Effect of Protein Kinase C delta (PKC-δ) Inhibition on the Transcriptome of Normal and Systemic Sclerosis Human Dermal Fibroblasts In Vitro

Previous studies demonstrated that protein kinase C- δ (PKC-δ) inhibition with the selective inhibitor, rottlerin, resulted in potent downregulation of type I collagen expression and production in normal human dermal fibroblasts and abrogated the exaggerated type I collagen production and expression in fibroblasts cultured from affected skin from patients with the fibrosing disorder systemic sclerosis (SSc). To elucidate the mechanisms involved in the ability of PKC-δ to regulate collagen production in fibroblasts, we examined the effects of PKC-δ inhibition on the transcriptome of normal and SSc human dermal fibroblasts. Normal and SSc human dermal fibroblasts were incubated with rottlerin (5 µM), and their gene expression was analyzed by microarrays. Pathway analysis and gene ontology analysis of differentially expressed genes in each comparison were performed. Identification of significantly overrepresented transcriptional regulatory elements (TREs) was performed using the Promoter Analysis and Interaction Network Toolset (PAINT) program. PKC-δ activity was also inhibited using RNA interference (siRNA) and by treating fibroblasts with a specific PKC-δ inhibitory cell permeable peptide. Differential gene expression of 20 genes was confirmed using real time PCR. PKC-δ inhibition caused a profound change in the transcriptome of normal and SSc human dermal fibroblasts in vitro. Pathway and gene ontology analysis identified multiple cellular and organismal pathways affected by PKC-δ inhibition. Furthermore, both pathway and PAINT analyses indicated that the transcription factor NFκB played an important role in the transcriptome changes induced by PKC-δ inhibition. Multiple genes involved in the degradation of the extracellular matrix components were significantly reduced in SSc fibroblasts and their expression was increased by PKC-δ inhibition. These results indicate that isoform-specific inhibition of PKC-δ profibrotic effects may represent a novel therapeutic approach for SSc and other fibrotic diseases

A central role for G9a and EZH2 in the epigenetic silencing of cyclooxygenase-2 in idiopathic pulmonary fibrosis

Selective silencing of the cyclooxygenase-2 (COX-2) gene with the loss of the antifibrotic mediator PGE2 contributes to the fibrotic process in idiopathic pulmonary fibrosis (IPF). This study explored the role of G9a- and EZH2-mediated methylation of histone H3 lysine 9 (H3K9me3) and 27 (H3K27me3) in COX-2 silencing in IPF. Chromatin immunoprecipitation (ChIP) and Re-ChIP assays demonstrated marked increases in H3K9me3, H3K27me3 and DNA methylation, together with their respective modifying enzymes G9a, EZH2 and DNA methyltransferases (Dnmts) and respective binding proteins heterochromatin protein 1 (HP1), polycomb protein complex 1 (PRC1) and MeCP2, at the COX-2 promoter in lung fibroblasts from IPF patients (F-IPF) compared with fibroblasts from non-fibrotic lungs (F-NL). HP1, EZH2 and MeCP2 in turn were associated with additional repressive chromatin modifiers in F-IPF. G9a and EZH2 inhibitors and siRNAs and Dnmt1 inhibitor markedly reduced H3K9me3 (49-79%), H3K27me3 (44-81%) and DNA methylation (61-97%) at the COX-2 promoter. This was correlated with increased histone H3 and H4 acetylation, resulting in COX-2 mRNA and protein re-expression in F-IPF. Our results support a central role for G9a- and EZH2-mediated histone hypermethylation and a model of bidirectional, mutually reinforcing and interdependent crosstalk between histone hypermethylation and DNA methylation in COX-2 epigenetic silencing in IPF

The Membrane-Associated Adaptor Protein DOK5 Is Upregulated in Systemic Sclerosis and Associated with IGFBP-5-Induced Fibrosis

