Systemic sclerosis (SSc) is a multisystem autoimmune disease of unknown etiology characterized by inflammation, autoantibody production, and fibrosis. It predominantly affects women, this suggesting that female sex hormones such as estrogens may play a role in disease pathogenesis. However, up to date, the role of estrogens in SSc has been scarcely explored. The activity of estrogens is mediated either by transcription activity of the intracellular estrogen receptors (ER), ERα and ERβ, or by membrane-associated ER. Since the presence of autoantibodies to ERα and their role as estrogen agonists interfering with T lymphocyte homeostasis were demonstrated in other autoimmune diseases, we wanted to ascertain whether anti-ERα antibodies were detectable in sera from patients with SSc. We detected anti-ERα antibody serum immunoreactivity in 42% of patients with SSc (30 out of 71 analyzed). Importantly, a significant association was found between anti-ERα antibody values and key clinical parameters of disease activity and severity. Fittingly, anti-ERα antibody levels were also significantly associated with alterations of immunological features of SSc patients, including increased T cell apoptotic susceptibility and changes in T regulatory cells (Treg) homeostasis. In particular, the percentage of activated Treg (CD4+CD45RA− FoxP3brightCD25bright) was significantly higher in anti-ERα antibody positive patients than in anti-ERα antibody negative patients. Taken together our data clearly indicate that anti-ERα antibodies, probably via the involvement of membrane-associated ER, can represent: i) promising markers for SSc progression but, also, ii) functional modulators of the SSc patients’ immune system.
We previously described a primitive cell population derived from human circulating CD14+ monocytes, named monocyte-derived multipotential cells (MOMCs), which are capable of differentiating into mesenchymal and endothelial lineages. To generate MOMCs in vitro, monocytes are required to bind to fibronectin and be exposed to soluble factor(s) derived from circulating CD14− cells. The present study was conducted to identify factors that induce MOMC differentiation.
We cultured CD14+ monocytes on fibronectin in the presence or absence of platelets, CD14− peripheral blood mononuclear cells, platelet-conditioned medium, or candidate MOMC differentiation factors. The transformation of monocytes into MOMCs was assessed by the presence of spindle-shaped adherent cells, CD34 expression, and the potential to differentiate in vitro into mesenchymal and endothelial lineages.
The presence of platelets or platelet-conditioned medium was required to generate MOMCs from monocytes. A screening of candidate platelet-derived soluble factors identified stromal cell-derived factor (SDF)-1 as a requirement for generating MOMCs. Blocking an interaction between SDF-1 and its receptor CXCR4 inhibited MOMC generation, further confirming SDF-1′s critical role in this process. Finally, circulating MOMC precursors were found to reside in the CD14+CXCR4high cell population.
The interaction of SDF-1 with CXCR4 is essential for the transformation of circulating monocytes into MOMCs.
The potential for amniotic fluid stem cell (AFSC) treatment to inhibit the progression of fibrotic lung injury has not been described. We have previously demonstrated that AFSC can attenuate both acute and chronic-fibrotic kidney injury through modification of the cytokine environment. Fibrotic lung injury, such as in Idiopathic Pulmonary Fibrosis (IPF), is mediated through pro-fibrotic and pro-inflammatory cytokine activity. Thus, we hypothesized that AFSC treatment might inhibit the progression of bleomycin-induced pulmonary fibrosis through cytokine modulation. In particular, we aimed to investigate the effect of AFSC treatment on the modulation of the pro-fibrotic cytokine CCL2, which is increased in human IPF patients and is correlated with poor prognoses, advanced disease states and worse fibrotic outcomes. The impacts of intravenous murine AFSC given at acute (day 0) or chronic (day 14) intervention time-points after bleomycin injury were analyzed at either day 3 or day 28 post-injury. Murine AFSC treatment at either day 0 or day 14 post-bleomycin injury significantly inhibited collagen deposition and preserved pulmonary function. CCL2 expression increased in bleomycin-injured bronchoalveolar lavage (BAL), but significantly decreased following AFSC treatment at either day 0 or at day 14. AFSC were observed to localize within fibrotic lesions in the lung, showing preferential targeting of AFSC to the area of fibrosis. We also observed that MMP-2 was transiently increased in BAL following AFSC treatment. Increased MMP-2 activity was further associated with cleavage of CCL2, rendering it a putative antagonist for CCL2/CCR2 signaling, which we surmise is a potential mechanism for CCL2 reduction in BAL following AFSC treatment. Based on this data, we concluded that AFSC have the potential to inhibit the development or progression of fibrosis in a bleomycin injury model during both acute and chronic remodeling events.
