Matrilin-3 supports neuroprotection in ischemic stroke by suppressing astrocyte-mediated neuroinflammation.

In the brain, the role of matrilin-3, an extracellular matrix component in cartilage, is unknown. Here, we identify that matrilin-3 decreased in reactive astrocytes but was unchanged in neurons after ischemic stroke in animals. Importantly, it declined in serum of patients with acute ischemic stroke. Genetic or pharmacological inhibition or supplementation of matrilin-3 aggravates or reduces brain injury, astrocytic cell death, and glial scar, respectively, but has no direct effect on neuronal cell death. RNA sequencing demonstrates that Matn3-/- mice display an increased inflammatory response profile in the ischemic brain, including the nuclear factor κB (NF-κB) signaling pathway. Both endogenous and exogenous matrilin-3 reduce inflammatory mediators. Mechanistically, extracellular matrilin-3 enters astrocytes via caveolin-1-mediated endocytosis. Cytoplasmic matrilin-3 translocates into the nucleus by binding to NF-κB p65, suppressing inflammatory cytokine transcription. Extracellular matrilin-3 binds to BMP-2, blocking the BMP-2/Smads pathway. Thus, matrilin-3 is required for astrocytes to exert neuroprotection, at least partially, by suppressing astrocyte-mediated neuroinflammation.

Low expression of TGF-ß2 and matrilin2 in human aqueous humour with acute primary angle closure.

Primary angle-closure glaucoma (PACG) is the leading cause of irreversible blindness in the world. Angle closure induced by pupil block and secondary iris synechia is the fundamental pathology of the PACG. The molecular mechanisms of angle closure have not yet been clearly illustrated. This study was designed to investigate the protein difference in the aqueous humour and explore new biomarker of the PACG. Aqueous humour (AH) was collected from patients with acute primary angle closure (APAC) and cataract (n = 10 in APAC group) and patients with cataract only (n = 10 in control group). Samples were pooled and measured using label-free proteome technology. Then, the differentially expressed proteins (DEPs) were verified by ELISA using independent AH samples (n = 20 each group). More than 400 proteins were revealed in both groups through proteomics. Comparing the two groups, there were 91DEPs. These proteins participate in biological activities such as inflammation, fibrosis, nerve growth and degeneration and metabolism. We found that the expression of transforming growth factor-ß2 and matrilin2 was downregulated in the APAC group. The two proteins are related to inflammation and extracellular matrix formation, which might be involved in angle closure. This study characterized DEPs in AH of the APAC and found a downregulated protein matrilin2.

Health consequences of mutant cartilage oligomeric matrix protein and its relationship to abnormal growth and joint degeneration.

Cartilage oligomeric matrix protein (COMP), an extracellular matrix protein, has been shown to enhance proliferation and mechanical integrity in the matrix, supporting functions of the growth plate and articular cartilage. Mutations in COMP cause pseudoachondroplasia (PSACH), a severe dwarfing condition associated with premature joint degeneration and significant lifelong joint pain. The MT (mutant)-COMP mouse mimics PSACH with decreased limb growth, early joint degeneration and pain. Ablation of endoplasmic reticulum stress CHOP signaling eliminated pain and prevented joint degeneration. The health effects of mutant COMP are discussed in relation to cellular/chondrocyte stress in the growth plate, articular cartilage and nearby tissues, and the implications for therapeutic approaches. There are many similarities between osteoarthritis and mutant-COMP protein-induced joint degeneration, suggesting that the relevance of findings in the joints may extend beyond PSACH to idiopathic primary OA.

Curcumin Reduces Pathological Endoplasmic Reticulum Stress through Increasing Proteolysis of Mutant Matrilin-3.

