Endothelial cells derived extracellular vesicles promote diabetic arterial calcification via circ_0008362/miR-1251-5p/Runx2 axial.

INTRODUCTION: Arterial calcification, an independent predictor of cardiovascular events, increases morbidity and mortality in patients with diabetes mellitus (DM), but its mechanisms remain unclear. Extracellular vesicles (EVs) play an important role in intercellular communication. The study investigates the role and potential mechanisms of EVs derived from endothelial cells (ECs) in regulating vascular smooth muscle cell (VSMC) calcification under high glucose (HG) condition, with a goal of developing effective prevention and treatment strategies for diabetic arterial calcification. RESULTS: The results showed that EVs derived from HG induced ECs (ECHG-EVs) exhibited a bilayer structure morphology with a mean diameter of 74.08 ± 31.78 nm, expressing EVs markers including CD9, CD63 and TSG101, but not express calnexin. ECHG-EVs was internalized by VSMCs and induced VSMC calcification by increasing Runx2 expression and mineralized nodule formation. The circ_0008362 was enriched in ECHG-EVs, and it can be transmitted to VSMCs to promote VSMC calcification both in vitro and in vivo. Mechanistically, miR-1251-5p might be one of the targets of circ_0008362 and they were co-localization in the cytoplasm of VSMCs. Runx2 was identified as the downstream target of miR-1251-5p, and circ_0008362 acted as a sponge, enhancing Runx2 expression and then promoted VSMC calcification. Besides, circ_0008362 could directly interact with Runx2 to aggravate VSMC calcification. Notably, DiR-labelled ECHG-EVs was detected in the vessels of mice. Meanwhile, the level of circ_0008362 and Runx2 were increased significantly, while the expression of miR-1251-5p was decreased significantly in calcified artery tissues of mice. However, inhibiting the release of EVs by GW4869 attenuated arterial calcification in diabetic mice. Finally, the level of circulation of plasma EVs circ_0008362 was significantly higher in patients with DM compared with normal controls. Elevated levels of plasma EVs circ_0008362 were associated with more severe coronary and aorta artery calcification in patients with DM. CONCLUSIONS: Our findings suggested that circ_0008362 was enriched in EVs derived from ECs and promoted VSMC calcification under HG conditions, both by sponging miR-1251-5p to upregulate Runx2 expression and through direct interaction with Runx2. Furthermore, elevated levels of plasma EVs circ_0008362 were associated with more severe coronary and aorta artery calcification in patients with DM. These results may serve as a potential prevention and therapeutic target for diabetic arterial calcification.

Shuanglongjiegu pill promoted bone marrow mesenchymal stem cell osteogenic differentiation by regulating the miR-217/RUNX2 axis to activate Wnt/ß-catenin pathway.

This study aimed to investigate the effects of Shuanglongjiegu pill (SLJGP) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and explore its mechanism based on miR-217/RUNX2 axis. Results found that drug-containing serum of SLJGP promoted BMSCs viability with a dose-dependent effect. Under osteogenic differentiation conditions, SLJGP promoted the expression of ALP, OPN, BMP2, RUNX2, and the osteogenic differentiation ability of BMSCs. In addition, SLJGP significantly reduced miR-217 expression, and miR-217 directly targeted RUNX2. After treatment with miR-217 mimic, the promoting effects of SLJGP on proliferation and osteogenic differentiation of BMSCs were significantly inhibited. MiR-217 mimic co-treated with pcDNA-RUNX2 further confirmed that the miR-217/RUNX2 axis was involved in SLJGP to promote osteogenic differentiation of BMSCs. In addition, analysis of Wnt/ß-catenin pathway protein expression showed that SLJGP activated the Wnt/ß-catenin pathway through miR-217/RUNX2. In conclusion, SLJGP promoted osteogenic differentiation of BMSCs by regulating miR-217/RUNX2 axis and activating Wnt/ß-catenin pathway.

SLAMF8 regulates osteogenesis and adipogenesis of bone marrow mesenchymal stem cells via S100A6/Wnt/ß-catenin signaling pathway.

