Critical Role of LMCD1 in Promoting Profibrotic Characteristics of Lung Myofibroblasts in Experimental and Scleroderma-Associated Lung Fibrosis.

OBJECTIVE: Interstitial lung disease (ILD) is a serious complication and leading cause of mortality in patients with systemic sclerosis (SSc). In this study, we explored the role of LIM and cysteine-rich domains protein 1 (LMCD1) as a novel factor in the pathogenesis of SSc-related ILD (SSc-ILD). METHODS: The expression and effects of LMCD1 were studied in lung tissue samples and fibroblasts from SSc-ILD patients and control subjects as well as in lung tissue samples from animal models. RESULTS: LMCD1 was consistently elevated in lung tissue samples and in fibroblasts isolated from SSc-ILD patients as compared to controls. Additionally, LMCD1 was found to be highly expressed in the lung in the fibroblast-specific protein (FSP)-driven, constitutively active transforming growth factor ß receptor type I (TGFßR1) transgenic mouse model of ILD and the bleomycin-induced mouse model of ILD. In lung fibroblasts from SSc-ILD patients, LMCD1 is an essential factor for the TGFß-induced generation of type I collagen, fibronectin, and α-smooth muscle actin (α-SMA). Depletion of LMCD1 by small interfering RNA reduced the expression of extracellular matrix proteins and lowered transcriptional activity and expression of α-SMA, as well as decreased the proliferation and contractile activity of SSc-ILD lung fibroblasts. In dense fibrotic areas of affected lung tissue, lung LMCD1 colocalized with α-SMA. In cultured scleroderma lung fibroblasts, LMCD1 colocalized and interacted with serum response factor which mediates LMCD1-induced contractile activity of lung fibroblasts. CONCLUSION: Our study identifies LMCD1 as a profibrotic molecule contributing to the activation of myofibroblasts and the persistent fibroproliferation observed in SSc-ILD. Thus, LMCD1 may be a potential novel therapeutic target for patients with SSc-ILD.

Nodular Regenerative Hyperplasia of the Liver in Rheumatic Disease: Cases and Review of the Literature.

Nodular regenerative hyperplasia (NRH) is a rare disease that is characterized by benign transformation of the hepatic parenchyma into small nodules with little to no fibrosis. Nodular regenerative hyperplasia is a cause of noncirrhotic portal hypertension. Symptoms can range from asymptomatic disease to more serious complications of portal hypertension such as esophageal varices and ascites. Nodular regenerative hyperplasia has been described in association with a variety of different rheumatologic, hematologic, and oncologic diseases, as well as in immune deficiency states and with exposures to certain toxins. Diagnosis is made by histology, and the treatment involves addressing the underlying disease. The first description of this rare disease was actually described in a patient with rheumatoid arthritis, neutropenia, and splenomegaly (Felty's Syndrome). We describe 2 cases of NRH associated with underlying rheumatic disorders, in one of which NRH was actually the presenting feature of the patient's underlying autoimmune condition. Subsequently, we provide a brief review of the literature of NRH in autoimmune disease with respect to epidemiology, cause, clinical manifestations, diagnosis, and treatment.

Riociguat in patients with early diffuse cutaneous systemic sclerosis (RISE-SSc): randomised, double-blind, placebo-controlled multicentre trial.

