The transforming growth factor-β (TGF-β) signalling pathway is a key mediator of fibroblast activation that drives the aberrant synthesis of extracellular matrix in fibrotic diseases. Here we demonstrate a novel link between transforming growth factor-β and the canonical Wnt pathway. TGF-β stimulates canonical Wnt signalling in a p38-dependent manner by decreasing the expression of the Wnt antagonist Dickkopf-1. Tissue samples from human fibrotic diseases show enhanced expression of Wnt proteins and decreased expression of Dickkopf-1. Activation of the canonical Wnt pathway stimulates fibroblasts in vitro and induces fibrosis in vivo. Transgenic overexpression of Dickkopf-1 ameliorates skin fibrosis induced by constitutively active TGF-β receptor type I signalling and also prevents fibrosis in other TGF-β-dependent animal models. These findings demonstrate that canonical Wnt signalling is necessary for TGF-β-mediated fibrosis and highlight a key role for the interaction of both pathways in the pathogenesis of fibrotic diseases.
Aberrant activation of the TGF-β pathway leads to fibrotic disease. Distler and colleagues show that TGF-β-mediated fibrosis requires the decrease of Dickkopf-1, an antagonist of canonical Wnt signalling, suggesting that the two pathways interact for the manifestation of this disease.
We recently show that CCN3 is a counter-regulatory molecule for the pro-fibrotic protein CCN2, and a potentially novel fibrosis therapy. The goal of this study was to assess the role of CCN3 in fibroproliferative/fibrotic responses in human dermal fibroblasts exposed to Omniscan, one of the gadolinium-based contrast agents associated with development of nephrogenic systemic fibrosis (NSF) a rare but life-threatening disease thought to be complication of NMR diagnostics in renal impaired patients. Human dermal fibroblasts were exposed to Omniscan; or to platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) as controls. Proliferation was assessed along with matrix metalloproteinase-1, tissue inhibitor of metalloproteinases-1 and type 1 procollagen in the absence and presence of CCN3. In parallel, CCN3 production was assessed in control and Omniscan-treated cells. The results showed that PDGF stimulated fibroblast proliferation, production of Timp-1 and MMP-1 whereas exogenous CCN3 inhibited, in a dose response manner, cell proliferation (approx. 50 % max.) and production of MMP-1 (approx 35 % max.) but had little effect on TIMP-1. TGF-β stimulated type 1 procollagen production but not proliferation, Timp-1 or MMP-1 compared to non-TGF-ß treated control cells, and CCN3 treatment blocked (approx. 80 % max.) this up-regulation. Interestingly, untreated, control fibroblasts produced high constitutive levels of CCN3 and concentrations of Omniscan that induced fibroproliferative/fibrogenic changes in dermal fibroblasts correspondingly suppressed CCN3 production. The use of PDGF and TGF-β as positive controls, and the study of differential responses, including that to Omniscan itself, provide the first evidence for a role of fibroblast-derived CCN3 as an endogenous regulator of pro-fibrotic changes, elucidating possible mechanism(s). In conclusion, these data support our hypothesis of a role for fibroblast-derived CCN3 as an endogenous regulator of pro-fibrotic changes in these cells, and suggest that CCN3 may be an important regulatory molecule in NSF and a target for treatment in this and other fibrotic diseases.
Nephroblastoma overexpressed gene (NOV) [CCN3]; Gadolinium-based contrast agent (GBCA); Matrix metalloproteinase-1 (MMP-1); Platelet-derived growth factor (PDGF); Tissue inhibitor of metalloprotienases-1 (TIMP-1); Nephrogenic systemic fibrosis (NSF)
Purpose of review
The lung in systemic sclerosis (scleroderma) is susceptible to fibrosis and the ensuing respiratory insufficiency contributes to significant morbidity and mortality in this disease. The lack of effective therapies for pulmonary fibrosis has spurred a re-evaluation of pathobiological paradigms and therapeutic strategies in scleroderma-associated interstitial lung disease and in idiopathic pulmonary fibrosis. The purpose of this review is to examine emerging new therapeutic targets that modulate pro-fibrotic phenotypes of tissue-resident cells and the associated aberrant tissue remodeling responses in fibrotic disorders.
