BMP-2 mRNA Research Articles

Investigation of the Mechanism of SEMA5A and Its Associated Autophagy-Related Genes in Gastric Cancer.

Semaphorin 5A (SEMA5A) and autophagy-related genes (ARGs) are pivotal in the pathogenesis of gastric cancer (GC). However, the potential regulatory role of SEMA5A in autophagy via its associated ARGs and the underlying molecular mechanisms remain unresolved. GC-related datasets from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were analyzed to identify differentially expressed genes (DEGs) between GC and control samples. The intersection of DEGs with ARGs produced candidate genes, which were further analyzed using Spearman correlation with SEMA5A to identify signature genes. Stratification of GC samples based on signature gene expression, followed by Kaplan-Meier survival analysis, identified key genes. Subsequent analyses, including gene set enrichment analysis (GSEA), immune infiltration, and immune checkpoint evaluation, were conducted on the key genes and SEMA5A. The mRNA expression level was quantified using real-time quantitative polymerase chain reaction (RT-qPCR). Ninety candidate genes were identified for Spearman correlation with SEMA5A, revealing TNFSF11, BMP6, ITPR1, and DLC1 with correlation coefficients exceeding 0.3. Survival analysis underscored DLC1 and BMP6 as key genes due to significant prognostic differences. GSEA implicated SEMA5A, BMP6, and DLC1 in the ECM receptor interaction pathway. Immune infiltration analysis indicated a negative correlation of SEMA5A and BMP6 with M1 macrophages, while DLC1 exhibited the strongest association with the immune checkpoint PDCD1LG2 (p < 0.05, cor = 0.43). The mRNA expression level of SEMA5A was significantly upregulated in AGS parental cells compared to GES-1 cells (p < 0.01), whereas DLC1 and BMP6 mRNA levels were markedly downregulated in AGS parental cells relative to GES-1 (p < 0.0001). ARGs BMP6 and DLC1, associated with SEMA5A, were identified, and their prognostic significance in GC was demonstrated. Additionally, their regulatory mechanisms were further elucidated through immune infiltration analysis and molecular network construction, providing a theoretical foundation for future research on the molecular mechanisms in patients with GC.

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.

Effect of Huoxue Jiegu compound capsule on osteoblast differentiation and fracture healing by regulating the PI3K/Akt/mTOR signaling pathway in rabbits

ObjectiveTo examine and talk about the mechanism of the Huoxue Jiegu compound capsule's effects on osteoblasts and the PI3K/Akt/mTOR signal pathway in rabbits suffering from tibial fractures. MethodIn vitro, CCK8 was used to assess the survival rates. Alizarinred staining was used to evaluate mineralized nodules. ALP staining was used to observe the osteoblasts. qRT-PCR was used to determine the mRNA expression of the bone formation-related factors BMP-2, bFGF, and TGF-β. In vivo, three groups of nine male rabbits each were randomly assigned to three groups: the Model group, the Huoxue Jiegu compound capsule group (HXJGC group), and the inhibitor group (HXJGC+3-MA), four weeks following the intervention. HE staining was employed to examine the rabbits' bone histology. immunohistochemistry was employed to examine the relative expression of the proteins VEGF and LC3-II. Western Blot was utilized to examine the relative expression of the proteins Beclin-1, LC3-II/Ⅰ, p62, p-PI3K, p-AKT, and p-mTOR. ResultsCompared to the control group, the medium- and high-dose groups exhibited considerably higher survival rates (P < 0.05), as well as enhanced cell proliferation and differentiation (P < 0.05) and more pronounced mineralized nodules. (P < 0.05), but the low-dose groups showed no appreciable variation. In the low, medium, and high-dose groups, there was a substantial reduction in the expression of bFGF mRNA, whereas the levels of BMP-2 and TGF-β mRNA were considerably higher than in the control group (P < 0.05). In vivo, after four weeks of treatment, the model control group and inhibito group had a large amount of fibrous hyperplasia accompanied by bleeding and a small amount of inflammatory cell infiltration. But in the HXJGC group, new cartilage appeared, and the surface of the cartilage was smooth and flat. Beclin-1 and LC3-II/I expression in the HXJGC+3 MA group was significantly lower than in the HXJGC and Model groups (P < 0.05). The HXJGC group showed lower p62 expression than the HXJGC+3 MA and model groups (P < 0.05). The HXJGC group exhibited significantly reduced levels of p-PI3K, p-AKT, and p-mTOR expression in comparison to HXJGC+3 MA groups (P < 0.05). ConclusionRabbits with tibial fractures can be treated with HXJGC, which can control the expression of the PI3K/Akt/mTOR signal pathway. It can promote the differentiation and maturation of osteoblasts at the fracture end of rabbits, accelerate the recovery of fractures, and achieve the purpose of treating the disease.

