome of chronic renal disease and help physicians to select therapeutic choices. More than a disease activity indicator. myofibroblasts appear to be a pivotal target for future therapies in progressive renal disease.

Trans-resveratrol, a grapevine-derived polyphenol, blocks hepatocyte growth factor-induced invasion of hepatocellular carcinoma cells.

We have shown that liver myofibroblasts stimulate in vitro invasion of hepatocellular carcinoma cell lines through a hepatocyte growth factor/urokinase-dependent mechanism. Resveratrol, a grapevine-derived polyphenol, has been shown to inhibit cellular events associated with tumor initiation, promotion and progression. The aim of this study was to evaluate the effects of trans-resveratrol on invasion of the human hepatoma cell line HepG2. Cell invasion was assessed using a Boyden chamber assay. Activation of the HGF signal transduction pathways was evaluated by Western blot with phospho-specific antibodies. Urokinase expression was measured by RT-PCR and zymography. Trans-resveratrol decreased hepatocyte growth factor-induced cell scattering and invasion. It also decreased cell proliferation without evidence for cytotoxicity or apoptosis. Trans-resveratrol did not decrease the level of the hepatocyte growth factor receptor c-met and did not impede the hepatocyte growth factor-induced increase in c-met precursor synthesis. Moreover, trans-resveratrol did not decrease hepatocyte growth factor-induced c-met autophosphorylation, or Akt-1 or extracellular-regulated kinases-1 and -2 activation. Finally, it did not decrease urokinase expression and did not block the catalytic activity of urokinase. In conclusion, our results demonstrate that trans-resveratrol decreases hepatocyte growth factor-induced HepG2 cell invasion by an as yet unidentified post-receptor mechanism.

Expression and cellular localization of fibrillin-1 in normal and pathological human liver.

BACKGROUND: The expression and the distribution of fibrillin-1 and elastin were studied in normal and pathological human liver samples. METHODS: As controls, histologically normal/subnormal liver samples (n = 24) were used. Pathological samples corresponded to seven cirrhosis and eight hepatocellular carcinomas (HCC) developed on cirrhotic (four) or noncirrhotic (four) liver. RESULTS: In normal liver, fibrillin-1 and elastin co-localized in vessel walls and portal tract connective tissue. Fibrillin-1 alone was detected along sinusoids and in portal spaces at the interface with the limiting hepatocytic plates and close to the basement membrane of bile ducts. By transmission electron microscopy, typical bundles of microfibrils were detected both in Disse space and in portal zones. Cirrhotic nodules were usually rich in fibrillin-1 along sinusoids; fibrillin-1 and elastin were co-localized in fibrotic septa surrounding nodules. In HCC, fibrillin-1 was present between tumoral hepatocytes; stromal reaction around the tumors contained both fibrillin-1 and elastin. CONCLUSIONS: Fibrillin-1 was associated with elastin in portal mesenchyme and vessel walls of normal liver, in fibrotic septa around cirrhotic nodules and stromal reaction around HCC, but was expressed alone in the perisinusoidal space. The functional roles for fibrillin-1 in non-elastic tissues, such as the liver, remain to be elucidated.

Expression of hepatocyte growth factor in human hepatocellular carcinoma.

BACKGROUND/AIMS: We have shown that hepatocyte growth factor, secreted by human liver myofibroblasts, promoted in vitro invasion of human hepatocellular carcinoma cell lines. The aim of this work was to measure hepatocyte growth factor expression in 29 human hepatocellular carcinomas and the corresponding peri-tumoral livers. METHODS: We used reverse transcription-polymerase chain reaction, in situ hybridization, ELISA and Western blot. RESULTS: Sixty-two of tested hepatocellular carcinomas were positive by reverse transcription-polymerase chain reaction. With in situ hybridization, a signal was found in every sample. In many cases, the signal was localized in cells labeled with an anti-smooth muscle alpka-actin antibody, while hepatocytes were mostly non-labeled. ELISA, performed in 15 pairs of hepatocellular carcinomas and surrounding livers, detected hepatocyte growth factor in every sample with wide variations. Hepatocellular carcinomas that had developed in non-cirrhotic livers contained essentially the same amount of hepatocyte growth factor as the matching non-tumoral liver. In cirrhotic livers, the hepatocyte growth factor content of the tumors was significantly lower than that of the surrounding cirrhotic livers. CONCLUSIONS: These data indicate that hepatocyte growth factor is expressed at significant levels in every hepatocellular carcinoma tested and that its expression takes place in the stromal myofibroblasts.

