Role Of Extracellular Matrix Proteins Research Articles

Deciphering the spatiotemporal transcriptional landscape of intestinal diseases (Review).

The intestines are the largest barrier organ in the human body. The intestinal barrier plays a crucial role in maintaining the balance of the intestinal environment and protecting the intestines from harmful bacterial invasion. Single‑cell RNA sequencing technology allows the detection of the different cell types in the intestine in two dimensions and the exploration of cell types that have not been fully characterized. The intestinal mucosa is highly complex in structure, and its proper functioning is linked to multiple structures in the proximal‑distal intestinal and luminal‑mucosal axes. Spatial localization is at the core of the efforts to explore the interactions between the complex structures. Spatial transcriptomics (ST) is a method that allows for comprehensive tissue analysis and the acquisition of spatially separated genetic information from individual cells, while preserving their spatial location and interactions. This approach also prevents the loss of fragile cells during tissue disaggregation. The emergence of ST technology allows us to spatially dissect enzymatic processes and interactions between multiple cells, genes, proteins and signals in the intestine. This includes the exchange of oxygen and nutrients in the intestine, different gradients of microbial populations and the role of extracellular matrix proteins. This regionally precise approach to tissue studies is gaining more acceptance and is increasingly applied in the investigation of disease mechanisms related to the gastrointestinal tract. Therefore, this review summarized the application of ST in gastrointestinal diseases.

A role for vessel-associated extracellular matrix proteins in multiple sclerosis pathology.

Multiple sclerosis (MS) is unsurpassed for its clinical and pathological hetherogeneity, but the biological determinants of this variability are unknown. HLA-DRB1*15, the main genetic risk factor for MS, influences the severity and distribution of MS pathology. This study set out to unravel the molecular determinants of the heterogeneity of MS pathology in relation to HLA-DRB1*15 status. Shotgun proteomics from a discovery cohort of MS spinal cord samples segregated by HLA-DRB*15 status revealed overexpression of the extracellular matrix (ECM) proteins, biglycan, decorin, and prolargin in HLA-DRB*15-positive cases, adding to established literature on a role of ECM proteins in MS pathology that has heretofore lacked systematic pathological validation. These findings informed a neuropathological characterisation of these proteins in a large autopsy cohort of 41 MS cases (18 HLA-DRB1*15-positive and 23 HLA-DRB1*15-negative), and seven non-neurological controls on motor cortical, cervical and lumbar spinal cord tissue. Biglycan and decorin demonstrate a striking perivascular expression pattern in controls that is reduced in MS (-36.5%, p = 0.036 and - 24.7%, p = 0.039; respectively) in lesional and non-lesional areas. A concomitant increase in diffuse parenchymal accumulation of biglycan and decorin is seen in MS (p = 0.015 and p = 0.001, respectively), particularly in HLA-DRB1*15-positive cases (p = 0.007 and p = 0.046, respectively). Prolargin shows a faint parenchymal pattern in controls that is markedly increased in MS cases where a perivascular deposition pattern is observed (motor cortex +97.5%, p = 0.001; cervical cord +49.1%, p = 0.016). Our findings point to ECM proteins and the vascular interface playing a central role in MS pathology within and outside the plaque area. As ECM proteins are known potent pro-inflammatory molecules, their parenchymal accumulation may contribute to disease severity. This study brings to light novel factors that may contribute to the heterogeneity of the topographical variation of MS pathology.

Genetic Variants in Matrix Metalloproteinases MMP3 (rs3025058) and MMP9 (rs3918242) Associated with Colonic Diverticulosis.