OBJECTIVES: Riociguat is approved for pulmonary arterial hypertension and has antiproliferative, anti-inflammatory and antifibrotic effects in animal models of tissue fibrosis. We evaluated the efficacy and safety of riociguat in patients with early diffuse cutaneous systemic sclerosis (dcSSc) at high risk of skin fibrosis progression. METHODS: In this randomised, double-blind, placebo-controlled, phase IIb trial, adults with dcSSc of <18 months' duration and a modified Rodnan skin score (mRSS) 10-22 units received riociguat 0.5 mg to 2.5 mg orally three times daily (n=60) or placebo (n=61). The primary endpoint was change in mRSS from baseline to week 52. RESULTS: At week 52, change from baseline in mRSS units was -2.09±5.66 (n=57) with riociguat and -0.77±8.24 (n=52) with placebo (difference of least squares means -2.34 (95% CI -4.99 to 0.30; p=0.08)). In patients with interstitial lung disease, forced vital capacity declined by 2.7% with riociguat and 7.6% with placebo. At week 14, average Raynaud's condition score had improved ≥50% in 19 (41.3%)/46 patients with riociguat and 13 (26.0%)/50 patients with placebo. Safety assessments showed no new signals with riociguat and no treatment-related deaths. CONCLUSIONS: Riociguat did not significantly benefit mRSS versus placebo at the predefined p<0.05. Secondary and exploratory analyses showed potential efficacy signals that should be tested in further trials. Riociguat was well tolerated.

Systemic sclerosis-associated interstitial lung disease.

Systemic sclerosis is an autoimmune connective tissue disease, which is characterised by immune dysregulation and progressive fibrosis that typically affects the skin, with variable internal organ involvement. It is a rare condition that affects mostly young and middle-aged women, resulting in disproportionate morbidity and mortality. Currently, interstitial lung disease is the most common cause of death among patients with systemic sclerosis, with a prevalence of up to 30% and a 10-year mortality of up to 40%. Interstitial lung disease is more common among African Americans and in people with the diffuse cutaneous form of systemic sclerosis or anti-topoisomerase 1 antibodies. Systemic sclerosis-associated interstitial lung disease most commonly presents with dyspnoea, cough, and a non-specific interstitial pneumonia pattern on CT scan, with a minority of cases fulfilling the criteria for usual interstitial pneumonia. The standard therapy has traditionally been combinations of immunosuppressants, particularly mycophenolate mofetil or cyclophosphamide. These immunosuppressants can be supplemented by targeted biological and antifibrotic therapies, whereas autologous haematopoietic stem-cell transplantation and lung transplantation are reserved for refractory cases.

Was Paul Klee's scleroderma an occupational disease? A series of historical and clinical vignettes, part III.

Paul Klee (1879-1940), one of the most influential artists of the 20th century, died at 60 years of age from complications of systemic sclerosis (scleroderma). The precipitating event(s) of Klee's scleroderma, as in most cases, will never be known. Among various potential factors, exposure to heavy metals, crystalline silica, and organic solvents-acting alone or in combination-can now be considered potential factors in the onset of Klee's disease. By altering and modulating epigenetic determinants in a genetically susceptible host, these and other environmental factors may have led to perturbations of self-tolerance and inflammation culminating in Klee's scleroderma.

HLA and autoantibodies define scleroderma subtypes and risk in African and European Americans and suggest a role for molecular mimicry.

Systemic sclerosis (SSc) is a clinically heterogeneous autoimmune disease characterized by mutually exclusive autoantibodies directed against distinct nuclear antigens. We examined HLA associations in SSc and its autoantibody subsets in a large, newly recruited African American (AA) cohort and among European Americans (EA). In the AA population, the African ancestry-predominant HLA-DRB1*08:04 and HLA-DRB1*11:02 alleles were associated with overall SSc risk, and the HLA-DRB1*08:04 allele was strongly associated with the severe antifibrillarin (AFA) antibody subset of SSc (odds ratio = 7.4). These African ancestry-predominant alleles may help explain the increased frequency and severity of SSc among the AA population. In the EA population, the HLA-DPB1*13:01 and HLA-DRB1*07:01 alleles were more strongly associated with antitopoisomerase (ATA) and anticentromere antibody-positive subsets of SSc, respectively, than with overall SSc risk, emphasizing the importance of HLA in defining autoantibody subtypes. The association of the HLA-DPB1*13:01 allele with the ATA+ subset of SSc in both AA and EA patients demonstrated a transancestry effect. A direct correlation between SSc prevalence and HLA-DPB1*13:01 allele frequency in multiple populations was observed (r = 0.98, P = 3 × 10-6). Conditional analysis in the autoantibody subsets of SSc revealed several associated amino acid residues, mostly in the peptide-binding groove of the class II HLA molecules. Using HLA α/ß allelic heterodimers, we bioinformatically predicted immunodominant peptides of topoisomerase 1, fibrillarin, and centromere protein A and discovered that they are homologous to viral protein sequences from the Mimiviridae and Phycodnaviridae families. Taken together, these data suggest a possible link between HLA alleles, autoantibodies, and environmental triggers in the pathogenesis of SSc.