BACKGROUND: The inflammatory microenvironment plays an essential role in bone healing after fracture. The signaling lymphocytic activation molecule family (SLAMF) members deeply participate in inflammatory response and make a vast difference. METHODS: We identified SLAMF8 in GEO datasets (GSE129165 and GSE176086) and co-expression analyses were performed to define the relationships between SLAMF8 and osteogenesis relative genes (RUNX2 and COL1A1). In vitro, we established SLAMF8 knockdown and overexpression mouse bone marrow mesenchymal stem cells (mBMSCs) lines. qPCR, Western blot, ALP staining, ARS staining, Oil Red O staining and Immunofluorescence analyses were performed to investigate the effect of SLAMF8 in mBMSCs osteogenesis and adipogenesis. In vivo, mice femoral fracture model was performed to explore the function of SLAMF8. RESULTS: SLAMF8 knockdown significantly suppressed the expression of osteogenesis relative genes (RUNX2, SP7 and COL1A1), ALP activity and mineral deposition, but increased the expression of adipogenesis relative genes (PPARγ and C/EBPα). Additionally, SLAMF8 overexpression had the opposite effects. The role SLAMF8 played in mBMSCs osteogenic and adipogenic differentiation were through S100A6 and Wnt/ß-Catenin signaling pathway. Moreover, SLAMF8 overexpression mBMSCs promoted the healing of femoral fracture. CONCLUSIONS: SLAMF8 promotes osteogenesis and inhibits adipogenesis of mBMSCs via S100A6 and Wnt/ß-Catenin signaling pathway. SLAMF8 overexpression mBMSCs effectively accelerate the healing of femoral fracture in mice.

The Therapeutic Potential of Adipose-Derived Mesenchymal Stem Cell Secretome in Osteoarthritis: A Comprehensive Study.

Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation and inflammation. This study investigates the therapeutic potential of secretome derived from adipose tissue mesenchymal stem cells (ASCs) in mitigating inflammation and promoting cartilage repair in an in vitro model of OA. Our in vitro model comprised chondrocytes inflamed with TNF. To assess the therapeutic potential of secretome, inflamed chondrocytes were treated with it and concentrations of pro-inflammatory cytokines, metalloproteinases (MMPs) and extracellular matrix markers were measured. In addition, secretome-treated chondrocytes were subject to a microarray analysis to determine which genes were upregulated and which were downregulated. Treating TNF-inflamed chondrocytes with secretome in vitro inhibits the NF-κB pathway, thereby mediating anti-inflammatory and anti-catabolic effects. Additional protective effects of secretome on cartilage are revealed in the inhibition of hypertrophy markers such as RUNX2 and COL10A1, increased production of COL2A1 and ACAN and upregulation of SOX9. These findings suggest that ASC-derived secretome can effectively reduce inflammation, promote cartilage repair, and maintain chondrocyte phenotype. This study highlights the potential of ASC-derived secretome as a novel, non-cell-based therapeutic approach for OA, offering a promising alternative to current treatments by targeting inflammation and cartilage repair mechanisms.

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 ß1, 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 ß1 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 ß1-FAK-ERK1/2 signaling. Altogether, the overlay angle of aligned core-shell nanofiber membranes regulates the osteogenic differentiation of hBMSCs via integrin ß1-FAK-ERK1/2 signaling, unveiling the effects of anisotropic fibers on bone tissue formation.

The efficacy of infrared diode laser in enhancing the regenerative potential of human periodontal ligament stem cells (hPDLSCs).