A similar immune response is implicated in the pathogenesis of pulmonary fibrosis and allergic disorders. We investigated the potential therapeutic efficacy and mechanism of rupatadine, a dual antagonist of histamine and platelet-activation factor (PAF), in bleomycin- (BLM-) and silica-induced pulmonary fibrosis. The indicated dosages of rupatadine were administered in rodents with bleomycin or silica-induced pulmonary fibrosis. The tissue injury, fibrosis, inflammatory cells and cytokines, and lung function were examined to evaluate the therapeutic efficacy of rupatadine. The anti-fibrosis effect of rupatadine was compared with an H1 or PAF receptor antagonist, and efforts were made to reveal rupatadine’s anti-fibrotic mechanism. Rupatadine promoted the resolution of pulmonary inflammation and fibrosis in a dose-dependent manner, as indicated by the reductions in inflammation score, collagen deposition and epithelial-mesenchymal transformation, and infiltration or expression of inflammatory cells or cytokines in the fibrotic lung tissue. Thus, rupatadine treatment improved the declined lung function and significantly decreased animal death. Moreover, rupatadine was able not only to attenuate silica-induced silicosis but also to produce a superior therapeutic efficacy compared to pirfenidone, histamine H1 antagonist loratadine, or PAF antagonist CV-3988. The anti-fibrotic action of rupatadine might relate to its attenuation of BLM- or PAF-induced premature senescence because rupatadine treatment protected against the in vivo and in vitro activation of the p53/p21-dependent senescence pathway. Our studies indicate that rupatadine promotes the resolution of pulmonary inflammation and fibrosis by attenuating the PAF-mediated senescence response. Rupatadine holds promise as a novel drug to treat the devastating disease of pulmonary fibrosis.
Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease of unknown etiology with few current treatment options. Recently, we determined an important role of prostaglandin F2α (PGF2α) in pulmonary fibrosis by using a bleomycin-induced pulmonary fibrosis model and found an abundance of PGF2α in bronchoalveolar lavage fluid of IPF patients. We investigated the role of PGF2α in human IPF by assessing plasma concentrations of 15-keto-dihydro PGF2α, a stable metabolite of PGF2α.
We measured plasma concentrations of 15-keto-dihydro PGF2α in 91 IPF patients and compared these values with those of controls (n = 25). We further investigated the relationships of plasma 15-keto-dihydro PGF2α concentrations with disease severity and mortality.
Plasma concentrations of 15-keto-dihydro PGF2α were significantly higher in IPF patients than controls (p<0.001). Plasma concentrations of this metabolite were significantly correlated with forced expiratory volume in 1 second (Rs [correlation coefficient] = −0.34, p = 0.004), forced vital capacity (Rs = −0.33, p = 0.005), diffusing capacity for carbon monoxide (Rs = −0.36, p = 0.003), the composite physiologic index (Rs = 0.40, p = 0.001), 6-minute walk distance (Rs = −0.24, p = 0.04) and end-exercise oxygen saturation (Rs = −0.25, p = 0.04) when patients with emphysema were excluded. Multivariate analysis using stepwise Cox proportional hazards model showed that a higher composite physiologic index (relative risk = 1.049, p = 0.002) and plasma 15-keto-dihydro PGF2α concentrations (relative risk = 1.005, p = 0.002) were independently associated with an increased risk of mortality.
We demonstrated significant associations of plasma concentrations of PGF2α metabolites with disease severity and prognosis, which support a potential pathogenic role for PGF2α in human IPF.
Endothelin 1 (ET-1) is a key regulator of vascular homeostasis. We have recently reported that the presence of Human antigen class I, HLA-B35, contributes to human dermal microvascular endothelial cell (HDMEC) dysfunction by upregulating ET-1 and proinflammatory genes. Likewise, a Toll-like receptor 3 (TLR3) ligand, Poly(I:C), was shown to induce ET-1 expression in HDMECs. The goal of this study was to determine the molecular mechanism of ET-1 induction by these two agonists. Because HLA-B35 expression correlated with induction of Binding Immunoglobulin Protein (BiP/GRP78) and several heat shock proteins, we first focused on ER stress and unfolded protein response (UPR) as possible mediators of this response. ER stress inducer, Thapsigargin (TG), HLA-B35, and Poly(I:C) induced ET-1 expression with similar potency in HDMECs. TG and HLA-B35 activated the PERK/eIF2α/ATF4 branch of the UPR and modestly increased the spliced variant of XBP1, but did not affect the ATF6 pathway. Poly(I:C) also activated eIF2α/ATF4 in a protein kinase R (PKR)-dependent manner. Depletion of ATF4 decreased basal expression levels of ET-1 mRNA and protein, and completely prevented upregulation of ET-1 by all three agonists. Additional experiments have demonstrated that the JNK and NF-κB pathways are also required for ET-1 upregulation by these agonists. Formation of the ATF4/c-JUN complex, but not the ATF4/NF-κB complex was increased in the agonist treated cells. The functional role of c-JUN in responses to HLA-B35 and Poly(I:C) was further confirmed in ET-1 promoter assays. This study identified ATF4 as a novel activator of the ET-1 gene. The ER stress/UPR and TLR3 pathways converge on eIF2α/ATF4 during activation of endothelial cells.
