Runx2 Levels Research Articles

Exosomes derived from platelet-rich plasma present a novel potential in repairing knee articular cartilage defect combined with cyclic peptide-modified β-TCP scaffold

BackgroundThe aim of this study was to investigate the therapeutic effects and mechanisms of PRP-exos combined with cyclic peptide-modified β-TCP scaffold in the treatment of rabbit knee cartilage defect.MethodsPRP-exos were extracted and characterized by TEM, NTA and WB. The therapeutic effects were evaluated by ICRS score, HE staining, Immunohistochemistry, qRT-PCR and ELISA. The repair mechanism of PRP-exos was estimated and predicted by miRNA sequencing analysis and protein–protein interaction network analysis.ResultsThe results showed that PRP-exos had a reasonable size distribution and exhibited typical exosome morphology. The combination of PRP-exos and cyclic peptide-modified β-TCP scaffold improved ICRS score and the expression level of COL-2, RUNX2, and SOX9. Moreover, this combination therapy reduced the level of MMP-3, TNF-α, IL-1β, and IL-6, while increasing the level of TIMP-1. In PRP-exos miRNA sequencing analysis, the total number of known miRNAs aligned across all samples was 252, and a total of 91 differentially expressed miRNAs were detected. The results of KEGG enrichment analysis and the protein–protein interaction network analysis indicated that the PI3K/AKT signaling pathway could impact the function of chondrocytes by regulating key transcription factors to repair cartilage defect.ConclusionPRP-exos combined with cyclic peptide-modified β-TCP scaffold effectively promoted cartilage repair and improved chondrocyte function in rabbit knee cartilage defect. Based on the analysis and prediction of PRP-exos miRNAs sequencing, PI3K/AKT signaling pathway may contribute to the therapeutic effect. These findings provide experimental evidence for the application of PRP-exos in the treatment of cartilage defect.

Functional Mechanism and Clinical Implications of lncRNA LINC-PINT in Delayed Fracture Healing

Background Fracture healing can be impeded or even compromised by various factors, resulting in a growing number of patients suffering. The lncRNA LINC-PINT has garnered attention for its latent role in enhancing fracture healing, but its specific functions in this process remain unclear. Objectives The primary objective of this study is to investigate the clinical relevance and underlying molecular mechanisms of LINC-PINT in delayed fracture healing (DFH), while also assessing its potential as an early diagnostic biomarker. Materials and methods The expression levels of LINC-PINT were measured in the serum of DFH patients and those with normal fracture healing using RT-qPCR. In MC3T3-E1 cells, the study investigated the influence on the expression of differentiation-related protein, cell viability, and apoptosis through the modulation of LINC-PINT and miR-324-3p. To elucidate the targeting relationship between LINC-PINT, miR-324-3p, and BMP2, a dual-luciferase reporter assay was employed. Results The findings revealed a significant downregulation of LINC-PINT expression in DFH patients. LINC-PINT showed high sensitivity and specificity as a diagnostic marker for DFH. In MC3T3-E1 cells, LINC-PINT overexpression markedly enhanced the expression levels of ALP, OCN, Runx2, and OPN, improved cell viability, and inhibited apoptosis. LINC-PINT negatively regulated miR-324-3p, and the effects of LINC-PINT were counteracted by miR-324-3p. LINC-PINT was found to regulate BMP2 by targeting miR-324-3p. Conclusion LINC-PINT could serve as an early diagnostic biomarker for DFH and slow the progression of DFH by modulating BMP2 through the targeted regulation of miR-324-3p. This research presents new molecular targets for the diagnosis and treatment of DFH.

The novel role of complement 3 in Wnt5a-mediated vascular smooth muscle cell calcification

Abstract Background Although vascular calcification (VC) is a risk factor for cardiovascular and all-cause mortality, a therapy is not yet available. VC involves the transdifferentiation of vascular smooth muscle cells (VSMC) towards an osteochondrogenic lineage that results in calcium phosphate salts deposition in the arterial wall. We have previously shown an association between plasma WNT5A levels and new onset and progression of coronary artery calcification. Purpose We aimed to study the poorly understood role of Wnt5a in VSMC calcification. Methods Study 1 included 18 patients with coronary artery disease (CAD) undergoing cardiac surgery; VSMC were isolated from their saphenous veins to perform in vitro assays and microarrays. Study 2 included 11 patients with CAD; IMA were treated ex vivo with Wnt5a recombinant protein (rWnt5a) to perform western blot (WB) analyses. Study 3 included 647 patients with CAD; RNA sequencing was performed on samples from their internal mammary arteries (IMA), IMA perivascular adipose (PVAT), and thoracic adipose tissue (ThAT). VSMC and human aortic smooth muscle cells (HASMC) from healthy donors were grown in osteogenic medium in the presence or absence of rWnt5a with or without specific protein inhibitors. Calcium deposition and expression of VC markers (i.e. RUNX2, BMP2, and MSX2) were assessed with a colorimetric assay and by RT-qPCR, respectively. Phosphorylation/activation of non-canonical Wnt pathway effectors was evaluated by WB analyses. Results rWnt5a treatment significantly increased calcium deposition in both VSMC and HASMC at a concentration that activates both JNK and CaMKII, that are effectors of the non-canonical planar cell polarity pathway and the calcium-dependent pathway, respectively. Inhibition of either JNK or CaMKII could not counteract rWnt5a-mediated increase in VSMC calcium deposition; whereas, inhibition of both non-canonical pathway effectors (JNK-i and CaMKII-i) prevented rWnt5a-mediated increase in calcium deposition by VSMC (A) and HASMC. Inhibition of JNK and CaMKII also prevented rWnt5a-induced gene expression of VC markers, such as RUNX2 (B). rWnt5a treatment in IMA increased the activation of JNK and CaMKII. Patients with higher WNT5A levels in IMA had significantly higher RUNX2 levels. By intersecting RNA sequencing and microarray data complement 3 (C3) resulted as the most upregulated gene in rWnt5a-treated VSMC among those genes whose levels positively correlated in IMA with both WNT5A and RUNX2 expression (C). Patients with higher WNT5A levels in IMA, their PVAT, and ThAT had significantly higher C3 levels (D), and patients with higher levels of C3AR1, that is the gene coding for the C3a receptor, had higher VC markers expression. C3 inhibition (C3-i) with a C3AR1 antagonist counteracted Wnt5a-induced VSMC calcium deposition (E). Conclusions Non-canonical Wnt signalling promotes VSMC calcification through C3. Wnt5a may be a therapeutic target in VC.Abstract Figure

