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The extracellular matrix (ECM) is a highly dynamic network of proteins, glycoproteins, and proteoglycans. Numerous diseases result from mutation in genes coding for ECM proteins, but only recently it has been reported that mutations in the fibronectin (FN) gene were associated with a human disorder. FN is one of the main components of the ECM. It generates protein diversity through alternative splicing of a single pre-mRNA, having at least 20 different isoforms in humans. The precise function of these protein isoforms has remained obscure in most cases. Only in the recent few years, it was possible to shed light on the multiple roles of the alternatively spliced FN isoforms. This substantial progress was achieved basically with the knowledge derived from engineered mouse models bearing subtle mutations in specific FN domains. These data, together with a recent report associating mutations in the FN gene to a form of glomerulopathy, clearly show that mutations in constitutive exons or misregulation of alternatively spliced domains of the FN gene may have nonlethal pathological consequences. In this review, we focus on the pathological consequences of mutations in the FN gene, by connecting the function of alternatively spliced isoforms of fibronectin to human diseases. © 2011 IUBMB IUBMB Life, 63(7): 538-546, 2011 [ABSTRACT FROM AUTHOR]

Abstract: Fibronectin (FN), a ubiquitous glycoprotein that plays critical roles in physiologic and pathologic conditions, undergoes alternative splicing which distinguishes plasma FN (pFN) from cellular FN (cFN). Although both pFN and cFN can be incorporated into the extracellular matrix, a distinguishing feature of cFN is the inclusion of an alternatively spliced exon termed EDA (for extra type III domain A). The molecular steps involved in EDA splicing are well-characterized, but pathways influencing EDA splicing are less clear. We have previously found an obligate role for inhibition of the tumor suppressor phosphatase and tensin homologue on chromosome 10 (PTEN), the primary regulator of the PI3K/Akt pathway, in fibroblast activation. Here we show TGF-β, a potent inducer of both EDA splicing and fibroblast activation, inhibits PTEN expression and activity in mesenchymal cells, corresponding with enhanced PI3K/Akt signaling. In pten −/− fibroblasts, which resemble activated fibroblasts, inhibition of Akt attenuated FN production and decreased EDA alternative splicing. Moreover, inhibition of mammalian target of rapamycin (mTOR) in pten −/− cells also blocked FN production and EDA splicing. This effect was due to inhibition of Akt-mediated phosphorylation of the primary EDA splicing regulatory protein SF2/ASF. Importantly, FN silencing in pten −/− cells resulted in attenuated proliferation and migration. Thus, our results demonstrate that the PI3K/Akt/mTOR axis is instrumental in FN transcription and alternative splicing, which regulates cell behavior. [ABSTRACT FROM AUTHOR]

Although the lipid mediator prostaglandin E2 (PGE2) exerts antifibrotic effects by inhibiting multiple fibroblast functions, its ability to regulate fibroblast survival is unknown. Here, we examined the effects of this prostanoid on apoptosis and apoptosis pathways in normal and fibrotic lung fibroblasts. As compared to medium alone, 24 h of treatment with PGE2 increased apoptosis of normal lung fibroblasts in a dose-dependent manner (EC50 ~ 50 nM), as measured by annexin V staining, caspase 3 activity, cleavage of poly-ADP-ribose polymerase, and single-stranded DNA levels. PGE also potentiated apoptosis elicited by Fas ligand plus cycloheximide. These proapoptotic actions were dependent on signaling through the EP2/EP4 receptors and by downstream activation of both caspases 8 and 9. Silencing and gene deletion of PTEN demonstrated that the effects of PGE involved decreased activity of the prosurvival molecule Akt. PGE2 also down-regulated expression of survivin, an inhibitor of apoptosis, and increased expression of Fas. Fibroblasts from patients with pulmonary fibrosis exhibited resistance to the apoptotic effects of PGE2. These findings show for the first time that, in contrast to its effects on many other cell types, PGE promotes apoptosis in lung fibroblasts through diverse pathways. They provide another dimension by which PGE2 may inhibit, and perhaps even reverse, fibrogenesis in patients with interstitial lung disease. [ABSTRACT FROM AUTHOR]

Wegenerʼs granulomatosis (WG) is the most common pulmonary granulomatous vasculitis and was a uniformly fatal disease prior to the identification of efficacious pharmacological regimens. The pathogenesis of WG remains elusive but proteinase 3-specific anti-neutrophil cytoplasmic antibodies may be involved. Histologically, WG is defined by the triad of small vessel necrotising vasculitis, ‘geographic’ necrosis and granulomatous inflammation. Organ involvement characteristically includes the upper and lower respiratory tracts and kidney, but virtually any organ can be involved. The severity of the disease varies, ranging from asymptomatic disease to fulminant, fatal vasculitis. Similarly, the degree of organ involvement is highly variable; WG may be limited to a single organ (typically the lungs or upper respiratory tract), or may be systemic. Currently, a regimen consisting of daily cyclophosphamide and corticosteroids, which induces complete remission in the majority of patients, is considered standard therapy. Since approximately 50% of patients experience a relapse following discontinuation of therapy, alternative regimens designed to maintain remissions after using cyclophosphamide and corticosteroids are usually necessary. This ‘induction maintenance’ approach to treatment has emerged as a central premise in planning therapy for patients with WG.A number of trials have evaluated the efficacy of less toxic immunosuppressants (e.g. methotrexate, azathioprine, mycophenolate mofetil) and antibacterials (i.e. cotrimoxazole [trimethoprim/sulfamethoxazole]) for treating patients with WG, resulting in the identification of effective alternative regimens to induce or maintain remissions in certain sub-populations of patients. Given the efficacy of methotrexate (for early systemic WG) and cotrimoxazole (in WG limited solely to the upper airways) to induce remissions, and the relatively decreased associated morbidity compared with cyclophosphamide, these alternative regimens are preferred in appropriate patients. Similarly, therapeutic options to maintain disease remission that are less toxic than cyclophosphamide should be offered following induction of remission unless a specific contraindication exists. By following this premise, the development of cyclophosphamide-induced morbidities (e.g. haemorrhagic cystitis, uroepithelial cancers and prolonged myelosuppression) may be minimised. Recent investigation has focussed on other immunomodulatory agents (tumour necrosis factor-α inhibitors [infliximab and etanercept] and anti-CD20 antibodies [rituximab]) for treating patients with WG. However, the current data are conflicting and difficult to interpret. As a result, these newer agents cannot be recommended for routine use until vigorous clinical study confirms their efficacy. [ABSTRACT FROM AUTHOR]

Tissue injury evokes highly conserved, tightly regulated inflammatory responses and less well-understood host repair responses. Both inflammation and repair involve the recruitment, activation, apoptosis, and eventual clearance of key effector cells. In this review, we propose the concept of pulmonary fibrosis as a dysregulated repair process that is perpetually "turned on" even though classical inflammatory pathways may be dampened or "switched off". Significant regional heterogeneity, with varied histopathological patterns of inflammation and fibrosis, has been observed in individual patients with idiopathic pulmonary fibrosis. We discuss environmental factors and host response factors, such as genetic susceptibility and age, that may influence these varied manifestations. Better understanding of the mechanisms of lung repair, which include alveolar reepithelialization, myofibroblast differentiation/activation, and apoptosis, should offer more effective therapeutic options for progressive pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

Reports on the case of a man with diplopia and pain over the left eye. Evidence of paralysis with hypalgesia in the right lumbar nerve root; Diagnosis of sarcoidosis; Administration of oral corticosteroids.

