Swati Bhattacharyya

Understanding fibrosis in systemic sclerosis: shifting paradigms, emerging opportunities.

Fibrosis in multiple organs is a prominent pathological finding and distinguishing hallmark of systemic sclerosis (SSc). Findings during the past 5 years have contributed to a more complete understanding of the complex cellular and molecular underpinning of fibrosis in SSc. Fibroblasts, the principal effector cells, are activated in the profibrotic cellular milieu by cytokines and growth factors, developmental pathways, endothelin 1 and thrombin. Innate immune signaling via Toll-like receptors, matrix-generated biomechanical stress signaling via integrins, hypoxia and oxidative stress seem to be implicated in perpetuating the process. Beyond chronic fibroblast activation, fibrosis represents a failure to terminate tissue repair, coupled with an expanded population of mesenchymal cells originating from bone marrow and transdifferentiation of epithelial cells, endothelial cells and pericytes. In addition, studies have identified intrinsic alterations in SSc fibroblasts resulting from epigenetic changes, as well as altered microRNA expression that might underlie the cell-autonomous, persistent activation phenotype of these cells. Precise characterization of the deregulated extracellular and intracellular signaling pathways, mediators and cellular differentiation programs that contribute to fibrosis in SSc will facilitate the development of selective, targeted therapeutic strategies. Effective antifibrotic therapy will ultimately involve novel compounds and repurposing of drugs that are already approved for other indications.

Fibrosis in systemic sclerosis: Emerging concepts and implications for targeted therapy

Systemic sclerosis (SSc) is a complex and incompletely understood disease associated with fibrosis in multiple organs. Recent findings identify transforming growth factor-ß (TGF-ß), Wnt ligands, toll-like receptor-mediated signaling, hypoxia, type I interferon, type 2 immune responses and mechanical stress as extracellular cues that modulate fibroblast function and differentiation, and as potential targets for therapy. Moreover, fibrillin-1 has a major role in storing and regulating the bioavailability of TGF-ß and other cytokines, and fibrillin-1 mutations are implicated in a congenital form of scleroderma called stiff skin syndrome. Fibrosis is due not only to the activation of tissue-resident fibroblasts and their transdifferentiation into myofibroblasts, but also the differentiation of bone marrow-derived fibrocytes, and transition of endothelial and epithelial cells, pericytes and adipocytes into activated mesenchymal cells. These responses are modulated by signaling mediators and microRNAs that amplify or inhibit TGF-ß and Wnt signaling. Gain-of-function and loss-of-function abnormalities of these mediators may account for the characteristic activated phenotype of SSc fibroblasts. The nuclear orphan receptor PPAR-γ plays a particularly important role in limiting the duration and intensity of fibroblast activation and differentiation, and impaired PPAR-γ expression or function in SSc may underlie the uncontrolled progression of fibrosis. Identifying the perturbations in signaling pathways, mediators and differentiation programs that are responsible for SSc tissue damage allows their selective targeting. This in turn opens the door for therapies utilizing novel compounds, or drug repurposing by innovative uses of already-approved drugs. In view of the heterogeneous clinical presentation and unpredictable course of SSc, as well as its complex pathogenesis, only robust clinical trials incorporating the judicious application of biomarkers will be able to clarify the clinical utility of these innovative approaches.

FibronectinEDA Promotes Chronic Cutaneous Fibrosis Through Toll-Like Receptor Signaling