Long non-coding TRPM2-AS regulates fracture healing by targeting miR-545-3p/Bmp2

ObjectiveDelayed fracture healing increases the suffering of patients. An in-depth investigation of the pathogenesis of delayed fracture healing may offer new direction for the prevention and treatment.MethodsThe study included 63 normal healing tibial fractures and 58 delayed healing tibial fractures patients. Long non-coding RNA (lncRNA)TRPM2-AS, microRNA-545-3p (miR-545-3p), bone morphogenetic protein 2 (Bmp2) mRNA and osteogenic differentiation markers, including runt-related transcription factor 2 (Runx2), osteocalcin (Ocn), and alkaline phosphatase (Alp) mRNA expression were determined by Real-time quantitative reverse transcription-polymerase chain reaction in serum and MC3T3-E1 cells. The prediction potential of TRPM2-AS in delayed healing fracture patients was verified by receiver operating characteristic curves. The binding relationship of TRPM2-AS/miR-545-3p/Bmp2 was evaluated by dual luciferase reporter gene assay. Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry.ResultsTRPM2-AS was remarkably down-regulated in patients with delayed fracture healing and could better predict the fracture healing status. TRPM2-AS downregulation inhibited osteogenic markers mRNA expression, restrained proliferation, and promoted apoptosis of MC3T3-E1 cells (p < 0.05). In delayed fracture healing, miR-545-3p was dramatically up-regulated and was negatively regulated by TRPM2-AS. Reducing miR-545-3p eliminate the negative effect of TRPM2-AS down-regulation on osteoblast proliferation and differentiation (p < 0.05). miR-545-3p targets Bmp2, which plays a positive role in osteoblast differentiation (p < 0.05).ConclusionThis study found that TRPM2-AS has the potential to be a diagnostic marker for delayed fracture healing and revealed that the TRPM2-AS/miR-545-3p/Bmp2 axis affects fracture healing by regulating osteoblast.

Mangiferin Induces Post-Implant Osteointegration in Male Diabetic Rats.

Background and Objectives: Hyperglycemia is known to undermine the osteointegration process of implants. In this study, the effects of mangiferin (MF) on the post-implant osteointegration process in a type-II diabetes model were investigated molecularly and morphologically. Materials and Methods: Sprague Dawley male rats were divided into three groups: control, diabetes, and diabetes + MF. All animals were implanted in their tibia bones on day 0. At the end of the 3-month experimental period, the animals' blood and the implant area were isolated. Biochemical measurements were performed on blood samples and micro-CT, qRT-PCR, histological, and immunohistochemical measurements were performed on tibia samples. Results: MF significantly improved the increased glucose, triglyceride-VLDL levels, and liver enzymes due to diabetes. By administering MF to diabetic rats, the osteointegration percentage and bone volume increased while porosity decreased. DKK1 and BMP-2 mRNA expressions and OPN, OCN, and OSN mRNA-protein expressions increased by MF administration in diabetic rats. Additionally, while osteoblast and osteoid surface areas increased with MF, osteoclast and eroded surface areas decreased. Conclusions: The findings of our study indicate that MF will be beneficial to the bone-repairing process and osteointegration, which are impaired by type-II diabetes.