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial lung disease characterised by decline in lung function. We evaluated trajectories of forced vital capacity (FVC) and diffusing capacity (DLco) in a cohort of patients with IPF. Methods: Patients with IPF that was diagnosed or confirmed at the enrolling centre in the previous 6 months were enrolled into the IPF-PRO Registry between June 2014 and October 2018. Patients were followed prospectively, with lung function data collected as part of routine clinical care. Mean trajectories of FVC and DLco % predicted in all patients and in subgroups by characteristics assessed at enrolment were estimated using a joint model that accounted for factors such as disease severity and visit patterns. Results: Of 1002 patients in the registry, 941 had ≥ 1 FVC and/or DLco measurement after enrolment. The median (Q1, Q3) follow-up period was 35.1 (18.9, 47.2) months. Overall, mean estimated declines in FVC and DLco % predicted were 2.8% and 2.9% per year, respectively. There was no evidence that the mean trajectories of FVC or DLco had a non-linear relationship with time at the population level. Patients who were male, white, had a family history of ILD, were using oxygen, or had prior/current use of antifibrotic therapy at enrolment had greater rates of decline in FVC % predicted. Patients who were male or white had greater rates of decline in DLco % predicted. Conclusions: Data from the IPF-PRO Registry suggest a constant rate of decline in lung function over a prolonged period, supporting the inexorably progressive nature of IPF. A graphical abstract summarising the data in this manuscript is available at: https://www.usscicomms.com/respiratory/IPF-PRORegistry%5fLungFunctionTrajectories. Trial registration: NCT01915511. [ABSTRACT FROM AUTHOR]

Differentiation of lung fibroblasts into contractile protein-expressing myofibroblasts by transforming growth factor-β1 (TGF-β1) is a critical event in the pathogenesis of pulmonary fibrosis. Transcription of the contractile protein α-smooth muscle actin (α-SMA) is mediated by the transcription factor serum-response factor (SRF) along with its co-activator, myocardin-related transcription factor-A (MRTF-A). The endogenous lipid mediator prostaglandin E2 (PGE2) exerts anti-fibrotic effects, including the inhibition of myofibroblast differentiation. However, the mechanism by which PGE2 inhibits α-SMA expression is incompletely understood. Here, we show in normal lung fibroblasts that PGE2 reduced the nuclear accumulation of MRTF-A • SRF complexes and consequently inhibited α-SMA promoter activation. It did so both by independently inhibiting SRF gene expression and nuclear import of MRTF-A. We identified that p38 MAPK is critical for TGF-β1-induced SRF gene expression and that PGE2 inhibition of SRF expression is associated with its ability to inhibit p38 activation. Its inhibition of MRTF-A import occurs via activation of cofilin 1 and inactivation of vasodilator-stimulated phosphoprotein. Similar effects of PGE2 on SRF gene expression were observed in fibroblasts from the lungs of patients with idiopathic pulmonary fibrosis. Thus,PGE2 is the first substance described to prevent myofibroblast differentiation by disrupting, via distinct mechanisms, the actions of both SRF and MRTF-A. [ABSTRACT FROM AUTHOR]

Background: Interstitial lung abnormalities (ILA) may represent undiagnosed early-stage or subclinical interstitial lung disease (ILD). ILA are often observed incidentally in patients who subsequently develop clinically overt ILD. There is limited information on consensus definitions for, and the appropriate evaluation of, ILA. Early recognition of patients with ILD remains challenging, yet critically important. Expert consensus could inform early recognition and referral.Research Question: Can consensus-based expert recommendations be identified to guide clinicians in the recognition, referral, and follow-up of patients with or at risk of developing early ILDs?Study Design and Methods: Pulmonologists and radiologists with expertise in ILD participated in two iterative rounds of surveys. The surveys aimed to establish consensus regarding ILA reporting, identification of patients with ILA, and identification of populations that might benefit from screening for ILD. Recommended referral criteria and follow-up processes were also addressed. Threshold for consensus was defined a priori as ≥ 75% agreement or disagreement.Results: Fifty-five experts were invited and 44 participated; consensus was reached on 39 of 85 questions. The following clinically important statements achieved consensus: honeycombing and traction bronchiectasis or bronchiolectasis indicate potentially progressive ILD; honeycombing detected during lung cancer screening should be reported as potentially significant (eg, with the Lung CT Screening Reporting and Data System "S-modifier" [Lung-RADS; which indicates clinically significant or potentially significant noncancer findings]), recommending referral to a pulmonologist in the radiology report; high-resolution CT imaging and full pulmonary function tests should be ordered if nondependent subpleural reticulation, traction bronchiectasis, honeycombing, centrilobular ground-glass nodules, or patchy ground-glass opacity are observed on CT imaging; patients with honeycombing or traction bronchiectasis should be referred to a pulmonologist irrespective of diffusion capacity values; and patients with systemic sclerosis should be screened with pulmonary function tests for early-stage ILD.Interpretation: Guidance was established for identifying clinically relevant ILA, subsequent referral, and follow-up. These results lay the foundation for developing practical guidance on managing patients with ILA. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is a disease of progressive scarring caused by excessive extracellular matrix (ECM) deposition and activation of α‐SMA‐expressing myofibroblasts. Human antigen R (HuR) is an RNA binding protein that promotes protein translation. Upon translocation from the nucleus to the cytoplasm, HuR functions to stabilize messenger RNA (mRNA) to increase protein levels. However, the role of HuR in promoting ECM production, myofibroblast differentiation, and lung fibrosis is unknown. Human lung fibroblasts (HLFs) treated with transforming growth factor β1 (TGF‐β1) showed a significant increase in translocation of HuR from the nucleus to the cytoplasm. TGF‐β‐treated HLFs that were transfected with HuR small interfering RNA had a significant reduction in α‐SMA protein as well as the ECM proteins COL1A1, COL3A, and FN1. HuR was also bound to mRNA for ACTA2, COL1A1, COL3A1, and FN. HuR knockdown affected the mRNA stability of ACTA2 but not that of the ECM genes COL1A1, COL3A1, or FN. In mouse models of pulmonary fibrosis, there was higher cytoplasmic HuR in lung structural cells compared to control mice. In human IPF lungs, there was also more cytoplasmic HuR. This study is the first to show that HuR in lung fibroblasts controls their differentiation to myofibroblasts and consequent ECM production. Further research on HuR could assist in establishing the basis for the development of new target therapy for fibrotic diseases, such as IPF. [ABSTRACT FROM AUTHOR]

Background: Hospitalizations are common among patients with idiopathic pulmonary fibrosis (IPF). We investigated the impact of hospitalizations on outcomes in patients with IPF.Methods: The IPF-PRO Registry is an observational US registry that enrolled patients with IPF that was diagnosed or confirmed at the enrolling center in the previous 6 months. Associations between patient characteristics and hospitalization, and between hospitalization and mortality, were analyzed using Cox regression models.Results: A total of 1002 patients with IPF were enrolled into the IPF-PRO Registry. Over a median follow-up time of 23.7 months (maximum: 67.0 months), 568 patients (56.7%) had at least one hospitalization. Of these patients, 319 (56.2%) had at least one respiratory-related hospitalization and 120 (21.1%) had at least one hospitalization with ventilatory support. Younger age (HR 0.68 [95% CI 0.55, 0.84] per 5-year increase for patients < 62 years), lower BMI (0.96 [0.93, 0.98] per 1-point increase), lower FVC % predicted (0.90 [0.83, 0.97] per 10% increase), oxygen use at rest (2.85 [2.18, 3.72]) and history of pulmonary hypertension (2.02 [1.37, 2.96]) at enrollment were associated with an increased risk of respiratory-related hospitalization during follow-up. In a multivariable model, there was an eightfold increase in the risk of mortality during hospitalization or within 90 days of discharge compared with outside of this period. The risk of mortality associated with a respiratory hospitalization or a hospitalization with ventilatory support was even greater.Conclusions: Data from the IPF-PRO Registry demonstrate that hospitalizations are common among patients with IPF. The risk of mortality during hospitalization or within 90 days of discharge was high, particularly among patients who were hospitalized for a respiratory cause or received ventilatory support. Trial registration ClinicalTrials.gov, NCT01915511. Registered 5 August 2013, https://clinicaltrials.gov/ct2/show/NCT01915511. [ABSTRACT FROM AUTHOR]

Fibroblast differentiation is an essential step during wound healing and fibrosis. Fibronectin (FN) is a major component of the extracellular matrix and occurs in two main forms: plasma and cellular FN. The latter includes the alternatively spliced domain A (EDA). Although EDA-containing cellular fibronectin (EDA-FN) is associated with fibroblast differentiation, how EDA-FN promotes differentiation is incompletely understood. In this study, we investigate the mechanism by which EDA-FN contributes to fibroblast differentiation with emphasis on the characterization of the EDA-FN receptor. We show that EDA-FN increases α-SMA expression (immunofluorescence), collagen deposition, cell contractility, and focal adhesion kinase (FAK) activation (immunoblotting); whereas plasma FN, a form lacking EDA, shows no effect. Primary lung fibroblasts constitutively express α4β7 integrin receptor (FACS and RT-PCR). Blocking of α4β7 reduces fibroblast adhesion to EDA-FN and inhibits α-SMA expression, collagen deposition, and FAK activation induced by EDA-FN. Using recombinant EDA-containing peptides, we demonstrate that the EDA segment is sufficient to induce fibroblast differentiation via binding to α4β7. EDA-FN induces MAPK-Erkl/2 activation and inhibition of MEK1/2 attenuates EDA-FN-induced α-SMA expression. Our findings demonstrate that EDA-FN induces fibroblast differentiation by a mechanism that involves binding of EDA to α4β7 integrin followed by activation of FAK and MAPK-associated signaling pathways. [ABSTRACT FROM AUTHOR]