Antifibrotic efficacy of nintedanib in a cellular model of systemic sclerosis-associated interstitial lung disease.

OBJECTIVES: Nintedanib is approved for the treatment of idiopathic pulmonary fibrosis (IPF) and was demonstrated to slow disease progression in patients with IPF by reducing decline in forced vital capacity by 50%. Recently, nintedanib has been reported to exert anti-fibrotic activity on systemic sclerosis (scleroderma, SSc) skin fibroblasts and to diminish skin and lung fibrosis in mouse models. The goal of the present study was to determine the effects of nintedanib on a cellular model of SSc-associated interstitial lung disease (ILD). METHODS: Study was performed using lung fibroblasts (LF) isolated from five patients with SSc-ILD and from three control subjects. RESULTS: Nintedanib inhibited LF proliferation and migration in a concentration- and time-dependent manner. The proliferation rate of LF stimulated with PDGF in the presence of nintedanib was reduced 1.9-fold within 24 h as compared to cells stimulated with PDGF alone. Migration of SSc-ILD LF incubated with 100 nM nintedanib was reduced from 62.8±12.5% to 39.1±9.0% in the presence of PDGF and from 38.2±7.9% to 26.6±7.2% in serum-free medium. Nintedanib attenuated PDGF-induced Ca2+ efflux, reduced α-SMA promoter activity and α-SMA protein expression. Furthermore, nintedanib blocked PDGF-induced differentiation of normal LF to myofibroblasts, reduced production of collagen and fibronectin, and decreased contractility of SSc-ILD LF in both floating and fixed collagen gels. CONCLUSIONS: Our data demonstrate significant antifibrotic efficacy of nintedanib in SSc-ILD LF suggesting that nintedanib has the potential not only to prevent but also to reverse the increased activity of LF consequently attenuating excessive lung fibrosis observed in SSc-ILD.

Voriconazole-associated periostitis presenting as hypertrophic osteoarthropathy following lung transplantation report of two cases and review of the literature.

BACKGROUND: Hypertrophic osteoarthropathy (HOA) is a syndrome characterized by abnormal proliferation of skin and osseous tissue frequently associated with underlying pulmonary disorders. Cardinal features include digital clubbing, periostitis and significant joint and bone pain. A number of recent reports have emerged of HOA and periostitis occurring in association with the antifungal agent voriconazole. METHODS: We present two additional cases of voriconazole-induced HOA and periostitis in lung transplant recipients with a review the medical literature. RESULTS: In both cases, symptoms were painful and severe enough to require opioid medication. Rapid improvement occurred within days of voriconazole cessation. A review of existing literature revealed an additional 17 cases of voriconazole-induced HOA and periostitis in lung transplant patients. CONCLUSION: We highlight the importance of recognizing the association of voriconazole with painful HOA and periostitis in lung transplant patients receiving antifungal therapy. Management of this painful condition involves cessation of voriconazole therapy, which may necessitate alternative anti-fungal drug therapies as well as adjustment of immunosuppressive drug dosage since voriconazole is a strong drug-inducer.

Brief Report: Whole-Exome Sequencing to Identify Rare Variants and Gene Networks That Increase Susceptibility to Scleroderma in African Americans.

