YTHDF2-regulated matrilin-3 mitigates post-reperfusion hemorrhagic transformation in ischemic stroke via the PI3K/AKT pathway.

Hemorrhagic transformation can complicate ischemic strokes after recanalization treatment within a time window that requires early intervention. To determine potential therapeutic effects of matrilin-3, rat cerebral ischemia-reperfusion was produced using transient middle cerebral artery occlusion (tMCAO); intracranial hemorrhage and infarct volumes were assayed through hemoglobin determination and 2,3,5-triphenyltetrazoliumchloride (TTC) staining, respectively. Oxygen-glucose deprivation (OGD) modeling of ischemia was performed on C8-D1A cells. Interactions between matrilin-3 and YTH N6-methyladenosine RNA binding protein F2 (YTHDF2) were determined using RNA immunoprecipitation assay and actinomycin D treatment. Reperfusion after tMCAO modeling increased hemorrhage, hemoglobin content, and infarct volumes; these were alleviated by matrilin treatment. Matrilin-3 was expressed at low levels and YTHDF2 was expressed at high levels in ischemic brains. In OGD-induced cells, matrilin-3 was negatively regulated by YTHDF2. Matrilin-3 overexpression downregulated p-PI3K/PI3K, p-AKT/AKT, ZO-1, VE-cadherin and occludin, and upregulated p-JNK/JNK in ischemic rat brains; these effects were reversed by LY294002 (a PI3K inhibitor). YTHDF2 knockdown inactivated the PI3K/AKT pathway, inhibited inflammation and decreased blood-brain barrier-related protein levels in cells; these effects were reversed by matrilin-3 deficiency. These results indicate that YTHDF2-regulated matrilin-3 protected ischemic rats against post-reperfusion hemorrhagic transformation via the PI3K/AKT pathway and that matrilin may have therapeutic potential in ischemic stroke.

Interleukin 38 alleviates aortic valve calcification by inhibition of NLRP3.

Calcific aortic valve disease (CAVD) is common in people over the age of 65. Progressive valvular calcification is a characteristic of CAVD and due to chronic inflammation in aortic valve interstitial cells (AVICs) resulting in CAVD progression. IL-38 is a naturally occurring anti-inflammatory cytokine; here, we report lower levels of endogenous IL-38 in AVICs isolated from patients' CAVD valves compared to AVICs from non-CAVD valves. Recombinant IL-38 suppressed spontaneous inflammatory activity and calcium deposition in cultured AVICs. In mice, knockdown of IL-38 enhanced the production of inflammatory mediators in murine AVICs exposed to the proinflammatory stimulant matrilin-2. We also observed that in cultured AVICs matrilin-2 stimulation activated the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome with procaspase-1 cleavage into active caspase-1. The addition of IL-38 to matrilin-2-treated AVICs suppressed caspase-1 activation and reduced the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, runt-related transcription factor 2, and alkaline phosphatase. Aged IL-38-deficient mice fed a high-fat diet exhibited aortic valve lesions compared to aged wild-type mice fed the same diet. The interleukin-1 receptor 9 (IL-1R9) is the putative receptor mediating the anti-inflammatory properties of IL-38; we observed that IL-1R9-deficient mice exhibited spontaneous aortic valve thickening and greater calcium deposition in AVICs compared to wild-type mice. These data demonstrate that IL-38 suppresses spontaneous and stimulated osteogenic activity in aortic valve via inhibition of the NLRP3 inflammasome and caspase-1. The findings of this study suggest that IL-38 has therapeutic potential for prevention of CAVD progression.

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.

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.

Extraction and characterization of matrix protein from pacific oyster (Crassostrea gigs) shell and its anti-osteoporosis properties in vitro and in vivo.