Systemic sclerosis (SSc) is more prevalent in women. Our goal is to determine the effects of 17β-estradiol (E2) on the development of fibrosis and to compare circulating levels of estrogens in SSc patients and healthy controls.
Using primary human dermal fibroblasts, we evaluated the effect of E2 on fibronectin (FN) expression with and without the estrogen receptor (ER) antagonist ICI 182,780, inhibitors of signaling, propyl-pyrazole-triol, an ERα specific ligand, and genistein, an ERβ selective ligand, to identify the signaling pathways mediating E2's effect. We confirmed the fibrotic effect of E2 in human skin using an ex vivo organ culture model. Lastly, we measured levels of E2 and estrone in serum samples from SSc patients with diffuse cutaneous involvement and healthy controls using mass spectrometry.
E2 increased expression of FN in dermal fibroblasts. ICI 182,780, inositol-1,4,5-triphosphate inhibitor, and p38 mitogen-activated protein kinase inhibitor blocked the effects of E2 on FN. Propyl-pyrazole-triol, but not genistein, significantly increased FN expression. Ex vivo, E2 induced fibrosis of human skin. The effects of E2 were abrogated by ICI 182,780. Circulating levels of E2 and estrone were significantly increased in sera of patients with diffuse cutaneous SSc.
Our findings implicate estrogens in the fibrotic process and may explain the preponderance of SSc in women. ICI 182,780 or other ER signaling antagonists may be effective agents for the treatment of fibrosis.
Tenascin (TN)-C is an extracellular matrix protein associated with injury and remodeling. Since Transforming Growth Factor (TGF)-β induces both TN-C and Insulin-Like Growth Factor Binding Protein (IGFBP)-3, we sought to determine the role of IGFBP-3 in mediating TGF-β’s effects on TN-C production and to assess the levels of TN-C in vivo in SSc-associated pulmonary fibrosis (PF).
Primary human lung fibroblasts were stimulated with TGF-β or IGFBP-3 in the presence or absence of specific siRNAs and chemical signaling cascade inhibitors. TN-C levels were examined in lung tissues of patients with Systemic Sclerosis (SSc)-associated pulmonary fibrosis using immunohistochemistry (IHC) and compared to those of normal donors. TN-C levels were quantified in serum from normal donors and patients with SSc with or without PF using ELISA.
IGFBP-3 mediated TGF-β induction of TN-C. Direct induction of TN-C by IGFBP-3 occurred in a p38K-dependent manner. TN-C levels were abundant in SSc lung tissues and localized to subepithelial layers of the distal airways. No TN-C was detectable around proximal airways. Patients with SSc-associated pulmonary fibrosis had significantly greater levels of circulating TN-C compared to patients without this complication. Longitudinal samples obtained from patients with SSc before and after the onset of PF showed increased levels post-PF.
IGFBP-3, which is overexpressed in fibrotic lungs, induces production of TN-C by subepithelial fibroblasts. The increased lung tissue levels of TN-C parallel levels detected in sera of patients with SSc and lung fibrosis, suggesting that TN-C may be a useful biomarker for SSc-PF.
Reports of low sexual activity rates and high impairment rates among women with chronic diseases have not included comparisons to general population data. The objective of this study was to compare sexual activity and impairment rates of women with systemic sclerosis (SSc) to general population data and to identify domains of sexual function driving impairment in SSc.
Canadian women with SSc were compared to women from a UK population sample. Sexual activity and, among sexually active women, sexual impairment were evaluated with a 9-item version of the Female Sexual Function Index (FSFI).
Among women with SSc (mean age = 57.0 years), 296 of 730 (41%) were sexually active, 181 (61%) of whom were sexually impaired, resulting in 115 of 730 (16%) who were sexually active without impairment. In the UK population sample (mean age = 55.4 years), 956 of 1,498 women (64%) were sexually active, 420 (44%) of whom were impaired, with 536 of 1,498 (36%) sexually active without impairment. Adjusting for age and marital status, women with SSc were significantly less likely to be sexually active (OR = 0.34, 95%CI = 0.28–0.42) and, among sexually active women, significantly more likely to be sexually impaired (OR = 1.88, 95%CI = 1.42–2.49) than general population women. Controlling for total FSFI scores, women with SSc had significantly worse lubrication and pain scores than general population women.