Background and Objectives: Diverticulosis affects a significant portion of the elderly population, with age and lifestyle being established risk factors. Additionally, genetic predisposition is gaining recognition as a contributing factor. This pilot study sought to explore the frequency of genetic variants in matrix metalloproteinases (MMPs) 3, 9, and 12 in a population of colonic diverticulosis patients. Materials and Methods: The study encompassed 134 participants: 59 diagnosed with colon diverticulosis during colonoscopy and 75 healthy controls. The cases and controls were meticulously matched in terms of age and gender. We assessed the distribution of genetic variants MMP3 rs3025058, MMP9 rs3918242, and MMP12 rs2276109 using the polymerase chain reaction-restriction fragments length polymorphism technique. Results: The MMP9 rs3918242 allele T was notably more frequent in individuals with diverticulosis when compared with the control group (p < 0.03). Furthermore, it was associated with dominant (OR = 2.62; 95% CI: 1.24-5.56; p < 0.01) and co-dominant (OR = 2.10; 95% CI: 1.06-4.13; p < 0.03) genetic models. The MMP3 rs3025058 5A/5A genotype was nearly twice as frequent in patients with diverticulosis, while the 6A/6A genotype was only half as common in this group. Conversely, no significant correlation was established between MMP12 rs2276109 and colonic diverticulosis. Conclusions: Our study offers the first insight into a potential connection between genetic variants in MMPs and colon diverticulosis. Specifically, allele T of MMP9 rs3918242 and allele 5A of MMP3 rs3025058 appear to be linked to this condition. These findings indirectly suggest a role for extracellular matrix proteins in the pathogenesis of diverticulosis.

Citri Reticulatae Pericarpium-Reynoutria japonica Houtt. herb pair suppresses breast cancer liver metastasis by targeting ECM1-mediated cholesterol biosynthesis pathway

BackgroundLiver metastasis is a frequent event in breast cancer that causes low survival rate and poor prognosis. Citri Reticulatae Pericarpium-Reynoutria japonica Houtt. (CR), a traditional Chinese herb pair, is used for the treatment of breast cancer liver metastasis or cholesterol gallstone disease in clinics. PurposeThis study attempted to investigate the potential therapeutic target and mechanism of CR herb pair on breast cancer liver metastasis. MethodsThe anti-metastatic and cholesterol-lowering activities of CR extract were evaluated in triple-negative breast cancer (TNBC) cell lines and an experimental liver metastasis model. The role of extracellular matrix protein 1 (ECM1) in the cholesterol biosynthesis pathway was determined by the knockdown and overexpression of ECM1 gene of TNBC cells. Changes in the gene and protein expression levels of ECM1 and the cholesterol biosynthesis pathway after CR treatment were detected in vitro and in vivo by real-time PCR and Western blot. ResultsThe invasive and metastatic potentials and hypercholesterol levels of TNBC cells were positively associated with ECM1 expression. ECM1 knockdown reduced tumor cholesterol levels via downregulating cholesterol biosynthesis genes, including ACAT2, HMGCS1, HMGCR, MVK, and MVD, whereas ECM1 overexpression elicited the opposite effects. CR herb pair exerts the potential therapeutic effects on TNBC liver metastasis, which is partially mediated by disrupting ECM1-activated cholesterol biosynthesis process in TNBC cells. ConclusionThis study reveals that ECM1 is a novel target for the activation of cholesterol biosynthesis to promote TNBC liver metastasis occurrence. CR herb pair, an ECM1 inhibitor, maybe be considered to serve as an adjuvant therapeutic drug for liver metastasis in clinical practice.

The role of extracellular matrix proteins on placental angiogenesis in severe fetal growth restriction

Impaired vascular development is a central finding in severe fetal growth restriction (FGR) with abnormal Doppler velocimetry. Angiogenesis involves complex interactions between endothelial cells (ECs) and extracellular matrix (ECM). Our laboratory previously found that FGR ECs exhibit deficient angiogenic properties, a deficit that is further worsened in the presence of ECM generated from FGR placentae but partially rescued by control ECM. Our objective was to identify the effects of individual ECM substrates on EC function to better characterize the impaired angiogenic pathway characteristic of FGR. ECs were isolated from term control (n=8) or severe FGR (n=8) human placentae and plated on six individual ECM proteins: fibronectin; collagens I, III, and IV; laminin; and thrombospondin. Adhesion was assessed by colorimetric staining. Live cell microscopy was used to determine proliferation. Migration was analyzed via relative wound density following wound scratch. Apoptosis was evaluated by immunoblotting for cleaved caspase 3. T tests, ANOVAs, and nonlinear regressions were performed. There was no difference between adhesion of control and FGR ECs to individual ECM substrates. Control ECs proliferated more rapidly than FGR ECs (p< 0.01; figure 1). Deficient growth of FGR ECs was not rescued by any ECM substrate. Migration of FGR ECs was impaired relative to control ECs when accounting for all substrates (p=0.02). This finding persisted specifically in the presence of collagen I (p=0.03; figure 2) but not other substrates. Apoptosis was not detected for either type of ECs on any substrate. ECs from placentae affected by severe FGR display impaired proliferation and migration. This growth deficit appears to be independent of individual ECM substrates. FGR migratory defects persist on collagen I but not other substrates. Our data suggest that no singular ECM protein is responsible for the aberrant angiogenesis seen in severe FGR and rather there are likely multiple mechanisms underlying this pathology.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Evaluation of serum neopterin, periostin, Tenascin-C, tissue inhibitor of metalloproteinase-1 and matrix metalloproteinase-2 levels in obese pregnant women

