Effect of human amniotic epithelial cells on pro‐fibrogenic resident hepatic cells in a rat model of liver fibrosis

Myofibroblasts are key fibrogenic cells responsible for excessive extracellular matrix synthesis characterizing the fibrotic lesion. In liver fibrosis, myofibroblasts derive either from activation of hepatic stellate cells (HSC) and portal fibroblasts (PF), or from the activation of fibroblasts that originate from ductular epithelial cells undergoing epithelial–mesenchymal transition. Ductular cells can also indirectly promote myofibroblast generation by activating TGF‐β, the main fibrogenic growth factor, through αvβ6 integrin. In addition, after liver injury, liver sinusoidal cells can lose their ability to maintain HSC quiescence, thus favouring HSC differentiation towards myofibroblasts. The amniotic membrane and epithelial cells (hAEC) derived thereof have been shown to decrease hepatic myofibroblast levels in rodents with liver fibrosis. In this study, in a rat model of liver fibrosis, we investigated the effects of hAEC on resident hepatic cells contributing to myofibroblast generation. Our data show that hAEC reduce myofibroblast numbers with a consequent reduction in fibronectin and collagen deposition. Interestingly, we show that hAEC strongly act on specific myofibroblast precursors. Specifically, hAEC reduce the activation of PF rather than HSC. In addition, hAEC target reactive ductular cells by inhibiting their proliferation and αvβ6 integrin expression, with a consequent decrease in TGF‐β activation. Moreover, hAEC counteract the transition of ductular cells towards fibroblasts, while it does not affect injury‐induced and fibrosis‐promoting sinusoidal alterations. In conclusion, among the emerging therapeutic applications of hAEC in liver diseases, their specific action on PF and ductular cells strongly suggests their application in liver injuries involving the expansion and activation of the portal compartment.     

How to model the events in cutaneous fibrosis?

Skin fibrosis is classically seen as the consequence of chronic inflammation and altered healing response that is characterized by the differentiation of fibroblasts into secretory myofibroblasts and accumulation of connective tissue. Although fibrosis severely affects organ function and causes esthetic defects, no effective therapy is currently available to attenuate the fibrogenic process probably because the fibrogenic process is more complex than previously thought. Indeed, it might involve several interacting and mutually dependent cell types (fibroblasts, keratinocytes, endothelial cells, inflammatory cells), numerous paracrine factors, bio-active molecules and micro-environmental stimuli (growth factors, vasoactive peptides, balance between pro- and anti-inflammatory cytokines, coagulation system, reactive oxygen species, extracellular matrix...). In this perspective, the traditional approach that model individual cell response in simple cell culture system is probably inadequate and too simplistic. This article reviews the new models used to study skin fibrosis in vitro, in organotypic culture systems and in vivo and examines how these different models might be used to identify new molecular pathways involved in fibrogenesis. The monolayer cultures allow the study of fibrogenic signals induced by a single factor on a single cell type. Isolation of cells from fibrotic tissue allows to define the fibrogenic differentiation acquired in vivo. The organotypic models allow cell to cell and cell to matrix interaction and the experimental models in pigs and mice allowed studies in integrated physiological systems. These various and complementary models would also provide new tools to develop and test new drugs and treatments.     

Urokinase receptor cleavage: a crucial step in fibroblast-to-myofibroblast differentiation

Fibroblasts migrate into and repopulate connective tissue wounds. At the wound edge, fibroblasts differentiate into myofibroblasts, and they promote wound closure. Regulated fibroblast-to-myofibroblast differentiation is critical for regenerative healing. Previous studies have focused on the role in fibroblasts of urokinase plasmingen activator/urokinase plasmingen activator receptor (uPA/uPAR), an extracellular protease system that promotes matrix remodeling, growth factor activation, and cell migration. Whereas fibroblasts have substantial uPA activity and uPAR expression, we discovered that cultured myofibroblasts eventually lost cell surface uPA/uPAR. This led us to investigate the relevance of uPA/uPAR activity to myofibroblast differentiation. We found that fibroblasts expressed increased amounts of full-length cell surface uPAR (D1D2D3) compared with myofibroblasts, which had reduced expression of D1D2D3 but increased expression of the truncated form of uPAR (D2D3) on their cell surface. Retaining full-length uPAR was found to be essential for regulating myofibroblast differentiation, because 1) protease inhibitors that prevented uPAR cleavage also prevented myofibroblast differentiation, and 2) overexpression of cDNA for a noncleavable form of uPAR inhibited myofibroblast differentiation. These data support a novel hypothesis that maintaining full-length uPAR on the cell surface regulates the fibroblast to myofibroblast transition and that down-regulation of uPAR is necessary for myofibroblast differentiation.     