The intracellular retention of mutant cartilage matrix proteins and pathological endoplasmic reticulum (ER) stress disrupts ossification and has been identified as a shared disease mechanism in a range of skeletal dysplasias including short limbed-dwarfism, multiple epiphyseal dysplasia type 5 (EDM5). Although targeting ER stress is an attractive avenue for treatment and has proven successful in the treatment of a related skeletal dysplasia, to date no drugs have proven successful in reducing ER stress in EDM5 caused by the retention of mutant matrilin-3. Our exciting findings show that by using our established luciferase ER stress screening assay, we can identify a "natural" chemical, curcumin, which is able to reduce pathological ER stress in a cell model of EDM5 by promoting the proteasomal degradation mutant matrilin-3. Therefore, this is an important in vitro study in which we describe, for the first time, the success of a naturally occurring chemical as a potential treatment for this currently incurable rare skeletal disease. As studies show that curcumin can be used as a potential treatment for range of diseases in vitro, current research is focused on developing novel delivery strategies to enhance its bioavailability. This is an important and exciting area of research that will have significant clinical impact on a range of human diseases including the rare skeletal disease, EDM5.

Cartilage Oligomeric Matrix Protein, Diseases, and Therapeutic Opportunities.

Cartilage oligomeric matrix protein (COMP) is an extracellular matrix (ECM) glycoprotein that is critical for collagen assembly and ECM stability. Mutations of COMP cause endoplasmic reticulum stress and chondrocyte apoptosis, resulting in rare skeleton diseases. The bouquet-like structure of COMP allows it to act as a bridging molecule that regulates cellular phenotype and function. COMP is able to interact with many other ECM components and binds directly to a variety of cellular receptors and growth factors. The roles of COMP in other skeleton diseases, such as osteoarthritis, have been implied. As a well-established biochemical marker, COMP indicates cartilage turnover associated with destruction. Recent exciting achievements indicate its involvement in other diseases, such as malignancy, cardiovascular diseases, and tissue fibrosis. Here, we review the basic concepts of COMP and summarize its novel functions in the regulation of signaling events. These findings renew our understanding that COMP has a notable function in cell behavior and disease progression as a signaling regulator. Interestingly, COMP shows distinct functions in different diseases. Targeting COMP in malignancy may withdraw its beneficial effects on the vascular system and induce or aggravate cardiovascular diseases. COMP supplementation is a promising treatment for OA and aortic aneurysms while it may induce tissue fibrosis or cancer metastasis.

Fabrication and Characterization of Layer-by-Layer Janus Base Nano-Matrix to Promote Cartilage Regeneration.

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo uses. The addition of growth factors into these biomaterial scaffolds is generally done to provide an optimal cell culture environment, mediating cell differentiation and its subsequent functions. However, the growth factors in a conventional biomaterial scaffold are typically designed to be released upon implantation, which could result in unintended side effects on surrounding tissue or cells. Here, the DNA-inspired Janus base nano-matrix (JBNm) has successfully achieved a highly localized microenvironment with a layer-by-layer structure for self-sustainable cartilage tissue constructs. JBNms are self-assembled from Janus base nanotubes (JBNts), matrilin-3, and transforming growth factor beta-1 (TGF-ß1) via bioaffinity. The JBNm was assembled at a TGF-ß1:matrilin-3:JBNt ratio of 1:4:10, as this has been the determined ratio at which proper assembly into the layer-by-layer structure could occur. First, the TGF-ß1 solution was added to the matrilin-3 solution. Then, this mixture was pipetted several times to ensure sufficient homogeneity before the addition of the JBNt solution. This formed the layer-by-layer JBNm, after pipetting several times again. A variety of experiments were performed to characterize the layer-by-layer JBNm structure, JBNts alone, matrilin-3 alone, and TGF-ß1 alone. The formation of JBNm was studied with UV-Vis absorption spectra, and the structure of the JBNm was observed with transmission electron microscopy (TEM). As the innovative layer-by-layer JBNm scaffold is formed on a molecular scale, the fluorescent dye-labeled JBNm could be observed. The TGF-ß1 is confined within the inner layer of the injectable JBNm, which can prevent the release of growth factors to surrounding areas, promote localized chondrogenesis, and promote an anti-hypertrophic microenvironment.

Valproic acid modulates collagen architecture in the postoperative conjunctival scar.