Systemic sclerosis (SSc) is characterized by excessive fibrosis of the skin and internal organs due to fibroblast proliferation and excessive production of extracellular matrix (ECM). We have shown that insulin-like growth factor binding protein (IGFBP)-5 plays an important role in the development of fibrosis in vitro, ex vivo, and in vivo. We identified a membrane-associated adaptor protein, downstream of tyrosine kinase/docking protein (DOK)5, as an IGFBP-5-regulated target gene using gene expression profiling of primary fibroblasts expressing IGFBP-5. DOK5 is a tyrosine kinase substrate associated with intracellular signaling. Our objective was to determine the role of DOK5 in the pathogenesis of SSc and specifically in IGFBP-5-induced fibrosis. DOK5 mRNA and protein levels were increased in vitro by endogenous and exogenous IGFBP-5 in primary human fibroblasts. DOK5 upregulation required activation of the mitogen-activated protein kinase (MAPK) signaling cascade. Further, IGFBP-5 triggered nuclear translocation of DOK5. DOK5 protein levels were also increased in vivo in mouse skin and lung by IGFBP-5. To determine the effect of DOK5 on fibrosis, DOK5 was expressed ex vivo in human skin in organ culture. Expression of DOK5 in human skin resulted in a significant increase in dermal thickness. Lastly, levels of DOK5 were compared in primary fibroblasts and lung tissues of patients with SSc and healthy donors. Both DOK5 mRNA and protein levels were significantly increased in fibroblasts and skin tissues of patients with SSc compared with those of healthy controls, as well as in lung tissues of SSc patients. Our findings suggest that IGFBP-5 induces its pro-fibrotic effects, at least in part, via DOK5. Furthermore, IGFBP-5 and DOK5 are both increased in SSc fibroblasts and tissues and may thus be acting in concert to promote fibrosis

TGF-β Isoform Specific Regulation of Airway Inflammation and Remodelling in a Murine Model of Asthma

The TGF-β family of mediators are thought to play important roles in the regulation of inflammation and airway remodelling in asthma. All three mammalian isoforms of TGF-β, TGF-β1–3, are expressed in the airways and TGF-β1 and -β2 are increased in asthma. However, there is little information on the specific roles of individual TGF-β isoforms. In this study we assess the roles of TGF-β1 and TGF-β2 in the regulation of allergen-induced airway inflammation and remodelling associated with asthma, using a validated murine model of ovalbumin sensitization and challenge, and isoform specific TGF-β neutralising antibodies. Antibodies to both isoforms inhibited TGF-β mediated Smad signalling. Anti-TGF-β1 and anti-TGF-β2 inhibited ovalbumin-induced sub-epithelial collagen deposition but anti-TGF-β1 also specifically regulated airway and fibroblast decorin deposition by TGF-β1. Neither antibody affected the allergen-induced increase in sub-epithelial fibroblast-like cells. Anti- TGF-β1 also specifically inhibited ovalbumin-induced increases in monocyte/macrophage recruitment. Whereas, both TGF-β1 and TGF-β2 were involved in regulating allergen-induced increases in eosinophil and lymphocyte numbers. These data show that TGF-β1 and TGF-β2 exhibit a combination of specific and shared roles in the regulation of allergen-induced airway inflammation and remodelling. They also provide evidence in support of the potential for therapeutic regulation of specific subsets of cells and extracellular matrix proteins associated with inflammation and remodelling in airway diseases such as asthma and COPD, as well as other fibroproliferative diseases

Smoking Is Associated with Shortened Airway Cilia

BACKGROUND:Whereas cilia damage and reduced cilia beat frequency have been implicated as causative of reduced mucociliary clearance in smokers, theoretically mucociliary clearance could also be affected by cilia length. Based on models of mucociliary clearance predicting that cilia length must exceed the 6-7 microm airway surface fluid depth to generate force in the mucus layer, we hypothesized that cilia height may be decreased in airway epithelium of normal smokers compared to nonsmokers. METHODOLOGY/PRINCIPAL FINDINGS:Cilia length in normal nonsmokers and smokers was evaluated in aldehyde-fixed, paraffin-embedded endobronchial biopsies, and air-dried and hydrated samples were brushed from human airway epithelium via fiberoptic bronchoscopy. In 28 endobronchial biopsies, healthy smoker cilia length was reduced by 15% compared to nonsmokers (p<0.05). In 39 air-dried samples of airway epithelial cells, smoker cilia length was reduced by 13% compared to nonsmokers (p<0.0001). Analysis of the length of individual, detached cilia in 27 samples showed that smoker cilia length was reduced by 9% compared to nonsmokers (p<0.05). Finally, in 16 fully hydrated, unfixed samples, smoker cilia length was reduced 7% compared to nonsmokers (p<0.05). Using genome-wide analysis of airway epithelial gene expression we identified 6 cilia-related genes whose expression levels were significantly reduced in healthy smokers compared to healthy nonsmokers. CONCLUSIONS/SIGNIFICANCE:Models predict that a reduction in cilia length would reduce mucociliary clearance, suggesting that smoking-associated shorter airway epithelial cilia play a significant role in the pathogenesis of smoking-induced lung disease