To investigate the role of extracellular matrix proteins in the molecular mechanism of inflammatory response in obese pregnant women by comparing serum levels of neopterin, periostin, Tenascin-C, tissue inhibitor of metalloproteinase-1, and matrix metalloproteinase-2 between obese and normal weight pregnant women in the third trimester. A prospective cross-sectional study was conducted between April 2021 and December 2021. A total of 84 pregnant women were included and three groups were formed with 28 participants in each group. Serum levels of neopterin, periostin, Tenascin-C and tissue inhibitor of metalloproteinase-1 were significantly higher in class II-III obese pregnant women than in class I obese and normal-weight women (p=0.002, p<0.001, p<0.001, and p<0.001, respectively). There was no significant difference in serum matrix metalloproteinase-2 levels between the groups (p=0.769). Receiver operating characteristic curve analysis showed that Tenascin-C and periostin were effective in predicting pre-eclampsia [area under the curve (AUC)=0.82, 95% confidence interval (CI), 0.72-0.90, p<0.001 and AUC=0.71, 95% CI, 0.60-0.80, p=0.007, respectively]. This study demonstrated that class II-III obese pregnant women had significantly higher serum levels of neopterin, periostin, Tenascin-C, and tissue inhibitor of metalloproteinase-1 in the third trimester. These higher serum levels may be associated with the adverse perinatal effects of obesity during pregnancy.

The Role of Extracellular Matrix Proteins in Breast Cancer.

The extracellular matrix is a structure composed of many molecules, including fibrillar (types I, II, III, V, XI, XXIV, XXVII) and non-fibrillar collagens (mainly basement membrane collagens: types IV, VIII, X), non-collagenous glycoproteins (elastin, laminin, fibronectin, thrombospondin, tenascin, osteopontin, osteonectin, entactin, periostin) embedded in a gel of negatively charged water-retaining glycosaminoglycans (GAGs) such as non-sulfated hyaluronic acid (HA) and sulfated GAGs which are linked to a core protein to form proteoglycans (PGs). This highly dynamic molecular network provides critical biochemical and biomechanical cues that mediate the cell–cell and cell–matrix interactions, influence cell growth, migration and differentiation and serve as a reservoir of cytokines and growth factors’ action. The breakdown of normal ECM and its replacement with tumor ECM modulate the tumor microenvironment (TME) composition and is an essential part of tumorigenesis and metastasis, acting as key driver for malignant progression. Abnormal ECM also deregulate behavior of stromal cells as well as facilitating tumor-associated angiogenesis and inflammation. Thus, the tumor matrix modulates each of the classically defined hallmarks of cancer promoting the growth, survival and invasion of the cancer. Moreover, various ECM-derived components modulate the immune response affecting T cells, tumor-associated macrophages (TAM), dendritic cells and cancer-associated fibroblasts (CAF). This review article considers the role that extracellular matrix play in breast cancer. Determining the detailed connections between the ECM and cellular processes has helped to identify novel disease markers and therapeutic targets.

The Functional Role of Extracellular Matrix Proteins in Cancer.