Sexual functioning is a problem for many women with scleroderma and is associated with pain and poor lubrication. Evidence-based interventions to support sexual activity and function in women with SSc are needed.
Heightened production of collagen and other matrix proteins underlies the fibrotic phenotype of systemic sclerosis (SSc). Roscovitine is an inhibitor of cyclin-dependent kinases that promote cell cycling (CDK1, 2), neuronal development (CDK5) and control transcription (CDK7,9). In an in vivo glomerulonephritis model, roscovitine treatment decreased mesangial cell proliferation and matrix proteins . We investigated whether roscovitine could regulate fibrotic protein production directly rather than through cell cycling. Our investigations revealed that roscovitine coordinately inhibited the expression of collagen, fibronectin, and connective tissue growth factor (CTGF) in normal and SSc fibroblasts. This effect occurred on a transcriptional basis and did not result from roscovitine-mediated cell cycle inhibition. Roscovitine-mediated suppression of matrix proteins could not be reversed by the exogenous profibrotic cytokines TGF-β or IL-6. To our knowledge, we are the first to report that roscovitine modulates matrix protein transcription. Roscovitine may thus be a viable treatment option for SSc and other fibrosing diseases.
Genetic analysis of TP63 implicates ΔNp63 isoforms in preservation of replicative capacity and cellular lifespan within adult stem cells. ΔNp63α is also an oncogene and survival factor that mediates therapeutic resistance in squamous carcinomas. These diverse activities are the result of genetic and functional interactions between TP63 and an array of morphogenic and morphostatic signals that govern tissue and tumor stasis, mitotic polarity, and cell fate; however the cellular signals that account for specific functions of TP63 are incompletely understood. To address this we sought to identify signaling pathways that regulate expression, stability or activity of ΔNp63α. An siRNA-based screen of the human kinome identified the Type 1 TGFβ receptor, ALK5, as the kinase required for phosphorylation of ΔNp63α at Serine 66/68 (S66/68). This activity is TGFβ-dependent and sensitive to either ALK5-directed siRNA or the ALK5 kinase inhibitor A83-01. Mechanistic studies support a model in which ALK5 is proteolytically cleaved at the internal juxtamembrane region resulting in the translocation of the C-terminal ALK5-intracellular kinase domain (ALK5IKD). In this study, we demonstrate that ALK5-mediated phosphorylation of ΔNp63α is required for the anti-clonogenic effects of TGFΒ and ectopic expression of ALK5IKD mimics these effects. Finally, we present evidence that ultraviolet irradiation-mediated phosphorylation of ΔNp63α is sensitive to ALK5 inhibitors. These findings identify a non-canonical TGFβ-signaling pathway that mediates the anti-clonogenic effects of TGFβ and the effects of cellular stress via ΔNp63α phosphorylation.
Pulmonary fibrosis is a disease that results in loss of normal lung architecture, but the signaling events that drive tissue destruction are incompletely understood. Wnt/β-catenin signaling is important in normal lung development, but whether abnormal signaling occurs in lung fibrosis due to systemic sclerosis and the consequences of β-catenin signaling toward the fibrogenic phenotype remain poorly defined. In this study, we show nuclear β-catenin accumulation in fibroblastic foci from lungs of patients with systemic sclerosis–associated advanced pulmonary fibrosis. Forced activation of β-catenin signaling in three independently derived sources of normal human lung fibroblasts promotes proliferation and migratory activities but is not sufficient to activate classic markers of fibroblast activation, such as TGF-β, type 1 collagen, α-smooth muscle actin, and connective tissue growth factor. These findings indicate that activation of β-catenin signaling in pulmonary fibroblasts may be a common feature of lung fibrosis, contributing to fibroproliferative and migratory activities associated with the disease.