Vanadium (β-(Dimethylamino)ethyl)cyclopentadienyl Complexes with Diphenylacetylene Ligands

Reduction of the V(III) (β-(dimethylamino)ethyl)cyclopentadienyl dichloride complex [η5:η1-C5H4(CH2)2NMe2]VCl2(PMe3) with 1 equiv of Na/Hg yielded the V(II) dimer {[η5:η1-C5H4(CH2)2NMe2]V(µ-Cl)}2 (2). This compound reacted with diphenylacetylene in THF to give the V(II) alkyne adduct [η5:η1-C5H4(CH2)2NMe2]VCl(η2-PhC≡CPh). Further reduction of 2 with Mg in the presence of diphenylacetylene resulted in oxidative coupling of two diphenylacetylene groups to yield the diamagnetic, formally V(V), bent metallacyclopentatriene complex [η5:η1-C5H4(CH2)2NMe2]V(C4Ph4).

Immature Blood Vessels in Rheumatoid Synovium Are Selectively Depleted in Response to Anti-TNF Therapy

BACKGROUND:Angiogenesis is considered an important factor in the pathogenesis of Rheumatoid Arthritis (RA) where it has been proposed as a therapeutic target. In other settings, active angiogenesis is characterized by pathologic, immature vessels that lack periendothelial cells. We searched for the presence of immature vessels in RA synovium and analyzed the dynamics of synovial vasculature along the course of the disease, particularly after therapeutic response to TNF antagonists. METHODOLOGY/PRINCIPAL FINDINGS:Synovial arthroscopic biopsies from RA, osteoarthritis (OA) and normal controls were analyzed by double labeling of endothelium and pericytes/smooth muscle mural cells to identify and quantify mature/immature blood vessels. To analyze clinicopathological correlations, a cross-sectional study on 82 synovial biopsies from RA patients with variable disease duration and severity was performed. A longitudinal analysis was performed in 25 patients with active disease rebiopsied after anti-TNF-alpha therapy. We found that most RA synovial tissues contained a significant fraction of immature blood vessels lacking periendothelial coverage, whereas they were rare in OA, and inexistent in normal synovial tissues. Immature vessels were observed from the earliest phases of the disease but their presence or density was significantly increased in patients with longer disease duration, higher activity and severity, and stronger inflammatory cell infiltration. In patients that responded to anti-TNF-alpha therapy, immature vessels were selectively depleted. The mature vasculature was similarly expanded in early or late disease and unchanged by therapy. CONCLUSION/SIGNIFICANCE:RA synovium contains a significant fraction of neoangiogenic, immature blood vessels. Progression of the disease increases the presence and density of immature but not mature vessels and only immature vessels are depleted in response to anti-TNFalpha therapy. The different dynamics of the mature and immature vascular fractions has important implications for the development of anti-angiogenic interventions in RA

The HLA class II allele DRB1*1501 is over-represented in patients with idiopathic pulmonary fibrosis

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and medically refractory lung disease with a grim prognosis. Although the etiology of IPF remains perplexing, abnormal adaptive immune responses are evident in many afflicted patients. We hypothesized that perturbations of human leukocyte antigen (HLA) allele frequencies, which are often seen among patients with immunologic diseases, may also be present in IPF patients. Methods/Principal Findings: HLA alleles were determined in subpopulations of IPF and normal subjects using molecular typing methods. HLA-DRB1*15 was over-represented in a discovery cohort of 79 Caucasian IPF subjects who had lung transplantations at the University of Pittsburgh (36.7%) compared to normal reference populations. These findings were prospectively replicated in a validation cohort of 196 additional IPF subjects from four other U.S. medical centers that included both ambulatory patients and lung transplantation recipients. High-resolution typing was used to further define specific HLA-DRB1*15 alleles. DRB1*1501 prevalence in IPF subjects was similar among the 143 ambulatory patients and 132 transplant recipients (31.5% and 34.8%, respectively, p = 0.55). The aggregate prevalence of DRB1*1501 in IPF patients was significantly greater than among 285 healthy controls (33.1% vs. 20.0%, respectively, OR 2.0; 95%CI 1.3-2.9, p = 0.0004). IPF patients with DRB1*1501 (n = 91) tended to have decreased diffusing capacities for carbon monoxide (DLCO) compared to the 184 disease subjects who lacked this allele (37.8±1.7% vs. 42.8±1.4%, p = 0.036). Conclusions/Significance: DRB1*1501 is more prevalent among IPF patients than normal subjects, and may be associated with greater impairment of gas exchange. These data are novel evidence that immunogenetic processes can play a role in the susceptibility to and/or manifestations of IPF. Findings here of a disease association at the HLA-DR locus have broad pathogenic implications, illustrate a specific chromosomal area for incremental, targeted genomic study, and may identify a distinct clinical phenotype among patients with this enigmatic, morbid lung disease