Simple SummaryExtracellular matrix is a three-dimensional network of macromolecules that provide structural and biochemical support to surrounding cells. Extracellular matrix plays a critical role in the development and progression of cancer. The extracellular matrix of the tumor is very different from the matrix of the normal tissue. Mainly fibroblasts produce and regulate matrix remodeling, but in cancer, the tumor matrix also originates from cancer cells. We describe the mechanisms of how the protein composition and structure of the extracellular matrix changes during cancer progression and how abnormal matrix deregulates the behavior of stromal cells and influences cancer progression.The extracellular matrix (ECM) is highly dynamic as it is constantly deposited, remodeled and degraded to maintain tissue homeostasis. ECM is a major structural component of the tumor microenvironment, and cancer development and progression require its extensive reorganization. Cancerized ECM is biochemically different in its composition and is stiffer compared to normal ECM. The abnormal ECM affects cancer progression by directly promoting cell proliferation, survival, migration and differentiation. The restructured extracellular matrix and its degradation fragments (matrikines) also modulate the signaling cascades mediated by the interaction with cell-surface receptors, deregulate the stromal cell behavior and lead to emergence of an oncogenic microenvironment. Here, we summarize the current state of understanding how the composition and structure of ECM changes during cancer progression. We also describe the functional role of key proteins, especially tenascin C and fibronectin, and signaling molecules involved in the formation of the tumor microenvironment, as well as the signaling pathways that they activate in cancer cells.

End-Truncated LAMB1 Causes a Hippocampal Memory Defect and a Leukoencephalopathy.

The majority of patients with a familial cerebral small vessel disease (CSVD) referred for molecular screening do not show pathogenic variants in known genes. In this study, we aimed to identify novel CSVD causal genes. We performed a gene-based collapsing test of rare protein-truncating variants identified in exome data of 258 unrelated CSVD patients of an ethnically matched control cohort and of 2 publicly available large-scale databases, gnomAD and TOPMed. Western blotting was used to investigate the functional consequences of variants. Clinical and magnetic resonance imaging features of mutated patients were characterized. We showed that LAMB1 truncating variants escaping nonsense-mediated messenger RNA decay are strongly overrepresented in CSVD patients, reaching genome-wide significance (p < 5 × 10-8 ). Using 2 antibodies recognizing the N- and C-terminal parts of LAMB1, we showed that truncated forms of LAMB1 are expressed in the endogenous fibroblasts of patients and trapped in the cytosol. These variants are associated with a novel phenotype characterized by the association of a hippocampal type episodic memory defect and a diffuse vascular leukoencephalopathy. These findings are important for diagnosis and clinical care, to avoid unnecessary and sometimes invasive investigations, and also from a mechanistic point of view to understand the role of extracellular matrix proteins in neuronal homeostasis. ANN NEUROL 2021;90:962-975.

Exploring the role of extracellular matrix proteins to develop biomarkers of plaque vulnerability and outcome.

Cardiovascular disease (CVD) is the most common cause of death in industrialized countries. One underlying cause is atherosclerosis, which is a systemic disease characterized by plaques of retained lipids, inflammatory cells, apoptotic cells, calcium and extracellular matrix (ECM) proteins in the arterial wall. The biologic composition of an atherosclerotic plaque determines whether the plaque is more or less vulnerable, that is prone to rupture or erosion. Here, the ECM and tissue repair play an important role in plaque stability, vulnerability and progression. This review will focus on ECM remodelling in atherosclerotic plaques, with focus on how ECM biomarkers might predict plaque vulnerability and outcome.

The role of extracellular matrix proteins in gastric cancer development via epithelial-mesenchymal transition.

To acquire a deeper perception of EMT, we evaluated the expression of some candidate extra cellular matrix (ECM) proteins including THBS2, OSMR and CHI3L1 which were collected from RNA-seq bioinformatic analyses. Gastric cancer (GC) is a major incident gastrointestinal cancer with a high rate of mortality. Metastasis is a challenging issue in gastric cancer treatment. Epithelial mesenchymal transition (EMT) of cancer cells is a complicated process controlled by different cells and molecular pathways regarded as an important step at the onset of metastasis. AGS gastric cancer cell line was cultured and treated by TGF-β. EMT induction was verified by measuring the expression of E-cadherin, Snail, β-catenin and Vimentin genes by real time PCR. Then, following our previous study, we evaluated the expression of THBS2, OSMR and CHI3L1 genes in EMT induced cells by real time PCR. Downregulation of E-cadherin and upregulation of Snail, β-catenin and Vimentin genes were verified in AGS treated cells in comparison with none-treated cells (P-value = 0.0355, P-value = 0.007, P-value = 0.0059, P-value = 0.0206 respectively). Also, upregulation of THBS2, OSMR and CHI3L1 were validated in these cells after EMT induction (P-value = 0.0147, P-value = 0.05, P-value = 0.05 respectively). Our morphological and molecular results validated EMT induction by TGF- β cytokine in AGS gastric cancer cell line. Furthermore, significant upregulation of candidate genes including THBS2, OSMR and CHI3L1 verified the role of these proteins in gastric cancer invasiveness. However, further studies are needed for the validation of prognostic value of these markers.