Wnt/β-catenin signaling; scleroderma; fibrosis
Extracellular matrix deposition and tissue scarring characterize the process of fibrosis. Transforming growth factor beta (TGFβ) and Insulin-like growth factor binding protein-3 (IGFBP-3) have been implicated in the pathogenesis of fibrosis in various tissues by inducing mesenchymal cell proliferation and extracellular matrix deposition. We identified Syndecan-2 (SDC2) as a gene induced by TGFβ in an IGFBP-3-dependent manner. TGFβ induction of SDC2 mRNA and protein required IGFBP-3. IGFBP-3 independently induced production of SDC2 in primary fibroblasts. Using an ex-vivo model of human skin in organ culture expressing IGFBP-3, we demonstrate that IGFBP-3 induces SDC2 ex vivo in human tissue. We also identified Mitogen-activated protein kinase-interacting kinase (Mknk2) as a gene induced by IGFBP-3. IGFBP-3 triggered Mknk2 phosphorylation resulting in its activation. Mknk2 independently induced SDC2 in human skin. Since IGFBP-3 is over-expressed in fibrotic tissues, we examined SDC2 levels in skin and lung tissues of patients with systemic sclerosis (SSc) and lung tissues of patients with idiopathic pulmonary fibrosis (IPF). SDC2 levels were increased in fibrotic dermal and lung tissues of patients with SSc and in lung tissues of patients with IPF. This is the first report describing elevated levels of SDC2 in fibrosis. Increased SDC2 expression is due, at least in part, to the activity of two pro-fibrotic factors, TGFβ and IGFBP-3.
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- β (TGF-β) 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-β-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.
Egr-1; TGF-β; fibrosis; Scleroderma (systemic sclerosis); fibroblast
Our previous studies have demonstrated increased expression of insulin-like growth factor binding protein-5 (IGFBP-5) in fibrotic tissues and IGFBP-5 induction of extracellular matrix (ECM) components. The mechanism resulting in increased IGFBP-5 in the extracellular milieu of fibrotic fibroblasts is unknown. Since Caveolin-1 (Cav-1) has been implicated to play a role in membrane trafficking and signal transduction in tissue fibrosis, we examined the effect of Cav-1 on IGFBP-5 internalization, trafficking, and secretion. We demonstrated that IGFBP-5 localized to lipid rafts in human lung fibroblasts and bound Cav-1. Cav-1 was detected in the nucleus in IGFBP-5-expressing fibroblasts, within aggregates enriched with IGFBP-5, suggesting a coordinate trafficking of IGFBP-5 and Cav-1 from the plasma membrane to the nucleus. This trafficking was dependent on Cav-1 as fibroblasts from Cav-1 null mice had increased extracellular IGFBP-5, and as fibroblasts in which Cav-1 was silenced or lipid raft structure was disrupted through cholesterol depletion also had defective IGFBP-5 internalization. Restoration of Cav-1 function through administration of Cav-1 scaffolding peptide dramatically increased IGFBP-5 uptake. Finally, we demonstrated that IGFBP-5 in the ECM protects fibronectin from proteolytic degradation. Taken together, our findings identify a novel role for Cav-1 in the internalization and nuclear trafficking of IGFBP-5. Decreased Cav-1 expression in fibrotic diseases likely leads to increased deposition of IGFBP-5 in the ECM with subsequent reduction in ECM degradation, thus identifying a mechanism by which reduced Cav-1 and increased IGFBP-5 concomitantly contribute to the perpetuation of fibrosis.
Caveolin-1; insulin-like growth factor binding protein-5; fibrosis; extracellular matrix
Pulmonary complications in systemic sclerosis (SSc), including pulmonary fibrosis (PF) and pulmonary arterial hypertension (PAH), are the leading cause of mortality. We compared the molecular fingerprint of SSc lung tissues and matching primary lung fibroblasts to those of normal donors, and patients with idiopathic pulmonary fibrosis (IPF) and idiopathic pulmonary arterial hypertension (IPAH).
Lung tissues were obtained from 33 patients with SSc who underwent lung transplantation. Tissues and cells from a subgroup of SSc patients with predominantly PF or PAH were compared to those from normal donors, patients with IPF, or IPAH. Microarray data was analyzed using Efficiency Analysis for determination of optimal data processing methods. Real time PCR and immunohistochemistry were used to confirm differential levels of mRNA and protein, respectively.
We identified a consensus of 242 and 335 genes that were differentially expressed in lungs and primary fibroblasts, respectively. Enriched function groups in SSc-PF and IPF lungs included fibrosis, insulin-like growth factor signaling and caveolin-mediated endocytosis. Functional groups shared by SSc-PAH and IPAH lungs included antigen presentation, chemokine activity, and IL-17 signaling.
Using microarray analysis on carefully phenotyped SSc and comparator lung tissues, we demonstrated distinct molecular profiles in tissues and fibroblasts of patients with SSc-associated lung disease compared to idiopathic forms of lung disease. Unique molecular signatures were generated that are disease- (SSc) and phenotype- (PF vs PAH) specific. These signatures provide new insights into pathogenesis and potential therapeutic targets for SSc lung disease.
Rationale: Pulmonary hypertension (PH) is a progressive disease with unclear etiology. The significance of autophagy in PH remains unknown.
Objectives: To determine the mechanisms by which autophagic proteins regulate tissue responses during PH.
