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1.
Study objectives
Nonspecific interstitial pneumonia (NSIP) has been identified as a distinct entity with a more favorable prognosis and better response to immunosuppressive therapies than usual interstitial pneumonia (UIP). However the inflammatory profile of NSIP has not been characterized.Design
Using immunohistochemistry techniques on open lung biopsy specimens, the infiltrate in NSIP was characterized in terms of T and B cells, and macrophages, and the T cell population further identified as either CD4 (helper) or CD8 (suppressor-cytotoxic) T cells. The extent of Th1 and Th2 cytokine producing cells was determined and compared to specimens from patients with UIP.Results
In ten NSIP tissue samples 41.4 ± 4% of mononuclear cells expressed CD3, 24.7 ± 1.8% CD4, 19.1 ± 2% CD8, 27.4 ± 3.9% CD20, and 14.3 ± 1.6% had CD68 expression. Mononuclear cells expressed INFγ 21.9 ± 1.9% of the time and IL-4 in 3.0 ± 1%. In contrast, biopsies from eight patients with UIP demonstrated substantially less cellular staining for either cytokine (INFγ; 4.6 ± 1.7% and IL-4; 0.6 ± 0.3%). Significant populations of CD20 positive B-cells were also identified.Conclusion
The lymphocytic infiltrate in NSIP is characterized by an elevated CD4/CD8 T-cell ratio, and is predominantly of Th1 type, with additional populations rich in B-cells. Such features are consistent with the favorable clinical course observed in patients with NSIP compared to UIP. 相似文献2.
Teng Moua Ana C Zamora Martinez Misbah Baqir Robert Vassallo Andrew H Limper Jay H Ryu 《Respiratory research》2014,15(1)
Background
Although usual interstitial pneumonia (UIP) appears to portend better survival when associated with connective tissue disease (CTD-UIP), little is known about the presenting clinical, radiologic, and pathologic features that differentiate pathologically confirmed UIP with CTD from idiopathic pulmonary fibrosis (IPF). In patients with atypical radiologic and clinical features, what specific findings predict underlying IPF vs. CTD-UIP diagnosis and their respective long term survival?Methods
A large retrospective cohort analysis was done of consecutive patients seen from 1995 through 2010 with biopsy confirmed UIP completed or reviewed at our institution. CTD-UIP was defined by independent rheumatology consultation with exclusion of all other secondary causes of lung fibrosis. Primary clinical data was collected and compared for IPF and CTD-UIP along with logistic regression performed for predictors of disease likelihood and Cox proportional hazards analysis for predictors of survival.Results
Six hundred and twenty five patients were included in the study of which 89 had diagnosed CTD-UIP representing 7 disease entities. Survival was better among those with CTD-UIP except in UIP associated with rheumatoid arthritis, which had similar presenting features and survival to IPF. Predictors of underlying CTD included female gender, younger age, positive autoimmune serology, and inconsistent presenting radiologic findings. Only age and forced vital capacity corrected for a priori covariates were predictive of survival in CTD-UIP.Conclusions
UIP pathology occurs frequently among patients with atypically presenting clinical and radiologic features, and may represent IPF or CTD-UIP with improved prognosis if underlying CTD is diagnosed. Presenting radiologic and pathologic features alone are not predictive of underlying secondary cause or survival between the two groups. 相似文献3.
Background
Toll-like receptor 4 (TLR4) is essential in lipopolysaccharide (LPS)-induced fibroblast activation and collagen secretion in vitro. However, its effects on the process of lung fibroblast activation and fibrosis initiation during LPS induced acute lung injury (ALI) remain unknown. The goal of the present study was to determine the effect of inhibiting TLR4 on LPS-induced ALI and fibrosis in vivo.Methods
The ALI model was established by intraperitoneal injection of LPS in mice. TLR4-small hairpin RNA (shRNA) lentivirus was injected intravenously into the mice to inhibit TLR4 expression. mRNA and protein levels were detected by real-time PCR and Western-blot analysis, respectively. The contents of the C-terminal propeptide of type I procollagen (PICP) in bronchoalveolar lavage fluid (BALF) were detected by ELISA, and the degree of fibrosis was detected by van Gieson collagen staining, the hydroxyproline assay, and alpha smooth muscle actin (α-SMA) immunohistochemical staining.Results
Overexpression of TLR4, type I procollagen, alpha-SMA, and p-AKT in murine pulmonary tissue after intraperitoneal injection of LPS at 72 hours and 28 days were detected. Moreover, the degree of fibrosis was shown to increase by ELISA analysis of PICP in BALF, van Gieson collagen staining, the hydroxyproline assay, and α-SMA immunohistochemical staining. All of these changes were alleviated by intravenous infection with TLR4-shRNA lentivirus.Conclusions
Inhibiting TLR4 signaling could ameliorate fibrosis at the early stage of ALI induced by LPS. 相似文献4.