Molecular analysis of a self-organizing signaling pathway for Xenopus axial patterning from egg to tailbud

Xenopus embryos provide a favorable material to dissect the sequential steps that lead to dorsal-ventral (D-V) and anterior-posterior (A-P) cell differentiation. Here, we analyze the signaling pathways involved in this process using loss-of-function and gain-of-function approaches. The initial step was provided by Hwa, a transmembrane protein that robustly activates early β-catenin signaling when microinjected into the ventral side of the embryo leading to complete twinned axes. The following step was the activation of Xenopus Nodal-related growth factors, which could rescue the depletion of β-catenin and were themselves blocked by the extracellular Nodal antagonists Cerberus-Short and Lefty. During gastrulation, the Spemann-Mangold organizer secretes a cocktail of growth factor antagonists, of which the BMP antagonists Chordin and Noggin could rescue simultaneously D-V and A-P tissues in β-catenin-depleted embryos. Surprisingly, this rescue occurred in the absence of any β-catenin transcriptional activity as measured by β-catenin activated Luciferase reporters. The Wnt antagonist Dickkopf (Dkk1) strongly synergized with the early Hwa signal by inhibiting late Wnt signals. Depletion of Sizzled (Szl), an antagonist of the Tolloid chordinase, was epistatic over the Hwa and Dkk1 synergy. BMP4 mRNA injection blocked Hwa-induced ectopic axes, and Dkk1 inhibited BMP signaling late, but not early, during gastrulation. Several unexpected findings were made, e.g., well-patterned complete embryonic axes are induced by Chordin or Nodal in β-catenin knockdown embryos, dorsalization by Lithium chloride (LiCl) is mediated by Nodals, Dkk1 exerts its anteriorizing and dorsalizing effects by regulating late BMP signaling, and the Dkk1 phenotype requires Szl.

BMP-2 regulates the expression of myosin Va via smad in melan-a melanocyte.

Myosin Va (Myo Va) is one of three protein complexes involved in melanosome transport. In this study, we identified BMP-2 as an up-regulator of Myo Va expression using 2-methyl-naphtho[1,2,3-de]quinolin-8-one (MNQO). Our results showed that MNQO reduced the mRNA and protein expression of Myo Va and BMP-2 in melanocytes. Knockdown of BMP-2 by siRNA also affected Myo Va mRNA and protein expression, confirming that MNQO regulates Myo Va through BMP-2. Furthermore, phosphorylation of Smad1/5/8 by BMP2 treatment confirmed that the BMP-2/Smad signaling pathway regulates Myo Va expression in Melan-a melanocytes. Smad-binding elements were found in the Myo Va promoter and phosphorylated Smad1/5/8 bind directly to the Myo Va promoter to activate Myo Va transcription and BMP-2 enhances this binding. These findings provide insight into a new role for BMP-2 in Melan-a melanocytes and a mechanism of regulation of Myo Va expression that may be beneficial in the treatment of albinism or hyperpigmentation disorders.

#1293 Endothelial calcification induced by high-phosphate is characterized by mesenchymal and osteoblastic transdifferentiation