Screening of active components of melastoma dodecandrum lour. against diabetic osteoporosis using cell membrane chromatography-mass spectrometry

BackgroundMelastoma dodecandrum Lour. (MD), a traditional botanical drug known for its hypoglycemic, antioxidant, and anti-inflammatory properties, is commonly used to treat diabetes, osteoarthritis, and osteoporosis. However, its specific active components against diabetic osteoporosis remain unclear.PurposeThis study aimed to identify the key active components in MD using cell membrane chromatography coupled with mass spectrometry and validate their effects in vitro.MethodsAn AGEs-induced osteoblast injury model was established. MTT assays measured cell viability, and ALP activity was assessed using a biochemical kit. Western blotting was employed to detect the expression levels of osteoblast-related proteins OCN and RUNX2 and the AGE receptor protein RAGE. ELISA was used to determine the levels of SOD, MDA, CAT, and GPx. PCR quantified TNF-α expression to evaluate the protective effects and potential mechanisms of MD. The AGEs-induced osteoblast cell membrane chromatography-mass spectrometry method facilitated the rapid identification of potentially active compounds based on their affinity for the osteoblast cell membrane. Cell experiments further validated the activity of the characteristic component isovitexin.ResultsMD significantly improved cell viability in AGEs-damaged osteoblasts, enhanced ALP, SOD, CAT, and GPx activities, reduced MDA levels, increased OCN and RUNX2 protein expression, and decreased TNF-α mRNA and RAGE protein expression. Cell membrane chromatography identified 20 chemical constituents, including 13 flavonoids, 4 organic acids, 1 phenylpropanoids, 1 terpenoids, and 1 alkaloid. Cell experiments have confirmed that isovitexin has significant protective activity against osteoblasts and can inhibit the expression of specific receptor RAGE on the osteoblast membrane, consistent with the effect of MD.ConclusionMD and its active component, isovitexin, provide protective effects against AGEs-induced osteoblast injury, offering a basis for subsequent drug development.

Alleviating vascular calcification with Bushen Huoxue formula in rats with chronic kidney disease by inhibiting the PTEN/PI3K/AKT signaling pathway through exosomal microRNA-32.

Vascular calcification (VC) significantly raises cardiovascular mortality in chronic kidney disease (CKD) patients. VC is characterized by the phenotypic transformation of vascular smooth muscle cells (VSMCs) to osteoblast-like cells, mediated by exosomes derived from calcified VSMCs and the exosomal microRNAs (miRNA) which may trigger some signals to recipient VSMCs. Bushen Huoxue (BSHX) formula has demonstrated its clinical efficacy in CKD and its protective role in CKD-VC rats has also been observed. However, little is known about its underlying mechanism. To establish a VC model, aortic VSMCs from rats were induced to osteogenic differentiation by high-level phosphate (HP) in vitro. The expression of exosome and calcification makers were analyzed by western blot, including CD9, CD63, α-SMA, BMP-2, and Runx2, respectively. Differential expression of exosomal miRNAs in normal and HP-induced VSMCs were identified by using whole miRNA microarray technology. GO and KEGG analyses were performed to determine the significant enrichment of functions and signaling pathways in the target genes. In vivo, the CKD-VC rat model was established by administering adenine gavage combined with a high phosphorus diet. The rats were divided into normal control, model, low-dose BSHX, medium-dose BSHX, high-dose BSHX groups, and sevelamer groups. The blood biochemical parameters were measured. Renal histopathology and aortic calcification were observed. Western blot detected the levels of the calcification markers. Quantitative real-time PCR (qPCR) assay detected exosomal microRNA-32 (miR-32) mRNA expression in the aorta, the most differentially expressed exosomal miRNA previously identified. Phosphatase and tensin homolog located on chromosome ten (PTEN)/phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) signaling pathway components were also tested by western blot. Exosomal miRNA-32 and PI3K/AKT signaling pathways were highly differentially expressed between normal and HP-induced VSMCs. In vivo, BSHX improved blood biochemical parameters, renal histopathology, and aortic calcification in CKD-VC rats. BSHX increased the expression level of α-SMA and decreased the level of BMP-2 and Runx2. BSHX also lowered the expression level of exosomal miR-32 mRNA, enhanced PTEN expression, therefore, reduced p-PI3K and p-AKT levels in the aorta. BSHX alleviated VC in CKD rats by downregulating exosomal miR-32 expression in the aorta, thereby promoting PTEN expression and inhibiting the PI3K/AKT signaling pathway.