Xu Shi-wen Shangxi Liu Mark Eastwood Sonali Sonnylal Christopher P. Denton David J. Abraham Andrew Leask 《PloS one》2009,4(10)
Background
Fibrosis, the excessive deposition of scar tissue by fibroblasts, is one of the largest groups of diseases for which there is no therapy. Fibroblasts from lesional areas of scleroderma patients possess elevated abilities to contract matrix and produce α−smooth muscle actin (α-SMA), type I collagen and CCN2 (connective tissue growth factor, CTGF). The basis for this phenomenon is poorly understood, and is a necessary prerequisite for developing novel, rational anti-fibrotic strategies.Methods and Findings
Compared to healthy skin fibroblasts, dermal fibroblasts cultured from lesional areas of scleroderma (SSc) patients possess elevated Rac activity. NSC23766, a Rac inhibitor, suppressed the persistent fibrotic phenotype of lesional SSc fibroblasts. NSC23766 caused a decrease in migration on and contraction of matrix, and α−SMA, type I collagen and CCN2 mRNA and protein expression. SSc fibroblasts possessed elevated Akt phosphorylation, which was also blocked by NSC23766. Overexpression of rac1 in normal fibroblasts induced matrix contraction and α−SMA, type I collagen and CCN2 mRNA and protein expression. Rac1 activity was blocked by PI3kinase/Akt inhibition. Basal fibroblast activity was not affected by NSC23766.Conclusion
Rac inhibition may be considered as a novel treatment for the fibrosis observed in SSc. 相似文献5.
Michael F Nyp Angels Navarro Mohammad H Rezaiekhaligh Ricardo E Perez Sherry M Mabry Ikechukwu I Ekekezie 《Respiratory research》2014,15(1):19
Background
Myofibroblasts are the critical effector cells in the pathogenesis of pulmonary fibrosis which carries a high degree of morbidity and mortality. We have previously identified Type II TGFβ receptor interacting protein 1 (TRIP-1), through proteomic analysis, as a key regulator of collagen contraction in primary human lung fibroblasts—a functional characteristic of myofibroblasts, and the last, but critical step in the process of fibrosis. However, whether or not TRIP-1 modulates fibroblast trans-differentiation to myofibroblasts is not known.Methods
TRIP-1 expression was altered in primary human lung fibroblasts by siRNA and plasmid transfection. Transfected fibroblasts were then analyzed for myofibroblast features and function such as α-SMA expression, collagen contraction ability, and resistance to apoptosis.Results
The down-regulation of TRIP-1 expression in primary human lung fibroblasts induces α-SMA expression and enhances resistance to apoptosis and collagen contraction ability. In contrast, TRIP-1 over-expression inhibits α-SMA expression. Remarkably, the effects of the loss of TRIP-1 are not abrogated by blockage of TGFβ ligand activation of the Smad3 pathway or by Smad3 knockdown. Rather, a TRIP-1 mediated enhancement of AKT phosphorylation is the implicated pathway. In TRIP-1 knockdown fibroblasts, AKT inhibition prevents α-SMA induction, and transfection with a constitutively active AKT construct drives collagen contraction and decreases apoptosis.Conclusions
TRIP-1 regulates fibroblast acquisition of phenotype and function associated with myofibroblasts. The importance of this finding is it suggests TRIP-1 expression could be a potential target in therapeutic strategy aimed against pathological fibrosis. 相似文献6.