Valproic acid (VPA), widely used for the treatment of neurological disorders, has anti-fibrotic activity by reducing collagen production in the postoperative conjunctiva. In this study, we investigated the capacity of VPA to modulate the postoperative collagen architecture. Histochemical examination revealed that VPA treatment was associated with the formation of thinner collagen fibers in the postoperative days 7 and 14 scars. At the micrometer scale, measurements by quantitative multiphoton microscopy indicated that VPA reduced mean collagen fiber thickness by 1.25-fold. At the nanometer scale, collagen fibril thickness and diameter measured by transmission electron microscopy were decreased by 1.08- and 1.20-fold, respectively. Moreover, delicate filamentous structures in random aggregates or closely associated with collagen fibrils were frequently observed in VPA-treated tissue. At the molecular level, VPA reduced Col1a1 but induced Matn2, Matn3, and Matn4 in the postoperative day 7 conjunctival tissue. Elevation of matrilin protein expression induced by VPA was sustained till at least postoperative day 14. In primary conjunctival fibroblasts, Matn2 expression was resistant to both VPA and TGF-ß2, Matn3 was sensitive to both VPA and TGF-ß2 individually and synergistically, while Matn4 was modulable by VPA but not TGF-ß2. MATN2, MATN3, and MATN4 localized in close association with COL1A1 in the postoperative conjunctiva. These data indicate that VPA has the capacity to reduce collagen fiber thickness and potentially collagen assembly, in association with matrilin upregulation. These properties suggest potential VPA application for the prevention of fibrotic progression in the postoperative conjunctiva. KEY MESSAGES: VPA reduces collagen fiber and fibril thickness in the postoperative scar. VPA disrupts collagen fiber assembly in conjunctival wound healing. VPA induces MATN2, MATN3, and MATN4 in the postoperative scar.

Monocytes augment inflammatory responses in human aortic valve interstitial cells via ß2-integrin/ICAM-1-mediated signaling.

OBJECTIVE: Inflammatory infiltration in aortic valves promotes calcific aortic valve disease (CAVD) progression. While soluble extracellular matrix (ECM) proteins induce inflammatory responses in aortic valve interstitial cells (AVICs), the impact of monocytes on AVIC inflammatory responses is unknown. We tested the hypothesis that monocytes enhance AVIC inflammatory responses to soluble ECM protein in this study. METHODS: Human AVICs isolated from normal aortic valves were cocultured with monocytes and stimulated with soluble ECM protein (matrilin-2). ICAM-1 and IL-6 productions were assessed. YAP and NF-κB phosphorylation were analyzed. Recombinant CD18, neutralizing antibodies against ß2-integrin or ICAM-1, and inhibitor of YAP or NF-κB were applied. RESULTS: AVIC expression of ICAM-1 and IL-6 was markedly enhanced by the presence of monocytes, although matrilin-2 did not affect monocyte production of ICAM-1 or IL-6. Matrilin-2 up-regulated the expression of monocyte ß2-integrin and AVIC ICAM-1, leading to monocyte-AVIC adhesion. Neutralizing ß2-integrin or ICAM-1 in coculture suppressed monocyte adhesion to AVICs and the expression of ICAM-1 and IL-6. Recombinant CD18 enhanced the matrilin-2-induced ICAM-1 and IL-6 expression in AVIC monoculture. Further, stimulation of coculture with matrilin-2 induced greater YAP and NF-κB phosphorylation. Inhibiting either YAP or NF-κB markedly suppressed the inflammatory response to matrilin-2 in coculture. CONCLUSION: Monocyte ß2-integrin interacts with AVIC ICAM-1 to augment AVIC inflammatory responses to soluble matrilin-2 through enhancing the activation of YAP and NF-κB signaling pathways. Infiltrated monocytes may promote valvular inflammation through cell-cell interaction with AVICs to enhance their sensitivity to damage-associated molecular patterns.

Literature Analysis Regarding the Combination of Substances: Glucosamine + Chondroitin in the Treatment of Osteoarthritis.