Matrix protein is a kind of secretory protein that regulates the biomineralization of the bivalve shell. In this study, a water-soluble matrix protein (WSMP) from Pacific oysters (Crassostrea gigs) shell was isolated, and its structure was analyzed in detail, in addition to its anti-osteoporosis activity in vitro and in vivo. Results showed that WSMP was an acidic protein with an apparent molecular mass of 47 and 79 kDa and contained a glycoprotein structure. In vitro, the reduction of Tartrate-resistant acid phosphatase (TRAP) and deoxypyridinoline (DPD) indicated that osteoclast activity was inhibited compared with the model group. Moreover, the increased osteocalcin (OCN) and BMD levels suggested that the high osteoblast activity and bone mineralization was improved. SEM analysis of the femur showed that there were fewer bone pits in experimental groups, which was consistent with the above results. In vivo, WSMP promoted the expression of alkaline phosphatase (ALP) and osteogenic differentiation factor BMP-2 in osteoblasts. In addition, the activity of osteoclasts was inhibited by regulating the process of osteoclast differentiation induced by RANKL. Both in vitro and in vivo studies showed that WSMP could promote osteogenesis and inhibit osteoclast absorption, thus demonstrating their potential applications in osteoporosis.

Matrilin-3-Primed Adipose-Derived Mesenchymal Stromal Cell Spheroids Prevent Mesenchymal Stromal-Cell-Derived Chondrocyte Hypertrophy.

Adipose-derived mesenchymal stromal cells (Ad-MSCs) are a promising tool for articular cartilage repair and regeneration. However, the terminal hypertrophic differentiation of Ad-MSC-derived cartilage is a critical barrier during hyaline cartilage regeneration. In this study, we investigated the role of matrilin-3 in preventing Ad-MSC-derived chondrocyte hypertrophy in vitro and in an osteoarthritis (OA) destabilization of the medial meniscus (DMM) model. Methacrylated hyaluron (MAHA) (1%) was used to encapsulate and make scaffolds containing Ad-MSCs and matrilin-3. Subsequently, the encapsulated cells in the scaffolds were differentiated in chondrogenic medium (TGF-ß, 1-14 days) and thyroid hormone hypertrophic medium (T3, 15-28 days). The presence of matrilin-3 with Ad-MSCs in the MAHA scaffold significantly increased the chondrogenic marker and decreased the hypertrophy marker mRNA and protein expression. Furthermore, matrilin-3 significantly modified the expression of TGF-ß2, BMP-2, and BMP-4. Next, we prepared the OA model and transplanted Ad-MSCs primed with matrilin-3, either as a single-cell suspension or in spheroid form. Safranin-O staining and the OA score suggested that the regenerated cartilage morphology in the matrilin-3-primed Ad-MSC spheroids was similar to the positive control. Furthermore, matrilin-3-primed Ad-MSC spheroids prevented subchondral bone sclerosis in the mouse model. Here, we show that matrilin-3 plays a major role in modulating Ad-MSCs' therapeutic effect on cartilage regeneration and hypertrophy suppression.

Matrilin-3 codelivery with adipose-derived mesenchymal stem cells promotes articular cartilage regeneration in a rat osteochondral defect model.

Matrilin-3 is an essential extracellular matrix component present only in cartilaginous tissues. Matrilin-3 exerts chondroprotective effects by regulating an anti-inflammatory function and extracellular matrix components. We hypothesized that the codelivery of matrilin-3 with infrapatellar adipose-tissue-derived mesenchymal stem cells (Ad-MSCs) may enhance articular cartilage regeneration. Matrilin-3 treatment of Ad-MSCs in serum-free media induced collagen II and aggrecan expression, and matrilin-3 in chondrogenic media also enhanced in vitro chondrogenic differentiation. Next, the in vivo effect of matrilin-3 codelivery with Ad-MSCs on cartilage regeneration was assessed in an osteochondral defect model in Sprague Dawley rats: Ad-MSCs and hyaluronic acid were implanted at the defect site with or without matrilin-3 (140, 280, and 700 ng). Safranin O staining revealed that matrilin-3 (140 and 280 ng) treatment significantly improved cartilage regeneration and glycosaminoglycan accumulation. In the animals treated with 140-ng matrilin-3, in particular, the defect site exhibited complete integration with surrounding tissue and a smooth glistening surface. The International Cartilage Repair Society macroscopic and O'Driscoll microscopic scores for regenerated cartilage were furthermore shown to be considerably higher for this group (matrilin-3; 140 ng) compared with the other groups. Furthermore, the defects treated with 140-ng matrilin-3 revealed significant hyaline-like cartilage regeneration in the osteochondral defect model; in contrast, the defects treated with 700-ng matrilin-3 exhibited drastically reduced cartilage regeneration with mixed hyaline-fibrocartilage morphology. Codelivery of matrilin-3 with Ad-MSCs significantly influenced articular cartilage regeneration, supporting the potential use of this tissue-specific protein for a cartilage-targeted stem cell therapy.