Background
Diaphragmatic dysfunction found in the patients with acute lung injury required prolonged mechanical ventilation. Mechanical ventilation can induce production of inflammatory cytokines and excess deposition of extracellular matrix proteins via up-regulation of transforming growth factor (TGF)-β1. Lumican is known to participate in TGF-β1 signaling during wound healing. The mechanisms regulating interactions between mechanical ventilation and diaphragmatic injury are unclear. We hypothesized that diaphragmatic damage by short duration of mechanical stretch caused up-regulation of lumican that modulated TGF-β1 signaling.Methods
Male C57BL/6 mice, either wild-type or lumican-null, aged 3 months, weighing between 25 and 30 g, were exposed to normal tidal volume (10 ml/kg) or high tidal volume (30 ml/kg) mechanical ventilation with room air for 2 to 8 hours. Nonventilated mice served as control groups.Results
High tidal volume mechanical ventilation induced interfibrillar disassembly of diaphragmatic collagen fiber, lumican activation, type I and III procollagen, fibronectin, and α-smooth muscle actin (α-SMA) mRNA, production of free radical and TGF-β1 protein, and positive staining of lumican in diaphragmatic fiber. Mechanical ventilation of lumican deficient mice attenuated diaphragmatic injury, type I and III procollagen, fibronectin, and α-SMA mRNA, and production of free radical and TGF-β1 protein. No significant diaphragmatic injury was found in mice subjected to normal tidal volume mechanical ventilation.Conclusion
Our data showed that high tidal volume mechanical ventilation induced TGF-β1 production, TGF-β1-inducible genes, e.g., collagen, and diaphragmatic dysfunction through activation of the lumican. 相似文献7.
Amiq Gazdhar Njomeza Susuri Katrin Hostettler Mathias Gugger Lars Knudsen Michael Roth Matthias Ochs Thomas Geiser 《PloS one》2013,8(6)
Background
Pulmonary fibrosis may result from abnormal alveolar wound repair after injury. Hepatocyte growth factor (HGF) improves alveolar epithelial wound repair in the lung. Stem cells were shown to play a major role in lung injury, repair and fibrosis. We studied the presence, origin and antifibrotic properties of HGF-expressing stem cells in usual interstitial pneumonia.Methods
Immunohistochemistry was performed in lung tissue sections and primary alveolar epithelial cells obtained from patients with usual interstitial pneumonia (UIP, n = 7). Bone marrow derived stromal cells (BMSC) from adult male rats were transfected with HGF, instilled intratracheally into bleomycin injured rat lungs and analyzed 7 and 14 days later.Results
In UIP, HGF was expressed in specific cells mainly located in fibrotic areas close to the hyperplastic alveolar epithelium. HGF-positive cells showed strong co-staining for the mesenchymal stem cell markers CD44, CD29, CD105 and CD90, indicating stem cell origin. HGF-positive cells also co-stained for CXCR4 (HGF+/CXCR4+) indicating that they originate from the bone marrow. The stem cell characteristics were confirmed in HGF secreting cells isolated from UIP lung biopsies. In vivo experiments showed that HGF-expressing BMSC attenuated bleomycin induced pulmonary fibrosis in the rat, indicating a beneficial role of bone marrow derived, HGF secreting stem cells in lung fibrosis.Conclusions
HGF-positive stem cells are present in human fibrotic lung tissue (UIP) and originate from the bone marrow. Since HGF-transfected BMSC reduce bleomycin induced lung fibrosis in the bleomycin lung injury and fibrosis model, we assume that HGF-expressing, bone-marrow derived stem cells in UIP have antifibrotic properties. 相似文献8.