P6296The role of extracellular matrix protein 1 (ECM1) - a novel link between inflammation and cardiac fibrosis

Abstract Introduction Cardiac fibrosis is a severe consequence of cardiovascular disease and aging, in which we currently have no effective treatments. The mechanisms underpinning the development of cardiac fibrosis remains poorly understood. Our preliminary data suggested extracellular matrix protein 1 (ECM1) is involved in cardiac fibrosis. We therefore aimed to investigate the role of ECM1 in several fibrotic cardiac diseases. Methods Young and ageing (3m/18m) male C57BL/6 mice, and primary mouse cardiac fibroblast (cFB) cultures, commercial human cardiac fibroblasts (Hu-cFB), human coronary artery endothelial cell (HCAEC)/smooth muscle cell (HCASMC), and human cardiac myocyte (HCM) cell lines were used. Young mice were subject to myocardial infarction (MI, 3-day/28-day, n=6/6), or pressure overload (TAC, 3-day/13-week, n=4/4). Left ventricle (LV) was collected at all time-points, and at 18m (ageing; n=3). Spleen and bone marrow was extracted from young control mice. Hu-cFB cells were treated with recombinant ECM1 (20ng/ml) for either 10, 30 or 50 min, or 48h. Immunoblotting was conducted on all samples, qPCR on LV tissue only, density gradient centrifugation and multicolour flow cytometry coupled with fluorescent ECM1 mRNA in-situ hybridisation (FISH-Flow) on bone marrow cells. Results ECM1 expression was upregulated in ageing LV (mRNA 2.2±0.1-fold, p=0.0002; protein 2.0-fold, p=0.0006), day-3 post-MI (mRNA, 4.9±2.0-fold, p=0.004; protein, 3.0-fold, p=0.004), a trend of ECM1 upregulation was observed at day-28 post-MI (mRNA, 13.2±12.0-fold, p=0.003; protein, 1.8-fold, p=0.2), but no change post-TAC. Both ERK1/2 and AKT phosphorylation was upregulated 10 min post-ECM1 treatment of Hu-cFBs (ERK1/2, 2.0-fold, p&lt;0.0001; AKT, 1.9-fold, p&lt;0.0001), and Collagen-I protein expression was upregulated 48h post-ECM1 treatment (1.9-fold, p=0.004). ECM1 protein was not expressed in cFB, Hu-cFB, HCAEC, HCASMC or HCM, however ECM1 protein was highly expressed in spleen and bone marrow; to a greater extent in granulocytes compared to monocytes (p=0.004). tSNE analysis of ECM1 mRNA FISH-Flow revealed ECM1+ are highly granular, moderate to large in size, and express (to varying levels) CD45, CD11b, CD11c, F4/80, Ly6-C, Ly-6G, and FcεrI-α. However ECM1+ cells did not express markers indicative of smaller cells (CD3 or MHC II). Conclusions These data demonstrate that ECM1 plays a role in ageing and post-MI fibrosis. Although ECM1 was not produced by resident cardiac cells, it was highly expressed in spleen and bone marrow; specifically, large, granular bone marrow cell sub-types such as granulocytes and/or macrophages. Our data suggest ECM1 is expressed by cardiac infiltrating leukocytes to provoke fibroblast collagen expression in a disease specific manner; potentially via the ERK1/2 and/or AKT pathway activation. Therefore, ECM1 warrants further investigation, and may be a promising target for the treatment of fibrotic cardiac diseases. Acknowledgement/Funding John hunter hospital charitable trust, Hunter medical research institute (HMRI) grants

Novel role of extracellular matrix protein 1 (ECM1) in cardiac aging and myocardial infarction.