Transforming growth factor-beta 1 specifically induce proteins involved in the myofibroblast contractile apparatus

Transforming growth factor-beta(1) (TGF-beta(1)) induces alpha-smooth muscle actin (alpha-SMA) and collagen synthesis in fibroblast both in vivo and in vitro and plays a significant role in tissue repair and the development of fibrosis. During these processes the fibroblasts differentiate into activated fibroblasts (so called myofibroblasts), characterized by increased alpha-SMA expression. Because TGF-beta(1) is considered the main inducer of the myofibroblast phenotype and cytoskeletal changes accompany this differentiation, the main objective of this investigation was to study how TGF-beta(1) alters protein expression of cytoskeletal-associated proteins. Metabolic labeling of cell cultures by [(35)S]methionine, followed by protein separation on two-dimensional gel electrophoresis, displayed approximately 2500 proteins in the pI interval of 3-10. Treatment of TGF-beta(1) led to specific spot pattern changes that were identified by mass spectrometry and represent specific induction of several members of the contractile apparatus such as calgizzarin, cofilin, and profilin. These proteins have not previously been shown to be regulated by TGF-beta(1), and the functional role of these proteins is to participate in the depolymerization and stabilization of the microfilaments. These results show that TGF-beta(1) induces not only alpha-SMA but a whole set of actin-associated proteins that may contribute to the increased contractile properties of the myofibroblast. These proteins accompany the induced expression of alpha-SMA and may participate in the formation of stress fibers, cell contractility, and cell spreading characterizing the myofibroblasts phenotype.     

Redirecting valvular myofibroblasts into dormant fibroblasts through light-mediated reduction in substrate modulus

Fibroblasts residing in connective tissues throughout the body are responsible for extracellular matrix (ECM) homeostasis and repair. In response to tissue damage, they activate to become myofibroblasts, which have organized contractile cytoskeletons and produce a myriad of proteins for ECM remodeling. However, persistence of myofibroblasts can lead to fibrosis with excessive collagen deposition and tissue stiffening. Thus, understanding which signals regulate de-activation of myofibroblasts during normal tissue repair is critical. Substrate modulus has recently been shown to regulate fibrogenic properties, proliferation and apoptosis of fibroblasts isolated from different organs. However, few studies track the cellular responses of fibroblasts to dynamic changes in the microenvironmental modulus. Here, we utilized a light-responsive hydrogel system to probe the fate of valvular myofibroblasts when the Young's modulus of the substrate was reduced from ~32 kPa, mimicking pre-calcified diseased tissue, to ~7 kPa, mimicking healthy cardiac valve fibrosa. After softening the substrata, valvular myofibroblasts de-activated with decreases in α-smooth muscle actin (α-SMA) stress fibers and proliferation, indicating a dormant fibroblast state. Gene signatures of myofibroblasts (including α-SMA and connective tissue growth factor (CTGF)) were significantly down-regulated to fibroblast levels within 6 hours of in situ substrate elasticity reduction while a general fibroblast gene vimentin was not changed. Additionally, the de-activated fibroblasts were in a reversible state and could be re-activated to enter cell cycle by growth stimulation and to express fibrogenic genes, such as CTGF, collagen 1A1 and fibronectin 1, in response to TGF-β1. Our data suggest that lowering substrate modulus can serve as a cue to down-regulate the valvular myofibroblast phenotype resulting in a predominantly quiescent fibroblast population. These results provide insight in designing hydrogel substrates with physiologically relevant stiffness to dynamically redirect cell fate in vitro.     