An essential component of joint quality is cartilage. Therefore, the protection of this is a prerequisite for maintaining the condition of each joint. The assessment of the presence of articular cartilage is shown by X-ray of both joints in the standing position. Cartilage protection is possible for 1, 2 and 3 degree of cartilage damage according to the Kellgren and Lawrence scale.The challenge for the physician is to identify the cause of OA in accordance with the principles of Evidence Based Orthopedics/Traumatology, and not merely treat symptomatically, which is usually ineffective.In order to objectively present treatment methods, indications and the period of their implementation, it is biologically reasonable to refer to the needs of cartilage tissue resulting from the analysis of the causes of its damage and indications for justified methods of its protection.Biomechanical and biological elements are important in the process of implementing articular cartilage protection.The biomechanical elements are: limb axis disorders, differences in length, distortions at the level of the support quadrilateral, pelvic triangle and shoulder triangle, as well as balance disorders resulting from disturbances in the segmental proportion of the Fi number according to Leonardo da Vinci.There are many biological elements of the discussed disorder and they concern: the state of articular cartilage structure, matrix structure, matrix biophysical elements, molecular sponge mechanism, chondrocytes, cartilage nutrition and the severity of osteoarthritis (OA).The improvement of the conditions of the biological elements of damaged articular cartilage is considered fundamental and concerns the positive impact on numerous cartilage matrix proteins by chondroprotection. This element of treatment consists in the use of chondroitin sulphate and glucosamine as a drug, administered together in the appropriate dose and for a long time depending on the degree of degradation of the articular cartilage, usually from several to several months. The combination of chondroitin sulfate with glucosamine causes the activation of a much larger number of matrix proteins than each of the preparations separately.The pharmacokinetics of chondroitin sulfate and glucosamine are positive and favor their chondroprotective effect.The pharmacoproteomics of chondroitin sulfate and glucosamine administered together result from the activation of as many joint cartilage matrix proteins as possible. The development of proteomic techniques creates completely new therapeutic possibilities and is used to study the action of individual molecules.A clinically significant fact is that both chondroitin and glucosamine are natural, endogenous components of bone tissue and articular cartilage, so the use of both drugs is biologically compatible and results in numerous elements of cartilage protection.

Recombinant Unique Cartilage Matrix-associated Protein Potentiates Osteogenic Differentiation and Mineralization of MC3T3-E1 Cells.

OBJECTIVE: The relative balance of osteoblasts in bone formation and osteoclasts in bone resorption is crucial for maintaining bone health. With age, this balance between osteoblasts and osteoclasts is broken, resulting in bone loss. Anabolic drugs are continuously being developed to counteract this low bone mass. Recombinant proteins are used as biotherapeutics due to being relatively easy to produce on a large scale and are cost-effective through various expression systems. This study aimed to develop a recombinant protein that would positively impact osteoblast differentiation and mineralized nodule formation using unique cartilage matrix-associated protein (UCMA). METHODS: A recombinant glutathione-S-transferase (GST)-UCMA fusion protein was generated in an E.coli system, and purified by affinity chromatography. MC3T3-E1 osteoblast cells and Osterix (Osx)-knockdown stable cells were cultured for 14 days to investigate osteoblast differentiation and nodule formation in the presence of the recombinant GST-UCMA protein. The differentiated cells were assessed by alizarin red S staining and quantitative PCR of the osteoblast differentiation marker osteocalcin. In addition, cell viability in the presence of the recombinant GST-UCMA protein was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell adhesion assay. RESULTS: The isolation of both purified recombinant GST-only and GST-UCMA proteins were confirmed at 26 kDa and 34 kDa, respectively, by Coomassie staining and western blot analysis. Neither dose-dependent nor time-dependent presence of recombinant GST-UCMA affected MC3T3-E1 cell viability. However, MC3T3-E1 cell adhesion to the recombinant GST-UCMA protein increased dose-dependently. Osteoblast differentiation and nodule formation were promoted in both MC3T3-E1 osteoblast cells and Osxknockdown stable cells when cultured in the presence of recombinant GST-UCMA protein. CONCLUSION: A recombinant GST-UCMA protein induces osteogenic differentiation and mineralization, suggesting its potential use as an anabolic drug to increase low bone mass in osteoporotic patients.