Effects of i-PRF, A-PRF+, and EMD on Osteogenic Potential of Osteoblasts on Titanium.

The study evaluates three biologically active substances with known bone-inductive potential on previously decontaminated titanium (Ti) discs. Rough and smooth Ti surfaces were contaminated with a multispecies biofilm and cleaned with a chitosan brush. Discs were treated either with injectable-platelet-rich fibrin (i-PRF), advanced platelet-rich fibrin (A-PRF+), or enamel matrix derivatives (EMDs) before osteoblast seeding. Biocompatibility, adhesion, migration, and gene expression of runt-related transcription factor 2 (RUNX2), collagen Type I Alpha 2 (COL1a2), alkaline phosphatase (ALP), osteocalcin (OC), and osteonectin (ON) were performed. All the tested biologic agents similarly increased cell viability. Specifically, osteoblasts seeded over i-PRF and EMD-treated surfaces showed improvement in adhesion and migration and significantly increased ALP, OC, ON, RUNX-2, and COL1a2 mRNA levels up to 2.8 fold (p < 0.05) with no differences between Ti surfaces. i-PRF and EMD possess beneficial bioactive properties that enhance tissue healing and promote regeneration on thoroughly sterilized surfaces. Biologically active materials may hold the potential to influence the process of implant re-osseointegration, which warrants more research since sterilization of the affected surfaces under clinical conditions is still not reliably possible and remains one of the greatest challenges.

Predictive value of preoperative Fried Frailty Phenotype assessment and serum biomarkers on the prognosis of elderly patients with femoral neck fracture under general anesthesia within 3 months after surgery.

Femoral neck fracture (FNF) seriously impact the health of the elderly and affect their long-term quality of life of the patients. This study aimed to determine whether combining the preoperative Fried Frailty Phenotype (FFP) with serum fibroblast growth factor receptor 3 (FGFR3) and run-related transcription factor 2 (RUNX2) could better predict the prognosis of elderly patients with FNF 3 months after surgery. A total of 150 elderly patients with FNF (60-89 years old) were enrolled and divided into a nonfrailty cohort and a frailty cohort based on preoperative FFP evaluation. The hip recovery of patients 3 months after surgery was evaluated using Harris Hip Score (HHS). Serum FGFR3 and RUNX2 levels were analyzed, and the relationship between HHS and serum FGFR3 and RUNX2 levels was evaluated. The specificity and sensitivity of FFP, serum FGFR3 and RUNX2 were evaluated using ROC curves before surgery. Potential prognostic factors were analyzed using multivariate logistic regression. Serum FGFR3 and RUNX2 levels were lower and hip recovery was poorer in the frailty cohort than in the nonfrailty cohort (p < 0.001). Within 3 months after surgery, there were 12 deaths (17.6%) in the frailty cohort and 1 in the nonfrailty cohort (1.2%) (p < 0.001). FFP assessment combined with serum FGFR3 and RUNX2 levels had a higher diagnostic significance. Readmission and preoperative frailty phenotype were independent factors affecting the prognosis of patients with FNF. HHS scores greater than 70 and higher levels of serum FGFR3 and RUNX2 cutoff values (7.85 ng/mL and 56.5 ng/mL, respectively) were identified as protective factors for prognosis. Assessing FFP alongside serum FGFR3 and RUNX2 levels may aid in evaluating the prognosis of elderly patients with FNF 3 months after surgery.

Comparative Study of Photobiomodulation Effects on Alveolar Socket Hard Tissue Healing in Rats: Application of 980 nm Versus 810 nm Lasers.

Background: This study aimed to explore the differential effects of photobiomodulation (PBM) via 980 nm and 810 nm lasers on the hard tissue healing of rat alveolar sockets, with a focus on a comparative analysis of hard tissue regeneration and osteogenic gene expression. Objective: This study aimed to explore the effects of PBM using 980 nm and 810 nm lasers on hard tissue healing of rat alveolar sockets, focusing on hard tissue regeneration and osteogenic gene expression. Materials and Methods: Thirty-six male Wistar rats (5 weeks old) had both right and left maxillary first molars extracted. Post extraction, the right alveolar sockets received PBM treatment with either 980 nm (0.3 W, 18 J/cm2) or 810 nm (0.1 W, 6 J/cm2) lasers for seven days, whereas the left sockets served as controls. Rats were euthanized on days 3, 7, 14, and 28 for histopathological, immunohistochemical, micro computed tomography (micro-CT), and quantitative polymerase chain reactionanalyses. Results: On day 3, early granulation tissue, neovascularization, and inflammatory cell aggregates were observed in all groups. By day 7, active osteoclasts and osteoblasts were noted, with a significant increase in CD31-positive cells in the 980 nm group (p < 0.05). Day 14 showed new bone formation, and by day 28, increased cancellous bone and collagen content were present in all groups, with no significant differences between them (p > 0.05). Gene expression analysis revealed higher BMP-2 and Runx-2 levels in laser-treated groups on day 14 (p < 0.05), with the 980 nm group having higher BMP-2 levels than the 810 nm group (p < 0.05). Bone sialoprotein expression was higher in laser-treated groups on days 14 and 28 (p < 0.05), and osteocalcin expression was highest in the 980 nm group on both days (p < 0.05). Micro-CT analysis showed no significant differences among groups in bone mineral density, bone surface (BS)/bone volume (BV), or bone volume (BV)/TV (total volume) indices. Conclusion: PBM with 980 nm and 810 nm lasers promotes early-stage hard tissue healing in extraction sockets, with the 980 nm laser more effectively enhancing osteogenic gene expression, suggesting its potential as an adjunctive therapy in dental and oral surgery.