Background
Extracellular heat shock protein 70 and peptide complexes (eHSP70/HSP70-PCs) regulate a variety of biological behaviors in tumor cells. Whether eHSP70/HSP70-PCs are involved in the epithelial-mesenchymal transition (EMT) of tumor cells remains unclear.Aims
To determine the effects of eHSP70/HSP70-PCs on EMT of hepatocarcinoma cells.Methods
The expressions of E-cadherin, HSP70, α-smooth muscle actin protein (α-SMA) and p-p38 were detected immunohistochemically in liver cancer samples. Immunofluorescence, western blotting and real-time RT-PCR methods were used to analyze the effects of eHSP70/HSP70-PCs on the expressions of E-cadherin, α-SMA and p38/MAPK in vivo.Results
HSP70, E-cadherin, α-SMA and p-p38 were elevated in hepatocellular carcinoma tissues. The expression of HSP70 was positively correlated with malignant differentiated liver carcinoma. The expressions of HSP70, α-SMA and p-p38 correlated with recurrence-free survival after resection. eHSP70/HSP70-PCs significantly promoted the expressions of α-SMA and p-p38 and reduced the expressions of E-cadherin in vivo. The effect was inhibited by SB203580.Conclusion
The expressions of HSP70, E-cadherin, α-SMA and p-p38 may represent indicators of malignant potential and could discriminate the malignant degree of liver cancer. eHSP70/HSP70-PCs play an important role in the EMT of hepatocellular carcinoma via the p38/MAPK pathway. 相似文献9.
Nobuho Tanaka Yasuko Ikeda Tetsuo Yamaguchi Hiroshi Furukawa Hiroyuki Mitomi Takumi Nakagawa Shigeto Tohma Naoshi Fukui 《Arthritis research & therapy》2013,15(5):R127
Introduction
Articular chondrocytes undergo an obvious phenotypic change when cultured in monolayers. During this change, or dedifferentiation, the expression of type I and type III procollagen is induced where normal chondrocytes express little type I and type III procollagen. In this study, we attempted to determine the mechanism(s) for the induction of such procollagen expression in dedifferentiating chondrocytes.Methods
All experiments were performed using primary-cultured human articular chondrocytes under approval of institutional review boards. Integrin(s) responsible for the induction of type I and type III procollagen expression were specified by RNAi experiments. The signal pathway(s) involved in the induction were determined by specific inhibitors and RNAi experiments. Adenovirus-mediated experiments were performed to identify a small GTPase regulating the activity of integrins in dedifferentiating chondrocytes. The effect of inhibition of integrins on dedifferentiation was investigated by experiments using echistatin, a potent disintegrin. The effect of echistatin was investigated first with monolayer-cultured chondrocytes, and then with pellet-cultured chondrocytes.Results
In dedifferentiating chondrocytes, α5β1 integrin was found to be involved in the induction of type I and type III procollagen expression. The induction was known to be mediated by v-akt murine thymoma viral oncogene homolog (AKT) signaling. Among the three AKT isoforms, AKT1 seemed to be most involved in the signaling. Elated RAS viral (r-ras) oncogene homolog (RRAS) was considered to regulate the progression of dedifferentiation by modulating the affinity and avidity of α5β1 integrin to ligands. Echistatin inhibited dedifferentiation of monolayer-cultured chondrocytes. Furthermore, the matrix formed by pellet-cultured chondrocytes more closely resembled that of normal cartilage compared with the controls.Conclusions
The result of this study has shown, for the first time, that α5β1 integrin may be responsible for the induction of non-cartilaginous collagen expression in chondrocytes undergoing dedifferentiation. Again, this study has shown that the inhibition of ligand ligation to integrins may be an effective strategy to inhibit phenotypic change of cultured chondrocytes, and to improve the quality of matrix synthesized by primary cultured chondrocytes. 相似文献10.