Myofibroblasts are pathogenic in pulmonary fibrotic disease due to their exuberant production of matrix rich iii collagen that interferes with gas exchange and the ability of these cells to contract and distort the alveolar space. Transforming growth factor-β1 (TGF-β1) is a well-known inducer of myofibroblast differentiation. TGF-β1-induced transformation of fibroblasts to apoptosis-resistant myofibroblasts is adhesion-dependent and focal adhesion kinase (FAK)-mediated. Prostaglandin E2 (PGE2) inhibits this differentiation via E prostanoid receptor 2 (EP2) signaling and cAMP elevation, but whether PGE2 does so by interfering with TGF-β1 signaling is unknown. Thus we examined the effects of PGE2 in the presence and absence of TGF-β1 stimulation on candidate signaling pathways in human lung fibroblasts. We now demonstrate that PGE2 does not interfere with TGF-β1-induced Smad phosphorylation or its translocation to the nucleus. Rather, PGE2 has dramatic effects on cell shape and cytoskeletal architecture and disrupts the formation of appropriate focal adhesions. PGE2 treatment diminishes TGF-I β1-induced phosphorylation of paxillin, STAT-3, and FAK and, in turn, limits activation of the protein kinase B (PKB/Akt) pathway. These alterations do not, however, result in increased apoptosis within the first 24 h of treatment. Interestingly, the effects of PGE2 stimulation alone do not always mirror the effects of PGE2 in the presence of TGF-β1, indicating that the context for EP2 signaling is different in the presence of TGF-β1. Taken together, our results demonstrate that PGE2 has the potential to limit TGF-β1-induced myofibroblast differentiation via adhesion-dependent, but Smad-independent, pathways. [ABSTRACT FROM AUTHOR]

Reports that the myofibroblast differentiation by transforming growth factor (TGF)-beta1 is dependent on cell adhesion and integrin signaling via focal adhesion kinase (FAK). Pharmacologic inhibition of FAK; Elevation of basal expression of alpha-smooth muscle actin in cells growth on fibronectin-coated dishes; Safer and effective therapeutic strategy for fibrotic diseases.

Idiopathic pulmonary fibrosis (IPF) is a progressive and heterogeneous interstitial lung disease of unknown origin with a low survival rate. There are few treatment options available due to the fact that mechanisms underlying disease progression are not well understood, likely because they arise from dysregulation of complex signaling networks spanning multiple tissue compartments. To better characterize these networks, we used systems-focused data-driven modeling approaches to identify cross-tissue compartment (blood and bronchoalveolar lavage) and temporal proteomic signatures that differentiated IPF progressors and non-progressors. Partial least squares discriminant analysis identified a signature of 54 baseline (week 0) blood and lung proteins that differentiated IPF progression status by the end of 80 weeks of follow-up with 100% cross-validation accuracy. Overall we observed heterogeneous protein expression patterns in progressors compared to more homogenous signatures in non-progressors, and found that non-progressors were enriched for proteomic processes involving regulation of the immune/defense response. We also identified a temporal signature of blood proteins that was significantly different at early and late progressor time points (p < 0.0001), but not present in non-progressors. Overall, this approach can be used to generate new hypothesis for mechanisms associated with IPF progression and could readily be translated to other complex and heterogeneous diseases. [ABSTRACT FROM AUTHOR]

Objective: Systemic sclerosis (SSc) is associated with interstitial lung disease (ILD) and pulmonary hypertension (PH). This study was undertaken to determine the prevalence, characteristics, treatment, and outcomes of PH in a cohort of patients with SSc‐associated ILD. Methods: Patients with SSc‐associated ILD on high‐resolution computed tomography (HRCT) were included in a prospective observational cohort. Patients were screened for PH based on a standardized screening algorithm and underwent right‐sided heart catheterization (RHC) if indicated. PH classification was based on hemodynamic findings and the extent of ILD on HRCT. Summary statistics and survival using the Kaplan‐Meier method were calculated. Results: Of the 93 patients with SSc‐associated ILD included in the study, 76% were women and 65.6% had diffuse cutaneous SSc. The mean age was 54.9 years, and the mean SSc disease duration was 8 years. Twenty‐nine patients (31.2%) had RHC‐proven PH; of those 29 patients, 24.1% had PAH, 55.2% had World Health Organization (WHO) Group III PH, 34.5% had WHO Group III PH with pulmonary vascular resistance >3.0 Wood units, 48.3% had a PH diagnosis within 7 years of SSc onset, 82.8% received therapy for ILD, and 82.8% received therapy for PAH. The survival rate 3 years after SSc‐associated ILD diagnosis for all patients was 97%. The survival rate 3 years after PH diagnosis for those with SSc‐associated ILD and PH was 91%. Conclusion: In a large cohort of patients with SSc‐associated ILD, a significant proportion of patients had coexisting PH, which often occurs early after SSc diagnosis. Most patients were treated with ILD and PAH therapies, and survival was good. Patients with SSc‐associated ILD should be evaluated for coexisting PH. [ABSTRACT FROM AUTHOR]

Background: Hypersensitivity pneumonitis (HP) is an interstitial lung disease with a better prognosis, on average, than idiopathic pulmonary fibrosis (IPF). We compare survival time and pulmonary function trajectory in patients with HP and IPF by radiologic phenotype.Methods: HP (n = 117) was diagnosed if surgical/transbronchial lung biopsy, BAL, and exposure history results suggested this diagnosis. IPF (n = 152) was clinically and histopathologically diagnosed. All participants had a baseline high-resolution CT (HRCT) scan and FVC % predicted. Three thoracic radiologists documented radiologic features. Survival time is from HRCT scan to death or lung transplant. Cox proportional hazards models identify variables associated with survival time. Linear mixed models compare post-HRCT scan FVC % predicted trajectories.Results: Subjects were grouped by clinical diagnosis and three mutually exclusive radiologic phenotypes: honeycomb present, non-honeycomb fibrosis (traction bronchiectasis and reticulation) present, and nonfibrotic. Nonfibrotic HP had the longest event-free median survival (> 14.73 years) and improving FVC % predicted (1.92%; 95% CI, 0.49-3.35; P = .009). HP with non-honeycomb fibrosis had longer survival than IPF (> 7.95 vs 5.20 years), and both groups experienced a significant decline in FVC % predicted. Subjects with HP and IPF with honeycombing had poor survival (2.76 and 2.81 years, respectively) and significant decline in FVC % predicted.Conclusions: Three prognostically distinct, radiologically defined phenotypes are identified among patients with HP. The importance of pursuing a specific diagnosis (eg, HP vs IPF) among patients with non-honeycomb fibrosis is highlighted. When radiologic honeycombing is present, invasive diagnostic testing directed at determining the diagnosis may be of limited value given a uniformly poor prognosis. [ABSTRACT FROM AUTHOR]

While the transcription factor forkhead box M1 (FOXM1) is well known as a proto-oncogene, its potential role in lung fibroblast activation has never been explored. Here, we show that FOXM1 is more highly expressed in fibrotic than in normal lung fibroblasts in humans and mice. FOXM1 was required not only for cell proliferation in response to mitogens, but also for myofibroblast differentiation and apoptosis resistance elicited by TGF-β. The lipid mediator PGE2, acting via cAMP signaling, was identified as an endogenous negative regulator of FOXM1. Finally, genetic deletion of FOXM1 in fibroblasts or administration of the FOXM1 inhibitor Siomycin A in a therapeutic protocol attenuated bleomycin-induced pulmonary fibrosis. Our results identify FOXM1 as a driver of lung fibroblast activation and underscore the therapeutic potential of targeting FOXM1 for pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

Dysregulated secretion and extracellular activation of TGF-ß1 stimulates myofibroblasts to accumulate disordered and stiff extracellular matrix (ECM) leading to fibrosis. Fibronectin immobilizes latent TGF-ß-binding protein-1 (LTBP-1) and thus stores TGF-ß1 in the ECM. Because the ED-A fibronectin splice variant is prominently expressed during fibrosis and supports myofibroblast activation, we investigated whether ED-A promotes LTBP-1-fibronectin interactions. Using stiffness-tuneable substrates for human dermal fibroblast cultures, we showed that high ECM stiffness promotes expression and colocalization of LTBP-1 and EDA-containing fibronectin. When rescuing fibronectin-depleted fibroblasts with specific fibronectin splice variants, LTBP-1 bound more efficiently to ED-A-containing fibronectin than to ED-Bcontaining fibronectin and fibronectin lacking splice domains. Function blocking of the ED-A domain using antibodies and competitive peptides resulted in reduced LTBP-1 binding to ED-Acontaining fibronectin, reduced LTBP-1 incorporation into the fibroblast ECM and reduced TGF-ß1 activation. Similar results were obtained by blocking the heparin-binding stretch FNIII12-13-14 (HepII), adjacent to the ED-A domain in fibronectin. Collectively, our results suggest that the ED-A domain enhances association of the latent TGF-ß1 by promoting weak direct binding to LTBP-1 and by enhancing heparin-mediated protein interactions through HepII in fibronectin. [ABSTRACT FROM AUTHOR]