Regulation of Osteogenic Differentiation of hBMSCs by the Overlay Angles of Bone Lamellae-like Matrices.

Oriented fibers in bone lamellae are recognized for their contribution to the anisotropic mechanical performance of the cortical bone. While increasing evidence highlights that such oriented fibers also exhibit osteogenic induction to preosteoblasts, little is known about the effect of the overlay angle between lamellae on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). In this study, bone lamellae-like fibrous matrices composed of aligned core-shell [core: polycaprolactone (PCL)/type I collagen (Col I) + shell: Col I] nanofibers were seeded with human BMSCs (hBMSCs) and then laid over on each other layer-by-layer (L-b-L) at selected angles (0 or 45°) to form three-dimensional (3D) constructs. Upon culture for 7 and 14 days, osteogenic differentiation of hBMSCs and mineralization within the lamellae assembly (LA) were characterized by real-time PCR, Western blot, immunofluorescent staining for osteogenic markers, and alizarin red staining for calcium deposition. Compared to those of random nanofibers (LA-RF) or aligned fibers with the overlay angle of 45° (LA-AF-45), the LA of aligned fibers at a 0° overlay angle (LA-AF-0) exhibited a noticeably higher osteogenic differentiation of hBMSCs, i.e., elevated gene expression of OPN, OCN, and RUNX2 and protein levels of ALP and RUNX2, while promoting mineral deposition as indicated by alizarin red staining and mechanical testing. Further analyses of hBMSCs within LA-AF-0 revealed an increase in both total and phosphorylated integrin β1, which subsequently increased total focal adhesion kinase (FAK), phosphorylated FAK (p-FAK), and phosphorylated extracellular signal kinase ERK1/2 (p-ERK1/2). Inhibition of integrin β1 and ERK1/2 activity effectively reduced the LA-AF-0-induced upregulation of p-FAK and osteogenic markers (OPN, OCN, and RUNX2), confirming the involvement of integrin β1-FAK-ERK1/2 signaling. Altogether, the overlay angle of aligned core-shell nanofiber membranes regulates the osteogenic differentiation of hBMSCs via integrin β1-FAK-ERK1/2 signaling, unveiling the effects of anisotropic fibers on bone tissue formation.

Impact of Polydeoxyribonucleotides on the Morphology, Viability, and Osteogenic Differentiation of Gingiva-Derived Stem Cell Spheroids

Background and Objectives: Polydeoxyribonucleotides (PDRN), composed of DNA fragments derived from salmon DNA, is widely recognized for its regenerative properties. It has been extensively used in medical applications, such as dermatology and wound healing, due to its ability to enhance cellular metabolic activity, stimulate angiogenesis, and promote tissue regeneration. In the field of dentistry, PDRN has shown potential in promoting periodontal healing and bone regeneration. This study aims to investigate the effects of PDRN on the morphology, survival, and osteogenic differentiation of gingiva-derived stem cell spheroids, with a focus on its potential applications in tissue engineering and regenerative dentistry. Materials and Methods: Gingiva-derived mesenchymal stem cells were cultured and formed into spheroids using microwells. The cells were treated with varying concentrations of PDRN (0, 25, 50, 75, and 100 μg/mL) and cultivated in osteogenic media. Cell morphology was observed over seven days using an inverted microscope, and viability was assessed with Live/Dead Kit assays and Cell Counting Kit-8. Osteogenic differentiation was evaluated by measuring alkaline phosphatase activity and calcium deposition. The expression levels of osteogenic markers RUNX2 and COL1A1 were quantified using real-time polymerase chain reaction. RNA sequencing was performed to assess the gene expression profiles related to osteogenesis. Results: The results demonstrated that PDRN treatment had no significant effect on spheroid diameter or cellular viability during the observation period. However, a PDRN concentration of 75 μg/mL significantly enhanced calcium deposition by Day 14, suggesting increased mineralization. RUNX2 and COL1A1 mRNA expression levels varied with PDRN concentration, with the highest RUNX2 expression observed at 25 μg/mL and the highest COL1A1 expression at 75 μg/mL. RNA sequencing further confirmed the upregulation of genes involved in osteogenic differentiation, with enhanced expression of RUNX2 and COL1A1 in PDRN-treated gingiva-derived stem cell spheroids. Conclusions: In summary, PDRN did not significantly affect the viability or morphology of gingiva-derived stem cell spheroids but influenced their osteogenic differentiation and mineralization in a concentration-dependent manner. These findings suggest that PDRN may play a role in promoting osteogenic processes in tissue engineering and regenerative dentistry applications, with specific effects observed at different concentrations.

