TLR2/1 and sphingosine 1-phosphate modulate inflammation,myofibroblast differentiation and cell migration in fibroblasts

Dermal fibroblasts are important regulators of inflammatory and immune responses in the skin. The aim of the present study was to elucidate the interaction between two key players in inflammation, Toll-like receptors (TLRs) and sphingosine 1-phosphate (S1P), in normal human fibroblasts in the context of inflammation, fibrosis and cell migration. We demonstrate that TLR2 ligation strongly enhances the production of the pro-inflammatory cytokines IL-6 and IL-8. S1P significantly induces pro-inflammatory cytokines time- and concentration-dependently via S1P receptor (S1PR)2 and S1PR3. The TLR2/1 agonist Pam₃CSK₄ and S1P (> 1 μM) or TGF-β markedly upregulate IL-6 and IL-8 secretion. Pam₃CSK₄ and S1P alone promote myofibroblast differentiation as assessed by significant increases of α-smooth muscle actin and collagen I expression. Importantly, costimulation with S1P (> 1 μM) induces differentiation into myofibroblasts. In contrast, Pam₃CSK₄ and low S1P concentrations (< 1 μM) accelerate cell migration. These results suggest that TLR2/1 signaling and S1P cooperate in pro-inflammatory cytokine production and myofibroblast differentiation and promote cell migration of skin fibroblasts in a S1P-concentration dependent manner. Our findings provide significant insights into how infectious stimuli or danger signals and sphingolipids contribute to dermal inflammation which may be relevant for skin tissue repair after injury or disease.     

Characterization of Smad3 knockout mouse derived skin cells

TGF-β plays an important role in skin wound healing process, in which Smad3 acts as a signaling molecule. Smad3 knockout mice exhibit enhanced wound healing and less inflammatory process, but the intrinsic properties of the mouse derived skin cells are generally unexplored. The purpose of this study is to characterize the biological behavior of skin cells derived from Smad3 knockout mice and thus to define the mechanism of this particular wound healing process. Keratinocytes and dermal fibroblasts were harvested from the skin of Smad3 knockout (Smad3 KO) and wild-type (WT) mice and in vitro cultured for one and two passages for various experiments. The results showed that KO mouse serum contained significantly higher levels of TGF-β1 and lower level of IL-6 and IL-10 than WT mouse serum (p < 0.05), which were also supported by the same findings of more TGF-β1 and less IL-6 and IL-10 in the supernatant of cultured KO dermal fibroblasts than those of WT cells (p < 0.05). At gene levels, IL-6, IL-10, and TGF-β1 were significantly less expressed in KO fibroblasts than in WT fibroblasts (p < 0.05). In addition, KO dermal fibroblasts also exhibited stronger migration and proliferation potentials than WT fibroblasts (p < 0.05). Moreover, both KO fibroblasts and keratinocytes showed higher colony-forming efficiency than WT counterparts with significant difference (p < 0.05). These findings indicate that both systemic factors and intrinsic properties of skin cells contribute to enhanced wound healing and less inflammatory reaction observed in Smad3 knock-out mice.     

Inhibition of myofibroblasts by skin grafts.

The myofibroblast population was studied by electron microscopy in rat wounds healing by (1) contraction of granulation tissue, (2) by coverage with split-skin grafts, and (3) by coverage with full-thickness skin grafts. In all 3 types of wounds, myofibroblasts appeared early and reached a peak number at two weeks after wounding. At this time, 40 to 50 percent of the wound fibroblasts had myofibroblast characteristics. The granulating wounds contracted rapidly and completely, and had long persistence of myofibroblasts. Split-skin grafted wounds contracted less and had a more rapid decrease in myofibroblasts. The wounds covered with full thickness skin grafts had a minimum of contraction with a very rapid decrease in the number of myofibroblasts until by 4 weeks no myofibroblasts were present. Full-thickness skin grafts thus appeared to influence contracting wounds not by preventing the formation of myofibroblasts, but by speeding up completion of their life cycle.     