FibronectinEDA is an endogenous TLR4 ligand in scleroderma. Scleroderma Takes Its Toll Scleroderma is a disease where the immune system attacks the connective tissues of the body, with notable hardening of the skin and fibrosis in multiple organs. However, what causes scleroderma—and the associated persistent fibrosis activation—remains unknown. Bhattacharyya et al. now report that the fibronectin extra domain A (FnEDA)—an endogenous damage-induced TLR4 ligand—may contribute to cutaneous fibrosis. The authors found that FnEDA is elevated in lesions and circulation both of patients with scleroderma and of a mouse model of fibrosis. FnEDA was up-regulated by TGF-β in healthy fibroblasts. In vitro, FnEDA increased mechanical stiffness of human skin equivalents. Indeed, the profibrotic responses induced by FnEDA were dependent on TLR4 signaling and could be blocked by genetic, RNA interference, and pharmacologic TLR4 inhibition. These data suggest that a damage-induced TLR4 signaling may contribute to fibrogenesis in scleroderma. Scleroderma is a progressive autoimmune disease affecting multiple organs. Fibrosis, the hallmark of scleroderma, represents transformation of self-limited wound healing into a deregulated self-sustaining process. The factors responsible for maintaining persistent fibroblast activation in scleroderma and other conditions with chronic fibrosis are not well understood. Toll-like receptor 4 (TLR4) and its damage-associated endogenous ligands are implicated in immune and fibrotic responses. We now show that fibronectin extra domain A (FnEDA) is an endogenous TLR4 ligand markedly elevated in the circulation and lesional skin biopsies from patients with scleroderma, as well as in mice with experimentally induced cutaneous fibrosis. Synthesis of FnEDA was preferentially stimulated by transforming growth factor–β in normal fibroblasts and was constitutively up-regulated in scleroderma fibroblasts. Exogenous FnEDA was a potent stimulus for collagen production, myofibroblast differentiation, and wound healing in vitro and increased the mechanical stiffness of human organotypic skin equivalents. Each of these profibrotic FnEDA responses was abrogated by genetic, RNA interference, or pharmacological disruption of TLR4 signaling. Moreover, either genetic loss of FnEDA or TLR4 blockade using a small molecule mitigated experimentally induced cutaneous fibrosis in mice. These observations implicate the FnEDA-TLR4 axis in cutaneous fibrosis and suggest a paradigm in which aberrant FnEDA accumulation in the fibrotic milieu drives sustained fibroblast activation via TLR4. This model explains how a damage-associated endogenous TLR4 ligand might contribute to converting self-limited tissue repair responses into intractable fibrogenesis in chronic conditions such as scleroderma. Disrupting sustained TLR4 signaling therefore represents a potential strategy for the treatment of fibrosis in scleroderma.

Peroxisome proliferator-activated receptor-γ abrogates Smad-dependent collagen stimulation by targeting the p300 transcriptional coactivator

Ligands of peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) abrogate the stimulation of collagen gene transcription induced by transforming growth factor‐beta (TGF‐β). Here, we delineate the mechanisms underlying this important novel physiological function for PPAR‐γ in connective tissue homeostasis. First, we demonstrated that antagonistic regulation of TGF‐β activity by PPAR‐γ ligands involves cellular PPAR‐γ, since 15‐deoxy‐A12,14‐prostaglandin J2 (15d‐PGJ2) failed to block TGF‐β‐induced responses in either primary cultures of PPAR‐γ‐null murine embryonic fibroblasts, or in normal human skin fibroblasts with RNAi‐mediated knockdown of PPAR‐γ. Next, we examined the molecular basis underlying the abrogation of TGF‐β signaling by PPAR‐γ in normal human fibroblasts in culture. The results demonstrated that Smad‐dependent transcriptional responses were blocked by PPAR‐γ without preventing Smad2/3 activation. In contrast, the interaction between activated Smad2/3 and the transcriptional coactivator and histone acetyltransferase p300 induced by TGF‐β, and the accumulation of p300 on consensus Smad‐binding DNA sequences and histone H4 hyperacetylation at the COL1A2 locus, were all prevented by PPAR‐γ. Wild‐type p300, but not a mutant form of p300 lacking functional histone acetyltransferase, was able to restore TGF‐β‐induced stimulation of COL1A2 in the presence of PPAR‐γ ligands. Collectively, these results indicate that PPAR‐γ blocked Smad‐mediated transcriptional responses by preventing p300 recruitment and histone H4 hyperacetylation, resulting in the inhibition of TGF‐β‐induced collagen gene expression. Pharmacological activation of PPAR‐γ thus may represent a novel therapeutic approach to target p300‐dependent TGF‐β profibrotic responses such as stimulation of collagen gene expression.—Ghosh, A. K., Bhattacharyya, S., Wei, J., Kim, S., Barak, Y., Mori, Y., and Varga, J. Peroxisome proliferator‐activated receptor‐γ abrogates Smad‐dependent collagen stimulation by targeting the p300 transcriptional coactivator. FASEB J. 23, 2968–2977 (2009). www.fasebj.org

A non-Smad mechanism of fibroblast activation by transforming growth factor-β via c-Abl and Egr-1: selective modulation by imatinib mesylate