Green Tea and Green Coffee Therapy for Aortic Calcification Prevention in Metabolic Syndrome Model Rats: Effects on Expression of AKT, mTOR, RUNX2, and Osteopontin Levels

Metabolic syndrome (METS) consists of several independent risk factors for cardiovascular disease (CVD), one of which is vascular calcification (VC). Increased oxidative stress is essential in the pathogenesis of CVD in METS. One of the pathways involved in the pathogenesis of VC is the AKT pathway. Green tea and green coffee have many health benefits, including treating METS risk factors. Although several benefits of green tea and green coffee are known, there is not much information regarding the effects of these 2 extracts for treating heart disease, which is often found in METS sufferers. This research explores the benefits of green tea and green coffee extracts in preventing VC in METS through the AKT/MTOR mechanism. This research focuses on the mechanism of CVD in the AKT-mTOR, RUNX activity, and osteopontin (OPN) expression as one of the downstream pathogenesis of CVD in METS. The research was conducted on METS model rat treated with metformin and green tea with green coffee extracts. Male Sprague-Dawley rats were given a diet high in fat and sugar until they met METS for 4 months, then given treatment for 9 weeks. After 9 weeks, the rat had their aortas isolated for staining with specific antibodies to AKT1, MTOR, RUNX2, and serum OPN levels were measured using ELISA-sandwich methods. There was an increase in the expression of AKT1 and MTOR and a decrease in RUNX2 and OPN2, which was better than rat not treated with green tea and green coffee (p &lt; 0.05). A significant reduction was found in the green tea and green coffee therapy group, which was better than when given metformin alone (p &lt; 0.05). The results of this research showed that there is good potential for therapy using green tea and green coffee extracts to prevent VC in metabolic syndrome models. HIGHLIGHTS Metabolic syndrome consists of several independent risk factors for cardiovascular disease, one of which is vascular calcification. Increased oxidative stress is essential in the pathogenesis of cardiovascular disease in metabolic syndrome. This study explores the benefits of green tea and green coffee extracts in preventing vascular calcification in the metabolic syndrome model through the AKT/MTOR mechanism. This research focuses on the mechanism of CVD in the AKT-mTOR, RUNX activity, and osteopontin (OPN) expression as one of the downstream patogenesis of CVD in METS. The research was conducted on METS model rats treated with metformin and green tea with green coffee extracts. In conclusion, this study showed the potential for therapeutic use of green tea and green coffee extracts to prevent vascular calcification in rat models of metabolic syndrome by increasing AKT1 expression, decreasing MTOR and RUNX2, with the main implication of reducing the circular protein product, osteopontin. GRAPHICAL ABSTRACT

Role of Pulsed Electromagnetic Field on Alveolar Bone Remodeling during Orthodontic Retention Phase in Rat Models.

Alveolar bone remodeling during the retention phase is essential for successful orthodontic treatment. Pulsed electromagnetic field (PEMF) therapy is an adjunctive therapy for bone-related diseases that induces osteogenesis and prevents bone loss. This study aimed to examine the role of PEMF exposure during the retention phase of orthodontic treatment in alveolar bone remodeling. A total of 36 male Wistar rats were divided into control, PEMF 7, and PEMF 14 groups; a 50 g force nickel-titanium closed-coil spring was inserted to create mesial movement in the first molar for 21 d. Furthermore, the spring was removed, and the interdental space was filled with glass ionomer cement. Concurrently, rats were exposed to a PEMF at 15 Hz with a maximum intensity of 2.0 mT 2 h daily, for 7 and 14 days. Afterwards, the cements were removed and the rats were euthanized on days 1, 3, 7, and 14 to evaluate the expression of Wnt5a mRNA and the levels of RANKL, OPG, ALP, and Runx2 on the tension side. The data were analyzed with ANOVA and post hoc tests, with p < 0.05 declared statistically significant. PEMF exposure significantly upregulated Wnt5a mRNA expression, OPG and ALP levels, and Runx2 expression, and decreased RANKL levels in the PEMF 7 and 14 groups compared to the control group (p < 0.05). This study showed that PEMF exposure promotes alveolar bone remodeling during the orthodontic retention phase on the tension side by increasing alveolar bone formation and inhibiting resorption.

Lysyl oxidase expression in smooth muscle cells determines the level of intima calcification in hypercholesterolemia-induced atherosclerosis

