Project description:To examine the effects of GSK199 treatment on antibody responses, a synovial antigen array analysis was performed using day 35 sera derived from mice with CIA treated with 30 mg/kg qd, 30 mg/kg bid GSK199 or saline. The synovial antigen arrays contain nearly 200 peptides and proteins, both citrullinated and uncitrullinated, representing candidate antigens in RA. Significance Analysis of Microarrays identified 7 and 14 autoantibody reactivities that were statistically decreased in CIA mice treated with 30 mg/kg qd or 30 mg/kg bid GSK199 respectively, compared to PBS treatment. By comparison, over 150 native and citrullinated epitopes on the arrays did not exhibit significant differences in reactivity. Sera from mice treated with 30 mg/kg GSK199 qd exhibited decreased IgG reactivity to native epitopes such as peptides from human fibrinogen alpha and beta chains and keratin while sera from mice treated with 30 mg/kg bid GSK199 demonstrated decreased IgG reactivity to native epitopes including collagen IV, heat shock protein 65, peptides from human fibrinogen alpha and beta chains (two of which match those that were decreased in the 30 mg/kg qd group), and vimentin as compared to saline controls (q < 0.1%). Decreased autoantibody reactivity to citrullinated peptides from vimentin and fibromodulin were also observed in sera from mice treated with 30 mg/kg bid GSK199 compared to controls treated with saline (q < 0.1%), but not by GSK199 30 mg/kg qd. There were no differences between treatment with GSK199 or saline alone in the levels of autoantibodies to 38 other citrullinated proteins in the array. These data demonstrated that GSK199 treatment decreases the development of autoantibodies in CIA and impairs epitope spreading to a degree similar to that observed with pan-PAD inhibition.
Project description:3D scaffolds collagen I-based were crosslinked with different percentages of 1, 4-butanediol diglycidyl ether (BDDGE) to mimic native tissue and tumour tissue. Normal fibroblasts (NFs) or cancer-associated fibroblasts (CAFs) were added to the system to assess how mechanical features influence stromal compartment in native or tumour-like systems.
Project description:Auto-antibody (Ab) profiles between acute-onset diffuse ILD (AoDILD) and stable states of 25 collagen disease patients were compared to screen biomarkers or pathogenic auto-Abs.
Project description:Collagen deposition is a key process during idiopathic pulmonary fibrosis (IPF); however, little is known about the dynamics of collagen formation during disease development. Tissue samples of early stages of human disease are not readily available and it is difficult to identify changes in collagen content, since standard collagen analysis does not distinguish between 'old' and 'new' collagen. Therefore, the current study aimed to (i) investigate the dynamics of new collagen formation in mice using bleomycin-induced lung fibrosis in which newly synthesized collagen was labelled with deuterated water and (ii) use this information to identify genes and processes correlated to new collagen formation from gene expression analysis. Lung fibrosis was induced in female C57BL/6 mice by bleomycin instillation and sacrificed. Animals were sacrificed at 1 to 5 weeks after fibrosis induction. Collagen synthesized during the week before sacrifice was labelled with deuterium by providing mice with deuterated drinking water. After sacrifice, lung tissue was collected for microarray analysis, determination of new collagen formation, and histology. Deuterated water labelling showed a strong increase in new collagen formation already during the first week after fibrosis induction and a complete return to baseline at five weeks. Correlation of new collagen formation data with gene expression data revealed fibrosis specific processes, of which proliferation was an unexpected one. This was confirmed by measuring cell proliferation and collagen synthesis simultaneously using deuterated water incorporation. Furthermore, new collagen formation strongly correlated with gene expression of e.g. elastin, tenascin C, MMP-14, lysyl oxidase, and type V collagen. These data demonstrate, using a novel combination of technologies, that proliferation and extracellular matrix production are correlated to the core process of fibrosis, i.e. the formation of new collagen. In addition, it identified genes directly correlated to fibrosis, thus providing more insight into the aetiology of IPF. Total RNA was obtained from mouse lungs at timepoint 0 as a control (n = 7) or timepoints 1 (n = 7), 2 (n = 6), 3 (n = 6), 4 (n = 6) or 5 (n = 6) weeks after bleomycin-instillation to induce lung fibrosis.