[Local injection therapy of damage of paraarticular tissue]. / Lokal'naia in''ektsionnaia terapiia porazhenii paraartikuliarnykh tkanei.

AIM: To evaluate the potential of local injection therapy with Alflutop in management of paraarticular tissue lesions (PTL). MATERIAL AND METHODS: 44 patients with various localization of PTL were examined and exposed to treatment. RESULTS: As a result of local therapy with Alflutop, the majority of patients with PTL experienced a decrease in pain syndrome and an increase in the functional capabilities. There was no negative dynamic of symptoms. CONCLUSION: Treatment with Alflutop was well tolerated by patients, easily applied, had a small number of side effects and was accompanied by a pronounced clinical effect.

Shark cartilage 14 kDa protein as a dendritic cells activator.

Low molecular weight components of shark cartilage are reported to have anti-tumor as well as immuno-stimulating effects. Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that have a key role in establishment of anti-cancer immune response. In this study, the effect of 14 kDa protein from shark cartilage was investigated on stimulation and maturation of dendritic cells. The isolated 14 kDa protein from shark cartilage extract was added to DCs medium during overnight culture and their maturation and T cells stimulation potential was investigated. The majority of shark-cartilage-treated DCs expressed higher levels of maturation markers and were more effective in stimulation of allogenic T cells compared with non-treated DCs (p < 0.05). Our results showed that shark cartilage 14 kDa protein can potentially be used in DC-mediated T-cells stimulation and induction of desirable immune responses in clinical trials such as cancer immunotherapy. However, further studies are required to examine this proposal.

Matrilin-1 is an inhibitor of neovascularization.

In the course of conducting a series of studies whose goal was to discover novel endogenous angiogenesis inhibitors, we have purified matrilin-1 (MATN-1) and have demonstrated, for the first time, that it inhibits neovascularization both in vitro and in vivo. Proteins were extracted from cartilage using a 2 m NaCl, 0.01 m HEPES buffer at 4 °C, followed by concentration of the extract. The concentrate was fractionated by size exclusion chromatography, and fractions were then screened for their ability to inhibit capillary endothelial cell (EC) proliferation in vitro. Fractions containing EC inhibitory activity were pooled and further purified by cation exchange chromatography. The resulting fractions from this step were then screened to isolate the antiangiogenic activity in vitro. This activity was identified by tandem mass spectrometry as being MATN-1. Human MATN-1 was cloned and expressed in Pichia pastoris and purified to homogeneity. Purified recombinant MATN-1, along with purified native protein, was shown to inhibit angiogenesis in vivo using the chick chorioallantoic membrane assay by the inhibition of capillary EC proliferation and migration. Finally, using a MATN-1-deficient mouse, we showed that angiogenesis during fracture healing was significantly higher in MATN-1(-/-) mice compared with the wild type mice as demonstrated by in vivo imaging and by elevated expression of angiogenesis markers including PECAM1, VEGFR, and VE-cadherin.

The matrilin-3 VWA1 domain modulates interleukin-6 release from primary human chondrocytes.