IntroductionThe prevalence of heart failure increases in the aging population and following myocardial infarction (MI), yet the extracellular matrix (ECM) remodeling underpinning the development of aging- and MI-associated cardiac fibrosis remains poorly understood. A link between inflammation and fibrosis in the heart has long been appreciated, but has mechanistically remained undefined. We investigated the expression of a novel protein, extracellular matrix protein 1 (ECM1) in the aging and infarcted heart.MethodsYoung adult (3-month old) and aging (18-month old) C57BL/6 mice were assessed. Young mice were subjected to left anterior descending artery-ligation to induce MI, or transverse aortic constriction (TAC) surgery to induce pressure-overload cardiomyopathy. Left ventricle (LV) tissue was collected early and late post-MI/TAC. Bone marrow cells (BMCs) were isolated from young healthy mice, and subject to flow cytometry. Human cardiac fibroblast (CFb), myocyte, and coronary artery endothelial & smooth muscle cell lines were cultured; human CFbs were treated with recombinant ECM1. Primary mouse CFbs were cultured and treated with recombinant angiotensin-II or TGF-β1. Immunoblotting, qPCR and mRNA fluorescent in-situ hybridization (mRNA-FISH) were conducted on LV tissue and cells.ResultsECM1 expression was upregulated in the aging LV, and in the infarct zone of the LV early post-MI. No significant differences in ECM1 expression were found late post-MI or at any time-point post-TAC. ECM1 was not expressed in any resident cardiac cells, but ECM1 was highly expressed in BMCs, with high ECM1 expression in granulocytes. Flow cytometry of bone marrow revealed ECM1 expression in large granular leucocytes. mRNA-FISH revealed that ECM1 was indeed expressed by inflammatory cells in the infarct zone at day-3 post-MI. ECM1 stimulation of CFbs induced ERK1/2 and AKT activation and collagen-I expression, suggesting a pro-fibrotic role.ConclusionsECM1 expression is increased in ageing and infarcted hearts but is not expressed by resident cardiac cells. Instead it is expressed by bone marrow-derived granulocytes. ECM1 is sufficient to induce cardiac fibroblast stimulation in vitro. Our findings suggest ECM1 is released from infiltrating inflammatory cells, which leads to cardiac fibroblast stimulation and fibrosis in aging and MI. ECM1 may be a novel intermediary between inflammation and fibrosis.

CONGENITAL MYASTHENIC SYNDROMES AND MYASTHENIA: P.72Clinical features of congenital myasthenic syndrome due to mutations in COL13A1

COL13A1 is a membrane protein expressed at the human neuromuscular junction and cleaved via ectodomain shedding into a soluble form that is part of the synaptic basal lamina. In recent years, we have identified that mutations in COL13A1 underlay a novel subtype of congenital myasthenic syndrome highlighting the importance of collagen XIII in the muscle endplate cytoarchitecture and neurotransmission, and the crucial role of extra-cellular matrix proteins, other than those in the agrin pathway, in the formation and maintenance of the synapse. Here we review 7 patients with COL13A1 mutations from 6 different kinships in order to identify the associated clinical, neurophysiological, pathological, and genetic features. A total of 6 different mutations were identified, including the first case homozygous for a missense variant. The results showed that age at presentation was variable and main clinical features included respiratory and feeding difficulties, facial dysmorphism, and ptosis with limited fatiguabilty but normal eye movements. Neurophysiological studies showed impaired neuromuscular transmission in all cases. There was no beneficial response to anticholinesterase medication but patients did improve on β2-adrenergic agonists consistent with the fact that COL13A1 mutations affect the maturation and maintenance of the synaptic structure. Treatment with 3,4-Diaminopyridine was also beneficial in keeping with the importance of COL13A1 for the integrity of the presynaptic terminal. This study will facilitate the recognition of this genetic disorder, which can be treated pharmacologically.

The Matricellular Protein R-Spondin 2 Promotes Midbrain Dopaminergic Neurogenesis and Differentiation.