IntroductionCardiovascular calcification is an important public health issue with an unmeet therapeutic need. We had previously shown that lysyl oxidase (LOX) activity critically influences vascular wall smooth muscle cells (VSMCs) and valvular interstitial cells (VICs) calcification by affecting extracellular matrix remodeling. We have delved into the participation of LOX in atherosclerosis and vascular calcification, as well as in the mineralization of the aortic valve. MethodsImmunohistochemical and expression studies were carried out in human atherosclerotic lesions and experimental models, valves from patients with aortic stenosis, VICs, and in a genetically modified mouse model that overexpresses LOX in CMLV (TgLOXCMLV). Hyperlipemia and atherosclerosis was induced in mice through the administration of adeno-associated viruses encoding a PCSK9 mutated form (AAV-PCSK9D374Y) combined with an atherogenic diet. ResultsLOX expression is increased in the neointimal layer of atherosclerotic lesions from human coronary arteries and in VSMC-rich regions of atheromas developed both in the brachiocephalic artery of control (C57BL/6 J) animals transduced with PCSK9D374Y and in the aortic root of ApoE−/− mice. In TgLOXCMLV mice, PCSK9D374Y transduction did not significantly alter the enhanced aortic expression of genes involved in matrix remodeling, inflammation, oxidative stress and osteoblastic differentiation. Likewise, LOX transgenesis did not alter the size or lipid content of atherosclerotic lesions in the aortic arch, brachiocephalic artery and aortic root, but exacerbated calcification. Among lysyl oxidase isoenzymes, LOX is the most expressed member of this family in highly calcified human valves, colocalizing with RUNX2 in VICs. The lower calcium deposition and decreased RUNX2 levels triggered by the overexpression of the nuclear receptor NOR-1 in VICs was associated with a reduction in LOX. ConclusionsOur results show that LOX expression is increased in atherosclerotic lesions, and that overexpression of this enzyme in VSMC does not affect the size of the atheroma or its lipid content, but it does affect its degree of calcification. Further, these data suggest that the decrease in calcification driven by NOR-1 in VICs would involve a reduction in LOX. These evidences support the interest of LOX as a therapeutic target in cardiovascular calcification.

Resveratrol alleviates thiram-induced tibial dyschondroplasia by regulating BMP-2, RUNX-2 and HIF-1α expressions in broiler chickens

Tetramethylthiuram disulfide (thiram) is a pesticide and fungicide, widely used in agriculture, and is a recognized cause of tibial dyschondroplasia (TD), a skeletal abnormality in fast-growing broiler chickens. This study aimed to investigate the effect of resveratrol (RSV) against TD induced by thiram in broilers. A total of 240 broilers were evenly divided into three groups (n = 80) i.e., Control, TD and RSV. All groups received feed and water ad libitum, whereas the TD and RSV groups received standard diet with an addition of thiram (50 mg/kg of feed) from 4–7 days to induce TD. After the cessation of thiram on day 8, the RSV group was administered RSV at 400 mg/kg body weight orally from 8–18 days. Histopathological changes in tibia were assessed using hematoxylin and eosin (H&E) staining. In addition, the mRNA and protein expressions of targeted genes were analyzed using RT-qPCR, immunohistochemistry, and western blotting. Thiram feeding resulted in an increased growth plate (GP) width, decreased vascularization, irregular shaped chondrocytes with pyknotic nuclei, lameness, poor production performance, reduced levels of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), alkaline phosphatase (ALP) and elevated levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and malondialdehyde (MDA). The mRNA and protein expressions of BMP-2 and RUNX-2 were downregulated, whereas HIF-1α was upregulated in TD affected broilers (p < 0.05). RSV administration resulted in a significant increase in mRNA and protein levels of BMP-2 and RUNX-2 (p < 0.05) and a decrease in the levels of HIF-1α. In addition, RSV improved vascularization, angiogenesis, alleviated lameness, and enhanced production efficiency. Furthermore, it restored the GP width, serum biochemical levels and liver antioxidant enzymes. In conclusion, this study indicated that resveratrol mitigated the adverse effects of thiram toxicity in broiler chickens.

Incorporation of calcium phosphate cement into decellularized extracellular matrix enhances its bone regenerative properties

Decellularized extracellular matrix (dECM) hydrogels are engineered constructs that are widely-used in the field of regenerative medicine. However, the development of ECM-based hydrogels for bone tissue engineering requires enhancement in its osteogenic properties. For this purpose, we initially employed bone-derived dECM hydrogel (dECM-Hy) in combination with calcium phosphate cement (CPC) paste to improve the biological and structural properties of the dECM hydrogel. A decellularization protocol for bovine bone was developed to prepare dECM-Hy, and the mechanically-tuned dECM/CPC-Hy was built based on both rheological and mechanical characteristics. The dECM/CPC-Hy displayed a double swelling ratio and compressive strength. An interconnected structure with distinct hydroxyapatite crystals was evident in dECM/CPC-Hy. The expression levels of Alp, Runx2 and Ocn genes were upregulated in dECM/CPC-Hy compared to the dECM-Hy. A 14-day follow-up of the rats receiving subcutaneous implanted dECM-Hy, dECM/CPC-Hy and mesenchymal stem cells (MSCs)-embedded (dECM/CPC/MSCs-Hy) showed no toxicity, inflammatory factor expression or pathological changes. Radiography and computed tomography (CT) of the calvarial defects revealed new bone formation and elevated number of osteoblasts-osteocytes and osteons in dECM/CPC-Hy and dECM/CPC/MSCs-Hy compared to the control groups. These findings indicate that the dECM/CPC-Hy has substantial potential for bone tissue engineering.