BACKGROUND: The present study aimed to investigate the effects of infrared diode laser irradiation on the proliferation and differentiation capacity of periodontal ligament stem cells (hPDLSCs), which are optimal cell sources for periodontal regeneration. METHODS: hPDLSCs were isolated and characterized by flow cytometric analysis of mesenchymal stem cell markers, and their trilineage differentiation capacity was tested. hPDLSCs were then cultured and irradiated with infrared diode laser (970 nm) at a power of 200 mW and a fluence of 4 J/cm2 for 3 s. MTT assay was performed to assess cellular proliferation. Cell cycle analysis was performed, and the impact of infrared diode laser irradiation on the stemness and osteogenic differentiation potential of hPDLSCs was evaluated via RT‒PCR. RESULTS: Infrared diode laser application enhanced the stemness, viability, proliferation, and differentiation of PDLSCs. Stem cell markers (OCT4, SOX2, and NANOG) were significantly upregulated in hPDLSCs exposed to laser irradiation. There was significant overexpression of RUNX2, ALP, OPN, and OCN on day 14 after laser application. CONCLUSIONS: These findings provide valuable insights into the specific applications of infrared diode lasers to effectively regenerate periodontal tissues. The results can aid in the development of precise clinical protocols aimed at enhancing osseointegration and promoting tissue regeneration. Ultimately, the combination of infrared diode laser with hPDLSCs is promising for stimulating periodontal regeneration.

Single-layer graphene oxide nanosheets induce proliferation and Osteogenesis of single-cell hBMSCs encapsulated in Alginate Microgels.

Microfluidics cell encapsulation offers a way to mimic a 3D microenvironment that supports cell growth and proliferation, while also protecting cells from environmental stress. This technique has found extensive applications in tissue engineering and cell therapies. Several studies have demonstrated the advantages of graphene oxide (GO) as an osteogenic inducer; however, the significance of GO on stem cell fate in the single-cell state is still unclear. Here, a microfluidics-based approach is developed for continuous encapsulation of mesenchymal stem cells (MSCs) at the single-cell level using alginate microgels. So, single-layer graphene oxide (slGO) nanosheet is used to be encapsulated inside the alginate droplets. The results of AFM and SEM show that slGO can increase the roughness and reduce the stiffness of alginate hydrogels. The Young's modulus of the alginate and alginate-slGO was obtained as 1414 kPa and 985.9 kPa, respectively. Live/dead assay and fluorescence microscopy images illustrate that slGO can maintain the viability and proliferation of microencapsulated hBMSCs. The obtained results show that slGO increases the mineralization of the encapsulated hBMSCs, so that microgels containing hBMSCs gradually became opaque during 21 days of culture. RT-qPCR results indicate that the expression of OCN, Runx2, and ALP in the alginate-slGO microgels is significantly higher than in the alginate microgels. The expression of OCN and Runx2 in the alginate-slGO microgels is 4.27 and 5.87-fold higher than in the alginate microgels, respectively. It can be concluded that low doses of slGO nanosheets have the potential to be utilized in the development of tissue engineering and bone regeneration. This finding offers a new method for creating injectable tissue transplants that are minimally invasive.

Bazi Bushen attenuates osteoporosis in SAMP6 mice by regulating PI3K-AKT and apoptosis pathways.

Osteoporosis (OP), a systemic skeletal disease, is characterized by low bone mass, bone tissue degradation and bone microarchitecture disturbance. Bazi Bushen, a Chinese patented medicine, has been demonstrated to be effective in attenuating OP, but the pharmacological mechanism remains predominantly unclear. In this study, the senescence-accelerated mouse prone 6 (SAMP6) model was used to explore bone homeostasis and treated intragastrically for 9 weeks with Bazi Bushen. In vivo experiments showed that Bazi Bushen treatment not only upregulated the levels of bone mineral density and bone mineral content but also increased the content of RUNX2 and OSX. Furthermore, the primary culture of bone mesenchymal stem cells (BMSCs) in SAMP6 mice was used to verify the effects of Bazi Bushen on the balance of differentiation between osteoblasts and adipocytes, as well as ROS and aging levels. Finally, the pharmacological mechanism of Bazi Bushen in attenuating OP was investigated through network pharmacology and experimental verification, and we found that Bazi Bushen could significantly orchestrate bone homeostasis and attenuate the progression of OP by stimulating PI3K-Akt and inhibiting apoptosis. In summary, our work sheds light on the first evidence that Bazi Bushen attenuates OP by regulating PI3K-AKT and apoptosis pathways to orchestrate bone homeostasis.