Background: Surgical lung biopsy (SLB) is invasive and not possible in all patients with undiagnosed interstitial lung disease (ILD). We hypothesized that transbronchial biopsy (TBB) findings combined with clinical and high-resolution CT (HRCT) data leads to a confident diagnosis congruent to SLB and therefore avoids the need for SLB in some patients.Methods: We evaluated 33 patients being investigated for suspected ILD who underwent HRCT, TBB, and SLB. First, clinicians, radiologists, and a pathologist reviewed the clinical information and HRCT and TBB findings. Clinicians were asked to provide a diagnosis and were also asked if SLB was needed for a more confident diagnosis. Subsequently, the clinical, HRCT, and SLB data were reviewed, and the same participants were asked to provide a final diagnosis. Clinician consensus and overall agreement between TBB- and SLB-based diagnoses were calculated.Results: Four patients had definite usual interstitial pneumonia (UIP) on HRCT and would not be considered for biopsy using current guidelines. Of the 29 patients without a definitive HRCT diagnosis, the clinicians felt confident of the diagnosis (ie, would not recommend SLB) in six cases. In these cases, there was 100% agreement between TBB and SLB diagnoses. UIP was the most common diagnosis (n = 3) and was associated with an HRCT diagnosis of possible UIP/nonspecific interstitial pneumonia-like. Agreement was poor (33%) between TBB and SLB diagnoses when confidence in the TBB diagnosis was low.Conclusions: Information from TBB, when combined with clinical and HRCT data, may provide enough information to make a confident and accurate diagnosis in approximately 20% to 30% of patients with ILD. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cell (hPSC) derived tissues often remain developmentally immature in vitro, and become more adult-like in their structure, cellular diversity and function following transplantation into immunocompromised mice. Previously we have demonstrated that hPSC-derived human lung organoids (HLOs) resembled human fetal lung tissue in vitro (Dye et al., 2015). Here we show that HLOs required a bioartificial microporous poly(lactide-co-glycolide) (PLG) scaffold niche for successful engraftment, long-term survival, and maturation of lung epithelium in vivo. Analysis of scaffold-grown transplanted tissue showed airway-like tissue with enhanced epithelial structure and organization compared to HLOs grown in vitro. By further comparing in vitro and in vivo grown HLOs with fetal and adult human lung tissue, we found that in vivo transplanted HLOs had improved cellular differentiation of secretory lineages that is reflective of differences between fetal and adult tissue, resulting in airway-like structures that were remarkably similar to the native adult human lung. [ABSTRACT FROM AUTHOR]

Rationale: Biomarkers in easily accessible compartments like peripheral blood that can predict disease progression in idiopathic pulmonary fibrosis (IPF) would be clinically useful regarding clinical trial participation or treatment decisions for patients. In this study, we used unbiased proteomics to identify relevant disease progression biomarkers in IPF. Methods: Plasma from IPF patients was measured using an 1129 analyte slow off-rate modified aptamer (SOMAmer) array, and patient outcomes were followed over the next 80 weeks. Receiver operating characteristic (ROC) curves evaluated sensitivity and specificity for levels of each biomarker and estimated area under the curve (AUC) when prognostic biomarker thresholds were used to predict disease progression. Both logistic and Cox regression models advised biomarker selection for a composite disease progression index; index biomarkers were weighted via expected progression-free days lost during follow-up with a biomarker on the unfavorable side of the threshold. Results: A six-analyte index, scaled 0 to 11, composed of markers of immune function, proteolysis and angiogenesis [high levels of ficolin-2 (FCN2), cathepsin-S (Cath-S), legumain (LGMN) and soluble vascular endothelial growth factor receptor 2 (VEGFsR2), but low levels of inducible T cell costimulator (ICOS) or trypsin 3 (TRY3)] predicted better progression-free survival in IPF with a ROC AUC of 0.91. An index score ≥ 3 (group ≥ 2) was strongly associated with IPF progression after adjustment for age, gender, smoking status, immunomodulation, forced vital capacity % predicted and diffusing capacity for carbon monoxide % predicted (HR 16.8, 95% CI 2.2–126.7, P = 0.006). Conclusion: This index, derived from the largest proteomic analysis of IPF plasma samples to date, could be useful for clinical decision making in IPF, and the identified analytes suggest biological processes that may promote disease progression. [ABSTRACT FROM AUTHOR]

Objective Fibrocytes are collagen-producing leukocytes that accumulate in patients with systemic sclerosis (SSc; scleroderma)-related interstitial lung disease (ILD) via unknown mechanisms that have been associated with altered expression of neuroimmune proteins. The extracellular matrix (ECM) influences cellular phenotypes. However, a relationship between the lung ECM and fibrocytes in SSc has not been explored. The aim of this study was to use a novel translational platform based on decellularized human lungs to determine whether the lung ECM of patients with scleroderma controls the development of fibrocytes from peripheral blood mononuclear cells. Methods We performed biomechanical evaluation of decellularized scaffolds prepared from lung explants from healthy control subjects and patients with scleroderma, using tensile testing and biochemical and proteomic analysis. Cells obtained from healthy controls and patients with SSc-related ILD were cultured on these scaffolds, and CD45+pro-ColIα1+ cells meeting the criteria for fibrocytes were quantified. The contribution of the neuromolecule netrin-1 to fibrosis was assessed using neutralizing antibodies in this system and by administering bleomycin via inhalation to netrin-1+/− mice. Results Compared with control lung scaffolds, lung scaffolds from patients with SSc-related ILD showed aberrant anatomy, enhanced stiffness, and abnormal ECM composition. Culture of control cells in lung scaffolds from patients with SSc-related ILD increased production of pro-ColIα1+ cells, which was stimulated by enhanced stiffness and abnormal ECM composition. Cells from patients with SSc-related ILD demonstrated increased pro-ColIα1 responsiveness to lung scaffolds from scleroderma patients but not enhanced stiffness. Enhanced detection of netrin-1-expressing CD14low cells in patients with SSc-related ILD was observed, and antibody-mediated netrin-1 neutralization attenuated detection of CD45+pro-ColIα1+ cells in all settings. Netrin-1+/− mice were protected against bleomycin-induced lung fibrosis and fibrocyte accumulation. Conclusion Factors present in the lung matrices of patients with scleroderma regulate fibrocyte accumulation via a netrin-1-dependent pathway. Netrin-1 regulates bleomycin-induced pulmonary fibrosis in mice. Netrin-1 might be a novel therapeutic target in SSc-related ILD. [ABSTRACT FROM AUTHOR]

Age-associated decline in organ function governs life span. We determined the effect of aging on lung function and cellular/molecular changes of 8- to 32-month old mice. Proteomic analysis of lung matrix indicated significant compositional changes with advanced age consistent with a profibrotic environment that leads to a significant increase in dynamic compliance and airway resistance. The excess of matrix proteins deposition was associated modestly with the activation of myofibroblasts and transforming growth factor-beta signaling pathway. More importantly, detection of senescent cells in the lungs increased with age and these cells contributed toward the excess extracellular matrix deposition observed in our aged mouse model and in elderly human samples. Mechanistic target of rapamycin (mTOR)/AKT activity was enhanced in aged mouse lungs compared with those from younger mice associated with the increased expression of the histone variant protein, MH2A, a marker for aging and potentially for senescence. Introduction in the mouse diet of rapamycin, significantly blocked the mTOR activity and limited the activation of myofibroblasts but did not result in a reduction in lung collagen deposition unless it was associated with prevention of cellular senescence. Together these data indicate that cellular senescence significantly contributes to the extracellular matrix changes associated with aging in a mTOR 1-dependent mechanism. [ABSTRACT FROM AUTHOR]