Bu-Sui-Dan Enhances Osteoblast Differentiation by Upregulating VGLL4 to Counteract TEAD4-Mediated RUNX2 Transcription Suppression in Ovariectomized Rats

Ethnopharmacological relevancePostmenopausal osteoporosis (PMOP) has been considered as a major causative factor for bone-joint pain and inducing pathologic fractures. Bu-Sui-Dan (BSD), a classic ancient herbal formula, has been shown to exhibit osteoprotective effects by promoting bone marrow development and bone growth. However, the exact mechanism of BSD are still unexplored. Aim of studyThe study aimed to investigate the protective effect of BSD against osteoporotic injury, and to explore whether BSD regulated BMSCs’ osteogenic differentiation by targeting VGLL4, which in turn improved PMOP. Materials and methodsThe anti-osteoporotic effect of BSD was studied in ovariectomized (OVX) rats and bone marrow mesenchymal stem cells (BMSCs). Micro-CT imaging and HE staining were performed, and the levels of osteogenic protein RUNX2 and osteogenesis-related factor VGLL4 were determined. Co-immunoprecipitation (Co-IP) was further employed to delve into the effects of BSD on the interactions between TEAD4 and RUNX2. The key osteogenic factors 1ALP, COLl1A1, and Osterix expression were detected by RT-qPCR. Co-IP and proximity ligation assay (PLA) were employed to scrutinize the influence of BSD on TEAD4 and RUNX2 inter-binding. Moreover, VGLL4 knockdown in BMSCs was conducted to confirm the role of VGLL4 in the therapeutic mechanism of BSD. ResultsBSD showed a dose-dependent protective effect against osteoporotic injury, as evidenced by improvement in bone volume, bone microarchitecture, and histomorphometry. Additionally, BSD treatment increased the levels of RUNX2 and its downstream target genes including ALP, COL1A1, and Osterix. Moreover, BSD upregulated VGLL4 expression and lessened TEAD4-RUNX2 interactions. In BMSCs experiment, BSD-containing serum could promote osteogenic differentiation of BMSCs, boosted the expression of osteogenesis-related factors and VGLL4 level. The knockdown of VGLL4 in BMSCs diminished the promotion effect of BSD in osteoblast differentiation, suggesting that VGLL4 play a vital role in the therapeutic effects exerted by BSD. ConclusionBSD ameliorated osteoporosis injury and promoted osteoblast differentiation through upregulation of VGLL4 levels, which in turn antagonized TEAD4-mediated RUNX2 transcriptional repression. Our study implied that BSD may be an osteoporosis therapeutic agent.

CDK9 phosphorylates RUNX1 to promote megakaryocytic fate in megakaryocytic-erythroid progenitors

AbstractThe specification of megakaryocytic (Mk) or erythroid (E) lineages from primary human megakaryocytic-erythroid progenitors (MEPs) is crucial for hematopoietic homeostasis, yet the underlying mechanisms regulating fate specification remain elusive. In this study, we identify RUNX1 as a key modulator of gene expression during MEP fate specification. Overexpression of RUNX1 in primary human MEPs promotes Mk specification, whereas pan-RUNX inhibition favors E specification. Although total RUNX1 levels do not differ between Mk progenitors (MkPs) and E progenitors (ErPs), there are higher levels of serine-phosphorylated RUNX1 in MkPs than ErPs, and mutant RUNX1 with phosphorylated-serine/threonine mimetic mutations (RUNX1-4D) significantly enhances the functional efficacy of RUNX1. To model the effects of RUNX1 variants, we use human erythroleukemia (HEL) cell lines expressing wild-type (WT), phosphomimetic (RUNX1-4D), and nonphosphorylatable (RUNX1-4A) mutants showing that the 3 forms of RUNX1 differentially regulate expression of 2625 genes. Both WT and RUNX1-4D variants increase expression in 40%, and decrease expression in another 40%, with lesser effects of RUNX1-4A. We find a significant overlap between the upregulated genes in WT and RUNX1-4D–expressing HEL cells and those upregulated in primary human MkPs vs MEPs. Although inhibition of known RUNX1 serine/threonine kinases does not affect phosphoserine RUNX1 levels in primary MEPs, specific inhibition of cyclin dependent kinase 9 (CDK9) in MEPs leads to both decreased RUNX1 phosphorylation and increased E commitment. Collectively, our findings show that serine/threonine phosphorylation of RUNX1 promotes Mk fate specification and introduce a novel kinase for RUNX1 linking the fundamental transcriptional machinery with activation of a cell type–specific transcription factor.

L-Carnitine relieves cachexia-related skeletal muscle fibrosis by inducing deltex E3 ubiquitin ligase 3L to negatively regulate the Runx2/COL1A1 axis.