Progressive forms of tissue fibrosis, including scleroderma, are characterized by an accumulation of activated mesenchymal cells and their secreted extracellular matrix proteins in association with dysrepair of epithelial and endothelial cells. Recent studies suggest that emergence of cellular phenotypes that perpetuate loss of cellular homeostasis is characteristic of many fibrosis-related clinical syndromes.
Therapeutic strategies that modulate the fate/phenotype of reparative structural cells, including epithelial, endothelial, and mesenchymal cells, offer new opportunities for the development of more effective drugs for the treatment of fibrosis.
Epithelial Cells; Mesenchymal Stem Cells; Fibroblasts; Apoptosis; Protein Kinase Inhibitors; PPAR gamma; Losartan; Bosentan; Hydroxymethylglutaryl CoA Reductase Inhibitors
Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology. A hallmark of SSc is fibrosis of the skin and internal organs. We recently demonstrated increased expression of IGFBP-3 and IGFBP-5 in primary cultures of fibroblasts from the skin of patients with SSc. In vitro, IGFBP-3 and IGFBP-5 induced a fibrotic phenotype and IGFBP-5 triggered dermal fibrosis in mice. To assess the ability of IGFBPs to trigger fibrosis, we used an ex vivo human skin organ culture model. Our findings demonstrate that IGFBP-3 and IGFBP-5, but not IGFBP-4, increase dermal and collagen bundle thickness in human skin explants, resulting in substantial dermal fibrosis and thickening. These fibrotic effects were sustained for at least two weeks. Our findings demonstrate that human skin ex vivo is an appropriate model to assess the effects of fibrosis-inducing factors such as IGFBPs, and for evaluating the efficacy of inhibitors/therapies to halt the progression of fibrosis and potentially reverse it.
Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic dysregulated response to alveolar epithelial injury with differentiation of epithelial cells and fibroblasts into matrix-secreting myofibroblasts resulting in lung scaring. The prognosis is poor and there are no effective therapies or reliable biomarkers. Galectin-3 is a β-galactoside binding lectin that is highly expressed in fibrotic tissue of diverse etiologies.
Objectives: To examine the role of galectin-3 in pulmonary fibrosis.
Methods: We used genetic deletion and pharmacologic inhibition in well-characterized murine models of lung fibrosis. Further mechanistic studies were performed in vitro and on samples from patients with IPF.
Measurements and Main Results: Transforming growth factor (TGF)-β and bleomycin-induced lung fibrosis was dramatically reduced in mice deficient in galectin-3, manifest by reduced TGF-β1–induced EMT and myofibroblast activation and collagen production. Galectin-3 reduced phosphorylation and nuclear translocation of β-catenin but had no effect on Smad2/3 phosphorylation. A novel inhibitor of galectin-3, TD139, blocked TGF-β–induced β-catenin activation in vitro and in vivo and attenuated the late-stage progression of lung fibrosis after bleomycin. There was increased expression of galectin-3 in the bronchoalveolar lavage fluid and serum from patients with stable IPF compared with nonspecific interstitial pneumonitis and controls, which rose sharply during an acute exacerbation suggesting that galectin-3 may be a marker of active fibrosis in IPF and that strategies that block galectin-3 may be effective in treating acute fibrotic exacerbations of IPF.
Conclusions: This study identifies galectin-3 as an important regulator of lung fibrosis and provides a proof of principle for galectin-3 inhibition as a potential novel therapeutic strategy for IPF.
fibrosis; epithelial cells; fibroblasts
Platelet aggregation may contribute to the pathogenesis of systemic sclerosis: following activation, platelets release significant amounts of serotonin – which promotes vasoconstriction and fibrosis, and further enhances aggregation. The C+1354T polymorphism in the exonic region of the serotonin 2A receptor gene determining the His452Tyr substitution was associated with blunted intracellular responses after serotonin stimulation, and may have a role in susceptibility to scleroderma.