A heterologous 3-D coculture model of breast tumor cells and fibroblasts to study tumor-associated fibroblast differentiation

The objective of our study was to establish spheroid cocultures as a valid 3-D in vitro model mimicking tumor-fibroblast interactions in scirrhous breast tumors. The experimental setup was designed to verify if in cocultures (a) adherence and migration reflect the invasive potential of breast tumor cells, (b) breast tumor cells induce tumor-associated fibroblast differentiation, and (c) tumor-derived fibroblasts better reflect the in vivo situation than normal skin fibroblasts. Only one (SK-BR-3) out of five tumor cell types showed extensive fibroblast infiltration, MCF-7 cells frequently invaded fibroblast spheroids; BT474, T47D, and ZR-75-1 were noninvasive. While tumor cell invasion was independent of fibroblast origin, tumor-associated myofibroblast differentiation defined by alpha-SMA expression was demonstrated for tumor-derived but not normal skin fibroblasts in coculture indicating that (a) tumor cell invasion and myofibroblast differentiation are autonomous processes and (b) cocultures with tumor-derived fibroblasts resemble advanced stages of desmoplastic carcinomas while cocultures with normal skin fibroblasts rather reflect the early tumor development. The latter is also implied by fibroblast-associated alterations in tumor cell morphology and ECM distribution in the system. By using RNA arbitrarily primed PCR and cells isolated from cocultures by fluorescence-activated and magnetic cell separation, peripheral myelin protein PMP22/SR13 has been identified as a novel candidate with potential relevance in the interaction between tumor cell and normal fibroblast since PMP22 mRNA was significantly reduced in normal skin fibroblasts in coculture with BT474 cells.     

Myofibroblasts cause heterogeneous Cx43 reduction and are unlikely to be coupled to myocytes in the healing canine infarct

Following myocardial infarction (MI) inflammatory responses transform cardiac fibroblasts to myofibroblasts, which in vitro studies show form heterocellular gap junctions with cardiac myocytes via Connexin43 (Cx43). The ability to form heterocellular junctions in the intact heart and the impact of these junctions on propagation is unclear. We used a canine model of MI and characterized the distribution and quantity of myofibroblasts in surviving epicardial cells [epicardial border zone (EBZ)]. We found a significant increase in myofibroblasts within the EBZ and no gap junction plaques between myofibroblasts and myocytes. Because myofibroblasts produce IL-1β, which downregulates Cx43, we asked whether myofibroblast proliferation causes loss of Cx43 near myofibroblast clusters. In vitro studies showed that IL-1β caused loss of Cx43 and reduced coupling. Western blot showed a significant increase of IL-1β in the EBZ, and immunohistochemistry showed a loss of Cx43 in regions of myofibroblasts in the intact heart. Additionally, dye studies in intact heart showed no coupling between myocytes and myofibroblasts. To quantify the effect of myofibroblasts on propagation we used a two-dimensional subcellular computer model of the EBZ, which showed that heterogeneities in myofibroblast density lead to conduction abnormalities. In conclusion, an increase of myofibroblasts in the infarcted heart causes heterogeneous Cx43 levels, possibly as a result of the release of IL-1β and decreased cell-cell communication, which leads to conduction abnormalities following MI.     

Alpha-smooth muscle actin is crucial for focal adhesion maturation in myofibroblasts

Cultured myofibroblasts are characterized by stress fibers, containing alpha-smooth muscle actin (alpha-SMA) and by supermature focal adhesions (FAs), which are larger than FAs of alpha-SMA-negative fibroblasts. We have investigated the role of alpha-SMA for myofibroblast adhesion and FA maturation. Inverted centrifugation reveals two phases of initial myofibroblast attachment: during the first 2 h of plating microfilament bundles contain essentially cytoplasmic actin and myofibroblast adhesion is similar to that of alpha-SMA-negative fibroblasts. Then, myofibroblasts incorporate alpha-SMA in stress fibers, develop mature FAs and their adhesion capacity is significantly increased. When alpha-SMA expression is induced in 5 d culture by TGFbeta or low serum levels, fibroblast adhesion is further increased correlating with a "supermaturation" of FAs. Treatment of myofibroblasts with alpha-SMA fusion peptide (SMA-FP), which inhibits alpha-SMA-mediated contractile activity, reduces their adhesion to the level of alpha-SMA negative fibroblasts. With the use of flexible micropatterned substrates and EGFP-constructs we show that SMA-FP application leads to a decrease of myofibroblast contraction, shortly followed by disassembly of paxillin- and beta3 integrin-containing FAs; alpha5 integrin distribution is not affected. FRAP of beta3 integrin-EGFP demonstrates an increase of FA protein turnover following SMA-FP treatment. We conclude that the formation and stability of supermature FAs depends on a high alpha-SMA-mediated contractile activity of myofibroblast stress fibers.     

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.     

Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-β1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.     