Abstract Background and Aims Chronic kidney disease (CKD) is a prevalent condition that affects millions of people. Patients with CKD have an increased cardiovascular risk, mostly due to vascular calcification (VC). By now, it's quite clear how, in vitro and in vivo, vascular smooth muscle cells (VSMCs) actively participate to VC. In contrast to the wide studies on VSMCs, the role of endothelial cells (ECs) has been poorly investigated. It's known that ECs have a dynamic nature and following particular stimuli they can trans-differentiate and acquire several different phenotypes through the endothelial-to-mesenchymal transition (EndMT). Methods Human aortic ECs (HAECs) were challenged, in an in vitro calcification model, with 2, 2.5, 3, 3.5 mM Pi for up to 7 days. Early morphological changes, gene expression and cell migration were evaluated. Afterwards calcium deposition, genes and protein expression and ultrastructural changes were analyzed. Results High-Pi, at a concentration of 3 mM, is able to induce EC calcium deposition. Calcification is dependent on calcium influx without influencing Pit-1 expression. During the first days of high-Pi treatment ECs change their morphology from cobblestone to spindle shape acquiring motility, as demonstrated by the 24% increased closure in the scratch wound test. EndMT master genes such as SNAIL and TWIST resulted up-regulated at day 3 together with an increased protein expression of mesenchymal markers such as N-Cadherin, α-SMA, SM22α, FSP-1 and Fibronectin. Starting from day 5 high-Pi induces osteoblastic differentiation with RUNX2 and BMP2 mRNA up-regulation up to day 7, osteopontin increased protein expression and positive Alcian Blu staining. In calcified ECs mesenchymal and endothelial markers, such as α-SMA, SM22α, FSP-1 and Fibronectin or vWF progressively decrease. Finally, uremic serum is able to exacerbate in vitro calcification, compared to healthy serum characterized by an increase in both mesenchymal and osteoblastic mastergene such as TWIST and RUNX2. Conclusions Our data demonstrate that in vitro high-Pi induces the EndMT process and similar osteoblastic trans-differentiation suggesting a potential role of endothelium in VC. The relevancy of endothelial calcification is suggested by the calcification exacerbation induced by uremic compared to healthy sera.

Evaluation of Cardiospermum halicacabum on Bone Morphogenetic Protein-2 (BMP2) mRNA Expression in Osteoblast Cells.

Introduction Maintaining bone health is crucial for overall well-being, with osteoblasts playing a vital role in bone formation. Bone morphogenetic protein-2 (BMP2) is a key regulator, stimulating bone matrix synthesis and osteoblast differentiation. Recognizing BMP2's significance, there's growing interest in natural compounds, such as Cardiospermum halicacabum. This study explores Cardiospermum halicacabum's potential influence on BMP2 mRNA expression in osteoblast cells for insights into bone health modulation. Materials and methods This research utilized Cardiospermum halicacabum to explore its impact on MG-63 cells, a human osteoblast cell line. Osteoblast cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum, and maintained at 37°C in a 5% CO2 and 95% air environment. Cell viability was evaluated by seeding osteoblast cells into 96-well plates and exposing them to different concentrations of Cardiospermum halicacabum (2.0 μg/ml and 20 μg/ml). The study observed both the promotion of osteoblast cell growth in MG-63 and morphological changes in thecells under an inverted light microscope at 10x magnification. Results were presented using one-way analysis of variance (ANOVA) conducted with IBM SPSS Statistics for Windows, Version 23 (Released 2015; IBM Corp., Armonk, New York, United States). Result The reverse transcription-polymerase chain (RT-PCR) results revealed an increased expression of BMP-2 mRNA fold change in comparison to the control group. A clear positive correlation was observed between the BMP-2 mRNA fold change and the notable increase in the concentration of Cardiospermum halicacabum. This investigation revealed a direct association of BMP-2 mRNA expressionwith the proliferation of osteoblast cells. Specifically, the BMP-2 mRNA fold change was recorded at 2.26±1.05 in Cardiospermum halicacabum at 2.0 μg/ml and 2.0 ± 0.84 at 20 μg/ml, with corresponding significances of 0.00, respectively. Conclusion Potential effects of Cardiospermum halicacabum on BMP-2 mRNA expression in osteoblast cellsand its role in bone health modulation revealed that Cardiospermum halicacabum may upregulate BMP-2 mRNA expression, suggesting its potential as a natural compound for enhancing bone formation. The observed positive correlation between Cardiospermum halicacabum concentration and BMP-2 mRNA fold change showed the significance of this botanical agent in promoting osteoblast cell proliferation. These results highlight the importance of further research to explore the applications of Cardiospermum halicacabum in managing bone disorders and improving overall bone health.