OBJECTIVE: Whole-exome sequencing (WES) studies in systemic sclerosis (SSc) patients of European American (EA) ancestry have identified variants in the ATP8B4 gene and enrichment of variants in genes in the extracellular matrix (ECM)-related pathway that increase SSc susceptibility. This study was undertaken to evaluate the association of the ATP8B4 gene and the ECM-related pathway with SSc in a cohort of African American (AA) patients. METHODS: SSc patients of AA ancestry were enrolled from 23 academic centers across the US under the Genome Research in African American Scleroderma Patients consortium. Unrelated AA individuals without serologic evidence of autoimmunity who were enrolled in the Howard University Family Study were used as unaffected controls. Functional variants in genes reported in the 2 WES studies in EA patients with SSc were selected for gene association testing using the optimized sequence kernel association test (SKAT-O) and pathway analysis by Ingenuity Pathway Analysis in 379 patients and 411 controls. RESULTS: Principal components analysis demonstrated that the patients and controls had similar ancestral backgrounds, with roughly equal proportions of mean European admixture. Using SKAT-O, we examined the association of individual genes that were previously reported in EA patients and none remained significant, including ATP8B4 (P = 0.98). However, we confirmed the previously reported association of the ECM-related pathway with enrichment of variants within the COL13A1, COL18A1, COL22A1, COL4A3, COL4A4, COL5A2, PROK1, and SERPINE1 genes (corrected P = 1.95 × 10-4 ). CONCLUSION: In the largest genetic study in AA patients with SSc to date, our findings corroborate the role of functional variants that aggregate in a fibrotic pathway and increase SSc susceptibility.

Myeloablative Autologous Stem-Cell Transplantation for Severe Scleroderma.

BACKGROUND: Despite current therapies, diffuse cutaneous systemic sclerosis (scleroderma) often has a devastating outcome. We compared myeloablative CD34+ selected autologous hematopoietic stem-cell transplantation with immunosuppression by means of 12 monthly infusions of cyclophosphamide in patients with scleroderma. METHODS: We randomly assigned adults (18 to 69 years of age) with severe scleroderma to undergo myeloablative autologous stem-cell transplantation (36 participants) or to receive cyclophosphamide (39 participants). The primary end point was a global rank composite score comparing participants with each other on the basis of a hierarchy of disease features assessed at 54 months: death, event-free survival (survival without respiratory, renal, or cardiac failure), forced vital capacity, the score on the Disability Index of the Health Assessment Questionnaire, and the modified Rodnan skin score. RESULTS: In the intention-to-treat population, global rank composite scores at 54 months showed the superiority of transplantation (67% of 1404 pairwise comparisons favored transplantation and 33% favored cyclophosphamide, P=0.01). In the per-protocol population (participants who received a transplant or completed ≥9 doses of cyclophosphamide), the rate of event-free survival at 54 months was 79% in the transplantation group and 50% in the cyclophosphamide group (P=0.02). At 72 months, Kaplan-Meier estimates of event-free survival (74% vs. 47%) and overall survival (86% vs. 51%) also favored transplantation (P=0.03 and 0.02, respectively). A total of 9% of the participants in the transplantation group had initiated disease-modifying antirheumatic drugs (DMARDs) by 54 months, as compared with 44% of those in the cyclophosphamide group (P=0.001). Treatment-related mortality in the transplantation group was 3% at 54 months and 6% at 72 months, as compared with 0% in the cyclophosphamide group. CONCLUSIONS: Myeloablative autologous hematopoietic stem-cell transplantation achieved long-term benefits in patients with scleroderma, including improved event-free and overall survival, at a cost of increased expected toxicity. Rates of treatment-related death and post-transplantation use of DMARDs were lower than those in previous reports of nonmyeloablative transplantation. (Funded by the National Institute of Allergy and Infectious Diseases and the National Institutes of Health; ClinicalTrials.gov number, NCT00114530 .).

Establishment of an indirect ELISA for detection of the novel antifibrotic peptide M10.