Differentiation potential of CD14+ monocytes into myofibroblasts in patients with systemic sclerosis
Background
Circulating monocytes are a highly plastic and functionally heterogeneic cell type with an activated phenotype in patients with systemic sclerosis (SSc). CD14+ monocytes have the potential to differentiate into extra-cellular matrix (ECM) producing cells, possibly participating in fibrogenesis.Aim
To study the effect of GM-CSF, IL-4 and endothelin -1 (ET-1) alone or in combination on monocyte differentiation into myofibroblasts.Methods
CD14+ cells were isolated from peripheral blood from 14 SSc patients and healthy controls by positive selection and incubated with different combinations of GM-CSF, IL-4 and ET-1 for 14 days. Type-1 collagen and α-SMA were detected by Western blot, qPCR and confocal microscopy. HLA-DR, CD11c and CD14 expression was analysed by flow cytometry. A collagen gel contraction assay was performed for functional myofibroblast assessment.Results
GM-CSF both induced collagen and α-SMA expression after 14 days. ET-1 further increased GM-CSF-induced collagen expression in a dose dependent manner up to 30-fold. IL-4/GM-CSF combination leads to a more DC-like phenotype of monocytes associated with reduced collagen and α-SMA expression compared to GM-CSF alone. Collagen and α-SMA expression was higher in monocytes from SSc patients and monocytes were more prone to obtain a spindle form. In contrast to controls, ET-1 and IL-4 alone were sufficient to induce α-SMA expression in monocytes from SSc patients. Despite the induction of α-SMA expression, monocyte-derived myofibroblasts only had a moderate capability of contraction in functional analyses.Conclusion
SSc monocytes display increased maturation towards myofibroblasts demonstrated by their phenotype and α-SMA expression when compared to monocytes from healthy controls, however only with minor functional contraction properties. 相似文献11.
Stephen R Reeves Tessa Kolstad Tin-Yu Lien Sarah Herrington-Shaner Jason S Debley 《Respiratory research》2015,16(1)
Background
Airway remodeling is a proposed mechanism that underlies the persistent loss of lung function associated with childhood asthma. Previous studies have demonstrated that human lung fibroblasts (HLFs) co-cultured with primary human bronchial epithelial cells (BECs) from asthmatic children exhibit greater expression of extracellular matrix (ECM) components compared to co-culture with BECs derived from healthy children. Myofibroblasts represent a population of differentiated fibroblasts that have greater synthetic activity. We hypothesized co-culture with asthmatic BECs would lead to greater fibroblast to myofibroblast transition (FMT) compared to co-culture with healthy BECs.Methods
BECs were obtained from well-characterized asthmatic and healthy children and were proliferated and differentiated at an air-liquid interface (ALI). BEC-ALI cultures were co-cultured with HLFs for 96 hours. RT-PCR was performed in HLFs for alpha smooth muscle actin (α-SMA) and flow cytometry was used to assay for α-SMA antibody labeling of HLFs. RT-PCR was also preformed for the expression of tropomyosin-I as an additional marker of myofibroblast phenotype. In separate experiments, we investigated the role of TGFβ2 in BEC-HLF co-cultures using monoclonal antibody inhibition.Results
Expression of α-SMA by HLFs alone was greater than by HLFs co-cultured with healthy BECs, but not different than α-SMA expression by HLFs co-cultured with asthmatic BECs. Flow cytometry also revealed significantly less α-SMA expression by healthy co-co-cultures compared to asthmatic co-cultures or HLF alone. Monoclonal antibody inhibition of TGFβ2 led to similar expression of α-SMA between healthy and asthmatic BEC-HLF co-cultures. Expression of topomyosin-I was also significantly increased in HLF co-cultured with asthmatic BECs compared to healthy BEC-HLF co-cultures or HLF cultured alone.Conclusion
These findings suggest dysregulation of FMT in HLF co-cultured with asthmatic as compared to healthy BECs. Our results suggest TGFβ2 may be involved in the differential regulation of FMT by asthmatic BECs. These findings further illustrate the importance of BEC-HLF cross-talk in asthmatic airway remodeling. 相似文献12.