Regulation of Skeletal Development and Maintenance by Runx2 and Sp7.

Runx2 (runt related transcription factor 2) and Sp7 (Sp7 transcription factor 7) are crucial transcription factors for bone development. The cotranscription factor Cbfb (core binding factor beta), which enhances the DNA-binding capacity of Runx2 and stabilizes the Runx2 protein, is necessary for bone development. Runx2 is essential for chondrocyte maturation, and Sp7 is partly involved. Runx2 induces the commitment of multipotent mesenchymal cells to osteoblast lineage cells and enhances the proliferation of osteoprogenitors. Reciprocal regulation between Runx2 and the Hedgehog, fibroblast growth factor (Fgf), Wnt, and parathyroid hormone-like hormone (Pthlh) signaling pathways and Dlx5 (distal-less homeobox 5) plays an important role in these processes. The induction of Fgfr2 (Fgf receptor 2) and Fgfr3 expression by Runx2 is important for the proliferation of osteoblast lineage cells. Runx2 induces Sp7 expression, and Runx2+ osteoprogenitors become Runx2+Sp7+ preosteoblasts. Sp7 induces the differentiation of preosteoblasts into osteoblasts without enhancing their proliferation. In osteoblasts, Runx2 is required for bone formation by inducing the expression of major bone matrix protein genes, including Col1a1 (collagen type I alpha 1), Col1a2, Spp1 (secreted phosphoprotein 1), Ibsp (integrin binding sialoprotein), and Bglap (bone gamma carboxyglutamate protein)/Bglap2. Bglap/Bglap2 (osteocalcin) regulates the alignment of apatite crystals parallel to collagen fibrils but does not function as a hormone that regulates glucose metabolism, testosterone synthesis, and muscle mass. Sp7 is also involved in Co1a1 expression and regulates osteoblast/osteocyte process formation, which is necessary for the survival of osteocytes and the prevention of cortical porosity. SP7 mutations cause osteogenesis imperfecta in rare cases. Runx2 is an important pathogenic factor, while Runx1, Runx3, and Cbfb are protective factors in osteoarthritis development.

Nitrogen-containing bisphosphonate induces enhancement of OPG expression and inhibition of RANKL expression via inhibition of farnesyl pyrophosphate synthase to inhibit the osteogenic differentiation and calcification in vascular smooth muscle cells.

BACKGROUND: Nitrogen-containing bisphosphonate(N-BP)had been found to inhibit the osteogenic differentiation and calcification in vascular smooth muscle cells (VSMCs), but the mechanism is not clear. We intend to verify that N-BP induces enhancement of OPG expression and inhibition of RANKL expression via inhibition of farnesyl pyrophosphate synthase(FPPS) to inhibit the osteogenic differentiation and calcification in VSMCs. METHODS: ß-glycerophosphate (ß-GP) was used to induce the osteogenic differentiation and calcification in VSMCs. VSMCs were treated with N-BP or pretreated with downstream products of farnesyl pyrophosphate synthase(FPPS) in mevalonate pathway, such as farnesol (FOH) or geranylgeraniol (GGOH). Alizarin red S staining and determination of calcium content were used to detect calcium deposition.Western Blotting were used to detect expressions of proteins(OPG and RANKL ) and osteogenic marker proteins (Runx2 and OPN). RESULTS: ß-GP induced the osteogenic differentiation and calcification in VSMCs, increased RANKL protein expression and had no significant effect on OPG protein expression. With the treatment of N-BP, the expression of OPG protein was increased and expression of RANKL protein was decreased in VSMCs undergoing osteogenic differentiation and calcification. In addition, N-BP reduced the osteogenic marker proteins (Runx2 and OPN) expression and calcium deposition in VSMCs undergoing osteogenic differentiation and calcification. These effects of N-BP on the osteogenic differentiation and calcification in VSMCs were concentration-dependent, which could be reversed by the downstream products of FPPS, such as FOH or GGOH. CONCLUSION: N-BP increases OPG expression and decreases RANKL expression via inhibition of FPPS to inhibit the osteogenic differentiation and calcification in VSMCs.