OBJECTIVE: M10 is a ten amino acid peptide generated from the intracellular cytoplasmic tail of the hepatocyte growth factor (HGF) receptor c-Met following cleavage by caspase-3. Recently we reported that M10 interacts with Smad2 and demonstrates antifibrotic properties in vitro and in vivo and can be advanced into a novel antifibrotic remedy. The current study was undertaken to develop an immunoassay to measure M10 concentration in biological specimens. EXPERIMENTAL DESIGN: An Indirect Enzyme-Linked Immunosorbent Assay (ELISA) for detection of M10 in biological fluids was developed using pharmaceutical grade synthetic M10 as a calibrator and commercially available anti-c-Met C12 antibody. RESULTS: M10 ELISA specifically detected in plasma M10, but not a scrambled peptide, following a single intraperitoneal administration of M10 (1mg/kg) to mice. The detection limit was 9.6 ng/ml, and the measuring limit was between 15 ng/ml and 200 ng/ml. The recovery limits of M10 were between 80% and 120%; intra-assay coefficient of variation was between 5.3% and 6.3%; inter-assay coefficient of variation was between 5.0% and 8.0% over the buffer concentration tested in the range from 15 ng /ml to 250 ng /ml. The peak of M10 concentration following a single intraperitoneal injection (1mg/kg) was achieved within 6 hours and declined to minimal levels by 48 hours. The experimentally obtained half-life for M10 was comparable to the theoretically predicted half-life for M10. CONCLUSIONS: We have established a highly sensitive ELISA to detect the antifibrotic peptide M10 in plasma samples, which should prove to be a novel tool to study the pharmacokinetics and efficacy of M10 in the treatment of fibroproliferative disorders.

Transforming growth factor ß1 (TGFß1)-induced CD44V6-NOX4 signaling in pathogenesis of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive clinical syndrome of fatal outcome. The lack of information about the signaling pathways that sustain fibrosis and the myofibroblast phenotype has prevented the development of targeted therapies for IPF. Our previous study showed that isolated fibrogenic lung fibroblasts have high endogenous levels of the hyaluronan receptor, CD44V6 (CD44 variant containing exon 6), which enhances the TGFß1 autocrine signaling and induces fibroblasts to transdifferentiate into myofibroblasts. NADPH oxidase 4 (NOX4) enzyme, which catalyzes the reduction of O2 to hydrogen peroxide (H2O2), has been implicated in the cardiac and lung myofibroblast phenotype. However, whether CD44V6 regulates NOX4 to mediate tissue repair and fibrogenesis is not well-defined. The present study assessed the mechanism of how TGF-ß-1-induced CD44V6 regulates the NOX4/reactive oxygen species (ROS) signaling that mediates the myofibroblast differentiation. Specifically, we found that NOX4/ROS regulates hyaluronan synthesis and the transcription of CD44V6 via an effect upon AP-1 activity. Further, CD44V6 is part of a positive-feedback loop with TGFß1/TGFßRI signaling that acts to increase NOX4/ROS production, which is required for myofibroblast differentiation, myofibroblast differentiation, myofibroblast extracellular matrix production, myofibroblast invasion, and myofibroblast contractility. Both NOX4 and CD44v6 are up-regulated in the lungs of mice subjected to experimental lung injury and in cases of human IPF. Genetic (CD44v6 shRNA) or a small molecule inhibitor (CD44v6 peptide) targeting of CD44v6 abrogates fibrogenesis in murine models of lung injury. These studies support a function for CD44V6 in lung fibrosis and offer proof of concept for therapeutic targeting of CD44V6 in lung fibrosis disorders.

D1398G Variant of MET Is Associated with Impaired Signaling of Hepatocyte Growth Factor in Alveolar Epithelial Cells and Lung Fibroblasts.