Interferon-gamma inducible protein-10 as a potential biomarker in localized scleroderma

Introduction: The purpose of this study was to evaluate the presence and levels of interferon-gamma inducible protein-10 (IP-10) in the plasma and skin of pediatric localized scleroderma (LS) patients compared to those of healthy pediatric controls and to determine if IP-10 levels correlate to clinical disease activity measures.Methods: The presence of IP-10 in the plasma was analyzed using a Luminex panel in 69 pediatric patients with LS and compared to 71 healthy pediatric controls. Of these patients, five had available skin biopsy specimens with concurrent clinical and serological data during the active disease phase, which were used to analyze the presence and location of IP-10 in the skin by immunohistochemistry (IHC).Results: IP-10 levels were significantly elevated in the plasma of LS patients compared to that of healthy controls and correlated to clinical disease activity measures in LS. Immunohistochemistry staining of IP-10 was present in the dermal infiltrate of LS patients and was similar to that found in psoriasis skin specimens, the positive disease control.Conclusions: Elevation of IP-10 levels in the plasma compared to those of healthy controls and the presence of IP-10 staining in the affected skin of LS patients indicates that IP-10 is a potential biomarker in LS. Furthermore, significant elevation of IP-10 in LS patients with active versus inactive disease and correlations between IP-10 levels and standardized disease outcome measures of activity in LS strongly suggest that IP-10 may be a biomarker for disease activity in LS. © 2013 Magee et al.; licensee BioMed Central Ltd

miR-155 in the progression of lung fibrosis in systemic sclerosis

Background: MicroRNA (miRNA) control key elements of mRNA stability and likely contribute to the dysregulated lung gene expression observed in systemic sclerosis associated interstitial lung disease (SSc-ILD). We analyzed the miRNA gene expression of tissue and cells from patients with SSc-ILD. A chronic lung fibrotic murine model was used. Methods: RNA was isolated from lung tissue of 12 patients with SSc-ILD and 5 controls. High-resolution computed tomography (HRCT) was performed at baseline and 2-3 years after treatment. Lung fibroblasts and peripheral blood mononuclear cells (PBMC) were isolated from healthy controls and patients with SSc-ILD. miRNA and mRNA were analyzed by microarray, quantitative polymerase chain reaction, and/or Nanostring; pathway analysis was performed by DNA Intelligent Analysis (DIANA)-miRPath v2.0 software. Wild-type and miR-155 deficient (miR-155ko) mice were exposed to bleomycin. Results: Lung miRNA microarray data distinguished patients with SSc-ILD from healthy controls with 185 miRNA differentially expressed (q < 0.25). DIANA-miRPath revealed 57 Kyoto Encyclopedia of Genes and Genomes pathways related to the most dysregulated miRNA. miR-155 and miR-143 were strongly correlated with progression of the HRCT score. Lung fibroblasts only mildly expressed miR-155/miR-21 after several stimuli. miR-155 PBMC expression strongly correlated with lung function tests in SSc-ILD. miR-155ko mice developed milder lung fibrosis, survived longer, and weaker lung induction of several genes after bleomycin exposure compared to wild-type mice. Conclusions: miRNA are dysregulated in the lungs and PBMC of patients with SSc-ILD. Based on mRNA-miRNA interaction analysis and pathway tools, miRNA may play a role in the progression of the disease. Our findings suggest that targeting miR-155 might provide a novel therapeutic strategy for SSc-ILD