The nonreceptor protein tyrosine kinase c-Abl regulates cell proliferation and survival. Recent studies provide evidence that implicate c-Abl as a mediator for fibrotic responses induced by transforming growth factor-β (TGF-β), but the precise mechanisms underlying this novel oncogene function are unknown. Here, we report that when expressed in normal fibroblasts, a constitutively active mutant of Abl that causes chronic myelogenous leukemia (CML) stimulated the expression and transcriptional activity of the early growth response factor 1 (Egr-1). Mouse embryonic fibroblasts (MEFs), lacking c-Abl, were resistant to TGF-β stimulation. Responsiveness of these MEFs to TGF-β could be rescued by wild-type c-Abl, but not by a kinase-deficient mutant form of c-Abl. Furthermore, Abl kinase activity was necessary for the induction of Egr-1 by TGF-β in normal fibroblasts, and Egr-1 was required for stimulation of collagen by Bcr-Abl. Lesional skin fibroblasts in mice with bleomycin-induced fibrosis of skin displayed evidence of c-Abl activation in situ, and elevated phospho-c-Abl correlated with increased local expression of Egr-1. Collectively, these results position Egr-1 downstream of c-Abl in the fibrotic response, delineate a novel Egr-1-dependent intracellular signaling mechanism that underlies the involvement of c-Abl in certain TGF-β responses, and identify Egr-1 as a target of inhibition by imatinib. Furthermore, the findings show in situ activation of c-Abl paralleling the upregulated tissue expression of Egr-1 that accompanies fibrosis. Pharmacological targeting of c-Abl and its downstream effector pathways may, therefore, represent a novel therapeutic approach to blocking TGF-β-dependent fibrotic processes.

Essential Roles for Early Growth Response Transcription Factor Egr-1 in Tissue Fibrosis and Wound Healing

The early growth response gene (Egr-1) codes for a zinc finger transcription factor that has important roles in the regulation of cell growth, differentiation, and survival. Aberrant Egr-1 expression is implicated in carcinogenesis, inflammation, atherosclerosis, and ischemic injury. We reported previously that normal fibroblasts stimulated by transforming growth factor-ss showed rapid and transient induction of Egr-1. Moreover, we observed that tissue expression of Egr-1 was elevated in patients with scleroderma, which suggests that Egr-1 may be involved in tissue repair and fibrosis. Here, we investigated matrix remodeling and wound healing in mice harboring gain of function or loss of function mutations of Egr-1. Using the model of bleomycin-induced scleroderma, we found that the early influx of inflammatory cells into the skin and lungs, and the subsequent development of fibrosis in these organs, were markedly attenuated in Egr-1 null mice. Furthermore, full-thickness incisional skin wound healing was impaired, and skin fibroblasts lacking Egr-1 showed reduced migration and myofibroblast transdifferentiation in vitro. In contrast, transgenic mice with fibroblast-specific Egr-1 overexpression showed exuberant tissue repair, with enhanced collagen accumulation and increased tensile strength of incisional wounds. Together, these results point to the fundamental role that Egr-1 plays in the regulation of transforming growth factor-ss-dependent physiological and pathological matrix remodeling.

Smad-Independent Transforming Growth Factor-β Regulation of Early Growth Response-1 and Sustained Expression in Fibrosis: Implications for Scleroderma

Transforming growth factor-beta (TGF-beta) plays a key role in scleroderma pathogenesis. The transcription factor early growth response-1 (Egr-1) mediates the stimulation of collagen transcription elicited by TGF-beta and is necessary for the development of pulmonary fibrosis in mice. Here, we report that TGF-beta causes a time- and dose-dependent increase in Egr-1 protein and mRNA levels and enhanced transcription of the Egr-1 gene via serum response elements in normal fibroblasts. The ability of TGF-beta to stimulate Egr-1 was preserved in Smad3-null mice and in explanted Smad3-null fibroblasts. The response was blocked by a specific mitogen-activated protein kinase kinase 1 (MEK1) inhibitor but not by an ALK5 kinase inhibitor. Furthermore, MEK1 was phosphorylated by TGF-beta, which was sufficient to drive Egr-1 transactivation. Stimulation by TGF-beta enhanced the transcriptional activity of Elk-1 via the MEK-extracellular signal-regulated kinase 1/2 pathway. Bleomycin-induced scleroderma in the mouse was accompanied by increased Egr-1 accumulation in lesional fibroblasts. Furthermore, biopsies of lesional skin and lung from patients with scleroderma showed increased Egr-1 levels, which were highest in early diffuse disease. Moreover, both Egr-1 mRNA and protein were elevated in explanted scleroderma skin fibroblasts in vitro. Together, these findings define a Smad-independent TGF-beta signal transduction mechanism that underlies the stimulation of Egr-1, demonstrate for the first time sustained Egr-1 up-regulation in fibrotic lesions and suggests that Egr-1 has a role in the induction and progression of fibrosis.

Egr-1: new conductor for the tissue repair orchestra directs harmony (regeneration) or cacophony (fibrosis).

