Extracellular Matrix Release Research Articles

Targeting A-kinase anchoring protein 12 phosphorylation in hepatic stellate cells regulates liver injury and fibrosis in mouse models.

Trans-differentiation of hepatic stellate cells (HSCs) to activated state potentiates liver fibrosis through release of extracellular matrix (ECM) components, distorting the liver architecture. Since limited antifibrotics are available, pharmacological intervention targeting activated HSCs may be considered for therapy. A-kinase anchoring protein 12 (AKAP12) is a scaffolding protein that directs protein kinases A/C (PKA/PKC) and cyclins to specific locations spatiotemporally controlling their biological effects. It has been shown that AKAP12's scaffolding functions are altered by phosphorylation. In previously published work, observed an association between AKAP12 phosphorylation and HSC activation. In this work, we demonstrate that AKAP12's scaffolding activity toward the endoplasmic reticulum (ER)-resident collagen chaperone, heat-shock protein 47 (HSP47) is strongly inhibited by AKAP12's site-specific phosphorylation in activated HSCs. CRISPR-directed gene editing of AKAP12's phospho-sites restores its scaffolding toward HSP47, inhibiting HSP47's collagen maturation functions, and HSC activation. AKAP12 phospho-editing dramatically inhibits fibrosis, ER stress response, HSC inflammatory signaling, and liver injury in mice. Our overall findings suggest a pro-fibrogenic role of AKAP12 phosphorylation that may be targeted for therapeutic intervention in liver fibrosis.

Enhanced Extracellular Matrix Deposition on Titanium Implant Surfaces: Cellular and Molecular Evidences.

The surface structure of the titanium dental implants can modulate the activity of mesenchymal stem cells in order to promote the upregulation of osteoblastic related genes and the release of extracellular matrix (ECM) components. The present work was focused on the in vitro evaluation of the interaction of human periodontal ligament stem cells (hPDLSCs) and two different implant titanium surfaces topography (CTRL and TEST). This study was aimed at analyzing the cytotoxicity of the dental implant surfaces, the cellular adhesion capacity, and the improvement in the release of ECM molecules in an in vitro model. These parameters were carried out by means of the microscopic evaluation, viability assays, immunofluorescence, Western blot and RT-PCR investigations. The knowledge of the cell/implant interaction is essential for implant healing in order to obtain a more performing surfaces that promote the ECM release and provide the starting point to initiate the osseointegration process.

Human Periodontal Ligament Stem Cells Response to Titanium Implant Surface: Extracellular Matrix Deposition.

Simple SummaryThe extracellular matrix (ECM) is a fundamental component of tissues and organs, providing the structural and non-structural support that leads to the bone osseointegration. Understanding the mechanisms of ECM components modulation is essential for developing novel strategies for tissue engineering and regenerative medicine and in particular for the dental implant surface design. The release of ECM components by cells is the first step that leads to the early stage of bone formation. The present work is focused on the modulation of ECM components expression by human periodontal ligament stem cells (hPDLSCs) cultured on two different dental implant surfaces, sandblasted (CTRL) and dual acid-etched (TEST). The effects were evaluated by means of the morphological observations and protein and gene expression. The results demonstrated that the rough surface of titanium dental implant can enhance the expression of ECM molecules and osteogenic markers in hPDLSCs. The surface topography is of vital importance as it regulates cell response. It can be considered one of the main factors that influence the success of a dental implant. The influence of surface topography on osseointegration could lead to short healing times and a better quality of oral rehabilitation and patient life.The major challenge for dentistry is to provide the patient an oral rehabilitation to maintain healthy bone conditions in order to reduce the time for loading protocols. Advancement in implant surface design is necessary to favour and promote the osseointegration process. The surface features of titanium dental implant can promote a relevant influence on the morphology and differentiation ability of mesenchymal stem cells, induction of the osteoblastic genes expression and the release of extracellular matrix (ECM) components. The present study aimed at evaluating the in vitro effects of two different dental implants with titanium surfaces, TEST and CTRL, to culture the human periodontal ligament stem cells (hPDLSCs). Expression of ECM components such as Vimentin, Fibronectin, N-cadherin, Laminin, Focal Adhesion Kinase (FAK) and Integrin beta-1 (ITGB1), and the osteogenic related markers, as runt related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP), were investigated. Human PDLSCs cultured on the TEST implant surface demonstrated a better cell adhesion capability as observed by Scanning Electron Microscopy (SEM) and immunofluorescence analysis. Moreover, immunofluorescence and Western blot experiments showed an over expression of Fibronectin, Laminin, N-cadherin and RUNX2 in hPDLSCs seeded on TEST implant surface. The gene expression study by RT-PCR validated the results obtained in protein assays and exhibited the expression of RUNX2, ALP, Vimentin (VIM), Fibronectin (FN1), N-cadherin (CDH2), Laminin (LAMB1), FAK and ITGB1 in hPDLSCs seeded on TEST surface compared to the CTRL dental implant surface. Understanding the mechanisms of ECM components release and its regulation are essential for developing novel strategies in tissue engineering and regenerative medicine. Our results demonstrated that the impact of treated surfaces of titanium dental implants might increase and accelerate the ECM apposition and provide the starting point to initiate the osseointegration process.

