Cell adhesion assay.

<p>Adherence of UCMD fibroblasts (n = 7) relative to control fibroblasts (n = 7) for fibronectin, vitronectin, laminin and collagen type I (experiments were performed in triplicate). To normalize adhesion values between experiments, we expressed the results as a ratio between the absorbance values for collagen type IV (which was the substrate that showed the smallest variability between individual cultures and experiments, data not shown) and each ECM protein, (student t-test * p < 0.05).</p

Ingenuity Pathway Analysis.

<p>Graphic representation of the network “cell cycle, skeletal and muscular system development”. Nodes represent genes and lines show the relationship between genes. The intensity of the node color indicates the degree of the up-regulation (red) or down-regulation (green) of significant genes in the P-C comparison. Non-color nodes are added by the tool. For a detailed legend refer to <a href="http://ingenuity.force.com/ipa/articles/Feature_Description/Legend" target="_blank">http://ingenuity.force.com/ipa/articles/Feature_Description/Legend</a>.</p

Co-variance analysis was used to plot the expression values of a selection of genes of interest in untreated patient cells (P), control cells (C) and patient cells treated with AA (P_AA).

<p>Median and ranges are represented. A. HLA-DRA, B. COL1A1, C. TNC, D. TNX.</p

miRNAs expression analysis.

<p>Real-time PCR was used to measure relative expression of miR-181a and miR-30c in skeletal muscle (A) and fibroblasts (B) from UCMD patients and in serum samples from UCMD, BM and DMD patients for miR-181a (C) and miR-30c (D). miRNA expression level was normalized against U6 miRNA. Results were calculated relative to control samples and are represented as mean and standard error.</p

Integrin-α3 expression in collagen VI deficient fibroblasts.

<p>A. Immunofluorescence for integrin-α3 in confluent fibroblast cultures. B. Representative western blot analysis. The intensity of the bands corresponding to integrin-α3 (ITGA3) was quantified by densitometry using α-tubulin (A-TUB) as a loading control and expressed as arbitrary units relative to the control samples.</p

Extracellular matrix gene correlation network.

<p>This network represents Pearson correlations (R >0.8, p-value<0.005) computed considering expression values of genes of interest according to the microarray data and using Cytoscape tool. We selected those genes on the ECM-receptor interaction KEGG pathway and COL6A genes. Continuous lines represent positive correlations and discontinuous lines negative ones. Those correlations that are significant in patients´cells only are represented in black lines. Orange lines represent those correlations that are present in both patients and control cells but are of different sign (positive or negative) whereas those that have the same sign are represented by lilac lines. Red lines around gene symbols represent significantly over-expressed genes and green lines those that were under-expressed in patients´ fibroblasts relative to control fibroblasts.</p

Gene Expression Profiling Identifies Molecular Pathways Associated with Collagen VI Deficiency and Provides Novel Therapeutic Targets

<div><p>Ullrich congenital muscular dystrophy (UCMD), caused by collagen VI deficiency, is a common congenital muscular dystrophy. At present, the role of collagen VI in muscle and the mechanism of disease are not fully understood. To address this we have applied microarrays to analyse the transcriptome of UCMD muscle and compare it to healthy muscle and other muscular dystrophies. We identified 389 genes which are differentially regulated in UCMD relative to controls. In addition, there were 718 genes differentially expressed between UCMD and dystrophin deficient muscle. In contrast, only 29 genes were altered relative to other congenital muscular dystrophies. Changes in gene expression were confirmed by real-time PCR. The set of regulated genes was analysed by Gene Ontology, KEGG pathways and Ingenuity Pathway analysis to reveal the molecular functions and gene networks associated with collagen VI defects. The most significantly regulated pathways were those involved in muscle regeneration, extracellular matrix remodelling and inflammation. We characterised the immune response in UCMD biopsies as being mainly mediated via M2 macrophages and the complement pathway indicating that anti-inflammatory treatment may be beneficial to UCMD as for other dystrophies. We studied the immunolocalisation of ECM components and found that biglycan, a collagen VI interacting proteoglycan, was reduced in the basal lamina of UCMD patients. We propose that biglycan reduction is secondary to collagen VI loss and that it may be contributing towards UCMD pathophysiology. Consequently, strategies aimed at over-expressing biglycan and restore the link between the muscle cell surface and the extracellular matrix should be considered. </p> </div

Characterization of inflammation found in UCMD muscle.

<p>HLA immunostaining in healthy muscle sections located in the endothelial cells of capillaries (<b>A</b>). Sarcolemmal and cytoplasmatic HLA staining on UCMD muscle sections including strong staining of mononucleated cells (<b>B</b>). Immunohistochemistry for CD68 demonstrate macrophage infiltration (<b>C</b>). Immunohistochemistry for CD206 identified M2-type macrophages (<b>D</b>). Arrows point to mononucleated cells CD68+ and CD206+ whereas arrowheads point to only CD68+ cells. Scale bar: 50 µm.</p

Secondary reduction of biglycan at the basal lamina of UCMD patients.

<p>The integrity of the basal lamina and the reduction of biglycan was further studied using confocal microscopy. An example for UCMD patient 6 at two different magnifications is shown. Arrows point to representative areas where biglycan (A, D, G) appears reduced relative to perlecan (B, E, H). Scale bar: 50 µm.</p

GO BP categories in UCMD vs control muscle.

<p>Pie chart representing GO BP categories up-regulated in UCMD vs control muscle.</p