SummaryThe development of midbrain dopaminergic (mDA) neurons is controlled by multiple morphogens and transcription factors. However, little is known about the role of extracellular matrix proteins in this process. Here we examined the function of roof plate-specific spondins (RSPO1-4) and the floor plate-specific, spondin 1 (SPON1). Only RSPO2 and SPON1 were expressed at high levels during mDA neurogenesis, and the receptor LGR5 was expressed by midbrain floor plate progenitors. Surprisingly, RSPO2, but not SPON1, specifically promoted the differentiation of mDA neuroblasts into mDA neurons in mouse primary cultures and embryonic stem cells (ESCs). In addition, RSPO2 was found to promote not only mDA differentiation, but also mDA neurogenesis in human ESCs. Our results thus uncover an unexpected function of the matricellular protein RSPO2 and suggest an application to improve mDA neurogenesis and differentiation in human stem cell preparations destined to cell replacement therapy or drug discovery for Parkinson disease.

PO-468 LAMININ332 (α3; ß3;γ2) genes and protein expression in cervical carcinomas

IntroductionLaminin332 (α3; ß3; γ2 chains) has been identified as an important macromolecule in cancer invasion and metastasis, influencing cell differentiation, migration, adhesion, cell proliferation and survival.The α3, β3, and γ2 chains of LAM-332 are encoded by three distinct genes, LAMA3, LAMB3, and LAMC2, respectively. LAMC2 is associated to the invasive and metastatic abilities of several tumour types such as colon and pancreas. However, the molecular role of LAMC2 in cervical carcinomas has not yet been fully elucidated.We investigated the functional significance of LAMC2 in cervical carcinomas: squamous cell carcinomas (SCC) and adenocarcinoma (ADC) as well as its role in SCC of the cervix.Material and methodsSiHa (SCC) and HeLa (ADC) cell lines were used. The expression of LAMA3, LAMB3, LAMC2 and EGFR was evaluated by qPCR in the presence/absence of EGF. Lamγ2 levels were evaluated by immunofluorescense. Lamγ2 role in migration and invasion was evaluated by wound healing and trans-well assays in control, EGF stimulated and LAMC2 silenced cells (shRNA). Immunohistochemical study evaluated lamα3, β3, and γ2 chains and EGFR, in formalin-fixed, paraffin-embedded in 122 cases, both in situ (n=15) and invasive cervical carcinomas: SCC (n=106) and ADC (n=41). Fisher’s exact test was performed for statistical analysis.Results and discussionsEGF stimulates the migration and invasion in SiHa cells. However knocking-down Lamγ2 decreases cell migration and invasion capacities in SiHa cells. Also, the knockdown of LAMC2 lead to an increased transcription of LAMA3 and LAMB3 genes only in SiHa. The immunohistochemical study demonstrated that lamγ2 chain in SCC cases was significantly associated with invasion (p=0.0001) and with that histological type (p=0.0183). In SCC cases, there was also a significant association of lamß3 and γ2 chains overexpression (p=0.008). Lamα3 was associate with ADC type (p<0.0001). EGFR immuno-overexpression was also associated with SCC (p<0.0001).ConclusionLaminin332 and EGFR are differently expressed in the two most common histological types of cervical carcinomas (SCC and ADC). Lamγ2 expression is associated with SCC in both in vitro and clinical context and Lamg2 -dependent invasion is probably mediated by EGFR dependent signalling pathways.Together, results demonstrate a role of extracellular matrix proteins in the progression of cervix carcinoma. Additionally, Siha and HeLa can be an effective in-vitro models for an overview of the processes involved in cervical cancer progression.

PO-134 Cytogenetic and molecular characterisation of new high-grade serous ovarian cancer cell line OVPA8