CA1 Modulates the Osteogenic Differentiation of Dental Follicle Stem Cells by Activating the BMP Signaling Pathway In Vitro.

Carbonic anhydrase 1 (CA1) has been found to be involved in osteogenesis and osteoclast in various human diseases, but the molecular mechanisms are not completely understood. In this study, we aim to use siRNA and lentivirus to reduce or increase the expression of CA1 in Dental follicle stem cells (DFSCs), in order to further elucidate the role and mechanism of CA1 in osteogenesis, and provide better osteogenic growth factors and stem cell selection for the application of bone tissue engineering in alveolar bone fracture transplantation. The study used RNA interference and lentiviral vectors to manipulate the expression of the CA1 gene in DFSCs during in vitro osteogenic induction. The expression of osteogenic marker genes was evaluated and changes in CA1, alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), and Bone morphogenetic proteins (BMP2) were measured using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB). The osteogenic effect was assessed through Alizarin Red staining. The mRNA and protein expression levels of CA1, ALP, RUNX2, and BMP2 decreased distinctly in the si-CA1 group than other groups (p < 0.05). In the Lentivirus-CA1 (LV-CA1) group, the mRNA and protein expressions of CA1, ALP, RUNX2, and BMP2 were amplified to varying degrees than other groups (p < 0.05). Apart from CA1, BMP2 (43.01%) and ALP (36.69%) showed significant upregulation (p < 0.05). Alizarin red staining indicated that the LV-CA1 group produced more calcified nodules than other groups, with a higher optical density (p < 0.05), and the osteogenic effect was superior. CA1 can impact osteogenic differentiation via BMP related signaling pathways, positioning itself upstream in osteogenic signaling pathways, and closely linked to osteoblast calcification and ossification processes.

A sericin hydrogel scaffold for sustained dexamethasone release modulates macrophage polarization to promote mandibular bone defect repair in rats

To evaluate the efficacy of a modified sericin hydrogel scaffold loaded with dexamethasone (SMH-CD/DEX) scaffold for promoting bone defect healing by stimulating anti-inflammatory macrophage polarization. The light-curable SMH-CD/DEX scaffold was prepared using dexamethasone-loaded NH2-β-cyclodextrin (NH2-β-CD) and sericin hydrogel and characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), biocompatibility assessment and drug release test. THP-1 macrophages incubated with the scaffold were examined for protein expressions of iNOS and Arg-1, mRNA expressions of IL-6, Il-10, Arg-1 and iNOS, and surface markers CD86 and CD206 using Western blotting, RT-qPCR, and flow cytometry. In a co-culture system of human periodontal ligament stem cells (HPDLSCs) and THP-1 macrophages, the osteogenic ability of the stem cells incubated with the scaffold was evaluated by detecting protein expressions of COL1A1 and Runx2 and expressions of ALP, Runx2, OCN and BMP2 mRNA, ALP staining, and alizarin red staining. In a rat model of mandibular bone defect, the osteogenic effect of the scaffold was assessed by observing bone regeneration using micro-CT and histopathological staining. In THP-1 macrophages, incubation with SMH-CD/DEX scaffold significantly enhanced protein expressions of Arg-1 and mRNA expressions of IL-10 and Arg-1 and lowered iNOS protein expression and IL-6 and iNOS mRNA expressions. In the co-culture system, SMH-CD/DEX effectively increased the protein expressions of COL1A1 and Runx2 and mRNA expressions of ALP and BMP2 in HPDLSCs and promoted their osteogenic differentiation. In the rat models, implantation of SMH-CD/DEX scaffold significantly promoted bone repair and bone regeneration in the bone defect. The SMH-CD/DEX scaffold capable of sustained dexamethasone release promotes osteogenic differentiation of stem cells and bone defect repair in rats by regulating M2 polarization.