Effects of the isothiocyanate sulforaphane on TGF-β1-induced rat cardiac fibroblast activation and extracellular matrix interactions

An important step in many pathological conditions, particularly tissue and organ fibrosis, is the conversion of relatively quiescent cells into active myofibroblasts. These are highly specialized cells that participate in normal wound healing but also contribute to pathogenesis. These cells possess characteristics of smooth muscle cells and fibroblasts, have enhanced synthetic activity secreting abundant extracellular matrix components, cytokines, and growth factors, and are capable of generating contractile force. As such, these cells have become potential therapeutic targets in a number of disease settings. Transforming growth factor β (TGF-β) is a potent stimulus of fibrosis and myofibroblast formation and likewise is an important therapeutic target in several disease conditions. The plant-derived isothiocyanate sulforaphane has been shown to have protective effects in several pathological models including diabetic cardiomyopathy, carcinogenesis, and fibrosis. These studies suggest that sulforaphane may be an attractive preventive agent against disease progression, particularly in conditions involving alterations of the extracellular matrix and activation of myofibroblasts. However, few studies have evaluated the effects of sulforaphane on cardiac fibroblast activation and their interactions with the extracellular matrix. The present studies were carried out to determine the potential effects of sulforaphane on the conversion of quiescent cardiac fibroblasts to an activated myofibroblast phenotype and associated alterations in signaling, expression of extracellular matrix receptors, and cellular physiology following stimulation with TGF-β1. These studies demonstrate that sulforaphane attenuates TGF-β1-induced myofibroblast formation and contractile activity. Sulforaphane also reduces expression of collagen-binding integrins and inhibits canonical and noncanonical TGF-β signaling pathways.     

Pirfenidone attenuates the profibrotic contractile phenotype of differentiated human dermal myofibroblasts

Dysregulated wound healing after burn injury frequently results in debilitating hypertrophic scarring and contractures. Myofibroblasts, the main effector cells for dermal fibrosis, develop from normal fibroblasts via transforming growth factor beta 1 (TGF-β1). During wound healing, myofibroblasts produce extracellular matrix (ECM) proteins, modulate ECM stability, and contract the ECM using alpha smooth muscle actin (α-SMA) in contractile stress fibers. The antifibrotic pirfenidone has previously been shown to inhibit the initial differentiation of fibroblasts into myofibroblasts in vitro and act as a prophylactic measure against hypertrophic scar development in a mouse burn model. To test whether pirfenidone affects differentiated myofibroblasts, we investigated the in vitro effects of pirfenidone treatment after three to five days of stimulation with TGF-β1. In assays for morphology, protein and gene expression, and contractility, pirfenidone treatment produced significant effects. Profibrotic gene expression returned to near-normal levels, further α-SMA protein expression was prevented, and cell contraction within a stressed collagen matrix was reduced. These in vitro results promote pirfenidone as a promising antifibrotic agent to treat existing scars and healing wounds by mitigating the effects of differentiated myofibroblasts.     

Mutational hotspots in the mitochondrial genome of lung cancer

In lung fibrosis tissue architecture and function is severely hampered by myofibroblasts due to excessive deposition of extracellular matrix and tissue contraction. Myofibroblasts differentiate from fibroblasts under the influence of transforming growth factor (TGF) β₁ but this process is also controlled mechanically by cytoskeletal tension. In healthy lungs, the cytoskeleton of fibroblasts is mechanically strained during breathing. In stiffer fibrotic lung tissue, this mechanical stimulus is reduced, which may influence fibroblast-to-myofibroblast differentiation. Therefore, we investigated the effect of cyclic mechanical stretch on fibroblast-to-myofibroblast differentiation.Primary normal human lung fibroblasts were grown on BioFlex culture plates and stimulated to undergo myofibroblast differentiation by 10 ng/ml TGFβ₁. Cells were either or not subjected to cyclic mechanical stretch (sinusoidal pattern, maximum elongation 10%, 0.2 Hz) for a period of 48 h on a Flexercell apparatus. mRNA expression was analyzed by real-time PCR.Cyclic mechanical loading reduced the mRNA expression of the myofibroblast marker α-smooth muscle actin and the extracellular matrix proteins type-I, type-III, and type-V collagen, and tenascin C. These outcomes indicate that fibroblast-to-myofibroblast differentiation is reduced. Cyclic mechanical loading did not change the expression of the fibronectin ED-A splice variant, but did decrease the paracrine expression of TGFβ₁, thereby suggesting a possible regulation mechanism for the observed effects. The data suggest that cyclic loading experienced by healthy lung cells during breathing may prevent fibroblasts from differentiating towards myofibroblasts.     