BMP4 micro-immunotherapy increases collagen deposition and reduces PGE2 release in human gingival fibroblasts and increases tissue viability of engineered 3D gingiva under inflammatory conditions.

BackgroundWe aimed to evaluate the effect of low doses (LD) bone morphogenetic protein‐2 (BMP2) and BMP4 micro‐immunotherapy (MI) in two in vitro models of periodontal wound healing/regeneration.MethodsWe first evaluated the effect of LD of BMP2 and BMP4 MI on a 2D cell culture using human gingival fibroblasts (hGF) under inflammatory conditions induced by IL1β. Biocompatibility, inflammatory response (Prostaglandin E2 (PGE2) release), collagen deposition and release of extracellular matrix (ECM) organization‐related enzymes (matrix metalloproteinase‐1 (MMP1) and metalloproteinase inhibitor 1 (TIMP1)) were evaluated after short (3 days) and long‐term (24 days) treatment with BMP2 or BMP4 MI. Then, given the results obtained in the 2D cell culture, LD BMP4 MI treatment was evaluated in a 3D cell culture model of human tissue equivalent of gingiva (GTE) under the same inflammatory stimulus, evaluating the biocompatibility, inflammatory response and effect on MMP1 and TIMP1 release.ResultsLD BMP4 was able to decrease the release of the inflammatory mediator PGE2 and completely re‐establish the impaired collagen metabolism induced by IL1β treatment. In the 3D model, LD BMP4 treatment improved tissue viability compared with the vehicle, with similar levels to 3D tissues without inflammation. No significant effects were observed on PGE2 levels nor MMP1/TIMP1 ratio after LD BMP4 treatment, although a tendency to decrease PGE2 levels was observed after 3 days.ConclusionsLD BMP4 MI treatment shows anti‐inflammatory and regenerative properties on hGF, and improved viability of 3D gingiva under inflammatory conditions. LD BMP4 MI treatment could be used on primary prevention or maintenance care of periodontitis.

High levels of biomarkers of collagen remodeling are associated with increased mortality in COPD – results from the ECLIPSE study