One hundred and fifteen consecutive systemic sclerosis patients and 140 well-matched healthy control individuals were genotyped by sequence-specific primer-PCR for the His452Tyr substitution of the serotonin 2A receptor gene, and associations were sought with scleroderma and its main clinical features. The functional relevance of the His452Tyr substitution was also assessed by evaluating the aggregation of platelet-rich plasma from His452/His452 and His452/Tyr452 healthy individuals after stimulation with adenosine diphosphate ± serotonin.
The T allele of the C+1354T polymorphism was underrepresented in scleroderma patients compared with control individuals (5.2% versus 12.4%, P < 0.001, chi-square test and 1,000-fold permutation test) and its carriage reduced the risk for systemic sclerosis (odds ratio = 0.39, 95% confidence interval = 0.19 to 0.85, P < 0.01). Platelets from His452/Tyr452 healthy subjects more weakly responded to serotonin stimulation compared with platelets from His452/His452 individuals (3.2 ± 2.6-fold versus 9.6 ± 8.6-fold increase in aggregation, P = 0.017 by Kolmogorov–Smirnov test and P = 0.003 after correction for baseline adenosine diphosphate-induced aggregation values).
The His452Tyr substitution may influence susceptibility to systemic sclerosis by altering platelet aggregation in response to serotonin.
There are no approved drugs for treating the fibrosis in scleroderma (systemic sclerosis, SSc). Myfibroblasts within connective tissue express the highly contractile protein α–smooth muscle actin (α–SMA) and are responsible for the excessive synthesis and remodeling of extracellular matrix (ECM) characterizing SSc. Drugs targeting myofibroblast differentiation, recruitment and activity are currently under consideration as anti-fibrotic treatments in SSc but thus far have principally focused on the transforming growth factor β (TGFβ), endothelin-1 (ET-1), connective tissue growth factor (CCN2/CTGF) and platelet derived growth factor (PDGF) pathways, which display substantial signaling crosstalk. Moreover, peroxisome proliferator-activated receptor (PPAR)γ also appears to act by intervening in TGFβ signaling. This review discusses these potential candidates for antifibrotic therapy in SSc.
PDGF; TGFβ; Endothelin; PPARγ
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 β (TGF-β) induces fibroblasts to produce and contract matrix. In this report, we show that TGF-β 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-β 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.
Fibrotic interstitial lung diseases are characterized by progressive decline in lung function and premature death from respiratory failure. Fibrocytes are circulating bone marrow-derived progenitor cell that traffic to the lungs and contribute to fibrosis and may represent novel therapeutic targets in these diseases. We have previously found the recruitment of fibrocytes to the lung to be dependent on the chemokine ligand CXCL12. Given that the expression of the CXCL12 receptor, CXCR4, can be modulated pharmacologically in other cell types, we tested the hypotheses that the regulation of CXCR4 expression on fibrocytes mediates their influx to the lung in the context of pulmonary fibrosis and that pharmacologic inhibition of this process results in attenuated disease severity. CXCR4 was the predominant chemokine receptor on human fibrocytes, and its expression on fibrocytes was enhanced by hypoxia and by growth factors including platelet-derived growth factor. Both hypoxia-induced and growth factor-induced CXCR4 expressions were attenuated by specific inhibition of PI3 kinase and mTOR. Finally, in the mouse model of bleomycin-induced pulmonary fibrosis, treatment with the mTOR inhibitor rapamycin resulted in reduced numbers of CXCR4-expressing fibrocytes in the peripheral blood and lung as well as reduced lung collagen deposition. Taken together, these experiments support the notion that pharmacologic inhibition of the CXCR4/CXCL12 biological axis is achievable in human fibrocytes and reduces the magnitude of pulmonary fibrosis in an animal model. This approach may hold promise in human fibrotic lung diseases.
chemokines; stem cells; lung; cell traffic; signal transduction
Peroxisome proliferator-activated receptor (PPAR)γ may be a key regulator of connective tissue deposition and remodeling in vivo. PPARγ expression is reduced in dermal fibroblasts isolated from fibrotic areas of scleroderma patients; PPARγ agonists suppress the persistent fibrotic phenotype of this cell type. Previously, we showed that loss of PPARγ expression in fibroblasts resulted in enhanced bleomycin-induced skin fibrosis. However, whether loss of PPARγ expression in skin fibroblasts affects cutaneous tissue repair or homeostasis is unknown.