Nicotine inhibits myofibroblast differentiation in human gingival fibroblasts

Cigarette smoking has been suggested as a risk factor for several periodontal diseases. It has also been found that smokers respond less favorably than non-smokers to periodontal therapy. Previous work in our lab has shown that nicotine inhibits human gingival cell migration. Since myofibroblasts play an important role in wound closure, we asked if nicotine affects gingival wound healing process by regulating myofibroblast differentiation. Human gingival fibroblasts (HGFs) from two patients were cultured in 10% fetal bovine serum cell culture medium. Cells were pretreated with different doses of nicotine (0, 0.01, 0.1, and 1 mM) for 2 h, and then incubated with transforming growth factor beta (TGF-beta1) (0, 0.25, 0.5, and 1 ng/ml) with or without nicotine for 30 h. The expression level of alpha-smooth muscle actin (alpha-SMA), a specific marker for myofibroblasts, was analyzed by Western blots, immunocytochemistry, and real-time polymerase chain reaction (real-time PCR). Phosphorylated p38 mitogen-activated protein kinase (Phospho-p38 MAPK) activity was analyzed by Western blots. TGF-beta1 induced an increase of alpha-SMA protein and mRNA expression, while nicotine (1 mM) inhibited the TGF-beta1-induced expression of alpha-SMA but not beta-actin. Nicotine treatment down-regulated TGF-beta1-induced p38 MAPK phosphorylation. Our results demonstrated for the first time that nicotine inhibits myofibroblast differentiation in human gingival fibroblasts in vitro; supporting the hypothesis that delayed wound healing in smokers may be due to decreased wound contraction by myofibroblasts.     

PPAR-gamma agonists inhibit profibrotic phenotypes in human lung fibroblasts and bleomycin-induced pulmonary fibrosis

Pulmonary fibrosis is characterized by alterations in fibroblast phenotypes resulting in excessive extracellular matrix accumulation and anatomic remodeling. Current therapies for this condition are largely ineffective. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear hormone receptor superfamily, the activation of which produces a number of biological effects, including alterations in metabolic and inflammatory responses. The role of PPAR-gamma as a potential therapeutic target for fibrotic lung diseases remains undefined. In the present study, we show expression of PPAR-gamma in fibroblasts obtained from normal human lungs and lungs of patients with idiopathic interstitial pneumonias. Treatment of lung fibroblasts and myofibroblasts with PPAR-gamma agonists results in inhibition of proliferative responses and induces cell cycle arrest. In addition, PPAR-gamma agonists, including a constitutively active PPAR-gamma construct (VP16-PPAR-gamma), inhibit the ability of transforming growth factor-beta1 to induce myofibroblast differentiation and collagen secretion. PPAR-gamma agonists also inhibit fibrosis in a murine model, even when administration is delayed until after the initial inflammation has largely resolved. These observations indicate that PPAR-gamma is an important regulator of fibroblast/myofibroblast activation and suggest a role for PPAR-gamma ligands as novel therapeutic agents for fibrotic lung diseases.     