Project description:Congenital aortic valve stenosis (CAVS) affects up to 10% of the world population without medical therapies to treat the disease. New molecular targets are continually being sought that can halt CAVS progression. Collagen deregulation is a hallmark of CAVS yet remains mostly undefined. Here, histological studies were paired with high resolution accurate mass (HRAM) collagen-targeting proteomics to investigate collagen fiber production with collagen regulation associated with human AV development and pediatric end-stage CAVS (pCAVS). Histological studies identified collagen fiber realignment and unique regions of high-density collagen in pCAVS. Proteomic analysis reported specific collagen peptides are modified by hydroxylated prolines (HYP), a post-translational modification critical to stabilizing the collagen triple helix. Quantitative data analysis reported significant regulation of collagen HYP sites across patient categories. Non-collagen type ECM proteins identified (26/44; 59%) have direct interactions in collagen synthesis, regulation, or modification. Network analysis identified BAMBI (BMP and Activin Membrane Bound Inhibitor) as a potential upstream regulator of the collagen interactome. This is the first study to detail the collagen types and HYP modifications associated with human AV development and pCAVS. We anticipate that this study will inform new therapeutic avenues that inhibit valvular degradation in pCAVS and engineered options for valve replacement.
Project description:Pathological fibrosis, an excessive accumulation of collagen, compromises tissue architecture, impedes its function and represents a medical complication. Properties of the amniotic membrane have demonstrated potential to regulate fibrotic processes. RNA sequencing analysis evaluated the effects of dehydrated human amnion/chorion membrane (DHACM) on fibrotic events using in vitro macromolecular crowding to mimic a physiologically relevant microenvironment. Functional enrichment with Reactome pathway analysis indicated that the function of a subset of these dysregulated genes was associated with the composition and function of the extracellular matrix. Further use of macromolecular crowding in vitro was used to evaluate the effects of DHACM and a lyophilized human amnion/chorion membrane (LHACM) on type 1 collagen production and deposition under profibrotic conditions (addition of TGFβ1). In the continued presence of TGFβ1, LHACM and DHACM regulated the intracellular production and extracellular deposition of type 1 collagen. In addition, results suggest that the effect on collagen deposition could in part be modulated through the regulation of proteins responsible for the extracellular post-translational modifications of collagen. These observations may support the use of DHACM and LHACM in the regulation collagen production/deposition in profibrotic environments.
Project description:Collagen deposition is a key process during idiopathic pulmonary fibrosis (IPF); however, little is known about the dynamics of collagen formation during disease development. Tissue samples of early stages of human disease are not readily available and it is difficult to identify changes in collagen content, since standard collagen analysis does not distinguish between 'old' and 'new' collagen. Therefore, the current study aimed to (i) investigate the dynamics of new collagen formation in mice using bleomycin-induced lung fibrosis in which newly synthesized collagen was labelled with deuterated water and (ii) use this information to identify genes and processes correlated to new collagen formation from gene expression analysis. Lung fibrosis was induced in female C57BL/6 mice by bleomycin instillation and sacrificed. Animals were sacrificed at 1 to 5 weeks after fibrosis induction. Collagen synthesized during the week before sacrifice was labelled with deuterium by providing mice with deuterated drinking water. After sacrifice, lung tissue was collected for microarray analysis, determination of new collagen formation, and histology. Deuterated water labelling showed a strong increase in new collagen formation already during the first week after fibrosis induction and a complete return to baseline at five weeks. Correlation of new collagen formation data with gene expression data revealed fibrosis specific processes, of which proliferation was an unexpected one. This was confirmed by measuring cell proliferation and collagen synthesis simultaneously using deuterated water incorporation. Furthermore, new collagen formation strongly correlated with gene expression of e.g. elastin, tenascin C, MMP-14, lysyl oxidase, and type V collagen. These data demonstrate, using a novel combination of technologies, that proliferation and extracellular matrix production are correlated to the core process of fibrosis, i.e. the formation of new collagen. In addition, it identified genes directly correlated to fibrosis, thus providing more insight into the aetiology of IPF.