Robert Matthew Kottmann Jesse Sharp Kristina Owens Peter Salzman Guang-Qian Xiao Richard P. Phipps Patricia J. Sime Edward B. Brown Seth W. Perry 《Respiratory research》2015,16(1)
Background
It is not understood why some pulmonary fibroses such as cryptogenic organizing pneumonia (COP) respond well to treatment, while others like usual interstitial pneumonia (UIP) do not. Increased understanding of the structure and function of the matrix in this area is critical to improving our understanding of the biology of these diseases and developing novel therapies. The objectives herein are to provide new insights into the underlying collagen- and matrix-related biological mechanisms driving COP versus UIP.Methods
Two-photon second harmonic generation (SHG) and excitation fluorescence microscopies were used to interrogate and quantify differences between intrinsic fibrillar collagen and elastin matrix signals in healthy, COP, and UIP lung.Results
Collagen microstructure was different in UIP versus healthy lung, but not in COP versus healthy, as indicated by the ratio of forward-to-backward propagating SHG signal (FSHG/BSHG). This collagen microstructure as assessed by FSHG/BSHG was also different in areas with preserved alveolar architecture adjacent to UIP fibroblastic foci or honeycomb areas versus healthy lung. Fibrosis was evidenced by increased col1 and col3 content in COP and UIP versus healthy, with highest col1:col3 ratio in UIP. Evidence of elastin breakdown (i.e. reduced mature elastin fiber content), and increased collagen:mature elastin ratios, were seen in COP and UIP versus healthy.Conclusions
Fibrillar collagen’s subresolution structure (i.e. “microstructure”) is altered in UIP versus COP and healthy lung, which may provide novel insights into the biological reasons why unlike COP, UIP is resistant to therapies, and demonstrates the ability of SHG microscopy to potentially distinguish treatable versus intractable pulmonary fibroses. 相似文献13.
Background
Injury and intestinal inflammation trigger wound healing responses that can restore mucosal architecture but if chronic, can promote intestinal fibrosis. Intestinal fibrosis is a major complication of Crohn’s disease. The cellular and molecular basis of mucosal healing and intestinal fibrosis are not well defined and better understanding requires well characterized mouse models.Methods
FVB-N wild type mice and C57BL6 procollagen α1(I)-GFP reporter mice were given one (DSS1) or two (DSS2) cycles of 3% DSS (5 days/cycle) followed by 7 days recovery. Histological scoring of inflammation and fibrosis were performed at DSS1, DSS1+3, DSS1+7, DSS2, DSS2+3, and DSS2+7. Procollagen α1(I)-GFP activation was assessed in DSS and also TNBS models by whole colon GFP imaging and fluorescence microscopy. Colocalization of GFP with α-smooth muscle actin (α-SMA) or vimentin was examined. GFP mRNA levels were tested for correlation with endogenous collagen α1(I) mRNA.Results
Males were more susceptible to DSS-induced disease and mortality than females. In FVB-N mice one DSS cycle induced transient mucosal inflammation and fibrosis that resolved by 7 days of recovery. Two DSS cycles induced transmural inflammation and fibrosis in a subset of FVB-N mice but overall, did not yield more consistent, severe or sustained fibrosis. In C57BL6 mice, procollagen α1(I)-GFP reporter was activated at the end of DSS1 and through DSS+7 with more dramatic and transmural activation at DSS2 through DSS2+7, and in TNBS treated mice. In DSS and TNBS models GFP reporter expression localized to vimentin+ cells and much fewer α-SMA+ cells. GFP mRNA strongly correlated with collagen α1(I) mRNA.Conclusions
One DSS cycle in FVB-N mice provides a model to study mucosal injury and subsequent mucosal healing. The procollagen α1(I)-GFP transgenic provides a useful model to study activation of a gene encoding a major extracellular matrix protein during acute or chronic experimental intestinal inflammation and fibrosis. 相似文献14.
Claire M Robinson Roisin Neary Ashleigh Levendale Chris J Watson John A Baugh 《Respiratory research》2012,13(1):74
Background
Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.Methods
CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.Results
Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2′-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.Conclusion
These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia. 相似文献15.