Fibroblasts and myofibroblasts are the key effector cells executing physiological tissue repair leading to regeneration on the one hand, and pathological fibrogenesis leading to chronic fibrosing conditions on the other. Recent studies identify the multifunctional transcription factor early growth response‐1(Egr‐1) as an important mediator of fibroblast activation triggered by diverse stimuli. Egr‐1 has potent stimulatory effects on fibrotic gene expression, and aberrant Egr‐1 expression or function is associated with animal models of fibrosis and human fibrotic disorders, including emphysema, pulmonary fibrosis, pulmonary hypertension and systemic sclerosis. Pharmacological suppression or genetic targeting of Egr‐1 blocks fibrotic responses in vitro and ameliorates experimental fibrosis in the skin and lung. In contrast, Egr‐1 appears to act as a negative regulator of hepatic fibrosis in mouse models, suggesting a context‐dependent role in fibrosis. The Egr‐1‐binding protein Nab2 is an endogenous inhibitor of Egr‐1‐mediated signalling and abrogates the stimulation of fibrotic responses induced by transforming growth factor‐β (TGFβ). Moreover, mice deficient in Nab2 show excessive collagen accumulation in the skin. These observations highlight a previously unsuspected fundamental physiological function for the Egr‐1–Nab2 signalling axis in regulating fibrogenesis, and suggest that Egr‐1 may be a potential novel therapeutic target in human diseases complicated by fibrosis. This review summarizes recent advances in understanding the regulation and complex functional role of Egr‐1 and its related proteins and inhibitors in pathological fibrosis.

Molecular signatures in skin associated with clinical improvement during mycophenolate treatment in systemic sclerosis.

Heterogeneity in systemic sclerosis/SSc confounds clinical trials. We previously identified ‘intrinsic’ gene expression subsets by analysis of SSc skin. Here we test the hypotheses that skin gene expression signatures including intrinsic subset are associated with skin score/MRSS improvement during mycophenolate mofetil (MMF) treatment. Gene expression and intrinsic subset assignment were measured in 12 SSc patients’ biopsies and ten controls at baseline, and from serial biopsies of one cyclophosphamide-treated patient, and nine MMF-treated patients. Gene expression changes during treatment were determined using paired t-tests corrected for multiple hypothesis testing. MRSS improved in four of seven MMF-treated patients classified as the inflammatory intrinsic subset. Three patients without MRSS improvement were classified as normal-like or fibroproliferative intrinsic subsets. 321 genes (FDR <5%) were differentially expressed at baseline between patients with and without MRSS improvement during treatment. Expression of 571 genes (FDR <10%) changed between pre- and post-MMF treatment biopsies for patients demonstrating MRSS improvement. Gene expression changes in skin are only seen in patients with MRSS improvement. Baseline gene expression in skin, including intrinsic subset assignment, may identify SSc patients whose MRSS will improve during MMF treatment, suggesting that gene expression in skin may allow targeted treatment in SSc.

Early growth response transcription factors: key mediators of fibrosis and novel targets for anti-fibrotic therapy

Fibrosis is a deregulated and ultimately defective form of tissue repair that underlies a large number of chronic human diseases, as well as obesity and aging. The pathogenesis of fibrosis involves multiple cell types and extracellular signals, of which transforming growth factor-ß (TGF-ß) is pre-eminent. The prevalence of fibrosis is rising worldwide, and to date no agents has shown clinical efficacy in the attenuating or reversing the process. Recent studies implicate the immediate-early response transcription factor Egr-1 in the pathogenesis of fibrosis. Egr-1 couples acute changes in the cellular environment to sustained alterations in gene expression, and mediates a broad spectrum of biological responses to injury and stress. In contrast to other ligand-activated transcription factors such as NF-κB, c-jun and Smad2/3 that undergo post-translational modification such as phosphorylation and nuclear translocation, Egr-1 activity is regulated via its biosynthesis. Aberrant Egr-1 expression or activity is implicated in cancer, inflammation, atherosclerosis, and ischemic injury and recent studies now indicate an important role for Egr-1 in TGF-ß-dependent profibrotic responses. Fibrosis in various animal models and human diseases such as scleroderma (SSc) and idiopathic pulmonary fibrosis (IPF) is accompanied by aberrant Egr-1 expression. Moreover Egr-1 appears to be required for physiologic and pathological connective tissue remodeling, and Egr-1-null mice are protected from fibrosis. As a novel profibrotic mediator, Egr-1 thus appears to be a promising potential target for the development of anti-fibrotic therapies.