Methods: Lungs from patients with PH, lungs from mice exposed to chronic hypoxia, and human pulmonary vascular cells were examined for autophagy using electron microscopy and Western analysis. Mice deficient in microtubule-associated protein-1 light chain-3B (LC3B−/−), or early growth response-1 (Egr-1−/−), were evaluated for vascular morphology and hemodynamics.
Measurements and Main Results: Human PH lungs displayed elevated lipid-conjugated LC3B, and autophagosomes relative to normal lungs. These autophagic markers increased in hypoxic mice, and in human pulmonary vascular cells exposed to hypoxia. Egr-1, which regulates LC3B expression, was elevated in PH, and increased by hypoxia in vivo and in vitro. LC3B−/− or Egr-1−/−, but not Beclin 1+/−, mice displayed exaggerated PH during hypoxia. In vitro, LC3B knockdown increased reactive oxygen species production, hypoxia-inducible factor-1α stabilization, and hypoxic cell proliferation. LC3B and Egr-1 localized to caveolae, associated with caveolin-1, and trafficked to the cytosol during hypoxia.
Conclusions: The results demonstrate elevated LC3B in the lungs of humans with PH, and of mice with hypoxic PH. The increased susceptibility of LC3B−/− and Egr-1−/− mice to hypoxia-induced PH and increased hypoxic proliferation of LC3B knockdown cells suggest adaptive functions of these proteins during hypoxic vascular remodeling. The results suggest that autophagic protein LC3B exerts a protective function during the pathogenesis of PH, through the regulation of hypoxic cell proliferation.
autophagy; hypoxia; hypertension, pulmonary
Evidence is lacking for direct pathogenicity of human anti-proteinase-3 (PR3) antibodies in development of systemic vasculitis and granulomatosis with polyangiitis (GPA, Wegener's granulomatosis). Progress in study of these antibodies in rodents has been hampered by lack of PR3 expression on murine neutrophils, and by different Fc-receptor affinities for IgG across species. Therefore, we tested whether human anti-PR3 antibodies can induce acute vasculitis in mice with a human immune system. Chimeric mice were generated by injecting human haematopoietic stem cells into irradiated NOD-scid-IL2Rγ−/− mice. Matched chimera mice were treated with human IgG from patients with: anti-PR3 positive renal and lung vasculitis; patients with non-vasculitic renal disease; or healthy controls. Six-days later, 39% of anti-PR3 treated mice had haematuria, compared with none of controls. There was punctate bleeding on the surface of lungs of anti-PR3 treated animals, with histological evidence of vasculitis and haemorrhage. Anti-PR3 treated mice had mild pauci-immune proliferative glomerulonephritis, with infiltration of human and mouse leukocytes. In 3 mice (17%) more severe glomerular injury was present. There were no glomerular changes in controls. Human IgG from patients with anti-PR3 autoantibodies is therefore pathogenic. This model of anti-PR3 antibody-mediated vasculitis may be useful in dissecting mechanisms of microvascular injury.
Fibrosis involves an orchestrated cascade of events including activation of fibroblasts, increased production and deposition of extracellular matrix components, and differentiation of fibroblasts into myofibroblasts. Epithelial-mesenchymal cross-talk plays an important role in this process, and current hypotheses of organ fibrosis liken it to an aberrant wound healing response in which epithelial-mesenchymal transition (EMT) and cellular senescence may also contribute to disease pathogenesis. The fibrotic response is associated with altered expression of growth factors and cytokines, including increased levels of transforming growth factor-β1 (TGF-β1) and the more recent observation that increased levels of several insulin-like growth factor binding proteins (IGFBPs) are associated with a number of fibrotic conditions. IGFBPs have been implicated in virtually every cell type and process associated with the fibrotic response, making the IGFBPs attractive targets for the development of novel anti-fibrotic therapies. In this review, the current state of knowledge regarding the classical IGFBP family in organ fibrosis will be summarized and the clinical implications considered.
Extracellular matrix; fibrosis; IGFBP; senescence; systemic sclerosis; idiopathic pulmonary fibrosis.
Fibrotic tissue in the liver is mainly composed of collagen. Fibronectin, which is also present in fibrotic matrices, is required for collagen matrix assembly in vitro. It also modulates the amount of growth factors and their release from the matrix. We therefore examined the effects of the absence of fibronectin on the development of fibrosis in mice.
Conditional deletion of fibronectin in the liver using the Mx promoter to drive cre expression resulted in increased collagen production and hence a more pronounced fibrosis in response to dimethylnitrosamine in mice. Exclusive deletion of fibronectin in hepatocytes or normalization of circulating fibronectin in Mx-cKO mice did not affect the development of fibrosis suggesting a role for fibronectin production by other liver cell types. The boosted fibrosis in fibronectin-deficient mice was associated with enhanced stellate cell activation and proliferation, elevated concentrations of active TGF-β, and increased TGF-β-mediated signaling.