Mice deleted for PPARγ in skin fibroblasts show an enhanced rate of dermal wound closure, concomitant with elevated phosphorylation of Smad3, Akt and ERK, and increased expression of proliferating cell nuclear antigen (PCNA), collagen, α-smooth muscle actin (α-SMA) and CCN2. Conversely, dermal homeostasis was not appreciably affected by loss of PPARγ expression.
PPARγ expression by fibroblasts suppresses cutaneous tissue repair. In the future, direct PPARγ antagonists and agonists might be of clinical benefit in controlling chronic wounds or scarring, respectively.
Data from our laboratory show that in vitro fibroblasts are exquisitely responsive to prostacyclin and the prostacyclin derivative Iloprost, which block their activation by TGFβ. A recent article by Zhu Y et al confirm these effects in vivo showing that Iloprost, given as a single intraperitoneal injection, blocks lung fibrosis in the bleomycin model of lung injury and fibrosis. These results are important because at present no effective clinical treatments are available to treat idiopathic lung fibrosis, which progresses and leads to respiratory failure. Limiting factors for the clinical use of prostacyclin derivatives as anti-fibrotics are failure to achieve therapeutic levels in the involved fibrotic tissues, and dose limiting side effects due to vasodilatation and binding to the IP receptor on vascular cells. Possible approaches include fibroblast directed gene therapies or amelioration of the vascular side effects.
Fibrosis; Iloprost; Prostacyclin
Angiotensin-converting enzyme (ACE) inhibitors reduce the progression of various fibrotic renal diseases both in humans and in animal models. Unilateral ureteral obstruction (UUO) is an animal model of accelerated renal tubulointerstitial fibrosis that is attenuated by ACE inhibition. Although ACE inhibitors increase bradykinin concentrations in addition to their effect on angiotensin II formation, the role of bradykinin in renal fibrosis has not been studied. We show here that genetic ablation (B2–/– mice) or pharmacological blockade of the bradykinin B2 receptor increases UUO-induced interstitial fibrosis in mice, whereas transgenic rats expressing increased endogenous bradykinin show reduced UUO-induced interstitial fibrosis. The increased interstitial fibrosis in B2–/– mice was accompanied by a decreased activity of plasminogen activators (PAs) and metalloproteinase-2 (MMP-2), enzymes involved in ECM degradation, suggesting that the protective effects of bradykinin involve activation of a B2 receptor/PA/MMP-2 cascade. This ability of bradykinin to increase PA activity was confirmed in primary culture proximal tubular cells. Thus, in both mice and rats, bradykinin B2 receptor activation reduces renal tubulointerstitial fibrosis in vivo, most likely by increasing ECM degradation.
Connective tissue growth factor (CTGF) is widely thought to promote the development of fibrosis in collaboration with transforming growth factor (TGF)-β; however, most of the evidence for its involvement comes from correlative and culture-based studies. In this study, the importance of CTGF in tissue fibrosis was directly examined in three murine models of fibrotic disease: a novel model of multiorgan fibrosis induced by repeated intraperitoneal injections of CTGF and TGF-β2; the unilateral ureteral obstruction (UUO) renal fibrosis model; and an intratracheal bleomycin instillation model of pulmonary fibrosis.
Intraperitoneal coadministration of CTGF and TGF-β2 elicited a profound fibrotic response that was inhibited by the human anti-CTGF antibody FG-3019, as indicated by the ability of FG-3019 to ameliorate the histologic signs of fibrosis and reduce the otherwise increased hydroxyproline:proline (Hyp:Pro) ratios by 25% in kidney (P < 0.05), 30% in liver (P < 0.01) and 63% in lung (P < 0.05). Moreover, administration of either cytokine alone failed to elicit a fibrotic response, thus demonstrating that CTGF is both necessary and sufficient to initiate fibrosis in the presence of TGF-β and vice versa. In keeping with this requirement for CTGF function in fibrosis, FG-3019 also reduced the renal Hyp:Pro response up to 20% after UUO (P < 0.05). In bleomycin-injured animals, a similar trend towards a FG-3019 treatment effect was observed (38% reduction in total lung Hyp, P = 0.056). Thus, FG-3019 antibody treatment consistently reduced excessive collagen deposition and the pathologic severity of fibrosis in all models.