Aucubin produces anti-osteoporotic effects under mechanical stretch stress and orthodontic tooth movement

Aucubin (AU), an iridoid glycoside extracted from Eucommia ulmoides, exerts anti-osteoporotic effects by promoting osteogenesis, as reported in previous studies. Here, we investigated the effects of AU under mechanical stretch stress. MC3T3-E1 cells were treated with dexamethasone (DEX) in vitro and subjected to mechanical stretch stress to establish an osteoporotic orthodontic force cell model. AU treatment increased the mRNA and protein expressions of BMP2, OPN, RUNX2, COL-1 and other osteogenic differentiation factors in MC3T3-E1 cells. Furthermore, we established an in vivo orthodontic tooth movement (OTM) model of osteoporosis. Serum parameter detection of ALP concentration, radiography of the femur, hematoxylin-eosin (HE) staining, and micro-CT of the maxilla confirmed that AU could partially reverse the damage induced by DEX. Immunohistochemical (IHC) analysis showed that AU increased the expression of COL-1, OCN, and OPN on the tension side of the periodontium. In conclusion, AU treatment promotes osteogenic differentiation under mechanical stretch stress and positively affects bone remodeling during OTM in DEX-induced osteoporosis.

Iron-triggered signaling via ETS1 and the p38/JNK MAPK pathway regulates Bmp6 expression.

BMP6 is an iron-sensing cytokine whose transcription in liver sinusoidal endothelial cells (LSECs) is enhanced by high iron levels, a step that precedes the induction of the iron-regulatory hormone hepcidin. While several reports suggested a cell-autonomous induction of Bmp6 by iron-triggered signals, likely via sensing of oxidative stress by the transcription factor NRF2, other studies proposed the dominant role of a paracrine yet unidentified signal released by iron-loaded hepatocytes. To further explore the mechanisms of Bmp6 transcriptional regulation, we used female mice aged 10-11 months, which are characterized by hepatocytic but not LSEC iron accumulation, and no evidence of systemic iron overload. We found that LSECs of aged mice exhibit increased Bmp6 mRNA levels as compared to young controls, but do not show a transcriptional signature characteristic of activated NFR2-mediated signaling in FACS-sorted LSECs. We further observed that primary murine LSECs derived from both wild-type and NRF2 knock-out mice induce Bmp6 expression in response to iron exposure. By analyzing transcriptomic data of FACS-sorted LSECs from aged versus young mice, as well as early after iron citrate injections, we identified ETS1 as a candidate transcription factor involved in Bmp6 transcriptional regulation. By performing siRNA-mediated knockdown, small-molecule treatments, and chromatin immunoprecipitation in primary LSECs, we show that Bmp6 transcription is regulated by iron via ETS1 and p38/JNK MAP kinase-mediated signaling, at least in part independently of NRF2. Thereby, these findings identify the new components of LSEC iron sensing machinery broadly associated with cellular stress responses.

Morphogenesis of fungiform papillae in developing miniature pigs

ObjectiveFungiform papillae contain taste buds and play a critical role in mastication and the gustatory system. In this study, we report a series of sequential observations of organogenesis of fungiform papillae in miniature pigs, as well as changes in the expression of BMP2, BMP4, Wnt5a, Sox2, and Notch1 signaling pathway components. DesignIn this study, we investigated the spatiotemporal expression patterns of BMP, Wnt, Sox2 and Notch in the fungiform papillae of miniature pigs at the bud stage (E40), cap stage (E50) and bell stage (E60). Pregnant miniature pigs were obtained, and the samples were processed for histological staining. Immunohistochemistry and real-time PCR were used to detect the mRNA and protein expression levels of BMP2, BMP4, Wnt5a, Sox2, and Notch1. ResultsAt E40, fungiform papillae were present on the anterior two-thirds of the tongue in a specific array and pattern. The fungiform papillae were enlarged and basically developed at E50 and were largest at the earlier stage (E60). Most of the BMP2 was concentrated in the epithelial layer and the connective tissue core of the fungal papilloma and gradually accumulated from E40-E60. BMP-4 was weakly expressed in the fungiform papillae epithelia, but BMP-4-positive cells were also observed in the developing tongue muscle at E50 and E60. Wnt5a-positive cells were observed in the fungiform papillae epithelia and developing tongue muscle at all three time points. Sox2-positive cells were observed only in fungiform papillae epithelial cells, and Notch1-positive cells could not be detected. ConclusionsThis study provides primary data regarding the morphogenesis and expression of developmental signals in the fungiform papillae of miniature pigs, establishing a foundation for further research in both this model and humans.