In vitro experiments revealed that collagen-type-I production by fibronectin-deficient hepatic stellate cells stimulated with TGF-β was more pronounced, and was associated with augmented Smad3-mediated signaling. Interfering with TGF-β signaling using SB431542 normalized collagen-type-I production in fibronectin-deficient hepatic stellate cells. Furthermore, precoating culture plates with fibronectin, but not collagen, or providing fibronectin fibrils unable to interact with RGD binding integrins via the RGD domain significantly diminished the amount of active TGF-β in fibronectin-deficient stellate cells and normalized collagen-type-I production in response to TGF-β stimulation. Thus, excessive stellate cell activation and production of collagen results from increased active TGF-β and TGF-β signaling in the absence of fibronectin.
In conclusion, our data indicate that fibronectin controls the availability of active TGF-β in the injured liver, which impacts the severity of the resulting fibrosis. We therefore propose a novel role for locally produced fibronectin in protecting the liver from an excessive TGF-β-mediated response.
Previous studies demonstrated that protein kinase C- δ (PKC-δ) inhibition with the selective inhibitor, rottlerin, resulted in potent downregulation of type I collagen expression and production in normal human dermal fibroblasts and abrogated the exaggerated type I collagen production and expression in fibroblasts cultured from affected skin from patients with the fibrosing disorder systemic sclerosis (SSc). To elucidate the mechanisms involved in the ability of PKC-δ to regulate collagen production in fibroblasts, we examined the effects of PKC-δ inhibition on the transcriptome of normal and SSc human dermal fibroblasts. Normal and SSc human dermal fibroblasts were incubated with rottlerin (5 µM), and their gene expression was analyzed by microarrays. Pathway analysis and gene ontology analysis of differentially expressed genes in each comparison were performed. Identification of significantly overrepresented transcriptional regulatory elements (TREs) was performed using the Promoter Analysis and Interaction Network Toolset (PAINT) program. PKC-δ activity was also inhibited using RNA interference (siRNA) and by treating fibroblasts with a specific PKC-δ inhibitory cell permeable peptide. Differential gene expression of 20 genes was confirmed using real time PCR. PKC-δ inhibition caused a profound change in the transcriptome of normal and SSc human dermal fibroblasts in vitro. Pathway and gene ontology analysis identified multiple cellular and organismal pathways affected by PKC-δ inhibition. Furthermore, both pathway and PAINT analyses indicated that the transcription factor NFκB played an important role in the transcriptome changes induced by PKC-δ inhibition. Multiple genes involved in the degradation of the extracellular matrix components were significantly reduced in SSc fibroblasts and their expression was increased by PKC-δ inhibition. These results indicate that isoform-specific inhibition of PKC-δ profibrotic effects may represent a novel therapeutic approach for SSc and other fibrotic diseases.
Longitudinal studies examining the baseline predictors of fatigue in SSc have not been reported. Our objectives were to examine the course of fatigue severity over time and to identify baseline clinical, demographic, and psychosocial predictors of sequentially obtained fatigue scores in early SSc. We also examined baseline predictors of change in fatigue severity over time.
We analyzed 1090 longitudinal Fatigue Severity Scale (FSS) scores belonging to 256 patients who were enrolled in the Genetics versus Environment in Scleroderma Outcomes Study (GENISOS). Predictive significance of baseline variables for sequentially obtained FSS scores was examined with generalized linear mixed models. Predictors of change in FSS over time were examined by adding an interaction term between the baseline variable and time-in-study to the model.
The patients' mean age was 48.6 years, 47% were Caucasians, and 59% had diffuse cutaneous involvement. The mean disease duration at enrollment was 2.5 years. The FSS was obtained at enrollment and follow-up visits (mean follow-up time = 3.8 years). Average baseline FSS score was 4.7(±0.96). The FSS was relatively stable and did not show a consistent trend for change over time (p = 0.221). In a multivariable model of objective clinical variables, higher Medsger Gastrointestinal (p = 0.006) and Joint (p = 0.024) Severity Indices, and anti-U1-RNP antibodies (p = 0.024) were independent predictors of higher FSS. In the final model, ineffective coping skills captured by higher Illness Behavior Questionnaire scores (p<0.001), higher self-reported pain (p = 0.006), and higher Medsger Gastrointestinal Severity Index (p = 0.009) at enrollment were independent predictors of higher longitudinal FSS scores. Baseline DLco % predicted was the only independent variable that significantly predicted a change in FSS scores over time (p = 0.013), with lower DLco levels predicting an increase in FSS over time.