Modulation of osteoblastogenesis by NRF2: NRF2 activation suppresses osteogenic differentiation and enhances mineralization in human bone marrow-derived mesenchymal stromal cells.

Mesenchymal stromal stem cells (MSCs) or skeletal stem cells (SSCs) play a major role in tissue repair due to their robust ability to differentiate into osteoblasts, chondrocytes, and adipocytes. Complex cell signaling cascades tightly regulate this differentiation. In osteogenic differentiation, Runt-related transcription factor 2 (RUNX2) and ALP activity are essential. Furthermore, during the latter stages of osteogenic differentiation, mineral formation mediated by the osteoblast occurs with the secretion of a collagenous extracellular matrix and calcium deposition. Activation of nuclear factor erythroid 2-related factor 2 (NRF2), an important transcription factor against oxidative stress, inhibits osteogenic differentiation and mineralization via modulation of RUNX2 function; however, the exact role of NRF2 in osteoblastogenesis remains unclear. Here, we demonstrate that NRF2 activation in human bone marrow-derived stromal cells (HBMSCs) suppressed osteogenic differentiation. NRF2 activation increased the expression of STRO-1 and KITLG (stem cell markers), indicating NRF2 protects HBMSCs stemness against osteogenic differentiation. In contrast, NRF2 activation enhanced mineralization, which is typically linked to osteogenic differentiation. We determined that these divergent results were due in part to the modulation of cellular calcium flux genes by NRF2 activation. The current findings demonstrate a dual role for NRF2 as a HBMSC maintenance factor as well as a central factor in mineralization, with implications therein for elucidation of bone formation and cellular Ca2+ kinetics, dystrophic calcification and, potentially, application in the modulation of bone formation.

Exploring the transcriptional cooperation between RUNX2 and its associated elncRNA RAIN.

Recent insights into the mechanisms controlling gene expression identified enhancer-associated long non-coding RNAs (elncRNAs) as master players of transcription in cancers. RUNX2, a mammalian RUNT-related transcription factor, is increasingly recognized in cancer biology for its role in supporting survival and progression also in thyroid cancer (TC). We recently identified, within the RUNX2 locus, a novel elncRNA that we named RAIN (RUNX2 associated intergenic lncRNA). We showed that RAIN and RUNX2 expression correlate in TC, both in vitro and in vivo, and that RAIN promotes RUNX2 expression by interacting with and affecting the activity of the RUNX2 P2 promoter through two distinct mechanisms. Here, we took forward these observations to explore the genome-wide transcriptional function of RAIN and its contribution to the RUNX2-dependent gene expression program in TC. By combining multiple omics data, we demonstrated that RAIN functionally cooperates with RUNX2 to the regulation of a subset of functionally related genes involved in promoting matrix remodeling, migration, and loss of differentiation. We showed that RAIN interacts with RUNX2 and its expression is required for the efficient recruitment of this TF to its target regulatory regions. In addition, our data revealed that besides RUNX2, RAIN governs a hierarchically organized complex transcriptional program by controlling a core of cancer-associated TFs that, in turn, orchestrate the expression of downstream genes. This evidence indicates that the functional cooperation observed between RAIN and RUNX2 can be a diffuse work mechanism for this elncRNA.

Key regulators of vascular calcification in chronic kidney disease: Hyperphosphatemia, BMP2, and RUNX2.