IntroductionBladder cancer (BC) is the ninth most common cancer worldwide. A quarter of all cases present as muscle invasive BC (MIBC). Treatment for this group has remained largely unchanged for the past 20 years limited mainly to cisplatin based therapy and radical cystectomy. Prognosis for these patients is poor, with 5 year survival <50% and identification of potential new therapeutic approaches is urgently needed. We have explored the possible role of extracellular matrix protein 1 (ECM1) overexpression in MIBC. ECM1 overexpression has been linked to carcinogenesis and progression to a more aggressive phenotype in a number of carcinomas, but its role in BC has not yet been examined. In a previous study, ECM1 was proposed to interact with and stabilise EGFR in a breast cancer cell line leading to increased resistance to ERBB targeting drugs. Despite repeated implication of EGFR in the progression of BC, EGFR targeting agents have so far had little success in the treatment of MIBC in part due to resistance. Improved understanding of the mechanisms of resistance to such agents is vital. The current study examined ECM1 overexpression and the relationship between ECM1 and EGFR in BC.Material and methodsECM1 expression was assessed at the mRNA and protein levels in 47 bladder tumour derived cell lines using qRT-PCR and western blot analysis. ECM1 knockdown cell lines were established using shRNA lentiviral transduction in a panel of cell lines which overexpress the protein. ECM1 knockdown cell lines were analysed for phenotypic effects. Phosphorylation of EGFR and downstream effectors was examined following recombinant ECM1 treatment. Publicly available microarray data was mined to assess the clinical significance of ECM1 expression in patients, relating high expression to tumour stage and overall survival.Results and discussionsECM1 knockdown had an inhibitory effect on wound-healing ability. Evidence suggests ECM1 initiates phosphorylation and thus activation of EGFR and downstream effectors in the pathway, however we have been unable to detect a direct interaction with EGFR as described in breast. Mining of publicly available data revealed a significant association between high ECM1 expression and reduced overall survival.ConclusionECM1 overexpression in MIBC appears to be associated with poorer prognosis in patients. While ECM1 has been shown to influence EGFR signalling, the mechanism of EGFR activation by ECM1 in BC may be different from that previously described in breast cancer.

The Neuromuscular Junction and Wide Heterogeneity of Congenital Myasthenic Syndromes.

Congenital myasthenic syndromes (CMS) are genetic disorders characterised by impaired neuromuscular transmission. This review provides an overview on CMS and highlights recent advances in the field, including novel CMS causative genes and improved therapeutic strategies. CMS due to mutations in SLC5A7 and SLC18A3, impairing the synthesis and recycling of acetylcholine, have recently been described. In addition, a novel group of CMS due to mutations in SNAP25B, SYT2, VAMP1, and UNC13A1 encoding molecules implicated in synaptic vesicles exocytosis has been characterised. The increasing number of presynaptic CMS exhibiting CNS manifestations along with neuromuscular weakness demonstrate that the myasthenia can be only a small part of a much more extensive disease phenotype. Moreover, the spectrum of glycosylation abnormalities has been increased with the report that GMPPB mutations can cause CMS, thus bridging myasthenic disorders with dystroglycanopathies. Finally, the discovery of COL13A1 mutations and laminin α5 deficiency has helped to draw attention to the role of extracellular matrix proteins for the formation and maintenance of muscle endplates. The benefit of β2-adrenergic agonists alone or combined with pyridostigmine or 3,4-Dyaminopiridine is increasingly being reported for different subtypes of CMS including AChR-deficiency and glycosylation abnormalities, thus expanding the therapeutic repertoire available.

The Role of Extracellular Matrix Protein 1 (ECM1) in Cardiac Fibrosis

The Role of Extracellular Matrix Protein 1 (ECM1) in Cardiac Fibrosis

Extracellular matrix proteins in healthy and retained placentas, comparing hemochorial and synepitheliochorial placentas

The placenta expresses structural and biologically active proteins. Their synthesis is mainly regulated by genomic or nongenomic signals and modulated by hormones. These protein profiles are altered during different stages of pregnancy. The biological properties of extracellular matrix (ECM) proteins were defined and described in a number of tissues including placenta. These properties enable them to be the main players in the processes of attachment or invasion into the endometrium during initial placenta formation and its timely separation after delivery and detachment. In this review, we focused on the role of ECM proteins during attachment of the placenta to the uterine wall, its timely separation, and the implications of this process on retained or pathologically attached placenta.Although the amount of published information in this area is relatively scant, some of the key proteins and processes are well defined. We focused on the available data detailing the ECM protein profiles of human (histologically thin; hemochorial) and bovine (histologically thick; epitheliochorial) placentas and compared the shared and unique ECM proteins that are relevant to placental attachment and separation.