This study identified potentially modifiable clinical and psychological factors that predict longitudinal fatigue severity in early SSc.
A subset of patients with Ewing's sarcoma responds to anti-insulin-like growth factor-1 receptor (IGF-1R) antibodies. Mechanisms of sensitivity and resistance are unknown. We investigated whether an anti-IGF-1R antibody acts via a pathway that could also be suppressed by small interfering (si) RNA against the EWS/FLI-1 fusion protein, the hallmark of Ewing's sarcoma. The growth of two Ewing's sarcoma cell lines (TC-32 and TC-71) was inhibited by the fully human anti-IGF-1R antibody, R1507 (clonogenic and MTT assays). TC-32 and TC-71 cells express high levels of IGF-2, while RD-ES and A4573 Ewing's cell lines, which were less responsive to R1507 in our assays, express low or undetectable IGF-2, respectively. TC-71 cells also expressed high levels of IGF-1R, and R1507 decreased steady-state levels of this receptor by internalization/degradation, an effect which was associated with a decrease in p-IGF-1R, p-IRS-1, and p-Akt. EWS/FLI-1 siRNA also decreased p-Akt, due to its ability to increase IGF-BP3 levels and subsequently decrease IGF-1 and IGF-2 levels, thus inhibiting signaling through p-IGF-1R. This inhibition correlated with growth suppression and apoptosis. The attenuation of Akt activation was confirmed in TC-71 and HEK-293 (human embryonic kidney) cells by transfecting them with IGF-1R siRNA. We conclude that antibodies and siRNA to IGF-1R, as well as siRNA to EWS/FLI-1, act via intersecting IGF/IGF-1R signals that suppress a common point in this pathway, namely the phosphorylation of Akt.
In rheumatoid arthritis, a significant proportion of cytokine and chemokine synthesis is attributed to innate immune mechanisms. TLR4 is a prominent innate receptor since several endogenous ligands known to activate the innate immune system bind to it and may thereby promote joint inflammation. We generated TLR4 deficient DBA1J mice by backcrossing the TLR4 mutation present in C3H/HeJ strain onto the DBA1J strain and investigated the course of collagen-induced arthritis in TLR4 deficient mice in comparison to wild type littermates. The incidence of collagen- induced arthritis was significantly lower in TLR4 deficient compared to wild type mice (59 percent vs. 100 percent). The severity of arthritis was reduced in the TLR4 deficient mice compared to wild type littermates (mean maximum score 2,54 vs. 6,25). Mice deficient for TLR4 were virtually protected from cartilage destruction, and infiltration of inflammatory cells was reduced compared to wt mice. In parallel to the decreased clinical severity, lower anti-CCP antibody concentrations and lower IL-17 concentrations were found in the TLR4 deficient mice. The study further supports the role of TLR4 in the propagation of joint inflammation and destruction. Moreover, since deficiency in TLR4 led to decreased IL-17 and anti-CCP antibody production, the results indicate a link between TLR4 stimulation and the adaptive autoimmune response. This mechanism might be relevant in human rheumatoid arthritis, possibly in response to activating endogenous ligands in the affected joints.
Idiopathic pulmonary fibrosis exhibits differential progression from the time of diagnosis but the molecular basis for varying progression rates is poorly understood. The aim of the present study was to ascertain whether differential miRNA expression might provide one explanation for rapidly versus slowly progressing forms of IPF.
Methodology and Principal Findings
miRNA and mRNA were isolated from surgical lung biopsies from IPF patients with a clinically documented rapid or slow course of disease over the first year after diagnosis. A quantitative PCR miRNA array containing 88 of the most abundant miRNA in the human genome was used to profile lung biopsies from 9 patients with rapidly progressing IPF, 6 patients with slowly progressing IPF, and 10 normal lung biopsies. Using this approach, 11 miRNA were significantly increased and 36 were significantly decreased in rapid biopsies compared with normal biopsies. Slowly progressive biopsies exhibited 4 significantly increased miRNA and 36 significantly decreased miRNA compared with normal lung. Among the miRNA present in IPF with validated mRNA targets were those with regulatory effects on epithelial-mesenchymal transition (EMT). Five miRNA (miR-302c, miR-423-5p, miR-210, miR-376c, and miR-185) were significantly increased in rapid compared with slow IPF lung biopsies. Additional analyses of rapid biopsies and fibroblasts grown from the same biopsies revealed that the expression of AGO1 and AGO2 (essential components of the miRNA processing RISC complex) were lower compared with either slow or normal lung biopsies and fibroblasts.
These findings suggest that the development and/or clinical progression of IPF might be the consequence of aberrant miRNA processing.