BackgroundThere is a need to identify individuals with COPD at risk for disease progression and mortality. Lung tissue remodeling is associated with the release of extracellular matrix (ECM) fragments into the peripheral circulation. We hypothesized that ECM remodeling was associated with mortality in COPD and measured neo-epitopes originating from ECM proteins associated with lung tissue remodeling.MethodsBiomarkers of ECM remodeling were assessed in a subpopulation (n = 1000) of the Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points (ECLIPSE) cohort. Validated immunoassays measuring serological neo-epitopes produced by proteolytic cleavage associated with degradation of collagen type I, III, IV, and VI, elastin, and biglycan, and formation of collagen type VI as well as fibrinogen and C-reactive protein were used. Multivariate models were used to assess the prognostic value of these biomarkers.ResultsThirty subjects (3.0 %) died during follow-up. Non-survivors were older, had reduced exercise capacity, increased dyspnea score, and included fewer current smokers. All collagen biomarkers were significantly elevated in non-survivors compared to survivors. Mortality risk was significantly increased for subjects with collagen remodeling biomarkers in the upper quartile, especially for the degradation fragment of collagen type IV C6M (hazard ratio 6.6 [95 % confidence interval 2.9-15.2], P < 0.0001) after adjusting for relevant confounders.ConclusionsSerological biomarkers of collagen remodeling were strongly associated with mortality in subjects with COPD indicating that assessment of tissue turnover in the parenchyma and small airways may be useful in the prognosis of COPD.Trial registration NCT00292552, GSK Study No. SCO104960.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-016-0440-6) contains supplementary material, which is available to authorized users.

Distinct miRNA profiles in normal and gastric cancer myofibroblasts and significance in Wnt signaling.

Stromal cells influence epithelial function in both health and disease. Myofibroblasts are abundant stromal cells that influence the cellular microenvironment by release of extracellular matrix (ECM) proteins, growth factors, proteases, cytokines, and chemokines. Cancer-associated myofibroblasts (CAMs) differ from adjacent tissue (ATMs) and normal tissue myofibroblasts (NTMs), but the basis of this is incompletely understood. We report now the differential expression of miRNAs in gastric cancer CAMs. MicroRNA arrays identified differences in the miRNA profile in gastric and esophageal NTMs and in CAMs from stomach compared with NTMs. miR-181d was upregulated in gastric CAMs. Analysis of differentially regulated miRNAs indicated an involvement in Wnt signaling. Examination of a microarray data set then identified Wnt5a as the only consistently upregulated Wnt ligand in gastric CAMs. Wnt5a stimulated miR-181d expression, and knockdown of miR-181d inhibited Wnt5a stimulation of CAM proliferation and migration. Analysis of miR-181d targets suggested a role in chemotaxis. Conditioned medium from CAMs stimulated gastric cancer cell (AGS) migration more than that from ATMs, and miR-181d knockdown reduced the effect of CAM-CM on AGS cell migration but had no effect on AGS cell responses to ATM conditioned media. The data suggest that dysregulation of miRNA expression in gastric CAMs, secondary to Wnt5a signaling, accounts at least in part for the effect of CAMs in promoting cancer cell migration.

The cell and molecular biology of bone fracture repair: role of the transforming growth factor-ß1 in activation reparative osteogenesis (review)

This paper describes the cellular and molecular mechanisms underlying in the basis of bone fractures healing. It is shown that there are two types of fracture healing. In the case of the primary type bone tissue directly form in zone of damage. In the case of instability of the bone fragments and considerable distance between them the process of restoration of the integrity of the bone goes through several stages including inflammation, migration and proliferation of cells, the formation of tissue-specific structures and the restructuring of the regenerate. At the same time at different stages certain factors affecting the proliferation, differentiation of osteogenic cells and synthesis of extracellular matrix release. Growth factors control the various stages of reparative osteogenesis. In recent years the regulatory role of transforming growth factor beta-1 (TGF-β1) and belonging to his family of bone morphogenetic protein (BMP) in the formation and development of bone regeneration was confirmed. According to modern scientific ideas BMP act as primary activators of differentiation of osteogenic progenitor cells, and mesenchymal cells, and are involved in the processes of bone remodeling and fracture healing. Initially it was thought that TGF-β1 stimulates the proliferation of undifferentiated mesenchymal cells and chondrocytes as well as the production of extracellular matrix during chondrogenesis and enchondral osteogenesis. As shown in recent studies optimizing effect of TGF-β1 on the processes of fracture healing is dose-dependent and requires its constant high concentration. In-depth understanding of the mechanisms of development reparative osteogenesis allows to develop a new strategy for optimization of the process of restoring the integrity of bone broken due to injury.