Cooperative interactions between CTGF and TGF-β signaling are required to elicit overt tissue fibrosis. This interdependence and the observed anti-fibrotic effects of FG-3019 indicate that anti-CTGF therapy may provide therapeutic benefit in different forms of fibroproliferative disease.
Platelets are thought to play a role in a variety of inflammatory conditions in the lung, some of which may lead to fibrosis. In the current study we tested the hypothesis that whole platelets and platelet lysate can mediate remodelling of extracellular matrix in vitro by affecting fibroblast-mediated contraction of a collagen gel. We also sought to determine to what extent platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) contribute to this effect.
Washed platelets, isolated from healthy blood donors, and platelet lysate (freezing and thawing), were cast together with human lung fibroblasts in three-dimensional collagen gels. The gels were then released and cultured for four days. PDGF and TGF-β1 concentrations were measured in culture supernatants by ELISA.
Both platelets and platelet lysate augmented fibroblast-mediated gel contraction in a time and concentration dependent manner (19.9% ± 0.1 (mean ± SEM) of initial area vs. 48.0% ± 0.4 at 48 hours; P < 0.001 and 41.5% ± 0.6 vs. 60.6% ± 0.3 at 48 hours; P < 0.001, respectively). Fixed platelets had no effect in the system. Both TGF-β1 and PDGF-AA/AB were released in co-culture. PDGF-AA/AB had a maximum release at 24 hours whereas TGF-β1 release increased with longer culture periods. Neutralising antibodies to these mediators partially inhibited platelet-induced gel contraction.
We conclude that platelets may promote remodelling of extracellular matrix in vitro and that PDGF and TGF-β partially mediate this effect, also indicating a role for other mediators. The findings may be an important mechanism in regulating repair processes after injury.
platelets; gel contraction; fibrosis; PDGF; TGF-β
The development of fibrosis involves a multitude of events and molecules. Until now the majority of these molecules were found to be proteins or peptides. But recent data show significant involvement of the phospholipid lysophosphatidic acid (LPA) in the development of pulmonary, liver and renal fibrosis. The latest data on the role of LPA and the G-protein-coupled LPA1 receptor in the development of renal fibrosis will be discussed. LPA1 receptor-activation was found to be associated with increased vascular leakage and increased fibroblast recruitment in pulmonary fibrosis. Furthermore, in renal fibrosis LPA1 receptor-activation stimulates macrophage recruitment and connective tissue growth factor expression. The observations make this receptor an interesting alternative and new therapeutic target in fibrotic diseases.
Tissue homeostasis requires an effective, limited wound-healing response to injury. In chronic disease, failure to regenerate parenchymal tissue leads to the replacement of lost cellular mass with a fibrotic matrix. The mechanisms that dictate the balance of cell regeneration and fibrogenesis are not well understood1. Here we report that fibrogenic hepatic stellate cells (HSCs) in the liver are negative regulators of hepatocyte regeneration. This negative regulatory function requires stimulation of the 5-hydroxytryptamine 2B receptor (5-HT2B) on HSCs by serotonin, which activates expression of transforming growth factor β1 (TGF-β1), a powerful suppressor of hepatocyte proliferation, through signaling by mitogen-activated protein kinase 1 (ERK) and the transcription factor JunD. Selective antagonism of 5-HT2B enhanced hepatocyte growth in models of acute and chronic liver injury. We also observed similar effects in mice lacking 5-HT2B or JunD or upon selective depletion of HSCs in wild-type mice. Antagonism of 5-HT2B attenuated fibrogenesis and improved liver function in disease models in which fibrosis was pre-established and progressive. Pharmacological targeting of 5-HT2B is clinically safe in humans and may be therapeutic in chronic liver disease.