Vascular calcification is quite common in patients with end-stage chronic kidney disease and is a major trigger for cardiovascular complications in these patients. These complications significantly impact the survival rate and long-term prognosis of individuals with chronic kidney disease. Numerous studies have demonstrated that the development of vascular calcification involves various pathophysiological mechanisms, with the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) being of utmost importance. High phosphate levels, bone morphogenetic protein 2 (BMP2), and runt-related transcription factor 2 (RUNX2) play crucial roles in the osteogenic transdifferentiation process of VSMCs. This article primarily reviews the molecular mechanisms by which high phosphate, BMP2, and RUNX2 regulate vascular calcification secondary to chronic kidney disease, and discusses the complex interactions among these factors and their impact on the progression of vascular calcification. The insights provided here aim to offer new perspectives for future research on the phenotypic switching and osteogenic transdifferentiation of VSMCs, as well as to aid in optimizing clinical treatment strategies for this condition, bearing significant clinical and scientific implications.

Effects of soluble Klotho and Wnt/ß-catenin signaling pathway in vascular calcification in chronic kidney disease model rats and the intervention of Shenyuan granules.

OBJECTIVE: This study aimed to investigate the effect of the soluble Klotho (sKlotho)/Wnt/ß-catenin signaling pathway on vascular calcification in rat models of chronic kidney disease (CKD) and the intervention effect of Shenyuan granules. METHODS: Rats with 5/6 nephrectomy and high phosphorus feeding were used to establish the vascular calcification model. The rats were given gradient doses of Shenyuan granules aqueous solution and calcitriol solution by gavage for 8 weeks, which were divided into experimental group and positive control group. RESULTS: The 5/6 nephrectomy combined with high phosphorus feeding induced thoracic aortic calcification in rats. Shenyuan granules intervention increased the serum sKlotho level, inhibited the mRNA and protein expression of Wnt1, ß-catenin, and Runx2 in the thoracic aorta, and alleviated thoracic aortic media calcification in rats. CONCLUSION: Shenyuan granules may partially regulate the Wnt/ß-catenin signaling pathway via serum sKl to interfere with the expression of Runx2, thereby improving vascular calcification in CKD.

Effect of photobiomodulation therapy with 660 and 980 nm diode lasers on differentiation of periodontal ligament mesenchymal stem cells.

This study aimed to compare the effects of photobiomodulation therapy (PBMT) with 660 and 980 nm diode lasers on differentiation of periodontal ligament mesenchymal stem cells (PDLMSCs). In this in vitro, experimental study, PDLMSCs were obtained from the Iranian Genetic Bank and cultured in osteogenic medium. They were then subjected to irradiation of 660 and 980 nm diode lasers, and their viability was assessed after one, two, and three irradiation cycles using the methyl thiazolyl tetrazolium (MTT) assay. The cells also underwent DAPI staining, cell apoptosis assay by using the Annexin V/PI, Alizarin Red staining, and real-time polymerase chain reaction (PCR) for assessment of the expression of osteogenic genes. Data were analyzed by two-way ANOVA. The two laser groups had no significant difference in cell apoptosis according to the results of DAPI staining. Both laser groups showed higher cell viability in the MTT assay at 4 and 6 days compared with the control group. Annexin V/PI results showed higher cell viability in both laser groups at 4 days compared with the control group. Rate of early and late apoptosis was lower in both laser groups than the control group at 4 days. Necrosis had a lower frequency in 980 nm laser group than the control group on day 6. Alizarin Red staining showed higher cell differentiation in both laser groups after 3 irradiation cycles than the control group. The highest expression of osteopontin (OPN), osteocalcin (OCN), and Runt-related transcription factor 2 (RUNX2) was noted in 660 nm laser group with 3 irradiation cycles at 14 days, compared with the control group. PBMT with 660 and 980 nm diode lasers decreased apoptosis and significantly increased PDLMSC differentiation after 3 irradiation cycles.

circRUNX2.2, highly expressed in Marek's disease tumor tissues, functions in cis to regulate parental gene RUNX2 expression.