Tong Liu Jian Zhang Jing Zhang Xin Mu Hui Su Xiaoding Hu Wenli Liu Enbing Zhao Weimin Li 《PloS one》2013,8(4)
Objective
To down-regulate expression of mRNA for the platelet-derived growth factor receptor (PDGFR)-α, block the signalling pathway of PDGF and its receptor, and study their influence on fibroblast transdifferentiation to myofibroblasts in systemic sclerosis (SSc).Methods
Fibroblasts from skin lesions of SSc patients and health adult controls were cultured in vitro, and α-smooth muscle actin (α-SMA) expression was determined by immunocytochemistry. Both groups of fibroblasts were stimulated with PDGF-AA, transforming growth factor β1 (TGF-β1), and costimulated with PDGF-AA and TGF-β1, then PDGFR-α and α-SMA mRNA and protein expression were detected with RT-PCR and WB respectively. Three pairs of siRNAs targeting different PDGFR-α mRNA sequences were synthesized for RNAi. SSc and control fibroblasts were transfected with PDGFR-α siRNA; stimulated with PDGF-AA; and assessed for PDGFR-α and α-SMA mRNA and protein expression.Results
Although the fibroblasts from both groups had similar morphology, the SSc skin lesions had significantly more myofibroblasts than control skin lesions. PDGF-AA stimulation, TGF-β1 stimulation, and costimulation significantly up-regulated PDGFR-α and α-SMA mRNA and protein expression in SSc fibroblasts compared to control (P<0.05), and costimulation had the strongest effects (P<0.05). All three pairs of siRNAs suppressed PDGFR-α mRNA and protein expression (P<0.05), but siRNA1495 had the highest gene-silencing efficiency (P<0.05). PDGFR-α siRNA attenuated the effects of PDGF-AA through up-regulating PDGFR-α and α-SMA mRNA and protein expression and inhibiting fibroblast transdifferentiation to myofibroblasts in SSc (P<0.05).Conclusions
PDGFR-α over-expression in SSc fibroblasts bound PDGF-AA more efficiently and promoted fibroblast transdifferentiation, which was enhanced by TGF-β1. PDGFR-α siRNA down-regulated PDGFR-α expression, blocked binding to PDGF-AA, and inhibited fibroblast transdifferentiation to myofibroblasts. 相似文献16.
Lei Chen Tao Wang Xun Wang Bei-Bei Sun Ji-Qiong Li Dai-Shun Liu Shang-Fu Zhang Lin Liu Dan Xu Ya-Juan Chen Fu-Qiang Wen 《Respiratory research》2009,10(1):55
Background
Advanced glycation end products (AGEs) have been proposed to be involved in pulmonary fibrosis, but its role in this process has not been fully understood. To investigate the role of AGE formation in pulmonary fibrosis, we used a bleomycin (BLM)-stimulated rat model treated with aminoguanidine (AG), a crosslink inhibitor of AGE formation.Methods
Rats were intratracheally instilled with BLM (5 mg/kg) and orally administered with AG (40, 80, 120 mg/kg) once daily for two weeks. AGEs level in lung tissue was determined by ELISA and pulmonary fibrosis was evaluated by Ashcroft score and hydroxyproline assay. The expression of heat shock protein 47 (HSP47), a collagen specific molecular chaperone, was measured with RT-PCR and Western blot. Moreover, TGFβ1 and its downstream Smad proteins were analyzed by Western blot.Results
AGEs level in rat lungs, as well as lung hydroxyproline content and Ashcroft score, was significantly enhanced by BLM stimulation, which was abrogated by AG treatment. BLM significantly increased the expression of HSP47 mRNA and protein in lung tissues, and AG treatment markedly decreased BLM-induced HSP47 expression in a dose-dependent manner (p < 0.05). In addition, AG dose-dependently downregulated BLM-stimulated overexpressions of TGFβ1, phosphorylated (p)-Smad2 and p-Smad3 protein in lung tissues.Conclusion
These findings suggest AGE formation may participate in the process of BLM-induced pulmonary fibrosis, and blockade of AGE formation by AG treatment attenuates BLM-induced pulmonary fibrosis in rats, which is implicated in inhibition of HSP47 expression and TGFβ/Smads signaling. 相似文献17.