Scleroderma (systemic sclerosis; SSc) is a clinically heterogeneous and often lethal acquired disorder of the connective tissue that is characterized by vascular, immune/inflammatory and fibrotic manifestations. Tissue fibrosis is the main cause of morbidity and mortality in SSc and an unmet medical challenge, mostly because of our limited understanding of the molecular factors and signalling events that trigger and sustain disease progression. Recent evidence has correlated skin fibrosis in SSc with stabilization of proto-oncogene Ha-Ras secondary to auto-antibody stimulation of reactive oxygen species production. The goal of the present study was to explore the molecular connection between Ha-Ras stabilization and collagen I production, the main read-out of fibrogenesis, in a primary dermal fibroblast culture system that replicates the early stages of disease progression in SSc.
Forced expression of proto-oncogene Ha-Ras in dermal fibroblasts demonstrated the promotion of an immediate collagen I up-regulation, as evidenced by enhanced activity of a collagen I-driven luciferase reporter plasmid and increased accumulation of endogenous collagen I proteins. Moreover, normal levels of Tgfβ transcripts and active transforming growth factor-beta (TGFβ) implied Ha-Ras stimulation of the canonical Smad2/3 signalling pathway independently of TGFβ production or activation. Heightened Smad2/3 signalling was furthermore correlated with greater Smad3 phosphorylation and Smad3 protein accumulation, suggesting that Ha-Ras may target both Smad2/3 activation and turnover. Additional in vitro evidence excluded a contribution of ERK1/2 signalling to improper Smad3 activity and collagen I production in cells that constitutively express Ha-Ras.
Our study shows for the first time that constitutively elevated Ha-Ras protein levels can directly stimulate Smad2/3 signalling and collagen I accumulation independently of TGFβ neo-synthesis and activation. This finding therefore implicates the Ha-Ras pathway with the early onset of fibrosis in SSc and implicitly identifies new therapeutic targets in SSc.
Autoimmunity, microangiopathy and tissue fibrosis are hallmarks of systemic sclerosis (SSc). Vascular alterations and reduced capillary density decrease blood flow and impair tissue oxygenation in SSc. Oxygen supply is further reduced by accumulation of extracellular matrix (ECM), which increases diffusion distances from blood vessels to cells. Therefore, severe hypoxia is a characteristic feature of SSc and might contribute directly to the progression of the disease. Hypoxia stimulates the production of ECM proteins by SSc fibroblasts in a transforming growth factor-β-dependent manner. The induction of ECM proteins by hypoxia is mediated via hypoxia-inducible factor-1α-dependent and -independent pathways. Hypoxia may also aggravate vascular disease in SSc by perturbing vascular endothelial growth factor (VEGF) receptor signalling. Hypoxia is a potent inducer of VEGF and may cause chronic VEGF over-expression in SSc. Uncontrolled over-expression of VEGF has been shown to have deleterious effects on angiogenesis because it leads to the formation of chaotic vessels with decreased blood flow. Altogether, hypoxia might play a central role in pathogenesis of SSc by augmenting vascular disease and tissue fibrosis.
Fibrosis is defined by the overgrowth, hardening, and/or scarring of various tissues and is attributed to excess deposition of extracellular matrix components including collagen. Fibrosis is the end result of chronic inflammatory reactions induced by a variety of stimuli including persistent infections, autoimmune reactions, allergic responses, chemical insults, radiation, and tissue injury. Although current treatments for fibrotic diseases such as idiopathic pulmonary fibrosis, liver cirrhosis, systemic sclerosis, progressive kidney disease, and cardiovascular fibrosis typically target the inflammatory response, there is accumulating evidence that the mechanisms driving fibrogenesis are distinct from those regulating inflammation. In fact, some studies have suggested that ongoing inflammation is needed to reverse established and progressive fibrosis. The key cellular mediator of fibrosis is the myofibroblast, which when activated serves as the primary collagen-producing cell. Myofibroblasts are generated from a variety of sources including resident mesenchymal cells, epithelial and endothelial cells in processes termed epithelial/endothelial-mesenchymal (EMT/EndMT) transition, as well as from circulating fibroblast-like cells called fibrocytes that are derived from bone-marrow stem cells. Myofibroblasts are activated by a variety of mechanisms, including paracrine signals derived from lymphocytes and macrophages, autocrine factors secreted by myofibroblasts, and pathogen-associated molecular patterns (PAMPS) produced by pathogenic organisms that interact with pattern recognition receptors (i.e. TLRs) on fibroblasts. Cytokines (IL-13, IL-21, TGF-β1), chemokines (MCP-1, MIP-1β), angiogenic factors (VEGF), growth factors (PDGF), peroxisome proliferator-activated receptors (PPARs), acute phase proteins (SAP), caspases, and components of the renin–angiotensin–aldosterone system (ANG II) have been identified as important regulators of fibrosis and are being investigated as potential targets of antifibrotic drugs. This review explores our current understanding of the cellular and molecular mechanisms of fibrogenesis.