Identification of MATN3 as a Novel Prognostic Biomarker for Gastric Cancer through Comprehensive TCGA and GEO Data Mining.

Gastric cancer (GC) is still a vital malignant cancer across the world with unsatisfactory prognostic results. Matrilin-3 (MATN3) is a member of the extracellular matrix (ECM) protein family. The present research intends to explore the expression level of MATN3 in patients with GC and to explore the prognosis significance of MATN3. In this study, we observed that the MATN3 expression was remarkably upregulated in GC samples in contrast to noncancer samples. Clinical analyses unveiled that high MATN3 expression was related to age, tumor status, and clinical stages. Survival analyses unveiled that patients with high MATN3 expression displayed a poorer overall survival and progression-free survival than those with low MATN3 expression. The AUC of the relevant ROC curve for 1 year, 3 years, and 5 years of survival is 0.571, 0.596, and 0.720, separately. Multivariate assays revealed that MATN3 expression and stage were independent predictors of poor prognosis of GC patients. A meta-analysis unveiled that high MATN3 expression was tightly associated with better overall survival. Overall, our data indicated that MATN3 may have a diagnostic and prognostic value for patients with advanced gastric cancer and assist to improve clinical outcomes for GC patients.

Differentiation of Hypertrophic Chondrocytes from Human iPSCs for the In Vitro Modeling of Chondrodysplasias.

Chondrodysplasias are hereditary diseases caused by mutations in the components of growth cartilage. Although the unfolded protein response (UPR) has been identified as a key disease mechanism in mouse models, no suitable in vitro system has been reported to analyze the pathology in humans. Here, we developed a three-dimensional culture protocol to differentiate hypertrophic chondrocytes from induced pluripotent stem cells (iPSCs) and examine the phenotype caused by MATN3 and COL10A1 mutations. Intracellular MATN3 or COL10 retention resulted in increased ER stress markers and ER size in most mutants, but activation of the UPR was dependent on the mutation. Transcriptome analysis confirmed a UPR with wide-ranging changes in bone homeostasis, extracellular matrix composition, and lipid metabolism in the MATN3 T120M mutant, which further showed altered cellular morphology in iPSC-derived growth-plate-like structures in vivo. We then applied our in vitro model to drug testing, whereby trimethylamine N-oxide led to a reduction of ER stress and intracellular MATN3.

Extracellular matrix changes in corneal opacification vary depending on etiology.

Purpose: The purpose of this study is to examine the expression of tenascin-C and matrilin-2 in three different disorders, which frequently require corneal transplantation. These pathological conditions include bullous keratopathy (BK), Fuchs' endothelial corneal dystrophy (FECD), and corneal scarring in herpetic keratitis. Methods: Histological sections of corneal buttons removed during keratoplasty were analyzed in BK (n = 20), FECD (n = 9), herpetic keratitis (n = 12), and cadaveric control (n = 10) groups with light microscopy following chromogenic immunohistochemistry. The sections were evaluated by three investigators, and semiquantitative scoring (0 to 3+) was applied according to standardized methods at 400X magnification. Each layer of the cornea was investigated; moreover, the stroma was subdivided into subepithelial, middle, and pre-Descemet's membrane areas for more detailed analysis. Results: Excessive epithelial and stromal expression of tenascin-C was identified in all investigated conditions; the results were most pronounced in the pre-Descemet's membrane. Regarding matrilin-2, when examined in BK, there was increased labeling intensity in the epithelium (p<0.001) and stromal layers (p<0.05), and a decrease in the endothelium (p<0.001). In the other investigated conditions, only a low degree of stromal localization (p<0.05) of matrilin-2 was detected. Conclusions: The expression of tenascin-C and matrilin-2 differs when examined in various corneal pathologies resulting in opacification. Both molecules seem to be involved in regeneration and wound healing of the corneal matrix in these diseases.

miR-107 affects cartilage matrix degradation in the pathogenesis of knee osteoarthritis by regulating caspase-1.