BMP4 up-regulated by 630 nm LED irradiation is associated with the amelioration of rheumatoid arthritis

Rheumatoid arthritis (RA) is caused by inflammatory response of joints with cartilage and damage of synovium and bone erosion. In our previous studies, it has showed that irradiation of 630 nm LED reduce inflammation of synovial fibroblasts and cartilage and bone destruction in RA. However, the key genes and mechanism in ameliorating RA by irradiation of 630 nm LED remains unknown. In this study, human fibroblast-like synoviocytes (FLS) cell line MH7A and primary human RA-FLSs were treated with TNF-α and 630 nm LED irradiation with the different energy density. The mRNA sequencing was performed to screen the differentially expressed genes (DEGs). In all datasets, 10 DEGs were identified through screening. The protein interaction network analysis showed that 8 out of the 10 DEGs interacted with each other including IL-6, CXCL2, CXCL3, MAF, PGF, IL-1RL1, RRAD and BMP4. This study focused on BMP4, which is identified as important morphogens in regulating the development and homeostasis. CCK-8 assay results showed that 630 nm LED irradiation did not affect the cell viability. The qPCR and ELISA results showed that TNF-α stimulation inhibited BMP4 mRNA and protein level and irradiation of 630 nm LED increased the BMP4 mRNA and protein level in MH7A cells. In CIA and transgenic hTNF-α mice models, H&E staining showed that irradiation of 630 nm LED decreased the histological scores assessed from inflammation and bone erosion, while BMP4 expression level was up-regulated after 630 nm LED irradiation. Pearson correlation analysis shown that BMP4 protein expression was negatively correlated with the histological score of CIA mice and transgenic hTNF-α mice. These results indicated that BMP4 increased by irradiation of 630 nm LED was associated with the amelioration of RA, which suggested that BMP4 may be a potential targeting gene for photobiomodulation.

Fgf9-Nolz-1-Wnt2 Axis Regulates Morphogenesis of the Lung.

Morphological development of the lung requires complex signal crosstalk between the mesenchymal and epithelial progenitors. Elucidating the genetic cascades underlying the signal crosstalk is essential to understanding lung morphogenesis. Here, we identified Nolz-1/Znf503 as a mesenchymal lineage-specific transcriptional regulator that plays a key role in lung morphogenesis. Nolz-1 null mutation resulted in a severe hypoplasia phenotype, including a decreased proliferation of mesenchymal cells, aberrant differentiation of epithelial cells, and defective growth of epithelial branches. Nolz-1 deletion also downregulated Wnt2, Lef1, Fgf10, Gli3 and Bmp4 mRNAs. Mechanistically, Nolz-1 regulated lung morphogenesis primarily through Wnt2 signaling. Loss of function and overexpression studies demonstrated that Nolz-1 transcriptionally activated Wnt2 and downstream β-catenin signaling to control mesenchymal cell proliferation and epithelial branching. Exogenous Wnt2 could causally rescue defective proliferation and epithelial branching in Nolz-1 knockout lungs. Finally, we identified Fgf9 as an upstream regulator of Nolz-1. Collectively, Fgf9-Nolz-1-Wnt2 signaling represents a novel axis in the control of lung morphogenesis. These findings are relevant to lung tumorigenesis in which a pathological function of Nolz-1 is implicated.