Autophagy fosters myofibroblast differentiation through MTORC2 activation and downstream upregulation of CTGF

Recent evidence suggests that autophagy may favor fibrosis through enhanced differentiation of fibroblasts in myofibroblasts. Here, we sought to characterize the mediators and signaling pathways implicated in autophagy-induced myofibroblast differentiation. Fibroblasts, serum starved for up to 4 d, showed increased LC3-II/-I ratios and decreased SQSTM1/p62 levels. Autophagy was associated with acquisition of markers of myofibroblast differentiation including increased protein levels of ACTA2/αSMA (actin, α 2, smooth muscle, aorta), enhanced gene and protein levels of COL1A1 (collagen, type I, α 1) and COL3A1, and the formation of stress fibers. Inhibiting autophagy with 3 different class I phosphoinositide 3-kinase and class III phosphatidylinositol 3-kinase (PtdIns3K) inhibitors or through ATG7 silencing prevented myofibroblast differentiation. Autophagic fibroblasts showed increased expression and secretion of CTGF (connective tissue growth factor), and CTGF silencing prevented myofibroblast differentiation. Phosphorylation of the MTORC1 target RPS6KB1/p70S6K kinase was abolished in starved fibroblasts. Phosphorylation of AKT at Ser473, a MTORC2 target, was reduced after initiation of starvation but was followed by spontaneous rephosphorylation after 2 d of starvation, suggesting the reactivation of MTORC2 with sustained autophagy. Inhibiting MTORC2 activation with long-term exposure to rapamycin or by silencing RICTOR, a central component of the MTORC2 complex abolished AKT rephosphorylation. Both RICTOR silencing and rapamycin treatment prevented CTGF and ACTA2 upregulation, demonstrating the central role of MTORC2 activation in CTGF induction and myofibroblast differentiation. Finally, inhibition of autophagy with PtdIns3K inhibitors or ATG7 silencing blocked AKT rephosphorylation. Collectively, these results identify autophagy as a novel activator of MTORC2 signaling leading to CTGF induction and myofibroblast differentiation.     

miR-145 Contributes to Hypertrophic Scarring of the Skin by Inducing Myofibroblast Activity

Hyperthrophic scarring of the skin is caused by excessive activity of skin myofibroblasts after wound healing and often leads to functional and/or aesthetic disturbance with significant impairment of patient quality of life. MicroRNA (miRNA) gene therapies have recently been proposed for complex processes such as fibrosis and scarring. In this study, we focused on the role of miR-145 in skin scarring and its influence in myofibroblast function. Our data showed not only a threefold increase of miR-145 levels in skin hypertrophic scar tissue but also in transforming growth factor β1 (TGF-β1)-induced skin myofibroblasts compared with healthy skin or nontreated fibroblasts (p < 0.001). Consistent with the upregulation of miR-145 induced by TGF-β1 stimulation of fibroblasts, the expression of Kruppel-like factor 4 (KLF4) was decreased by 50% and α-smooth muscle actin (α-SMA) protein expression showed a threefold increase. Both could be reversed by miR-145 inhibition (p < 0.05). Restoration of KLF4 levels equally abrogated TGF-β1–induced α-SMA expression. These data demonstrate that TGF-β1 induces miR-145 expression in fibroblasts, which in turn inhibits KLF4, a known inhibitor of α-SMA, hence upregulating α-SMA expression. Furthermore, treatment of myofibroblasts with a miR-145 inhibitor strongly decreased their α-1 type I collagen expression, TGF-β1 secretion, contractile force generation and migration. These data demonstrate that upregulation of miR-145 plays an important role in the differentiation and function of skin myofibroblasts. Additionally, inhibition of miR-145 significantly reduces skin myofibroblast activity. Taken together, these results suggest that miR-145 is a promising therapeutic target to prevent or reduce hypertrophic scarring of the skin.     

Antifibrotic action of Cu/Zn SOD is mediated by TGF-beta1 repression and phenotypic reversion of myofibroblasts

Skin fibrosis is characterized by the proliferation and accumulation of activated fibroblasts called myofibroblasts. They exhibit specific cytoskeletal differentiation, overexpress the fibrogenic cytokine TGF-beta1, synthesize excess extracellular matrix compounds and exhibit a depleted antioxidant metabolism. Recently, SOD was successfully used as an antifibrotic agent in vivo, thus challenging the postulate of established fibrosis irreversibility. We postulated that myofibroblasts could be a direct target for this therapeutic effect. To test this hypothesis, we used three-dimensional co-culture models of skin, in which specific phenotypes of normal fibroblasts versus myofibroblasts are retained. These 3-D models were treated with liposomal and carrier-free Cu/Zn SOD, and examined for their effects on cell number, cell death, and phenotypic differentiation. The results show that SOD did not induce myofibroblast cell death, whereas it significantly reduced TGF-beta1 expression, thus demonstrating that SOD might be proposed as a potent antagonist of this major fibrogenic growth factor. We also found that SOD significantly lowered the levels of the myofibroblast marker alpha-sm actin, of beta-actin, and of the extracellular matrix components alpha1(I) collagen and tenascin-C. In conclusion, our results suggest that SOD antifibrotic action occurred in vitro through the reversion of myofibroblasts into normal fibroblasts.     