BACKGROUND: Knee osteoarthritis (KOA) seriously affects the quality of life of KOA patients. This study aimed to investigate whether miR-107 could regulate KOA through pyroptosis to affect collagen protein secreted by chondrocytes through IL-1ß. METHODS: The proliferation of chondrocytes was detected by CCK-8 assay. RT-qPCR analysis was used to identify miR-107 expression and transfection effects. The expression of Col II, IL-1ß, IL-18, and MMP13 in supernatant of chondrocytes or chondrocytes was detected by ELISA assay and western blot analysis. The pyroptosis of chondrocytes was analyzed by TUNEL assay and the expression of pyroptosis-related proteins was analyzed by western blot. Luciferase reporter assay confirmed the relation of miR-107 to caspase-1. RESULTS: The proliferation of chondrocytes was decreased after LPS induction and further decreased by treatment of ATP. Single LPS treatment for chondrocytes downregulated the Col II expression while upregulated the expression of IL-1ß, IL-18, and MMP-13, which was further changed by ATP treatment. miR-107 expression was decreased in chondrocytes induced by LPS and further decreased in chondrocytes induced by LPS and ATP. In addition, miR-107 overexpression increased the proliferation and decreased the pyroptosis of chondrocytes induced by LPS and ATP. miR-107 overexpression upregulated the Col II expression while down-regulated the expression of IL-1ß, IL-18, and MMP-13 in supernatant of chondrocytes or chondrocytes induced by LPS and ATP. miR-107 overexpression down-regulated the expression of caspase-1, c-caspase-1, GSDMD-N, and TLR4 in chondrocytes induced by LPS and ATP. Furthermore, miR-107 directly targeted caspase-1. CONCLUSIONS: miR-107 can protect against KOA by downregulating caspase-1 to decrease pyroptosis, thereby promoting collagen protein secreted by chondrocytes by down-regulating IL-1ß.

Atelocollagen promotes chondrogenic differentiation of human adipose-derived mesenchymal stem cells.

Effective engineering approaches for cartilage regeneration involve a combination of cells and biomaterial scaffolds. Multipotent mesenchymal stem cells (MSCs) are important sources for cartilage regeneration. Atelocollagen provides a suitable substrate for MSC attachment and enhancing chondrogenic differentiation. Here, we assessed the chondrogenic potential of adipose tissue derived human MSCs (hMSCs) mixed with atelocollagen gel. We observed cell attachment, viability, and microstructures by electron microscopy over 21 days. The levels of Sox9, type II collagen, aggrecan, type I collagen, Runx2, type X collagen, ALP, Osterix, and MMP13 were measured by RT-qPCR. Cartilage matrix-related proteins were assessed by enzyme-linked immunosorbent assay (ELISA), histology, and immunohistochemistry. hMSCs of all groups exhibited well-maintained cell survival, distribution and morphology. Abundant type II collagen fibers developed on day 21; while Sox9, type II collagen, and aggrecan expression increased over time in the atelocollagen group. However, type I collagen, RUNX2, type X collagen (CoL10A1), Osterix, and ALP were not expressed. These results corroborated the protein expression detected by ELISA. Further, histological analysis revealed lacunae-like structures, while staining demonstrated glycosaminoglycan accumulation. Cumulatively, these results indicate that atelocollagen scaffolds improve hMSC chondrogenic differentiation and are a potential approach for cartilage regeneration.

Matrilin-3 alleviates extracellular matrix degradation of nucleus pulposus cells via induction of IL-1 receptor antagonist.