Collagen-producing myofibroblast transdifferentiation is considered a crucial determinant in the formation of scar tissue in the lungs of patients with idiopathic pulmonary fibrosis. Multiple resident pulmonary cell types and bone marrow-derived fibrocytes have been implicated as contributors to fibrotic lesions because of the transdifferentiation potential of these cells into myofibroblasts. In this study, we assessed the expression of Wilms tumor 1 (WT1), a known marker of mesothelial cells, in various cell types in normal and fibrotic lungs. We demonstrate that WT1 is expressed by both mesothelial and mesenchymal cells in idiopathicpulmonary fibrosis lungs but has limited or no expression in normal human lungs. We also demonstrate that WT1+ cells accumulate in fihrotic lung lesions, using two different mouse models of pulmonary fibrosis and WT1 promoter-driven fluorescent reporter mice. Reconstitution of bone marrow cells into a TGF-α transgenic mouse model demonstrated that fibrocytes do not transform into WT1+ mesenchymal cells, but they do augment accumulation of WT1+ cells in severe fibrotic lung disease. Importantly, the number of WT1+ cells in fibrotic lesions was correlated with severity of lung disease as assessed by changes in lung function, histology, and hydroxyproline levels in mice. Finally, inhibition of WT1 expression was sufficient to attenuate collagen and other extracellular matrix gene production by mesenchymal cells from both murine and human fibrotic lungs. Thus, the results of this study demonstrate a novel association between fibrocyte-driven WT1+ cell accumulation and severe fibrotic lung disease. [ABSTRACT FROM AUTHOR]

Progressive fibrosis is characterized by excessive deposition of extracellular matrix (ECM), resulting in gross alterations in tissue mechanics. Changes in tissue mechanics can further augment scar deposition through fibroblast mechanotransduction. In idiopathic pulmonary fibrosis, a fatal form of progressive lung fibrosis, previous work has shown that loss of Thy-1 (CD90) expression in fibroblasts correlates with regions of active fibrogenesis, thus representing a pathologically relevant fibroblast subpopulation. We now show that Thy-1 is a regulator of fibroblast rigidity sensing. Thy-1 physically couples to inactive OvP3 integrins via its RGD-like motif, altering baseline integrin avidity to ECM ligands and also facilitating preadhesion clustering of integrin and membrane rafts via Thy-1 's glycophosphatidylinositol tether. Disruption of Thy-1-avp3 coupling altered recruitment of Src family kinases to adhesion complexes and impaired mechanosensitive, force-induced Rho signaling, and rigidity sensing. Loss of Thy-1 was sufficient to induce myofibroblast differentiation in soft ECMs and may represent a physiological mechanism important in wound healing and fibrosis. [ABSTRACT FROM AUTHOR]

The article presents a model of in vitro generation of human lung organoids (HOLs) derived from human pluripotent stem cells (hPSCs), using RNA sequencing. Topics include development of manipulating developmental signaling pathways by hPSCs, and epithelial and mesenchymal compartments of the lung. The study infers that HLOs are a reliable model for the study of the development, disease and maturation of human lung.

Idiopathic pneumonia syndrome (IPS) is an acute, noninfectious lung disorder associated with high morbidity and mortality after hematopoietic cell transplantation. Previous studies have suggested a role for TNFα in the pathogenesis of IPS. We report a multicenter phase II trial investigating a soluble TNF-binding protein, etanercept (Enbrel, Amgen, Thousand Oaks, CA), for the treatment of pediatric patients with IPS. Eligible patients were < 18 years old, within 120 days after transplantation, and with radiographic evidence of a diffuse pneumonitis. All patients underwent a pretherapy broncho-alveolor lavage (BAL) to establish the diagnosis of IPS. Systemic corticosteroids (2.0 mg/kg/day) plus etanercept (.4 mg/kg twice weekly × 8 doses) were administered. Response was defined as survival and discontinuation of supplemental oxygen support by day 28 of study. Thirty-nine patients (median age, 11 years; range, 1 to 17) were enrolled, with 11 of 39 patients nonevaluable because of identification of pathogens from their pretherapy BAL. In the remaining 28 patients, the median fraction of inspired oxygen at study entry was 45%, with 17 of 28 requiring mechanical ventilation. Complete responses were seen in 20 (71%) patients, with a median time to response of 10 days (range, 1 to 24). Response rates were higher for patients not requiring mechanical ventilation at study entry (100% versus 53%, P = .01). Overall survival at 28 days and 1 year after therapy were 89% (95% confidence interval [CI], 70% to 96%) and 63% (95% CI, 42% to 79%), respectively. Plasma levels of proinflammatory cytokines were significantly increased at onset of therapy, subsequently decreasing in responding patients. The addition of etanercept to high-dose corticosteroids was associated with high response rates and survival in children with IPS. [ABSTRACT FROM AUTHOR]

Excessive fibroproliferation is a central hallmark of idiopathic pulmonary fibrosis (IPF), a chronic, progressive disorder that results in impaired gas exchange and respiratory failure. Fibroblasts are the key effector cells in IPF, and aberrant expression of multiple genes contributes to their excessive fibroproliferative phenotype. DNA methylation changes are critical to the development of many diseases, but the DNA methylome of IPF fibroblasts has never been characterized. Here, we utilized the HumanMethylation 27 array, which assays the DNA methylation level of 27,568 CpG sites across the genome, to compare the DNA methylation patterns of IPF fibroblasts (n = 6) with those of nonfibrotic patient controls (n = 3) and commercially available normal lung fibroblast cell lines (n = 3). We found that multiple CpG sites across the genome are differentially methylated (as defined by P value less than 0.05 and fold change greater than 2) in IPF fibroblasts compared to fibroblasts from nonfibrotic controls. These methylation differences occurred both in genes recognized to be important in fibroproliferation and extracellular matrix generation, as well as in genes not previously recognized to participate in those processes (including organ morphogenesis and potassium ion channels). We used bisulfite sequencing to independently verify DNA methylation differences in 3 genes (CDKN2B, CARD10, and MGMT); these methylation changes corresponded with differences in gene expression at the mRNA and protein level. These differences in DNA methylation were stable throughout multiple cell passages. DNA methylation differences may thus help to explain a proportion of the differences in gene expression previously observed in studies of IPF fibroblasts. Moreover, significant variability in DNA methylation was observed among individual IPF cell lines, suggesting that differences in DNA methylation may contribute to fibroblast heterogeneity among patients with IPF. These results demonstrate that IPF fibroblasts exhibit global differences in DNA methylation that may contribute to the excessive fibroproliferation associated with this disease. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is the most common and fatal form of idiopathic interstitial pneumonia. MicroRNAs (miRNAs), short, single-stranded RNAs that regulate protein expression in a post-transcriptional manner, have recently been demonstrated to contribute to IPF pathogenesis. We have previously identified WNT1-inducible signaling pathway protein 1 (WISP1) as a highly expressed pro-fibrotic mediator in IPF, but the underlying mechanisms resulting in increased WISP1 expression, remain elusive. Here, we investigated whether WISP1 is a target of miRNA regulation. We applied a novel supervised machine learning approach, which predicted miR-30a/d and miR-92a target sites in regions of the human WISP1 3′UTR preferentially bound by the miRNA ribonucleoprotein complex. Both miRNAs were decreased in IPF samples, whereas WISP1 protein was increased. We demonstrated further that transforming growth factor (TGF)-β1-induced WISP1 expression in primary lung fibroblasts in vitro and lung homogenates in vivo. Notably, miR-30a and miR-92a reversed TGF-β1-induced WISP1 mRNA expression in lung fibroblasts. Moreover, miR-92a inhibition increased WISP1 protein expression in lung fibroblasts. An inverse relationship for WISP1 and miR-92a was found in a TGF-β1 dependent lung fibrosis model in vivo. Finally, we found significantly increased WISP1 expression in primary IPF fibroblasts, which negatively correlated with miR-92a level ex vivo. Altogether, our findings indicate a regulatory role of miR-92a for WISP1 expression in pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

Abstract: Idiopathic pneumonia syndrome (IPS) is a diffuse, noninfectious lung injury that occurs acutely after allogeneic hematopoietic cell transplantation (HCT). IPS-related mortality has been historically high (>50%) despite treatment with systemic corticosteroids and supportive care measures. We have now examined the role of tumor necrosis factor inhibition in a randomized, double-blind, placebo-controlled trial of corticosteroids with etanercept or placebo. Thirty-four subjects (≥18 years) with IPS after HCT were randomized to receive methylprednisolone (2 mg/kg/day) plus etanercept (0.4 mg/kg twice weekly × 4 weeks; n = 16) or placebo (n = 18). No active infections and a pathogen-negative bronchoscopy were required at study entry. Response (alive, with complete discontinuation of supplemental oxygen support) and overall survival were examined. This study, originally planned to accrue 120 patients, was terminated prematurely due to slow accrual. In the limited number of patients examined, there were no differences in response rates at day 28 of study. Ten of 16 patients (62.5% [95% confidence interval {CI}, 35.4% to 84.8%]) receiving etanercept and 12 of 18 patients (66.7% [95% CI, 41.0% to 86.7%]) receiving placebo met the day 28 response definition (P = 1.00). The median survival was 170 days (95% CI, 11 to 362) with etanercept versus 64 days (95% CI, 26 to 209) with placebo (P = .51). Among responders, the median time to discontinuation of supplemental oxygen was 9 days (etanercept) versus 7 days (placebo). Therapy was well tolerated, with 1 toxicity-related death from infectious pneumonia in the placebo arm. The treatment of IPS with corticosteroids in adult HCT recipients was associated with high early response rates (>60%) compared with historical reports, with poor overall survival. The addition of etanercept did not lead to further increases in response, although the sample size of this truncated trial preclude a definitive conclusion. [Copyright &y& Elsevier]