fibrosis; myofibroblast; collagen; wound healing; liver; lung
Transforming growth factor-ß (TGF-ß) signaling is implicated in the pathogenesis of fibrosis in scleroderma or systemic sclerosis (SSc), but the precise mechanisms are poorly understood. The immediate-early gene Egr-1 is an inducible transcription factor with key roles in mediating fibrotic TGF-ß responses. To elucidate Egr-1 function in SSc-associated fibrosis, we examined change in gene expression induced by Egr-1 in human fibroblasts at the genome-wide level. Using microarray expression analysis, we derived a fibroblast “Egr-1-responsive gene signature” comprising over 600 genes involved in cell proliferation, TGF-ß signaling, wound healing, extracellular matrix synthesis and vascular development. The experimentally derived “Egr-1-responsive gene signature” was then evaluated in an expression microarray dataset comprising skin biopsies from 27 patients with localized and systemic forms of scleroderma and six healthy controls. We found that the “Egr-1 responsive gene signature” was substantially enriched in the “diffuse-proliferation” subset comprising exclusively of patients with diffuse cutaneous SSc (dcSSc) of skin biopsies. A number of Egr-1-regulated genes was also associated with the “inflammatory” intrinsic subset. Only a minority of Egr-1-regulated genes was concordantly regulated by TGF-ß. These results indicate that Egr-1 induces a distinct profibrotic/wound healing gene expression program in fibroblasts that is associated with skin biopsies from SSc patients with diffuse cutaneous disease. These observations suggest that targeting Egr-1 expression or activity might be a novel therapeutic strategy to control fibrosis in specific SSc subsets.
Patients with scleroderma receiving Iloprost as a treatment for severe Raynaud’s phenomenon report a reduction in skin tightness, suggesting that this drug inhibits skin fibrosis. Connective tissue growth factor (CTGF), a recently described profibrotic cytokine, acts downstream and in concert with TGF-β to stimulate the fibrotic process and is involved in the fibrosis seen in scleroderma. Here we show that Iloprost, acting by elevation of cAMP, blocks the induction of CTGF and the increase in collagen synthesis in fibroblasts exposed to TGF-β. The potency of Iloprost with respect to suppression of CTGF far exceeds that of other prostanoid receptor agonists, suggesting that its effect is mediated by the prostacyclin receptor IP. By sampling dermal interstitial fluid using a suction blister device, we show that CTGF levels are greatly elevated in the dermis of scleroderma patients compared with healthy controls and that Iloprost infusion causes a marked decrease in dermal CTGF levels. These studies suggest that Iloprost could be reducing the level of a key profibrotic cytokine in scleroderma patients and that endogenous production of eicosanoids may limit the fibrotic response to TGF-β.
Uncontrolled activation of the coagulation cascade contributes to the pathophysiology of several conditions, including acute and chronic lung diseases. Coagulation zymogens are considered to be largely derived from the circulation and locally activated in response to tissue injury and microvascular leak. Here we report that expression of coagulation factor X (FX) is locally increased in human and murine fibrotic lung tissue, with marked immunostaining associated with bronchial and alveolar epithelia. FXa was a potent inducer of the myofibroblast differentiation program in cultured primary human adult lung fibroblasts via TGF-β activation that was mediated by proteinase-activated receptor–1 (PAR1) and integrin αvβ5. PAR1, αvβ5, and α-SMA colocalized to fibrotic foci in lung biopsy specimens from individuals with idiopathic pulmonary fibrosis. Moreover, we demonstrated a causal link between FXa and fibrosis development by showing that a direct FXa inhibitor attenuated bleomycin-induced pulmonary fibrosis in mice. These data support what we believe to be a novel pathogenetic mechanism by which FXa, a central proteinase of the coagulation cascade, is locally expressed and drives the fibrotic response to lung injury. These findings herald a shift in our understanding of the origins of excessive procoagulant activity and place PAR1 central to the cross-talk between local procoagulant signaling and tissue remodeling.