OBJECTIVE: The intervertebral disc contains abundant extracellular matrix (ECM) imbued with proteoglycans, collagens, and water. With the development of intervertebral disc degeneration (IVDD), the ECM undergoes changes characterized by loss of water content, proteoglycans, and collagen content. The purpose of this study was to explore the vital role of Matrilin-3, an ECM protein involved in the progress of IVDD. MATERIALS AND METHODS: NP cells were isolated from the patients' disc samples and exposed to recombinant human (rh)-Matrilin-3 protein (MATN3), and IL-1ß is used as a reducer of nucleus pulposus (NP) cells degeneration. Matrilin-3 and IL-1 receptor antagonist (IL-1Ra) were knocked down by siRNA transfection. Messenger RNA expressions of IL-1Ra, Collagen II, aggrecan, MMP-13, and ADAMTS-5 were determined using Real-Time quantitative Polymerase Chain Reaction (RT-qPCR). Later, the protein levels of IL-Ra, Collagen II, and aggrecan were also detected by Western blot. The IL-1Ra, MMP-13, and ADAMTS-5 dose of the supernatants in the culture medium was determined by enzyme linked immunosorbent assay (ELISA). Finally, immunofluorescence was used to expose the expression of Collagen II, aggrecan, and Collagen X. RESULTS: It was found that the expression of IL-1Ra was markedly increased in the present of MATN3 or IL-1ß, especially these two at once. Besides, MATN3 could upregulate Collagen II and aggrecan expressions, as well as inhibit the MMP-13 and Collagen X production of NP cells. However, the protective effects of Collagen II and aggrecan were abolished after Matrilin-3 silenced. Furthermore, IL-1ß downregulated the Collagen II and aggrecan but promoted the MMP-13 and Collagen X levels of NP cells, which were antagonized by the action of MATN3. Surprisingly, silencing of IL-1Ra significantly abolished the MATN3-induced the protective effects of ECM in NP cells. CONCLUSIONS: This study provides a novel viewpoint of Matrilin-3 in the ECM stability of NP due to its ability to activate IL-1Ra. It is considered that MATN3 efficiently protects ECM degeneration of human NP cells related to maintain the content of Collagen II and aggrecan, as well as inflammatory inhibition.

Collagen IX deficiency leads to premature vascularization and ossification of murine femoral heads through an imbalance of pro- and antiangiogenic factors.

OBJECTIVE: The vascular invasion of cartilage is an essential process in the endochondral ossification of long bones. In contrast, vascularization of articular cartilage constitutes a pathological mechanism in the development of osteoarthritis. Polymorphisms of Col9a1 have been described as risk factors for hip osteoarthritis (OA) and the loss of collagen IX is known to lead to premature OA of the hip joint in mice but the underlying mechanism is so far unknown. DESIGN: To understand the contribution of collagen IX to OA development in the hip joint, we analyzed the early development of murine Col9a1-/- femoral heads between newborn stage and 16 weeks of age. RESULTS: We found significantly accelerated ossification of the femoral heads in the absence of collagen IX as well as premature vascular and osteoclast invasion, even though hypertrophic differentiation was delayed. The loss of collagen IX led to anatomically altered femoral heads lacking the epiphyseal tubercle. Interestingly, this region was found to contain highest levels of the antiangiogenic protein thrombospondin-1 (TSP-1). Hence, TSP-1 levels were strongly reduced in the Col9a1-/- femoral heads. In addition, antiangiogenic matrilin-1 was found to be decreased, while proangiogenic active MMP-9 levels were increased in the collagen IX deficient mice compared to wildtype controls. CONCLUSION: We conclude that collagen IX protects against premature vascularization and cartilage to bone transition in femoral heads by increasing the levels of antiangiogenic TSP-1 and matrilin-1 and decreasing the levels of proangiogenic active MMP-9.

Mediation of Cartilage Matrix Degeneration and Fibrillation by Decorin in Post-traumatic Osteoarthritis.