Constitutive ALK5-independent c-Jun N-terminal kinase activation contributes to endothelin-1 overexpression in pulmonary fibrosis: evidence of an autocrine endothelin loop operating through the endothelin A and B receptors

The signal transduction mechanisms generating pathological fibrosis are almost wholly unknown. Endothelin-1 (ET-1), which is up-regulated during tissue repair and fibrosis, induces lung fibroblasts to produce and contract extracellular matrix. Lung fibroblasts isolated from scleroderma patients with chronic pulmonary fibrosis produce elevated levels of ET-1, which contribute to the persistent fibrotic phenotype of these cells. Transforming growth factor beta (TGF-beta) induces fibroblasts to produce and contract matrix. In this report, we show that TGF-beta induces ET-1 in normal and fibrotic lung fibroblasts in a Smad-independent ALK5/c-Jun N-terminal kinase (JNK)/Ap-1-dependent fashion. ET-1 induces JNK through TAK1. Fibrotic lung fibroblasts display constitutive JNK activation, which was reduced by the dual ETA/ETB receptor inhibitor, bosentan, providing evidence of an autocrine endothelin loop. Thus, ET-1 and TGF-beta are likely to cooperate in the pathogenesis of pulmonary fibrosis. As elevated JNK activation in fibrotic lung fibroblasts contributes to the persistence of the myofibroblast phenotype in pulmonary fibrosis by promoting an autocrine ET-1 loop, targeting the ETA and ETB receptors or constitutive JNK activation by fibrotic lung fibroblasts is likely to be of benefit in combating chronic pulmonary fibrosis.     

Focal adhesion kinase (FAK)-related non-kinase inhibits myofibroblast differentiation through differential MAPK activation in a FAK-dependent manner

Transforming growth factor (TGF)-beta1 induces fibroblast transdifferentiation to myofibroblasts, a process that requires the involvement of integrin-mediated signaling and focal adhesion kinase (FAK). FAK-related non-kinase (FRNK) is known for its role in inhibiting integrin-mediated cell migration; however, its role in myofibroblast differentiation has not been defined. Here, we report that FRNK abrogates TGF-beta1-induced myofibroblast differentiation in vitro and in vivo. TGF-beta1 can induce alpha-smooth muscle actin (alpha-SMA) expression in the presence or absence of FAK; however, TGF-beta1-induced alpha-SMA expression is reduced (approximately 73%) in FAK-deficient fibroblasts. Although both ERK and p38 MAPK activation is required for maximal TGF-beta1-induced alpha-SMA expression, ERK is the major signaling intermediate in cells that express FAK. In contrast, p38 MAPK is the dominant mediator of TGF-beta1-induced alpha-SMA expression in FAK-deficient cells. FRNK overexpression blocks TGF-beta1-induced ERK or p38 MAPK activation in the presence, and surprisingly, in the absence of FAK. The loss of FRNK was tested in vivo during experimentally induced pulmonary fibrosis in mice. FRNK knock-out mice have a greater increase in alpha-SMA-expressing cells in response to a pulmonary fibrotic stimulus in vivo, as compared with congenic wild type mice. This is the first time that FRNK loss has been shown to modify the pathobiology in any animal disease model. Together, the data demonstrate that FRNK negatively regulates myofibroblast differentiation in vitro and in vivo. These data further suggest that modulation FRNK expression may be a novel avenue for therapeutic intervention in tissue fibrosis.     

Myofibroblasts. I. Paracrine cells important in health and disease

Myofibroblasts are aunique group of smooth-muscle-like fibroblasts that have a similarappearance and function regardless of their tissue of residence.Through the secretion of inflammatory and anti-inflammatory cytokines,chemokines, growth factors, both lipid and gaseous inflammatorymediators, as well as extracellular matrix proteins and proteases, theyplay an important role in organogenesis and oncogenesis, inflammation,repair, and fibrosis in most organs and tissues. Platelet-derivedgrowth factor (PDGF) and stem cell factor are two secreted proteinsresponsible for differentiating myofibroblasts from embryological stemcells. These and other growth factors cause proliferation ofmyofibroblasts, and myofibroblast secretion of extracellular matrix(ECM) molecules and various cytokines and growth factors causesmobility, proliferation, and differentiation of epithelial orparenchymal cells. Repeated cycles of injury and repair lead to organor tissue fibrosis through secretion of ECM by the myofibroblasts.Transforming growth factor- and the PDGF family of growth factorsare the key factors in the fibrotic response. Because of theirubiquitous presence in all tissues, myofibroblasts play important rolesin various organ diseases and perhaps in multisystem diseases as well.