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive disease of insidious onset, and is responsible for up to 30,000 deaths per year in the U.S. Excessive production of extracellular matrix by myofibroblasts has been shown to be an important pathological feature in IPF. TGF-β1 is expressed in fibrotic lung and promotes fibroblast to myofibroblast differentiation (FMD) as well as matrix deposition. Methods: To identify the mechanism of Arsenic trioxide's (ATO)'s anti-fibrotic effect in vitro, normal human lung fibroblasts (NHLFs) were treated with ATO for 24 hours and were then exposed to TGF-β1 (1 ng/ml) before harvesting at multiple time points. To investigate whether ATO is able to alleviate lung fibrosis in vivo, C57BL/6 mice were administered bleomycin by oropharyngeal aspiration and ATO was injected intraperitoneally daily for 14 days. Quantitative real-time PCR, western blotting, and immunofluorescent staining were used to assess the expression of fibrotic markers such as α-smooth muscle actin (α-SMA) and α-1 type I collagen. Results: Treatment of NHLFs with ATO at very low concentrations (10-20nM) inhibits TGF-β1-induced α-smooth muscle actin (α-SMA) and α-1 type I collagen mRNA and protein expression. ATO also diminishes the TGF-β1-mediated contractile response in NHLFs. ATO's down-regulation of profibrotic molecules is associated with inhibition of Akt, as well as Smad2/Smad3 phosphorylation. TGF-β1-induced H2O2 and NOX-4 mRNA expression are also blocked by ATO. ATO-mediated reduction in Smad3 phosphorylation correlated with a reduction of promyelocytic leukemia (PML) nuclear bodies and PML protein expression. PML-/- mouse embryonic fibroblasts (MEFs) showed decreased fibronectin and PAI-1 expression in response to TGF-β1. Daily intraperitoneal injection of ATO (1 mg/kg) in C57BL/6 mice inhibits bleomycin induced lung α-1 type I collagen mRNA and protein expression. Conclusions: In summary, these data indicate that low concentrations of ATO inhibit TGF-β1-induced fibroblast to myofibroblast differentiation and decreases bleomycin induced pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

Pathological remodeling of the extracellular matrix (ECM) by fibroblasts leads to organ failure. Development of idiopathic pulmonary fibrosis (IPF) is characterized by a progressive fibrotic scarring in the lung that ultimately leads to asphyxiation; however, the cascade of events that promote IPF are not well defined. Here, we examined how the interplay between the ECM and fibroblasts affects both the transcriptome and translatome by culturing primary fibroblasts generated from IPF patient lung tissue or nonfibrotic lung tissue on decellularized lung ECM from either IPF or control patients. Surprisingly, the origin of the ECM had a greater impact on gene expression than did cell origin, and differences in translational control were more prominent than alterations in transcriptional regulation. Strikingly, genes that were translationally activated by IPF-derived ECM were enriched for those encoding ECM proteins detected in IPF tissue. We determined that genes encoding IPF-associated ECM proteins are targets for miR-29, which was downregulated in fibroblasts grown on IPF-derived ECM, and baseline expression of ECM targets could be restored by overexpression of miR-29. Our data support a model in which fibroblasts are activated to pathologically remodel the ECM in IPF via a positive feedback loop between fibroblasts and aberrant ECM. Interrupting this loop may be a strategy for IPF treatment. [ABSTRACT FROM AUTHOR]

The use of induced pluripotent stem cells (iPSCs) has been postulated to be the most effective strategy for developing patient-specific respiratory epithelial cells, which may be valuable for lung-related cell therapy and lung tissue engineering. We generated a relatively homogeneous population of alveolar epithelial type II (AETII) and type I (AETI) cells from human iPSCs that had phenotypic properties similar to those of mature human AETII and AETI cells. We used these cells to explore whether lung tissue can be regenerated in vitro. Consistent with an AETII phenotype, we found that up to 97% of cells were positive for surfactant protein C, 95% for mucin-1, 93% for surfactant protein B, and 89% for the epithelial marker CD54. Additionally, exposing induced AETII to a Wnt/β-catenin inhibitor (IWR-1) changed the iPSC-AETII-like phenotype to a predominantly AETI-like phenotype. We found that of induced AET1 cells, more than 90% were positive for type I markers, T1a, and caveolin-1. Acellular lung matrices were prepared from whole rat or human adult lungs treated with decellularization reagents, followed by seeding these matrices with alveolar cells derived from human iPSCs. Under appropriate culture conditions, these progenitor cells adhered to and proliferated within the 3D lung tissue scaffold and displayed markers of differentiated pulmonary epithelium. [ABSTRACT FROM AUTHOR]

Background: Schistosomiasis affects nearly 40 million women of reproductive age, and is known to elicit a pro-inflammatory signature in the placenta. We have previously shown that antigens from schistosome eggs can elicit pro-inflammatory cytokine production from trophoblast cells specifically; however, the influence of these antigens on other characteristics of trophoblast function, particularly as it pertains to placentation in early gestation, is unknown. We therefore sought to determine the impact of schistosome antigens on key characteristics of first trimester trophoblast cells, including migration and invasion. Methods: First trimester HTR8/SVneo trophoblast cells were co-cultured with plasma from pregnant women with and without schistosomiasis or schistosome soluble egg antigens (SEA) and measured cytokine, cellular migration, and invasion responses. Results: Exposure of HTR8 cells to SEA resulted in a pro-inflammatory, anti-invasive signature, characterized by increased pro-inflammatory cytokines (IL-6, IL-8, MCP-1) and TIMP-1. Additionally, these cells displayed 62% decreased migration and 2.7-fold decreased invasion in vitro after treatment with SEA. These results are supported by increased IL-6 and IL-8 in the culture media of HTR8 cells exposed to plasma from Schistosoma japonica infected pregnant women. Conclusions: Soluble egg antigens found in circulation during schistosome infection increase pro-inflammatory cytokine production and inhibit the mobility and invasive characteristics of the first trimester HTR8/SVneo trophoblast cell line. This is the first study to assess the impact of schistosome soluble egg antigens on the behavior of an extravillous trophoblast model and suggests that schistosomiasis in the pre-pregnancy period may adversely impact placentation and the subsequent health of the mother and newborn. Author Summary: Approximately 40 million women of childbearing age suffer from schistosome infection globally at any given time. Multiple studies in rodent models, as well as a few reports in humans, suggest that schistosome infection results in poor pregnancy outcomes. We have previously shown that antigens released from schistosome eggs result in a pronounced pro-inflammatory response in syncytialized third trimester trophoblasts. Herein, we examine the effect of schistosome egg antigens on a first trimester trophoblast cell line, an accepted model for early placental development. Not only is the pro-inflammatory response recapitulated in this model system, but we also observed a decrease in migration and invasion of trophoblast cells after exposure to these antigens. Both migration and invasion are key aspects in early placental development, and inadequate invasion has been implicated in pregnancy-related diseases such as growth restriction and preeclampsia. This study is the first to examine the impact of schistosome antigens on early placental development, and may have implications for the subsequent health of both the pregnancy and the child. [ABSTRACT FROM AUTHOR]