OBJECTIVE: We previously demonstrated the ability of matrilin-3 to modulate the gene expression profile of primary human chondrocytes (PHCs) toward a state favoring cartilage catabolism. The structure within matrilin-3 responsible for the induction of these catabolic genes is unknown. Here, we investigated the potential of matrilin-3 (MATN3) and truncated matrilin-3 proteins, in both monomeric and oligomeric form, to stimulate interleukin (IL)-6 release in PHCs. METHODS: We expressed full-length matrilin-3 oligomers, matrilin-3 von Willebrand factor A (VWA) domain oligomers, matrilin-3 four epidermal growth factor (EGF) domain oligomers, matrilin-3 monomers without oligomerization domains, matrilin-3 VWA domain monomers, and matrilin-3 4EGF monomers. We then incubated PHCs in the absence or presence of full-length matrilin-3 or one of the truncated matrilin-3 proteins and finally determined the release of IL-6 in cell-culture supernatants. RESULTS: The addition of full-length matrilin-3 oligomers, matrilin-3 VWA domain oligomers, and, less pronounced, matrilin-3 monomers without oligomerization domains, and matrilin-3 4EGF-oligomers to the cell-culture medium led to a significant induction of IL-6 in PHCs. DISCUSSION: Based on recombinant expression of different matrilin-3 domains in both monomeric and oligomeric form, this work demonstrated that the VWA1 domain of matrilin-3 is primarily responsible for the induction of IL-6 release and that the oligomerization of the VWA1 domain markedly promotes its activity.

Matrilin-3 induction of IL-1 receptor antagonist is required for up-regulating collagen II and aggrecan and down-regulating ADAMTS-5 gene expression.

INTRODUCTION: Deletion or mutation of the gene encoding the cartilage extracellular matrix (ECM) protein matrilin-3 (MATN3) results in the early onset of osteoarthritis (OA), suggesting chondroprotective properties of MATN3. To understand the mechanisms underlying these properties, we determined the effects of MATN3 protein on the expression of several key anabolic and catabolic genes involved in chondrocyte homeostasis, and the dependence of such regulation on the anti-inflammatory cytokine: IL-1 receptor antagonist (IL-1Ra). METHODS: The effects of recombinant human (rh) MATN3 protein were examined in C28/I2 immortalized human chondrocytes, primary human chondrocytes (PHCs), and primary mouse chondrocytes (PMCs). Messenger RNA levels of IL-1Ra, COL2A1, ACAN, MMP-13, and ADAMTS-4 and -5 were determined using real-time RT-PCR. Knocking down IL-1Ra was achieved by siRNA gene silencing. IL-1Ra protein levels were quantified by ELISA and the Bio-Plex Suspension Array System. COL2A1 protein level was quantified using Western blot analysis. Statistic analysis was done using the two-tailed t-test or one-way ANOVA. RESULTS: rhMATN3 protein induced gene expression of IL-1Ra in C28/I2 cells, PHCs, and PMCs in a dose- and time-dependent manner. Treatment of C28/I2 cells and PHCs with MATN3 protein stimulated gene expression of COL2A1 and ACAN. Conversely, mRNA levels of COL2A1 and ACAN were decreased in MATN3 KO mice. MATN3 protein treatment inhibited IL-1ß-induced MMP-13, ADAMTS-4 and ADAMTS-5 in C28/I2 cells and PHCs. Knocking down IL-1Ra abolished the MATN3-mediated stimulation of COL2A1 and ACAN and inhibition of ADAMTS-5, but had no effect on MATN3 inhibition of MMP-13 mRNA. CONCLUSION: Our findings point to a novel regulatory role of MATN3 in cartilage homeostasis due to its capacity to induce IL-1Ra, to upregulate gene expression of the major cartilage matrix components, and to downregulate the expression of OA-associated matrix-degrading proteinases in chondrocytes. The chondroprotective properties of endogenous MATN3 depend partly on its induction of IL-1Ra. Our findings raise a possibility to use rhMATN3 protein for anti-inflammatory and chondroprotective therapy.