Marek's disease (MD), an immunosuppression disease induced by Marek's disease virus (MDV), is one of the significant diseases affecting the health and productive performance of poultry. The roles of circular RNAs (circRNAs) in MD development were poorly understood. In this study, we found a circRNA derived from exon 6 of RUNX family transcription factor 2 (RUNX2) gene, named circRUNX2.2, was highly expressed in chicken tumorous spleens (TS) induced by MDV. Through fluorescence in situ hybridization and nuclear-cytoplasmic separation assay, we determined circRUNX2.2 was mainly located in the nucleus. Knockout experiments confirmed that the flanking complementary sequences (RCMs) mediated its circularization. Gain of function assay and dual luciferase reporter gene assay revealed that circRUNX2.2 could promote the expression of RUNX2 via binding with its promoter region. RNA antisense purification assay and mass spectrometry assay showed circRUNX2.2 could recruit proteins such as CHD9 protein. Knocking down CHD9 expression decreased the expression of RUNX2 gene, which confirmed the positive regulation that circRUNX2.2 on RUNX2 expression was probably facilitated via recruiting CHD9 protein. Functional experiments showed that circRUNX2.2 promoted the proliferation of the MD lymphoma-derived chicken cell line, MDCC-MSB1, which confirmed the potential oncogenic role of circRNX2.2 in tumor development. In conclusion, we found that the RUNX2-derived circRUNX2.2 can positively regulate the transcription of the parental gene RUNX2 in a cis-acting manner. The high expression of circRUNX2.2 in MD tumor tissues indicated that it might mediate MD lymphoma progression.

Paracrine signals influence patterns of fibrocartilage differentiation in a lyophilized gelatin hydrogel for applications in rotator cuff repair.

Rotator cuff injuries present a clinical challenge for repair due to current limitations in functional regeneration of the native tendon-to-bone enthesis. A biomaterial that can regionally instruct unique tissue-specific phenotypes offers potential to promote enthesis repair. We have recently demonstrated the mechanical benefits of a stratified triphasic biomaterial made up of tendon- and bone-mimetic collagen scaffold compartments connected via a continuous hydrogel, and we now explore the potential of a biologically favorable enthesis hydrogel for this application. Here we report in vitro behavior of human mesenchymal stem cells (hMSCs) within thiolated gelatin (Gel-SH) hydrogels in response to chondrogenic stimuli as well as paracrine signals derived from MSC-seeded bone and tendon scaffold compartments. Chondrogenic differentiation media promoted upregulation of cartilage and entheseal fibrocartilage matrix markers COL2, COLX, and ACAN as well as the enthesis-associated transcription factors SCX, SOX9, and RUNX2 in hMSCs within Gel-SH. Similar effects were observed in response to TGF-ß3 and BMP-4, enthesis-associated growth factors known to play a role in entheseal development and maintenance. Conditioned media generated by hMSCs seeded in tendon- and bone-mimetic collagen scaffolds influenced patterns of gene expression regarding enthesis-relevant growth factors, matrix markers, and tendon-to-bone transcription factors for hMSCs within the material. Together, these findings demonstrate that a Gel-SH hydrogel provides a permissive environment for enthesis tissue engineering and highlights the significance of cellular crosstalk between adjacent compartments within a spatially graded biomaterial.

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

BACKGROUND: 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. METHODS: Female C57 mice (48 weeks old) were inoculated subcutaneously with MC38 cells to establish a cancer cachexia model. A 5 mg/kg dose of l-carnitine or an equivalent volume of water was administered for 14 days 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. RESULTS: 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 5 mg/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. CONCLUSIONS: 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.

PAR1 Activation, via LMBR1/BMP Axis, Promotes the Osteogenesis of Periodontal Ligament Stem Cells (PDLSCs) and Alleviates the Inhibitory Effect of Sodium Butyrate on PDLSCs Osteogenesis.