Background: Schistosomiasis affects nearly 40 million women of reproductive age, and is known to elicit a pro-inflammatory signature in the placenta. We have previously shown that antigens from schistosome eggs can elicit pro-inflammatory cytokine production from trophoblast cells specifically; however, the influence of these antigens on other characteristics of trophoblast function, particularly as it pertains to placentation in early gestation, is unknown. We therefore sought to determine the impact of schistosome antigens on key characteristics of first trimester trophoblast cells, including migration and invasion. Methods: First trimester HTR8/SVneo trophoblast cells were co-cultured with plasma from pregnant women with and without schistosomiasis or schistosome soluble egg antigens (SEA) and measured cytokine, cellular migration, and invasion responses. Results: Exposure of HTR8 cells to SEA resulted in a pro-inflammatory, anti-invasive signature, characterized by increased pro-inflammatory cytokines (IL-6, IL-8, MCP-1) and TIMP-1. Additionally, these cells displayed 62% decreased migration and 2.7-fold decreased invasion in vitro after treatment with SEA. These results are supported by increased IL-6 and IL-8 in the culture media of HTR8 cells exposed to plasma from Schistosoma japonica infected pregnant women. Conclusions: Soluble egg antigens found in circulation during schistosome infection increase pro-inflammatory cytokine production and inhibit the mobility and invasive characteristics of the first trimester HTR8/SVneo trophoblast cell line. This is the first study to assess the impact of schistosome soluble egg antigens on the behavior of an extravillous trophoblast model and suggests that schistosomiasis in the pre-pregnancy period may adversely impact placentation and the subsequent health of the mother and newborn. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective therapeutics. Periostin has been reported to be elevated in IPF patients relative to controls, but its sources and mechanisms of action remain unclear. We confirm excess periostin in lungs of IPF patients and show that IPF fibroblasts produce periostin. Blood was obtained from 54 IPF patients (all but 1 with 48 wk of follow-up). We show that periostin levels predict clinical progression at 48 wk (hazard ratio = 1.47, 95% confidence interval = 1.03-2.10, P < 0.05). Monocytes and fibrocytes are sources of periostin in circulation in IPF patients. Previous studies suggest that periostin may regulate the inflammatory phase of bleomycin-induced lung injury, but periostin effects during the fibroproliferative phase of the disease are unknown. Wild-type and periostin-deficient (periostin-/-) mice were anesthetized and challenged with bleomycin. Wild-type mice were injected with bleomycin and then treated with OC-20 Ab (which blocks periostin and integrin interactions) or control Ab during the fibroproliferative phase of disease, and fibrosis and survival were assessed. Periostin expression was upregulated quickly after treatment with bleomycin and remained elevated. Periostin-/- mice were protected from bleomycin-induced fibrosis. Instillation of OC-20 during the fibroproliferative phase improved survival and limited collagen deposition. Chimeric mouse studies suggest that hematopoietic and structural sources of periostin contribute to lung fibrogenesis. Periostin was upregulated by transforming growth factor-β in lung mesenchymal cells, and periostin promoted extracellular matrix deposition, mesenchymal cell proliferation, and wound closure. Thus periostin plays a vital role in late stages of pulmonary fibrosis and is a potential biomarker for disease progression and a target for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Subacute lung disease, manifested as either obstructive (OLD) or restrictive (RLD) lung dysfunction, is a common complication following allogeneic stem cell transplantation. In each case, therapeutic options are limited, morbidity remains high, and long-term survival is poor. Between 2001 and 2008, 34 patients with noninfectious, obstructive (25) or RLD restrictive lung dysfunction (nine) received etanercept (Enbrel®, Amgen Inc.) 0.4 mg/kg/dose, subcutaneously, twice weekly, for 4 (group A) or 12 weeks (group B). Corticosteroids (if present at study entry) were kept constant for the initial 4 weeks of therapy and then tapered as tolerated. Thirty-one of 34 (91%) subjects were evaluable for response, and 10 (32%) met primary response criteria. There was no difference in response based on the duration of treatment (29% group A versus 35% group B; P = .99), the presence of RLD or OLD (33% versus 32%; P = .73), or the severity of pulmonary disease at study onset. Estimated 5-year overall survival rates following therapy were 61% (95% confidence interval, 46%-80%) for all subjects and 90% (95% confidence level, 73%-100%) for the 10 who met the primary response criteria. Five-year survival estimates for subjects treated with RLD was 44%, compared with 67% for those treated for OLD (P = .19). Etanercept was well tolerated, with no bacteremia or viremia observed. Pathogens were noted on posttherapy bronchoalveolar lavage in two cases. These data support the development of expanded clinical trials to study etanercept as a therapeutic agent for subacute lung injury after allogeneic stem cell transplantation. [ABSTRACT FROM AUTHOR]

Abstract: Fibrosis of the lungs and other organs is characterized by the accumulation of myofibroblasts, effectors of wound-repair that are responsible for the deposition and organization of new extracellular matrix (ECM) in response to tissue injury. During the resolution phase of normal wound repair, myofibroblast apoptosis limits the continued deposition of ECM. Mounting evidence suggests that myofibroblasts from fibrotic wounds acquire resistance to apoptosis, but the mechanisms regulating this resistance have not been fully elucidated. Endothelin-1 (ET-1), a soluble peptide strongly associated with fibrogenesis, decreases myofibroblast susceptibility to apoptosis through activation of phosphatidylinositol 3′-OH kinase (PI3K)/AKT. Focal adhesion kinase (FAK) also promotes myofibroblast resistance to apoptosis through PI3K/AKT-dependent and -independent mechanisms, although the role of FAK in ET-1 mediated resistance to apoptosis has not been explored. The goal of this study was to investigate whether FAK contributes to ET-1 mediated myofibroblast resistance to apoptosis and to examine potential mechanisms downstream of FAK and PI3K/AKT by which ET-1 regulates myofibroblast survival. Here, we show that ET-1 regulates myofibroblast survival by Rho/ROCK-dependent activation of FAK. The anti-apoptotic actions of FAK are, in turn, dependent on activation of PI3K/AKT and the subsequent increased expression of Survivin, a member of the inhibitor of apoptosis protein (IAP) family. Collectively, these studies define a novel mechanism by which ET-1 promotes myofibroblast resistance to apoptosis through upregulation of Survivin. [Copyright &y& Elsevier]

Background. Schistosomes infect 200 million individuals annually and cause significant hepatic fibrosis in up to 20%. Little is known regarding the mechanisms of schistosome-associated hepatic fibrosis in humans, and few biomarkers for risk of fibrosis have been identified.Methods. We treated 611 Schistosoma japonicum–infected Filipinos with praziquantel (PZQ) and performed ultrasound to quantify hepatic fibrosis at baseline and 12 months after PZQ treatment. We developed a multiplexed assay (FibroPlex) that quantifies predictors and effect modifiers of fibrosis. We measured FibroPlex analytes produced by peripheral blood mononuclear cells stimulated with schistosome egg antigen 4 weeks after PZQ treatment and related these levels to risk of fibrosis 1 year after PZQ treatment.Results. After adjusting for potential confounders, including baseline grade of fibrosis, individuals with detectable tissue inhibitor of matrix-metalloprotease–1 (TIMP-1) had a 3.5-fold greater risk of fibrosis 1 year after PZQ treatment, compared with individuals with undetectable levels (odds ratio, 3.48; 95% confidence interval, 1.41–8.43; P = .007).Discussion Because TIMP-1 inhibits most matrix metalloproteases, which are responsible for collagen degradation, these data suggest that schistosome-associated hepatic fibrosis results, in part, from excessive inhibition of collagen remodeling. These data further suggest that TIMP-1 is a promising biomarker for assessing risk of hepatic fibrosis in schistosomiasis and, potentially, other infectious and noninfectious causes of liver disease. [ABSTRACT FROM PUBLISHER]

Background. Schistosomes infect 200 million individuals annually and cause significant hepatic fibrosis in up to 20%. Little is known regarding the mechanisms of schistosome-associated hepatic fibrosis in humans, and few biomarkers for risk of fibrosis have been identified.Methods. We treated 611 Schistosoma japonicum–infected Filipinos with praziquantel (PZQ) and performed ultrasound to quantify hepatic fibrosis at baseline and 12 months after PZQ treatment. We developed a multiplexed assay (FibroPlex) that quantifies predictors and effect modifiers of fibrosis. We measured FibroPlex analytes produced by peripheral blood mononuclear cells stimulated with schistosome egg antigen 4 weeks after PZQ treatment and related these levels to risk of fibrosis 1 year after PZQ treatment.Results. After adjusting for potential confounders, including baseline grade of fibrosis, individuals with detectable tissue inhibitor of matrix-metalloprotease–1 (TIMP-1) had a 3.5-fold greater risk of fibrosis 1 year after PZQ treatment, compared with individuals with undetectable levels (odds ratio, 3.48; 95% confidence interval, 1.41–8.43; P = .007).Discussion Because TIMP-1 inhibits most matrix metalloproteases, which are responsible for collagen degradation, these data suggest that schistosome-associated hepatic fibrosis results, in part, from excessive inhibition of collagen remodeling. These data further suggest that TIMP-1 is a promising biomarker for assessing risk of hepatic fibrosis in schistosomiasis and, potentially, other infectious and noninfectious causes of liver disease. [ABSTRACT FROM PUBLISHER]