The neuroendocrine phenotype of gastric myofibroblasts and its loss with cancer progression

Stromal cells influence cancer progression. Myofibroblasts are an important stromal cell type, which influence the tumour microenvironment by release of extracellular matrix (ECM) proteins, proteases, cytokines and chemokines. The mechanisms of secretion are poorly understood. Here, we describe the secretion of marker proteins in gastric cancer and control myofibroblasts in response to insulin-like growth factor (IGF) stimulation and, using functional genomic approaches, we identify proteins influencing the secretory response. IGF rapidly increased myofibroblast secretion of an ECM protein, TGFβig-h3. The secretory response was not blocked by inhibition of protein synthesis and was partially mediated by increased intracellular calcium (Ca(2+)). The capacity for evoked secretion was associated with the presence of dense-core secretory vesicles and was lost in cells from patients with advanced gastric cancer. In cells responding to IGF-II, the expression of neuroendocrine marker proteins, including secretogranin-II and proenkephalin, was identified by gene array and LC-MS/MS respectively, and verified experimentally. The expression of proenkephalin was decreased in cancers from patients with advanced disease. Inhibition of secretogranin-II expression decreased the secretory response to IGF, and its over-expression recovered the secretory response consistent with a role in secretory vesicle biogenesis. We conclude that normal and some gastric cancer myofibroblasts have a neuroendocrine-like phenotype characterized by Ca(2+)-dependent regulated secretion, dense-core secretory vesicles and expression of neuroendocrine marker proteins; loss of the phenotype is associated with advanced cancer. A failure to regulate myofibroblast protein secretion may contribute to cancer progression.

The Role of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in the Pathophysiology of Neurodegeneration: A Literature Study

Matrix metalloproteinases (MMPs) and their natural tissue inhibitors (TIMPs) are involved in cell signaling processes and the release of extracellular matrix (ECM) and non-ECM molecules. Nonregulated MMP activity and an imbalance between metalloproteinases and their inhibitors might contribute to various disorders, including neurodegenerative diseases such as Alzheimer's disease (AD), which is the most common cause of dementia. There is a complex relationship between MMPs and TIMPs with AD. It has been shown that MMPs and TIMPs are localized in neuritic senile plaques and neurofibrillary tangles in the postmortem brains of patients with AD. Some MMPs have also been shown to induce tau aggregation and the formation of neurofibrillary tangles in vitro. Moreover, MMPs contribute to AD pathogenesis via the disruption of the blood-brain barrier and promotion of neurodegeneration. However, MMPs can degrade both soluble and fibrillar forms of amyloid-β (Aβ). It has also been shown that Aβ enhances the expression of MMPs in neuroglial cultures and induces the release of TIMP-1 by brain cells. Inhibition of Aβ-induced MMP activity resulted in an improvement of performance tests in mice. Moreover, simultaneous examination of MMP-9, MMP-2, and TIMP-1 in the CSF contributed to the ability to differentiate between AD and other types of dementia. Thus, the aim of this literature study was to describe the role of MMPs and TIMPs in neurodegeneration, as well as their potential usefulness as CSF or plasma biomarkers in the diagnosis of AD as well as other neurodegenerative disorders and vascular dementia.

Matrix metalloproteinase‐3 in the central nervous system: a look on the bright side

Matrix metalloproteinases (MMPs) are a large family of proteases involved in many cell-matrix and cell-cell signalling processes through activation, inactivation or release of extracellular matrix (ECM) and non-ECM molecules, such as growth factors and receptors. Uncontrolled MMP activities underlie the pathophysiology of many disorders. Also matrix metalloproteinase-3 (MMP-3) or stromelysin-1 contributes to several pathologies, such as cancer, asthma and rheumatoid arthritis, and has also been associated with neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and multiple sclerosis. However, based on defined MMP spatiotemporal expression patterns, the identification of novel candidate molecular targets and in vitro and in vivo studies, a beneficial role for MMPs in CNS physiology and recovery is emerging. The main purpose of this review is to shed light on the recently identified roles of MMP-3 in normal brain development and in plasticity and regeneration after CNS injury and disease. As such, MMP-3 is correlated with neuronal migration and neurite outgrowth and guidance in the developing CNS and contributes to synaptic plasticity and learning in the adult CNS. Moreover, a strict spatiotemporal MMP-3 up-regulation in the injured or diseased CNS might support remyelination and neuroprotection, as well as genesis and migration of stem cells in the damaged brain.