     

CC-chemokine receptor 2 required for bleomycin-induced pulmonary fibrosis

MCP-1, which signals via the CC chemokine receptor 2 (CCR2), is induced in lung fibrosis that is accompanied by mononuclear cell recruitment and activation of lung fibroblasts. To evaluate the role of CCR2 in lung fibrosis, CCR2 knockout (ko) mice were used in a model of bleomycin-induced lung fibrosis. Wild type (wt) and ko mice were injected endotracheally with bleomycin to induce lung injury and fibrosis, and then analyzed for degree of lung fibrosis and cytokine expression. The results showed significantly reduced fibrosis in ko mice as evidenced by decreased lung type I collagen gene expression and hydroxyproline content relative to those in wt mice. Lung TNF-alpha and TGF-beta1 expression was significantly lower in ko vs. wt mice, while MCP-1 expression was unaffected. Interestingly, lung alpha-smooth muscle actin (alpha-SMA) expression, a marker for myofibroblast differentiation, was also decreased in ko mice, which was confirmed by analysis of isolated lung fibroblasts. Fibroblasts from ko mice exhibited decreased responsiveness to TGF-beta1 induced alpha-SMA expression, which was associated with reduced expression of TGF-beta receptor II (TbetaRII) and Smad3. These findings suggest that CCR2 signaling plays a key role in bleomycin-induced pulmonary fibrosis by regulating fibrogenic cytokine expression and fibroblast responsiveness to TGF-beta.     

Differential and combined effects of cardiotrophin-1 and TGF-beta1 on cardiac myofibroblast proliferation and contraction

Myofibroblasts respond to an array of signals from mitogens and cytokines during the course of wound healing following a myocardial infarction (MI), and these signals may coordinate ventricular myofibroblast proliferation. Furthermore, myofibroblasts are contractile and contribute to wound contraction by imparting mechanical tension on surrounding extracellular matrix. Although TGF-beta(1), CT-1, and PDGF-BB participate in various stages of post-MI wound healing, their combined net effect(s) on myofibroblast function is unknown. We investigated myofibroblast proliferation, expression of cell cycle proteins, and contractile function of cells treated with TGF-beta(1) and/or CT-1. We confirmed that TGF-beta(1) (10 ng/ml) suppresses proliferation of these cells, whereas CT-1 (10 ng/ml) and, for comparative purposes, PDGF-BB (1 ng/ml) treatments were associated with proliferation. Specific TGF-beta(1) treatment ablated CT-1-induced myofibroblast proliferation. TGF-beta(1) effects were specific, as they were suppressed by either TGF-beta-neutralizing antibody or viral Smad7 overexpression. TGF-beta(1) treatment also increased expression of p27 and decreased expression of cyclin E and Cdk2 in primary cells. CT-1 (10 ng/ml) treatment of myofibroblasts had no effect on collagen gel deformation versus controls, whereas TGF-beta(1) (10 ng/ml) and PDGF (10 ng/ml) treatments were associated with significant cell contraction; again, TGF-beta(1)-mediated contraction was unaffected by CT-1. Alone, CT-1 and TGF-beta(1) treatments exert opposing effects on myofibroblast function, whereas in combination TGF-beta(1)-mediated effects supersede those of CT-1 (and PDGF-BB). Thus TGF-beta(1) and CT-1 exert differential effects on myofibroblast proliferation and contraction in vitro, and we suggest that a balance of these effects may be important for the execution of normal cardiac wound healing.     