Sushovan Chowdhury Rajdeep Guha Ruchit Trivedi Uday B. Kompella Aditya Konar Sarbani Hazra 《PloS one》2013,8(8)
Purpose
To evaluate the effects of pirfenidone nanoparticles on corneal re-epithelialization and scarring, major clinical challenges after alkali burn.Methods
Effect of pirfenidone on collagen I and α-smooth muscle actin (α-SMA) synthesis by TGFβ induced primary corneal fibroblast cells was evaluated by immunoblotting and immunocytochemistry. Pirfenidone loaded poly (lactide-co-glycolide) (PLGA) nanoparticles were prepared, characterized and their cellular entry was examined in primary corneal fibroblast cells by fluorescence microscopy. Alkali burn was induced in one eye of Sprague Dawley rats followed by daily topical treatment with free pirfenidone, pirfenidone nanoparticles or vehicle. Corneal re-epithelialization was assessed daily by flourescein dye test; absence of stained area indicated complete re-epithelialization and the time for complete re-epithelialization was determined. Corneal haze was assessed daily for 7 days under slit lamp microscope and graded using a standard method. After 7 days, collagen I deposition in the superficial layer of cornea was examined by immunohistochemistry.Results
Pirfenidone prevented (P<0.05) increase in TGF β induced collagen I and α-SMA synthesis by corneal fibroblasts in a dose dependent manner. Pirfenidone could be loaded successfully within PLGA nanoparticles, which entered the corneal fibroblasts within 5 minutes. Pirfenidone nanoparticles but not free pirfenidone significantly (P<0.05) reduced collagen I level, corneal haze and the time for corneal re-epithelialization following alkali burn.Conclusion
Pirfenidone decreases collagen synthesis and prevents myofibroblast formation. Pirfenidone nanoparticles improve corneal wound healing and prevent fibrosis. Pirfenidone nanoparticles are of potential value in treating corneal chemical burns and other corneal fibrotic diseases. 相似文献18.
Fayong Luo Yan Zhuang Mark D Sides Cecilia G Sanchez Bin Shan Eric S White Joseph A Lasky 《Respiratory research》2014,15(1):51
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. 相似文献19.
Tomoyuki Kakugawa Shin-ichi Yokota Yuji Ishimatsu Tomayoshi Hayashi Shota Nakashima Shintaro Hara Noriho Sakamoto Yasuhiro Matsuoka Hiroshi Kubota Mariko Mine Hiroshi Mukae Kazuhiro Nagata Shigeru Kohno 《Respiratory research》2013,14(1):133
Background
Heat shock protein (HSP) 47 is a collagen-specific molecular chaperone that is required for molecular maturation of various types of collagens. We recently reported that HSP47 serum levels were markedly higher in patients with acute exacerbations of idiopathic pulmonary fibrosis (IPF) when compared with patients with stable IPF, suggesting that serum HSP47 levels correlate with interstitial pneumonia activity. The aim of this study was to evaluate serum HSP47 levels in patients with drug-induced lung disease (DILD).Methods
Findings from high-resolution computed tomographic chest scans of 47 patients with DILD were classified into one of four predominant patterns: organizing pneumonia (OP) (n = 4), nonspecific interstitial pneumonia (NSIP) (n = 24), hypersensitivity pneumonitis (HP) (n = 11), and diffuse alveolar damage (DAD) (n = 8). Serum levels of HSP47, Krebs von den Lungen-6 (KL-6), surfactant protein (SP)-A, and SP-D were measured in these patients.Results
The PaO2/fraction of inspired oxygen (FiO2) (P/F) ratios were significantly lower and the alveolar-arterial difference of oxygen (A-a DO2) was significantly higher in the DAD group than in the other groups. Patients with DAD had the worst outcomes among the different subgroups. Patients in the DAD group had significantly higher serum HSP47 levels than those in other groups. Receiver operating characteristic curves revealed that HSP47 was superior to KL-6, SP-A, and SP-D for discriminating between the DAD group and the other groups. The cut-off level for HSP47 that resulted in the highest diagnostic accuracy was 1711.5 pg/mL. The sensitivity, specificity, and diagnostic accuracy were 87.5%, 97.4%, and 95.7%, respectively. Serum levels of HSP47 in the group of patients requiring glucocorticoids were significantly higher than those in patients who experienced clinical improvement without glucocorticoid administration. Serum HSP47 levels also significantly correlated with various respiratory parameters.Conclusion
This study demonstrated that serum HSP47 levels were elevated in patients with DILD with a DAD pattern who had the worst outcomes among the different subgroups, and that this was correlated with P/F ratio and A-a DO2. 相似文献20.
Xiao-Yong Man Kenneth W Finnson Murray Baron Anie Philip 《Arthritis research & therapy》2012,14(3):R144