Autophagy fosters myofibroblast differentiation through MTORC2 activation and downstream upregulation of CTGF

Recent evidence suggests that autophagy may favor fibrosis through enhanced differentiation of fibroblasts in myofibroblasts. Here, we sought to characterize the mediators and signaling pathways implicated in autophagy-induced myofibroblast differentiation. Fibroblasts, serum starved for up to 4 d, showed increased LC3-II/-I ratios and decreased SQSTM1/p62 levels. Autophagy was associated with acquisition of markers of myofibroblast differentiation including increased protein levels of ACTA2/αSMA (actin, α 2, smooth muscle, aorta), enhanced gene and protein levels of COL1A1 (collagen, type I, α 1) and COL3A1, and the formation of stress fibers. Inhibiting autophagy with 3 different class I phosphoinositide 3-kinase and class III phosphatidylinositol 3-kinase (PtdIns3K) inhibitors or through ATG7 silencing prevented myofibroblast differentiation. Autophagic fibroblasts showed increased expression and secretion of CTGF (connective tissue growth factor), and CTGF silencing prevented myofibroblast differentiation. Phosphorylation of the MTORC1 target RPS6KB1/p70S6K kinase was abolished in starved fibroblasts. Phosphorylation of AKT at Ser473, a MTORC2 target, was reduced after initiation of starvation but was followed by spontaneous rephosphorylation after 2 d of starvation, suggesting the reactivation of MTORC2 with sustained autophagy. Inhibiting MTORC2 activation with long-term exposure to rapamycin or by silencing RICTOR, a central component of the MTORC2 complex abolished AKT rephosphorylation. Both RICTOR silencing and rapamycin treatment prevented CTGF and ACTA2 upregulation, demonstrating the central role of MTORC2 activation in CTGF induction and myofibroblast differentiation. Finally, inhibition of autophagy with PtdIns3K inhibitors or ATG7 silencing blocked AKT rephosphorylation. Collectively, these results identify autophagy as a novel activator of MTORC2 signaling leading to CTGF induction and myofibroblast differentiation.     

Myofibroblast phenotype and apoptosis in keloid and palmar fibroblasts in vitro

Keloid formation is a wound healing response, which fails to resolve and leads to formation of a raised collagen mass extending beyond the original wound margins. Keloids are typically excluded from palms and soles. Therefore we compared keloid and palmar fibroblasts in vitro using fibroblasts from nonaffected individuals as controls. Collagen I, alpha-smooth muscle actin and thrombospondin-1 were found at higher levels in keloid than in palmar fibroblasts. These differences were ameliorated by addition of TGFbeta1. The potential for resolution of the wound healing response was estimated analyzing apoptosis during serum starvation. Annexin V and TUNEL assays showed that palmar fibroblasts underwent faster apoptosis, than did the keloid fibroblasts, and started detaching. Addition of TGFbeta1 counteracted this effect. The weak expression of the myofibroblast phenotype and the advanced apoptosis of palmar fibroblasts suggest mechanisms for the exclusion of keloids from palmar sites.     

The effect of beta-carotene on the expression of interleukin-6 and heme oxygenase-1 in UV-irradiated human skin fibroblasts in vitro.

beta-Carotene is discussed as an anti-oxidant micronutrient and singlet oxygen quencher in human skin, protecting against UV light-induced damage. However, we recently demonstrated that beta-carotene has a pro-oxidant potential in cultured human skin fibroblasts because it enhances the UVA induction of heme oxygenase-1 (HO-1). Herein, we further show that beta-carotene also strongly promotes the UVA induction of pro-inflammatory interleukin-6 (IL-6) in skin fibroblasts in vitro. Singlet oxygen quencher sodium azide abrogated up-regulation of IL-6, and likewise also of HO-1. In UVB-irradiated cells, beta-carotene did not modulate levels of IL-6 and HO-1. The observed effects might be relevant for UV-induced inflammatory processes.     