Pulmonary fibrosis represents the terminal stage of a diverse group of lung diseases including scleroderma associated interstitial lung disease. The molecular mechanisms underlying the pathogenesis of lung fibrosis are not well understood and there is a great need for more effective treatment for this lethal disease. We recently discovered a small fragment of hepatocyte growth factor (HGF) receptor MET as a peptide designated "M10," with strong antifibrotic properties. Furthermore, we showed that aspartic acid at position 1398 of MET is essential for M10 generation. The current study was undertaken to investigate the D1398G variant of MET in which aspartic acid at position 1398 was mutated to glycine resulting in loss of M10. We demonstrate that lung fibroblasts, A549, and primary alveolar epithelial cells (AEC) expressing D1398G MET exhibit reduced auto-phosphorylation on tyrosine residues and reduced activation of Ras and MAPK. HGF treatment of scleroderma lung fibroblasts as well as HGF treatment of TGFß-treated normal lung fibroblasts transfected with wild type MET is associated with decreased collagen, connective tissue growth factor (CTGF, CCN2) and smooth muscle α-actin (SMA). However, HGF has no such effects in cells transfected with MET D1398G. Cisplatin- and FasL-induced apoptosis is significantly reduced in AEC transfected with MET wild type, but not in AEC transfected with MET D1398G. We conclude that the D1398G variant of MET is associated with compromised phosphorylation and impaired HGF signaling in lung fibroblasts and AEC, two cell types implicated in the pathogenesis of pulmonary fibrosis associated with scleroderma. Ongoing studies will explore the frequency of this variant and its relationship to pulmonary outcomes in scleroderma patients.

M10, a caspase cleavage product of the hepatocyte growth factor receptor, interacts with Smad2 and demonstrates antifibrotic properties in vitro and in vivo.

Hepatocyte growth factor receptor, also known as cellular mesenchymal-epithelial transition factor (c-MET, MET), is an important antifibrotic molecule that protects various tissues, including lung, from injury and fibrosis. The intracellular cytoplasmic tail of MET contains a caspase-3 recognition motif "DEVD-T" that on cleavage by caspase-3 generates a 10-amino acid peptide, TRPASFWETS, designated as "M10". M10 contains at its N-terminus the uncharged amino acid proline (P) directly after a cationic amino acid arginine (R) which favors the transport of the peptide through membranes. M10, when added to cell culture medium, remains in the cytoplasm and nuclei of cells for up to 24 hours. M10 effectively decreases collagen in both scleroderma and TGFß-stimulated normal lung and skin fibroblasts. M10 interacts with the Mad Homology 2 domain of Smad2 and inhibits TGFß-induced Smad2 phosphorylation, suggesting that the antifibrotic effects of M10 are mediated in part by counteracting Smad-dependent fibrogenic pathways. In the bleomycin murine model of pulmonary fibrosis, M10 noticeably reduced lung inflammation and fibrosis. Ashcroft fibrosis scores and lung collagen content were significantly lower in bleomycin-treated mice receiving M10 as compared with bleomycin-treated mice receiving scrambled peptide. We conclude that M10 peptide interacts with Smad2 and demonstrates strong antifibrotic effects in vitro and in vivo in an animal model of lung fibrosis and should be considered as a potential therapeutic agent for systemic sclerosis and other fibrosing diseases.

Fibrocytes in the fibrotic lung: altered phenotype detected by flow cytometry.