Uncontrolled extracellular matrix production by fibroblasts in response to tissue injury contributes to fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), a progressive and ultimately fatal process that currently has no cure. Although dysregulation of miRNAs is known to be involved in a variety of pathophysiologic processes, the role of miRNAs in fibrotic lung diseases is unclear. In this study, we found up-regulation of miR-21 in the lungs of mice with bleomycin-induced fibrosis and also in the lungs of patients with IPF. Increased miR-21 expression was primarily localized to myofibroblasts. Administration of miR-21 antisense probes diminished the severity of experimental lung fibrosis in mice, even when treatment was started 5–7 d after initiation of pulmonary injury. TGF-β1, a central pathological mediator of fibrotic diseases, enhanced miR-21 expression in primary pulmonary fibroblasts. Increasing miR-21 levels promoted, whereas knocking down miR-21 attenuated, the pro-fibrogenic activity of TGF-β1 in fibroblasts. A potential mechanism for the role of miR-21 in fibrosis is through regulating the expression of an inhibitory Smad, Smad7. These experiments demonstrate an important role for miR-21 in fibrotic lung diseases and also suggest a novel approach using miRNA therapeutics in treating clinically refractory fibrotic diseases, such as IPF.
Idiopathic pulmonary fibrosis (IPF) can lead to the development of secondary pulmonary hypertension (PH) and ultimately death. Despite this known association, the precise mechanism of disease remains unknown. Using a rat model of IPF, we explored the role of the proangiogenic and antiapoptotic growth factor VEGF in the vascular remodeling that underlies PH. In this model, adenoviral delivery of active TGF-β1 induces pulmonary arterial remodeling, loss of the microvasculature in fibrotic areas, and increased pulmonary arterial pressure (PAP). Immunohistochemistry and mRNA analysis revealed decreased levels of VEGF and its receptor, which were inversely correlated with PAP and endothelial cell apoptosis in both the micro- and macrovasculature. Treatment of IPF rats with adenoviral delivery of VEGF resulted in reduced endothelial apoptosis, increased vascularization, and improved PAP due to reduced remodeling but worsened PF. These data show that experimental pulmonary fibrosis (PF) leads to loss of the microvasculature through increased apoptosis and to remodeling of the pulmonary arteries, with both processes resulting in PH. As administration of VEGF ameliorated the PH in this model but concomitantly aggravated the fibrogenic process, VEGF-based therapies should be used with caution.
Ischemia resulting from myocardial infarction (MI) promotes VEGF expression, leading to vascular permeability (VP) and edema, a process that we show here contributes to tissue injury throughout the ventricle. This permeability/edema can be assessed noninvasively by MRI and can be observed at the ultrastructural level as gaps between adjacent endothelial cells. Many of these gaps contain activated platelets adhering to exposed basement membrane, reducing vessel patency. Following MI, genetic or pharmacological blockade of Src preserves endothelial cell barrier function, suppressing VP and infarct volume, providing long-term improvement in cardiac function, fibrosis, and survival. To our surprise, an intravascular injection of VEGF into healthy animals, but not those deficient in Src, induced similar endothelial gaps, VP, platelet plugs, and some myocyte damage. Mechanistically, we show that quiescent blood vessels contain a complex involving Flk, VE-cadherin, and β-catenin that is transiently disrupted by VEGF injection. Blockade of Src prevents disassociation of this complex with the same kinetics with which it prevents VEGF-mediated VP/edema. These findings define a molecular mechanism to account for the Src requirement in VEGF-mediated permeability and provide a basis for Src inhibition as a therapeutic option for patients with acute MI.