OBJECTIVE: To elucidate the role of decorin, a small leucine-rich proteoglycan, in the degradation of cartilage matrix during the progression of post-traumatic osteoarthritis (OA). METHODS: Three-month-old decorin-null (Dcn-/- ) and inducible decorin-knockout (Dcni KO ) mice were subjected to surgical destabilization of the medial meniscus (DMM) to induce post-traumatic OA. The OA phenotype that resulted was evaluated by assessing joint morphology and sulfated glycosaminoglycan (sGAG) staining via histological analysis (n = 6 mice per group), surface collagen fibril nanostructure via scanning electron microscopy (n = 4 mice per group), tissue modulus via atomic force microscopy-nanoindentation (n = 5 or more mice per group) and subchondral bone structure via micro-computed tomography (n = 5 mice per group). Femoral head cartilage explants from wild-type and Dcn-/- mice were stimulated with the inflammatory cytokine interleukin-1ß (IL-1ß) in vitro (n = 6 mice per group). The resulting chondrocyte response to IL-1ß and release of sGAGs were quantified. RESULTS: In both Dcn-/- and Dcni KO mice, the absence of decorin resulted in accelerated sGAG loss and formation of highly aligned collagen fibrils on the cartilage surface relative to the control (P < 0.05). Also, Dcn-/- mice developed more salient osteophytes, illustrating more severe OA. In cartilage explants treated with IL-1ß, loss of decorin did not alter the expression of either anabolic or catabolic genes. However, a greater proportion of sGAGs was released to the media from Dcn-/- mouse explants, in both live and devitalized conditions (P < 0.05). CONCLUSION: In post-traumatic OA, decorin delays the loss of fragmented aggrecan and fibrillation of cartilage surface, and thus, plays a protective role in ameliorating cartilage degeneration.

Mechanistic Roles of Matrilin-2 and Klotho in Modulating the Inflammatory Activity of Human Aortic Valve Cells.

BACKGROUND: Calcific aortic valve disease (CAVD) is a chronic inflammatory disease. Soluble extracellular matrix (ECM) proteins can act as damage-associated molecular patterns and may induce valvular inflammation. Matrilin-2 is an ECM protein and has been found to elevate the pro-osteogenic activity in human aortic valve interstitial cells (AVICs). Klotho, an anti-aging protein, appears to have anti-inflammatory properties. The effect of matrilin-2 and Klotho on AVIC inflammatory responses remains unclear. METHODS AND RESULTS: Isolated human AVICs were exposed to matrilin-2. Soluble matrilin-2 induced the production of ICAM-1, MCP-1, and IL-6. It also induced protein kinase R (PKR) activation via Toll-like receptor (TLR) 2 and 4. Pretreatment with PKR inhibitors inhibited NF-κB activation and inflammatory mediator production induced by matrilin-2. Further, recombinant Klotho suppressed PKR and NF-κB activation and markedly reduced the production of inflammatory mediators in human AVICs exposed to matrilin-2. CONCLUSIONS: This study revealed that soluble matrilin-2 upregulates AVIC inflammatory activity via activation of the TLR-PKR-NF-κB pathway and that Klotho is potent to suppress AVIC inflammatory responses to a soluble ECM protein through inhibiting PKR. These novel findings indicate that soluble matrilin-2 may accelerate the progression of CAVD by inducing valvular inflammation and that Klotho has the potential to suppress valvular inflammation.

MiR-202-5p/MATN2 are associated with regulatory T-cells differentiation and function in allergic rhinitis.

Regulatory T cells (Tregs) play a crucial role in allergic rhinitis (AR). However, the mechanism of how Tregs are regulated in AR is poorly understood. Here, we aimed to explore the role of Tregs in AR and how Tregs were regulated by miR-202-5P, which was demonstrated to be important in AR. Peripheral blood mononuclear cells (PBMC) were isolated from collected blood samples. Tregs were purified using Regulatory T Cell Isolation Kit, and differentiated from isolated CD4 T cells using recombinant human interleukin-2 (rhIL-2) and transforming growth factor beta (TGF-ß). mRNA expression levels of miR-202-5p, matrilin-2 (MATN2), TGF-ß1 and interleukin-10 (IL-10) were detected by real-time PCR. The concentrations of IL-4, interleukin-17 (IL-17), IL-10, interferon gamma (IFN-γ) and TGF-ß1 were detected by enzyme-linked immunosorbent assay (ELISA). MATN2 protein level was detected by Western blot. MiR-202-5p expression dramatically elevated in PBMCs, CD4+ T cells and Tregs of AR patients. In vitro, miR-202-5p promoted Tregs differentiation via targeting MATN2. MiR-202-5p/MATN2 axis mediated Tregs proliferation and functions. MiR-202-5p/MATN2 are associated with regulatory T-cells differentiation and function in allergic rhinitis.