Plasminogen activation to plasmin protects from lung fibrosis, but the mechanism underlying this antifibrotic effect remains unclear. We found that mice lacking plasminogen activation inhibitor-1 (PAI-1), which are protected from bleomycin-induced pulmonary fibrosis, exhibit lung overproduction of the antifibrotic lipid mediator prostaglandin E2 (PGE2). Plasminogen activation upregulated PGE2 synthesis in alveolar epithelial cells, lung fibroblasts, and lung fibrocytes from saline- and bleomycin-treated mice, as well as in normal fetal and adult primary human lung fibroblasts. This response was exaggerated in cells from Pai1-/- mice. Although enhanced PGE2 formation required the generation of plasmin, it was independent of proteinase-activated receptor 1 (PAR-1) and instead reflected proteolytic activation and release of HGF with subsequent induction of COX-2. That the HGF/COX-2/PGE2 axis mediates in vivo protection from fibrosis in Pai1-/- mice was demonstrated by experiments showing that a selective inhibitor of the HGF receptor c-Met increased lung collagen to WT levels while reducing COX-2 protein and PGE2 levels. Of clinical interest, fibroblasts from patients with idiopathic pulmonary fibrosis were found to be defective in their ability to induce COX-2 and, therefore, unable to upregulate PGE2 synthesis in response to plasmin or HGF. These studies demonstrate crosstalk between plasminogen activation and PGE2 generation in the lung and provide a mechanism for the well-known antifibrotic actions of the fibrinolytic pathway. [ABSTRACT FROM AUTHOR]

Abstract: Fibroblast migration plays an important role in the normal wound healing process; however, dysregulated cell migration may contribute to the progressive formation of fibrotic lesions in the diseased condition. To examine the role of focal-adhesion-kinase (FAK)-related non-kinase (FRNK) in regulation of fibrotic lung fibroblast migration, we examined cell migration, FRNK expression, and activation of focal adhesion kinase (FAK) and Rho GTPase (Rho and Rac) in primary lung fibroblasts derived from both idiopathic pulmonary fibrosis (IPF) patients and normal human controls. Fibrotic (IPF) lung fibroblasts have increased cell migration when compared to control human lung fibroblasts. FRNK expression is significantly reduced in IPF lung fibroblasts, while activation of FAK, Rho and Rac is increased in IPF lung fibroblasts. Endogenous FRNK expression is inversely correlated with FAK activation and cell migration rate in IPF lung fibroblasts. Forced exogenous FRNK expression abrogates the increased cell migration, and blocked the activation of FAK and Rho GTPase (Rho and Rac), in IPF lung fibroblasts. These data for the first time provide evidence that downregulation of endogenous FRNK plays a role in promoting cell migration through FAK and Rho GTPase in fibrotic IPF lung fibroblasts. [Copyright &y& Elsevier]

Prostaglandin E2 (PGE2) is an arachidonic acid metabolite that counters transforming growth factor-β-induced fibroblast activation via E prostanoid 2 (EP2) receptor binding. Phosphatase and tensin homologue on chromosome 10 (PTEN) is a lipid phosphatase that, by antagonizing the phosphoinositol 3-kinase (PI3K) pathway, also inhibits fibroblast activation. Here, we show that PTEN directly regulates PGE2 inhibition of fibroblast activation by augmenting EP2 receptor expression. The increase in collagen production and α-smooth muscle actin expression observed in fibroblasts in which PTEN is deficient was resistant to the usual suppressive effects of PGE2. This was due to marked down-regulation of EP2, a Gs protein-coupled receptor (GPCR) that mediates the inhibitory actions of this prostanoid via cAMP. pten-/- or PTEN-inhibited fibroblasts in which the P13K pathway was blocked demonstrated a restoration of EP2 receptor expression, due to augmented gene transcription and mRNA instability. Importantly, restoration of the balance between PI3K and PTEN reestablished the inhibitory effect of PGE2 on fibroblast activation. No such influence of PTEN was observed on alternative E prostanoid GPCRs. Moreover, our studies identified a positive feedback loop in which cAMP signaling enhanced EP2 receptor expression, independent of PTEN. Therefore, our findings indicate that PTEN regulates the antifibrotic effects of PGE2 by a specific and permissive effect on EP2 receptor expression. Further, our data imply that cAMP signaling circumvents EP2 down-regulation in pten-deficient cells to restore EP2 receptor expression. This is the first description, to our knowledge, of PI3K/PTEN balance directing GPCR expression, and provides a novel mechanism for cellular effects of PTEN. [ABSTRACT FROM AUTHOR]

Following a fibrogenic stimulus, the hepatic stellate cell (HSC) transforms from a quiescent to an activated cell type associated with increased proliferation, collagen and smooth muscle α-actin (αSMA) expression. Phosphatase and Tensin Homolog Deleted on Chromosome Ten (PTEN), a tumor suppressor phosphatase, has been shown to play a role in several nonmalignant diseases. Here, we investigated the role of PTEN during HSC activation. Rat HSCs 2 days after isolation were transduced with adenoviruses expressing either the wild-type (WT) or a dominant negative form of PTEN, and culture-associated activation of HSCs, including morphological changes, expression of αSMA and α1(I) collagen, and cell proliferation, were evaluated. Apoptosis of HSCs was detected by measuring activity of caspase 3/7. Phosphorylation status of Akt, p70S6K, and Erk was detected by Western blotting. Overexpression of WT-PTEN inhibited phenotypic changes were associated with HSC activation, including morphological changes, expression of αSMA and α1(I) collagen, and HSC proliferation, including cyclin D1 expression. WT-PTEN expression also induced apoptosis in HSCs with increased caspase 3/7 activity. Expression of WT-PTEN also caused decreased activation of Akt, p70S6K, and Erk signaling pathways. Taken together, these findings show that PTEN represents an important negative regulator for transactivation of HSCs. This may have important implications for the design of therapeutic strategies to prevent the progression of liver fibrosis. [ABSTRACT FROM AUTHOR]

Abstract: Transforming growth factor-β (TGF-β) is a prototypical tumour-suppressor cytokine with cytostatic and pro-apoptotic effects on most target cells; however, mechanisms of its pro-survival/anti-apoptotic signalling in certain cell types and contexts remain unclear. In human lung fibroblasts, TGF-β1 is known to induce myofibroblast differentiation in association with the delayed activation of focal adhesion kinase (FAK) and protein kinase B (PKB/AKT). Here, we demonstrate that FAK and AKT are independently regulated by early activation of SMAD3 and p38 MAPK, respectively. Pharmacologic or genetic approaches that disrupt SMAD3 signalling block TGF-β1-induced activation of FAK, but not AKT; in contrast, disruption of early p38 MAPK signalling abrogates AKT activation, but does not alter FAK activation. TGF-β1 is able to activate AKT in cells expressing mutant FAK or in cells treated with an RGD-containing peptide that interferes with integrin signalling, inhibits FAK activation and induces anoikis (apoptosis induced by loss of adhesion signalling). TGF-β1 protects myofibroblasts from anoikis, in part, by activation of the PI3K-AKT pathway. Thus, TGF-β1 co-ordinately and independently activates the FAK and AKT protein kinase pathways to confer an anoikis-resistant phenotype to myofibroblasts. Activation of these pro-survival/anti-anoikis pathways in myofibroblasts likely contributes to essential roles of TGF-β1 in tissue fibrosis and tumour-promotion. [Copyright &y& Elsevier]

Although chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) seem to be opposite entities from a clinical perspective, common initial pathogenic steps have been suggested in both lung diseases. Emphysema is caused by an elastase/anti-elastase imbalance leading to accelerated elastin degradation. Elastinolysis is however, also accelerated in the IPF patients' lungs. The amino acids desmosine and isodesmosine (DES) are unique to elastin. During the degradation process, elastases liberate DES from elastin fibers. Blood DES levels consequently reflect the rate of systemic elastinolysis and are increased in COPD. This is the first report describing elevated DES levels in IPF patients. We also demonstrated that the age-related increment of DES concentrations is enhanced in IPF. Our current study suggests that elastinolysis is a shared pathogenic step in both COPD and IPF. Further investigation is required to establish the relevance of accelerated elastin degradation in IPF and to determine whether decelerating this process leads to slower progression of lung fibrosis and better survival for patients with IPF. [ABSTRACT FROM AUTHOR]

This corrects the article DOI: 10.1038/srep46560 [ABSTRACT FROM AUTHOR]