Depressed IL-1 production by chronic GVHD dermal fibroblasts

Chronic Graft-versus-Host disease (GVHD) is characterized by overt immunosuppression. In addition, the skin is a major anatomical site affected in chronic GVHD for reasons not yet known. Increased collagen deposition, a mononuclear cell infiltrate in the dermis as well as loss of fat and appendages, are observed in the skin. The inflammatory cytokine IL-1 was shown to affect fibroblast proliferation and secretory activities. In the present study, IL-1 generation by dermal fibroblasts, of chronic GVHD or control mice, was assessed. It was shown that two sequential signals are needed for IL-1 generation by dermal fibroblasts; priming by lymphokines/cytokines followed by a challenge with LPS. A variety of recombinant lymphokines and cytokines (G/M-CSF, IL-2, TNF, IL-1 beta and IFNs alpha, beta and gamma) were shown to be efficient in priming dermal fibroblasts for IL-1 generation. IL-1 activity in dermal fibroblasts, most probably of the IL-1 alpha species, was located in frozen-thawed cell lysates or associated to the cell membrane, though not secreted into the culture fluids. Dermal fibroblasts from chronic GVHD mice manifested a pronounced depression in IL-1 generation upon stimulation with exogenous lymphokines/cytokines and LPS. This was observed over a wide range of concentrations of lymphokines/cytokines and LPS. The depressed ability of chronic GVHD fibroblasts to generate IL-1 was pronounced even after few passages of the cells in vitro, and upon stimulation in culture outside the suppressive milieu of the animal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel fibrogenic pathways are activated in response to endothelial apoptosis: implications in the pathophysiology of systemic sclerosis

Apoptosis of endothelial cells (EC) is appreciated as a primary pathogenic event in systemic sclerosis. Yet, how apoptosis of EC leads to fibrosis remains to be determined. We report that apoptosis of EC triggers the release of novel fibrogenic mediators. Medium conditioned by apoptotic EC (SSC) was found to inhibit apoptosis of fibroblasts, whereas medium conditioned by EC in which apoptosis was blocked (with either pan-caspase inhibition or Bcl-x(L) overexpression) did not. PI3K was activated in fibroblasts exposed to SSC. This was associated with downstream repression of Bim-EL and long-term up-regulation of Bcl-x(L) protein levels. RNA interference for Bim-EL in fibroblasts blocked apoptosis. SSC also induced PI3K-dependent myofibroblast differentiation with expression of alpha-smooth muscle actin, formation of stress fibers, and production of collagen I. A C-terminal fragment of the domain V of perlecan was identified as one of the fibrogenic mediators present in SSC. A synthetic peptide containing an EGF motif present on the perlecan fragment and chondroitin 4-sulfate, a glycosaminoglycan anchored on the domain V of perlecan, induced PI3K-dependent resistance to apoptosis in fibroblasts and myofibroblast differentiation. Human fibroblasts derived from sclerodermic skin lesions were more sensitive to the antiapoptotic activities of the synthetic peptide and chondroitin 4-sulfate than fibroblasts derived from normal controls. Hence, we propose that a chronic increase in endothelial apoptosis and/or increased sensitivity of fibroblasts to mediators produced by apoptotic EC could form the basis of a fibrotic response characterized by sustained induction of an antiapoptotic phenotype in fibroblasts and persistent myofibroblast differentiation.     

Thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via the proteolytically activated receptor-1 and a protein kinase C-dependent pathway

Myofibroblasts are ultrastructurally and metabolically distinctive fibroblasts that express smooth muscle (SM)-alpha actin and are associated with various fibrotic lesions. The present study was undertaken to investigate the myofibroblast phenotype that appears after activation of normal lung fibroblasts by thrombin. We demonstrate that thrombin induces smooth muscle-alpha actin expression and rapid collagen gel contraction by normal lung fibroblasts via the proteolytically activated receptor-1 and independent of transforming growth factor-beta pathway. Using antisense oligonucleotides we demonstrate that a decreased level of PKCepsilon abolishes SM-alpha actin expression and collagen gel contraction induced by thrombin in normal lung fibroblasts. Inhibition of PKCepsilon translocation also abolishes thrombin-induced collagen gel contraction, SM-alpha actin increase, and its organization by normal lung fibroblasts, suggesting that activation of PKCepsilon is required for these effects. In normal lung fibroblasts PKCepsilon binds to SM-alpha actin after thrombin treatment, but in activated fibroblasts derived from scleroderma lung they associate even in untreated cells. This suggests that SM-alpha actin may serve as a substrate for PKCepsilon in lung fibroblasts when activated by thrombin. We propose that thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via a PKC-dependent pathway. Thrombin-induced differentiation of normal lung fibroblasts to a myofibroblast phenotype resembles the phenotype observed in scleroderma lung fibroblasts. Therefore, we conclude that chronic exposure to thrombin after microvascular injury leads to activation of normal lung fibroblasts and to the appearance of a myofibroblast phenotype in vivo. Our study provides novel, compelling evidence that thrombin is an important mediator of the interstitial lung fibrosis associated with scleroderma.