Early activation of hepatic stellate cells and perisinusoidal extracellular matrix changes during ex vivo pig liver perfusion.

In previous works, we observed during liver transplantation procedure, the early activation of hepatic stellate cells (HSC) which acquire alpha-smooth muscle (SM) actin expression. In this study, we evaluated changes in HSC and in perisinusoidal extracellular matrix during ex vivo pig liver perfusion. Under general anesthesia, pig livers were flushed and removed, and then perfused ex vivo for 6 h with homologous blood. Liver biopsies were taken before and after washout, at 5 min perfusion, and then hourly. Tissues were processed for immunohistochemistry, immunofluorescence, confocal microscopy, in situ hybridization and electron microscopy. Before and after liver washout, alpha-SM actin was present in vessel walls but in very few lobular HSC. After 1 h perfusion, a strong reactivity for alpha-SM actin was present in HSC, particularly along dilated sinusoids. At the ultrastructural level, numerous microfilament bundles appeared in HSC cytoplasmic processes. During perfusion, type I and type IV collagens, type III procollagen, and fibronectin acquired a looser organisation in relation with the enlargement of perisinusoidal spaces; laminin appeared in perisinusoidal spaces around portal areas and fibrillin deposits increased. In situ hybridization studies showed an increase of the type I procollagen mRNA expression mainly in portal tracts and septa. Ex vivo liver perfusion induces: 1) an early activation of HSC which acquire the expression of alpha-SM actin, and 2) significant changes in the perisinusoidal extracellular matrix. These results are compatible with the view that HSC function as liver specific pericytes participating in the regulation of sinusoidal blood pressure.

Hepatocarcinoma cells stimulate hepatocyte growth factor secretion in human liver myofibroblasts.

Hepatocellular carcinoma (HCC), the main type of primary liver cancer, is characterized by a high rate of intra-hepatic invasion. The stroma of HCC is infiltrated by myofibroblasts. We have previously shown that hepatocyte growth factor (HGF) secreted by human liver myofibroblasts greatly increased the in vitro invasiveness of 3 human HCC cell lines. In this study we show that the conditioned medium (CM) from the same HCC cell lines dose-dependently stimulates HGF secretion by myofibroblasts. This effect was post-transcriptional as no increase in HGF mRNA was observed. We show that the effect of CM is not due to IL-1, IL-6, IGF-1, bFGF or PDGF, previously shown to stimulate HGF synthesis in other models. Our data demonstrate that HCC cells increase HGF secretion by liver myofibroblasts in a paracrine way that could act to enhance invasion.

Paradoxical pro-invasive effect of the serine proteinase inhibitor tissue factor pathway inhibitor-2 on human hepatocellular carcinoma cells.

We have previously shown that human liver myofibroblasts promote in vitro invasion of human hepatocellular carcinoma (HCC) cells through a hepatocyte growth factor (HGF)/urokinase/plasmin-dependent mechanism. In this study, we demonstrate that myofibroblasts synthesize the serine proteinase inhibitor tissue factor pathway inhibitor-2 (TFPI-2). Despite the fact that recombinant TFPI-2 readily inhibits plasmin, we show that it potentiates HGF-induced invasion of HCC cells and is capable of inducing invasion on its own. Furthermore, HCC cells stably transfected with a TFPI-2 expression vector became spontaneously invasive. HCC cells express tissue factor and specifically factor VII. Addition of an antibody to factor VII abolished the pro-invasive effect of TFPI-2. We suggest that TFPI-2 induces invasion following binding to a tissue factor-factor VIIa complex preformed on HCC cells. Our data thus demonstrate an original mechanism of cell invasion that may be specific for liver tumor cells.