Static magnetic field exposure promotes differentiation of osteoblastic cells grown on the surface of a poly-l-lactide substrate

This study investigated the effects of static magnetic fields on the differentiation of MG63 cells cultured on the surface of poly-L-lactide (PLLA) substrates. The cells were continuously exposed to a 4,000 Gauss-static magnetic field (SMF) for 5 days. The proliferation effects of the SMF were measured by MTT assay. Morphologic changes and extracellular matrix release were observed by scanning electron microscopy. The effects of the SMF on alkaline phosphatase activity levels were compared between exposed and unexposed cells. The SMF-exposed cells exhibited decreased MTT values after 1 and 3 days of culture. In addition, SMF exposure promoted the expression of extracellular matrix in MG63 cells on the PLLA substrate. After 1 day, the alkaline phosphatase-specific activity of SMF-exposed MG63 cells was significantly increased (P < 0.05) with a ratio of 1.5-fold. These results show that MG63 cells, seeded on a PLLA disc and treated with SMF, had a more differentiated phenotype.

Static Magnetic Fields Promote Osteoblast-Like Cells Differentiation Via Increasing the Membrane Rigidity

The aim of this study was to test the differentiative effects of osteoblasts after treatment with a static magnetic field (SMF). MG63 osteoblast-like cells were exposed to a 0.4-T SMF. The differentiation markers were assessed by observing the changes in alkaline phosphatase activity and electron microscopy images. Membrane fluidity was used to evaluate alterations in the biophysical properties of the cellular membranes after the SMF simulation. Our results show that SMF exposure increases alkaline phosphatase activity and extracellular matrix release in MG63 cells. On the other hand, MG63 cells exposed to a 0.4-T SMF exhibited a significant increase in fluorescence anisotropy at 6 h, with a significant reduction in the proliferation effects of growth factors noted at 24 h. Based on these findings, the authors suggest that one of the possible mechanisms that SMF affects osteoblastic maturation is by increasing the membrane rigidity and reducing the proliferation-promoting effects of growth factors at the membrane domain.

Is the upregulation of bradykinin B2 receptors by TGF-β1 one of the missing pieces in the “airway hyperresponsiveness” puzzle?

transforming growth factor (TGF)-β1 belongs to the TGF-β family of proteins corresponding to a set of pleiotropic secreted signaling molecules with unique and potent immunoregulatory properties ([10][1]). TGF-β1 is produced by every leukocyte lineage, including lymphocytes, macrophages, and

Release of the extracellular matrix from conidia of Blumeria graminis in relation to germination

The release of extracellular matrix (ECM) and the emergence of germ tubes from conidia ofBlumeria graminis were studied by light microscopy and micromanipulation. More prompt and frequent ECM release was confirmed on an artificial hydrophobic substratum than on an artificial hydrophilic substratum. Conidia initially incubated on the hydrophilic substratum were transferred by micromanipulation to either the hydrophobic or the hydrophilic substrata. Immediately after transfer onto the hydrophobic substratum, 75% of conidia released ECM, whereas only 16% did so upon transfer to the hydrophilic substratum. Conidia transferred onto the hydrophobic substratum produced a primary germ tube (PGT) more promptly and frequently than those transferred to the hydrophilic substratum. Thus, conidia recognize and respond to substratum hydrophobicity perhaps immediately after contact. When inoculated onto either isolated barley cuticle or the hydrophobic artificial substratum, 2/3 of the conidia produced a PGT from their polar regions. By contrast, on the hydrophilic substratum 2/3 of conidia did so from the side region. These results show that substratum hydrophobicity affects the location of PGT emergence from conidia. Furthermore, the study indicates that very rapid recognition of surface hydrophobicity by conidia promotes ECM release and this in turn may influence the location of PGT emergence.