Fibrocytes are bone marrow hematopoietic-derived cells that also express a mesenchymal cell marker (commonly collagen I) and participate in fibrotic diseases of multiple organs. Given their origin, they or their precursors must be circulating cells before recruitment into target tissues. While most previous studies focused on circulating fibrocytes, here we focus on the fibrocyte phenotype in fibrotic tissue. The study's relevance to human disease is heightened by use of a model in which bleomycin is delivered systemically, recapitulating several features of human scleroderma including multi-organ fibrosis not observed when bleomycin is delivered directly into the lungs. Using flow cytometry, we find in the fibrotic lung a large population of CD45(high) fibrocytes (called Region I) rarely found in vehicle-treated control mice. A second population of CD45+ fibrocytes (called Region II) is observed in both control and fibrotic lung. The level of CD45 in circulating fibrocytes is far lower than in either Region I or II lung fibrocytes. The chemokine receptors CXCR4 and CCR5 are expressed at higher levels in Region I than in Region II and are present at very low levels in all other lung cells including CD45+/collagen I- leucocytes. The collagen chaperone HSP47 is present at similar high levels in both Regions I and II, but at a higher level in fibrotic lung than in control lung. There is also a major population of HSP47(high)/CD45- cells in fibrotic lung not present in control lung. CD44 is present at higher levels in Region I than in Region II and at much lower levels in all other cells including CD45+/collagen I- leucocytes. When lung fibrosis is inhibited by restoring caveolin-1 activity using a caveolin-1 scaffolding domain peptide (CSD), a strong correlation is observed between fibrocyte number and fibrosis score. In summary, the distinctive phenotype of fibrotic lung fibrocytes suggests that fibrocyte differentiation occurs primarily within the target organ.

Bleomycin delivery by osmotic minipump: similarity to human scleroderma interstitial lung disease.

The interstitial lung diseases (ILD) include a large number of chronic, progressive, irreversible respiratory disorders involving pulmonary fibrosis, the most common of which are idiopathic pulmonary fibrosis and scleroderma lung disease (SSc ILD). Because bleomycin causes lung fibrosis when used in cancer chemotherapy, it is used to model human ILD in rodents. In most studies, bleomycin has been delivered directly into the lung by intratracheal or intraoral administration. Here we have compared the effects in mice of bleomycin delivered directly into the lungs (direct model) or systemically using osmotic minipumps (pump model) to determine which more closely resembles human ILD. The pump model is more similar to human SSc ILD in that: 1) lung injury/fibrosis is limited to the subpleural portion of the lung in the pump model and in SSc ILD, whereas the entire lung is affected in the direct model; 2) conversely, there is massive inflammation throughout the lung in the direct model, whereas inflammation is limited in the pump model and in SSc ILD; 3) hypertrophic type II alveolar epithelial cells are present at high levels in SSc ILD and in the pump model but not in the direct model; and 4) lung fibrosis is accompanied by dermal fibrosis. The pump model is also move convenient and humane than the direct model because there is less weight loss and mortality.

Thrombin increases lung fibroblast survival while promoting alveolar epithelial cell apoptosis via the endoplasmic reticulum stress marker, CCAAT enhancer-binding homologous protein.

Apoptosis of alveolar epithelial cells (AECs) and survival of lung fibroblasts are critical events in the pathogenesis of pulmonary fibrosis; however, mechanisms underlying the apoptosis of AECs and the resistance of lung fibroblasts to apoptosis remain obscure. Herein, we demonstrate that the fate of these two cell types depends on the expression of CCAAT enhancer-binding homologous protein (CHOP). We observed that thrombin, which is overexpressed in scleroderma (SSc; systemic sclerosis) and other interstitial lung diseases (ILDs), increases the expression of CHOP in primary AECs and in A549 cells via an Ets1-dependent pathway. In addition, thrombin activates caspase-3 in AECs and induces apoptosis of these cells in a CHOP-dependent manner. In contrast, thrombin decreases endoplasmic reticulum stress-induced CHOP in lung fibroblasts through Myc-dependent mechanisms and protects such cells from apoptosis. Furthermore, when lung fibroblasts are transfected with recombinant CHOP, they then undergo apoptosis, even in the presence of thrombin, suggesting that CHOP signaling pathways are downstream of thrombin. In accordance with the differential effects of thrombin on AECs and lung fibroblasts, we observed strong expression of CHOP in AECs in fibrotic lung tissue isolated from patients with SSc-associated ILD (SSc-ILD), but not in lung myofibroblasts nor in normal lung tissue. Expression of CHOP in SSc lung is accompanied by positive staining for the thrombin receptor, protease-activated receptor-1, and for terminal deoxynucleotidyl transferase dUTP nick end labeling, suggesting roles for both thrombin and CHOP in AEC apoptosis in SSc-ILD. We conclude that regulation of CHOP by thrombin directs AECs toward apoptosis while promoting survival of lung fibroblasts, ultimately contributing to the persistent fibroproliferation seen in SSc-ILD and other fibrosing lung diseases.