Establishment and characterization of a spontaneously immortalized myofibroblast cell line derived from a human liver angiosarcoma.

BACKGROUND/AIM: Fibrosis and/or cirrhosis are present in the precursor stages of most liver cancers. However, little is known about the reciprocal interactions of fibroblasts, mainly responsible for fibrosis, and the other liver cells. We report here the isolation of a new liver myofibroblast cell line from a human liver angiosarcoma and its characterization. METHODS: The cells were isolated by the explant technique and characterization was performed, on one hand, using immunohistochemical and ultrastructural analysis and, in the other hand, by determining their karyotype, ras and p53 status and their tumorigenic properties. RESULTS: To date, the cells have undergone approximately 170 population doublings and are still proliferating. Immunohistochemically, they were negative for desmin, smooth muscle myosin, cytokeratin 19 and von Willebrand factor, positive for vimentin and alpha-smooth muscle actin, with an important deposition of fibronectin around the cells. Ultrastructure showed particularly cytoplasmic microfilament bundles. Their chromosome number ranged from 38 to 168 with a bimodal population, near diploid and hypotetraploid. No mutations were found in codons 12, 13 or 61 of Ha-, Ki- and N-ras genes but a homozygous missense mutation in codon 179 (CAT-->CTT) was detected in the p53 gene. They were unable to form foci in soft agar or tumors in nude mice. CONCLUSIONS: Taken together, these results show that these cells, called BM 2.2.1, exhibited typical myofibroblast-like features. Although they contained a karyotype suggestive of tumoral cells and a homozygous mutated p53 gene, they were not tumorigenic. The nature of these cells and the abnormalities of the p53 gene and the karyotype, suggest that: i) they were a component of the tumor stroma, and ii) they could have been involved in angiosarcoma development. Thus, this cell line may be valuable for the study of cellular interactions in liver carcinogenesis.

Mycophenolate mofetil reduces myofibroblast infiltration and collagen III deposition in rat remnant kidney.

BACKGROUND: Myofibroblasts have been shown to play a pivotal role in the synthesis of extracellular matrix components in several animal models of renal fibrosis. The purpose of the present study was to investigate whether mycophenolate mofetil (MMF) reduces interstitial myofibroblast infiltration and collagen III deposition in 5/6 nephrectomized rats. METHODS: Forty-five Wistar rats underwent 5/6 renal ablation and received by daily oral gavage either vehicle (N = 20) or MMF (N = 25) during the 60 days following surgery. Groups of five treated and five untreated rats were killed at two, four, and eight weeks after subtotal nephrectomy. Four untreated and three treated rats were killed at week 12, one month after treatment withdrawal. At the time of sacrifice, proteinuria, plasma, and urine creatinine were determined. Immunohistochemistry was performed on renal tissue for alpha-smooth muscle actin (alpha-SMA), a cytoskeletal marker of myofibroblasts, for type III collagen, and for proliferating cell nuclear antigen (PCNA). Moreover, in order to study the in vitro effects of MMF on fibroblast proliferation, rat fibroblasts were cultured in the presence or absence of mycophenolic acid (MPA). RESULTS: At all periods studied, MMF treatment improved renal functional parameters and progressively decreased remnant kidney hypertrophy and glomerular volume increment. Proliferating cells in renal tubules, interstitium, and glomeruli, as well as interstitial myofibroblast infiltration and interstitial type III collagen deposition, were also significantly reduced by MMF treatment. In addition, MPA exhibited a dose-dependent inhibitory effect on in vitro proliferation of rat fibroblasts. CONCLUSION: Reduction of interstitial myofibroblast infiltration may be an important event by which MMF significantly prevents renal injury following subtotal renal ablation. Thus, our results suggest that MMF could be useful to limit the progression of chronic renal disease toward end-stage renal failure.