Cancer cachexia-induced skeletal muscle fibrosis (SMF) impairs muscle regeneration, alters the muscle structure and function, reduces the efficacy of anticancer drugs, diminishes the patient's quality of life and shortens overall survival. RUNX family transcription factor 2 (Runx2), a transcription factor, and collagen type I alpha 1 chain (COL1A1), the principal constituent of SMF, have been linked previously, with Runx2 shown to directly modulate COL1A1 mRNA levels. l-Carnitine, a marker of cancer cachexia, can alleviate fibrosis in liver and kidney models; however, its role in cancer cachexia-associated fibrosis and the involvement of Runx2 in the process remain unexplored. Female C57 mice (48weeks old) were inoculated subcutaneously with MC38 cells to establish a cancer cachexia model. A 5mg/kg dose of l-carnitine or an equivalent volume of water was administered for 14days via oral gavage, followed by assessments of muscle function (grip strength) and fibrosis. To elucidate the interplay between the deltex E3 ubiquitin ligase 3L(DTX3L)/Runx2/COL1A1 axis and fibrosis in transforming growth factor beta 1-stimulated NIH/3T3 cells, a suite of molecular techniques, including quantitative real-time PCR, western blot analysis, co-immunoprecipitation, molecular docking, immunofluorescence and Duolink assays, were used. The relevance of the DTX3L/Runx2/COL1A1 axis in the gastrocnemius was also explored in the in vivo model. l-Carnitine supplementation reduced cancer cachexia-induced declines in grip strength (>88.2%, P<0.05) and the collagen fibre area within the gastrocnemius (>57.9%, P<0.05). At the 5mg/kg dose, l-carnitine also suppressed COL1A1 and alpha-smooth muscle actin (α-SMA) protein expression, which are markers of SMF and myofibroblasts. Analyses of the TRRUST database indicated that Runx2 regulates both COL1A1 and COL1A2. In vitro, l-carnitine diminished Runx2 protein levels and promoted its ubiquitination. Overexpression of Runx2 abolished the effects of l-carnitine on COL1A1 and α-SMA. Co-immunoprecipitation, molecular docking, immunofluorescence and Duolink assays confirmed an interaction between DTX3L and Runx2, with l-carnitine enhancing this interaction to promote Runx2 ubiquitination. l-Carnitine supplementation restored DTX3L levels to those observed under non-cachectic conditions, both in vitro and in vivo. Knockdown of DTX3L abolished the effects of l-carnitine on Runx2, COL1A1 and α-SMA in vitro. The expression of DTX3L was negatively correlated with the levels of Runx2 and COL1A1 in untreated NIH/3T3 cells. This study revealed a previously unrecognized link between Runx2 and DTX3L in SMF and demonstrated that l-carnitine exerted a significant therapeutic impact on cancer cachexia-associated SMF, potentially through the upregulation of DTX3L.

Osteogenic Differential Expression of Rabbit Adipose-Derived Mesenchymal Stem Cells by PPARγ Transfection Coated with Nanoparticles

Adipose-derived stem cells (ADSCs) have advantages in utilization. Nanoparticles (NP) are degradable, nontoxic and have good biocompatibility, besides, they can encapsulate and transport water-soluble, fat-soluble and deoxyribonucleic acid. This study explored the influence of NP-coated PPARγ transfection on osteogenic differentiation of rabbit ADSCs. After preparing PPARγ transfection system coated with nanoparticles, Petri dishes containing the same amount of cells were divided at the beginning of cell culture into blank group (not transfected), control group and observation group (transfected with PPARγ coated NP). Alkaline phosphatase (ALP) staining and Western Blot were done to detect the contents of lipoprteinlipase (LPL), Runx2 and serum osteocalcin (BGP) protein. The levels of Runx2, BGP and PPARγ mRNA were measured by RT-PCR. After ADSCs cells were stained, a small number of cells adhered to each other, and the cells were mainly fibroblast-like and spindle-shaped, and proliferated in the form of colonies. The levels of Runx2 and BGP in the observation group were significantly lower than those in the control group (p &lt; 0.05) after the ADSCs cells were cultured for 7 and 14 days. The expression of LPL and PPARγ mRNA in the observation group was higher control group (p &lt; 0.05). Transfection of PPARγ coated with nanoparticles inhibited the osteogenic differentiation of rabbit ADSCs by inhibiting the expression of Runx2 and BGP and promoting the expression of LPL and PPARγ.

New Insights on the Therapeutic Potential of Runt-Related Transcription Factor2 for Osteoarthritis: Evidence from Mendelian Randomization.

Research has highlighted the role of runt-related transcription factor2 (Runx2) in the development of osteoarthritis (OA); however, its causal association remains unclear. This study aimed to explore whether Runx2 expression is causally associated with OA and assess its therapeutic potential for OA. Genetic proxy instruments for Runx2 expression were obtained from gene expression quantitative trait locus (eQTLs) study of eQTLGen Consortium (n = 31,684). Aggregated genome-wide association study (GWAS) data for OA (including all OA [177,517 cases and 649,173 controls], knee OA (KOA) [62,497 cases and 333,557 controls], and hip OA (HOA) [36,445 cases and 316,943 controls]) were extracted from the Genetics of Osteoarthritis Consortium. We integrated eQTLs data with OA GWAS data to estimate their causal association and to estimate the potential of Runx2 as a drug target in the treatment of OA using summary data-based Mendelian randomization (SMR) analysis. Furthermore, different OA GWAS data (including all OA [77,052 cases and 378,169 controls], KOA [24,955 cases and 378,169 controls], and HOA [15,704 cases and 378,169 controls]) derived from the GWAS Catalog database were used for replication study. SMR analysis showed that high expression levels of Runx2 were associated with an increased risk of all OA [odds ratio (OR) 1.044, 95% confidence interval (CI) 1.023-1.067; P = 5.03 × 10-5], KOA (OR1.040, 95%CI 1.006-1.075; P = 0.021), and HOA (OR1.067, 95%CI 1.022-1.113; P = 0.003). This suggests that Runx2 inhibitors may have promising potential for the treatment of OA. Notably, the causal effects of Runx2 with all OA (OR1.053, 95%CI 1.027-1.079; P = 3.95 × 10-5) and KOA (OR1.043, 95%CI 1.001-1.087; P = 0.045) were repeated in the replication study, but limited evidence supported the association of Runx2 expression levels with HOA (OR1.045, 95%CI 0.993-1.101; P = 0.094). Our analyses indicate a positive correlation between Runx2 expression and OA risk across all three phenotypes, suggesting the potential of Runx2 inhibitors in the treatment of OA and providing evidence from a genetic perspective.