Overexpression of c-Met and CD44v6 receptors contributes to autocrine TGF-ß1 signaling in interstitial lung disease.

The hepatocyte growth factor (HGF) and the HGF receptor Met pathway are important in the pathogenesis of interstitial lung disease (ILD). Alternatively spliced isoforms of CD44 containing variable exon 6 (CD44v6) and its ligand hyaluronan (HA) alter cellular function in response to interaction between CD44v6 and HGF. TGF-ß1 is the crucial cytokine that induces fibrotic action in ILD fibroblasts (ILDFbs). We have identified an autocrine TGF-ß1 signaling that up-regulates both Met and CD44v6 mRNA and protein expression. Western blot analysis, flow cytometry, and immunostaining revealed that CD44v6 and Met colocalize in fibroblasts and in tissue sections from ILD patients and in lungs of bleomycin-treated mice. Interestingly, cell proliferation induced by TGF-ß1 is mediated through Met and CD44v6. Further, cell proliferation mediated by TGF-ß1/CD44v6 is ERK-dependent. In contrast, action of Met on ILDFb proliferation does not require ERK but does require p38(MAPK). ILDFbs were sorted into CD44v6(+)/Met(+) and CD44v6(-)/Met(+) subpopulations. HGF inhibited TGF-ß1-stimulated collagen-1 and α-smooth muscle cell actin expression in both of these subpopulations by interfering with TGF-ß1 signaling. HGF alone markedly stimulated CD44v6 expression, which in turn regulated collagen-1 synthesis. Our data with primary lung fibroblast cultures with respect to collagen-1, CD44v6, and Met expressions were supported by immunostaining of lung sections from bleomycin-treated mice and from ILD patients. These results define the relationships between CD44v6, Met, and autocrine TGF-ß1 signaling and the potential modulating influence of HGF on TGF-ß1-induced CD44v6-dependent fibroblast function in ILD fibrosis.

Allogeneic mesenchymal stem cell transplantation in severe and refractory systemic lupus erythematosus: 4 years of experience.

Mesenchymal stem cells (MSCs) are multipotential nonhematopoietic progenitors and are capable of differentiating into several tissues of mesenchymal origin. We have shown that bone marrow-derived MSCs from both SLE patients and lupus-prone MRL/lpr mice are defective structurally and functionally. Here we observe the long-term safety and efficacy of allogeneic MSC transplantation (MSCT) in treatment-resistant SLE patients. Eighty-seven patients with persistently active SLE who were refractory to standard treatment or had life-threatening visceral involvement were enrolled. Allogeneic bone marrow or umbilical cord-derived MSCs were harvested and infused intravenously (1 × 10(6) cells/kg of body weight). Primary outcomes were rates of survival, disease remission and relapse, as well as transplantation-related adverse events. Secondary outcomes included SLE disease activity index (SLEDAI) and serologic features. During the 4-year follow-up and with a mean follow-up period of 27 months, the overall rate of survival was 94% (82/87). Complete clinical remission rate was 28% at 1 year (23/83), 31% at 2 years (12/39), 42% at 3 years (5/12), and 50% at 4 years (3/6). Rates of relapse were 12% (10/83) at 1 year, 18% (7/39) at 2 years, 17% (2/12) at 3 years, and 17% (1/6) at 4 years. The overall rate of relapse was 23% (20/87). Disease activity declined as revealed by significant changes in the SLEDAI score, levels of serum autoantibodies, albumin, and complements. A total of five patients (6%) died after MSCT from non-treatment-related events in the 4-year follow-up, and no transplantation-related adverse event was observed. Allogeneic MSCT resulted in the induction of clinical remission and improvement in organ dysfunction in drug-resistant SLE patients.