Aortic Carboxypeptidase-like Protein (ACLP) Enhances Lung Myofibroblast Differentiation through Transforming Growth Factor β Receptor-dependent and -independent Pathways

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease characterized by the overgrowth, hardening, and scarring of lung tissue. The exact mechanisms of how IPF develops and progresses are unknown. IPF is characterized by extracellular matrix remodeling and accumulation of active TGFβ, which promotes collagen expression and the differentiation of smooth muscle α-actin (SMA)-positive myofibroblasts. Aortic carboxypeptidase-like protein (ACLP) is an extracellular matrix protein secreted by fibroblasts and myofibroblasts and is expressed in fibrotic human lung tissue and in mice with bleomycin-induced fibrosis. Importantly, ACLP knockout mice are significantly protected from bleomycin-induced fibrosis. The goal of this study was to identify the mechanisms of ACLP action on fibroblast differentiation. As primary lung fibroblasts differentiated into myofibroblasts, ACLP expression preceded SMA and collagen expression. Recombinant ACLP induced SMA and collagen expression in mouse and human lung fibroblasts. Knockdown of ACLP slowed the fibroblast-to-myofibroblast transition and partially reverted differentiated myofibroblasts by reducing SMA expression. We hypothesized that ACLP stimulates myofibroblast formation partly through activating TGFβ signaling. Treatment of fibroblasts with recombinant ACLP induced phosphorylation and nuclear translocation of Smad3. This phosphorylation and induction of SMA was dependent on TGFβ receptor binding and kinase activity. ACLP-induced collagen expression was independent of interaction with the TGFβ receptor. These findings indicate that ACLP stimulates the fibroblast-to-myofibroblast transition by promoting SMA expression via TGFβ signaling and promoting collagen expression through a TGFβ receptor-independent pathway.     

Effect of nitric oxide on the differentiation of fibroblasts into myofibroblasts in the Peyronie’s fibrotic plaque and in its rat model

The myofibroblast shares phenotypic features of both fibroblasts and smooth muscle cells. It plays a critical role in collagen deposition and wound healing and disappears by apoptosis when the wound is closed. Its abnormal persistence leads to hypertrophic scar formation and other fibrotic conditions. Myofibroblasts are present in the fibrotic plaque of the tunica albuginea (TA) of the penis in men with Peyronie's disease (PD), a localized fibrosis that is accompanied by a spontaneous induction of the inducible nitric oxide synthase (iNOS), also observed in the TGFbeta1-elicited, PD-like lesion in the rat model. iNOS expression counteracts fibrosis, by producing nitric oxide (NO) that reduces collagen deposition in part by neutralization of profibrotic reactive oxygen species. In this study we investigated whether fibroblast differentiation into myofibroblasts is enhanced in the human and rat PD-like plaque and in cultures of human tissue fibroblasts. We also examined whether NO reduces this cell differentiation and collagen synthesis. The myofibroblast content in the fibroblast population was measured by quantitative immunohistochemistry as the ratio between alpha-smooth muscle actin (ASMA; myofibroblast marker) and vimentin (general fibroblast marker) levels. We found that myofibroblast content was considerably increased in the human and TGFbeta1-induced rat plaques as compared to control TA. Inhibition of iNOS activity by chronic administration of L-iminoethyl-L-lysine to rats with TGFbeta1-induced TA lesion increased myofibroblast abundance and collagen I synthesis measured in plaque and TA homogenates from animals injected with a collagen I promoter construct driving the expression of beta-galactosidase. Fibroblast differentiation into myofibroblasts occurred with passage in the cell cultures from the human PD plaque, but was minimal in cultures from the TA. Induction of iNOS in PD and TA cultures with a cytokine cocktail and a NO donor, S-nitroso-N-acetyl penicillamine (SNAP), was detected by immunohistochemistry. Both treatments reduced the total number of cells and the number of ASMA positive cells, whereas only SNAP decreased collagen I immunostaining. These results support the hypotheses that myofibroblasts play a role in the development of the PD plaque and that the antifibrotic effects of NO may be mediated at least in part by the reduction of myofibroblast abundance and lead to a reduction in collagen I synthesis.     

肌成纤维细胞在纤维化疾病中的研究进展

肌成纤维细胞是一种超微结构和生理功能介于平滑肌细胞和成纤维细胞之间的高度分化型细胞,具有很强的分泌细胞外基质及收缩的功能。本文就肌成纤维细胞的形态学特征,及其在肺、肝和肾纤维化疾病发生发展中的生物学行为进行综述。