BACKGROUND: Periodontitis is the leading cause of tooth loss and can exacerbate various systemic inflammatory conditions. Periodontal ligament stem cells (PDLSCs) stand out as prominent and favorable candidates for promoting periodontal tissue regeneration. This study aimed to investigate whether the protease-activated receptor type 1 (PAR1) can mitigate the sodium butyrate (NaB)-induced PDLSCs osteogenesis inhibition and unravel the underlying mechanism. METHODS: Public datasets from the Gene Expression Omnibus (GEO) were utilized to analyze differentially expressed genes (DEGs) in periodontitis and subsequent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. PDLSCs were cultured normally in control medium (CM) as the negative control or in osteogenic medium (OM) to induce osteogenesis. PAR1 was either activated or suppressed using a selective agonist or antagonist (OM+agonist and OM+antagonist). The evaluation of PDLSCs osteogenesis was based on the levels of osteogenesis-related markers, including runt-related transcription factor 2 (RUNX2), osterix (OSX), osteocalcin (OCN), and osteopontin (OPN), alkaline phosphatase (ALP) activity, and calcium concentration. Additionally, cell proliferation and osteogenic differentiation were measured through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Alizarin Red Staining. To determine the PAR1 targeting the limb development membrane protein 1 (LMBR1)/bone morphogenetic protein (BMP) pathway, LMBR1 was upregulated through cell transfection and BMP2 was inhibited using the selective inhibitor Noggin protein. Finally, NaB was introduced into PDLSCs to investigate the effect on NaB-induced inhibition of PDLSCs osteogenesis. RESULTS: PAR1, RUNX2, OSX, OCN, OPN, proliferation, ALP activity, calcium concentration, osteogenic differentiation, BMP2, and BMP4 exhibited significant increases in PDLSCs cultured in OM (p < 0.01). These parameters were further elevated by PAR1 agonist and conversely reduced by PAR1 antagonist (p < 0.01). Conversely, LMBR1 was decreased in PDLSCs cultured in OM (p < 0.001), with further reduction induced by PAR1 agonist and a reverse increase observed with PAR1 antagonist (p < 0.001). OE-LMBR1 transfection successfully elevated LMBR1 levels, subsequently inhibiting BMP2 and BMP4 (p < 0.001). Meanwhile, the Noggin protein effectively suppressed BMP2 and BMP4 (p < 0.001). All observed osteogenesis-related changes were reversed by the increased LMBR1 or inhibition of the BMP pathway (p < 0.001). Furthermore, NaB suppressed osteogenesis-related changes in OM-cultured PDLSCs (p < 0.001), and these effects were entirely reversed by PAR1 agonist (p < 0.001). Conversely, the increased LMBR1 or inhibited BMP pathway disrupted the osteogenesis reversion induced by PAR1 agonist (p < 0.001). CONCLUSION: The activation of PAR1, through suppressing LMBR1 signaling and activating BMP pathway, demonstrates the ability to enhance the osteogenesis of PDLSCs and mitigate the inhibitory effects on PDLSCs osteogenesis caused by NaB.

Indoxyl Sulfate Inhibits Osteogenesis in Bone Marrow Mesenchymal Stem Cells through the AhR/Hes1 Pathway.

Uremic toxins cause bone disorders in patients with chronic kidney disease (CKD). These disorders are characterized by low turnover osteodystrophy and impaired bone formation in the early stages of CKD. Evidence indicates that the aryl hydrocarbon receptor (AhR) mediates signals that suppress early osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). However, whether the AhR mediates the effects of indoxyl sulfate (IS), a uremic toxin, on BMSC osteogenesis remains unclear. We investigated whether IS affects osteogenesis through the AhR/Hes1 pathway. Expression levels of osteogenesis genes (Runx2, Bmp2, Alp, and Oc), AhR, and Hes1 were measured in mouse BMSCs (D1 cells). At concentrations of 2-50 µM, IS significantly reduced mineralization, particularly in the early stages of BMSC osteogenesis. Furthermore, IS significantly downregulated the expression of Runx2, Bmp2, Oc, and Alp. Notably, this downregulation could be prevented using an AhR antagonist and through Ahr knockdown. Mechanistically, IS induced the expression of Hes1 through AhR signaling, thereby suppressing the transcription of Runx2 and Bmp2. Our findings suggest that IS inhibits early osteogenesis of BMSCs through the AhR/Hes1 pathway, thus suppressing the transcription of Runx2 and Bmp2. Our findings may guide new therapeutic strategies against CKD-related bone disorders.