Circ_0001666 upregulation promotes intestinal epithelial cell fibrosis in pediatric Crohn's disease via the SRSF1/BMP7 axis.

The epithelial-mesenchymal transition (EMT) is closely associated with Crohn's disease (CD) related intestinal fibrosis, a condition whose prevalence is increasing annually among children. Recently, the CD marker gene microarray screening revealed an upregulation of circ_0001666 in the colon tissues of CD patients, but its underlying mechanisms remain unclear. In this study, we explored the molecular mechanism of circ_0001666 in regulating EMT-mediated fibrosis in CD in vitro. The levels of circ_0001666 and EMT-associated proteins were assessed in CD clinical samples, and a CD cell model was established using TGF-β1 to induce human intestinal epithelial cells (HIECs). Additionally, the expression levels of genes and proteins related to EMT and fibrosis were analyzed by quantitative real-time PCR and western blot, cell migration, and invasion were assessed via wound healing assay and transwell, respectively, and RNA pull-down and RNA immunoprecipitation assays were performed to verify the relationship between SRSF1 and BMP7 or circ_0001666. Circ_0001666 was overexpressed in the intestinal mucosal tissues of CD patients and was positively correlated with EMT. Silencing circ_0001666 inhibited the migration, invasion, EMT, and fibrosis of HIECs induced by TGF-β1. Mechanistically, circ_0001666 regulated BMP7 expression by interacting with SRSF1. Furthermore, the effects of inhibiting circ_0001666 on HIECs could be partially reversed by overexpressing SRSF1 or silencing BMP7. Collectively, circ_0001666 regulates TGF-β1-induced HIEC migration, invasion, EMT, and fibrosis. Circ_0001666 also promoted EMT-mediated fibrosis by interacting with SRSF1 to accelerate BMP7 mRNA decay. These findings provide new insights into the pathogenesis of CD and suggest that circ_0001666 might be a potential therapeutic target for CD.

Administration of mRNA-Nanomedicine-Augmented Calvarial Defect Healing via Endochondral Ossification.

Large-area craniofacial defects remain a challenge for orthopaedists, hastening the need to develop a facile and safe tissue engineering strategy; osteoconductive material and a combination of optimal growth factors and microenvironment should be considered. Faced with the unmet need, we propose that abundant cytokines and chemokines can be secreted from the bone defect, provoking the infiltration of endogenous stem cells to assist bone regeneration. We can provide a potent mRNA medicine cocktail to promptly initiate the formation of bone templates, osteogenesis, and subsequent bone matrix deposition via endochondral ossification, which may retard rapid fibroblast infiltration and prevent the formation of atrophic non-union. We explored the mutual interaction of BMP2 and TGFβ3 mRNA, both potent chondrogenic factors, on inducing endochondral ossification; examined the influence of in vitro the transcribed polyA tail length on mRNA stability; prepared mRNA nanomedicine using a PEGylated polyaspartamide block copolymer loaded in a gelatin sponge and grafted in a critical-sized calvarial defect; and evaluated bone regeneration using histological and μCT examination. The BMP2 and TGFβ3 composite mRNA nanomedicine resulted in over 10-fold new bone volume (BV) regeneration in 8 weeks than the BMP2 mRNA nanomedicine administration alone, demonstrating that the TGFβ3 mRNA nanomedicine synergistically enhances the bone's formation capability, which is induced by BMP2 mRNA nanomedicine. Our data demonstrated that mRNA-medicine-mediated endochondral ossification provides an alternative cell-free tissue engineering methodology for guiding craniofacial defect healing.

#4000 ROLE OF ENDOTHELIAL CELLS IN VASCULAR CALCIFICATION

Abstract Background and Aims Vascular calcification (VC) represents a common pathological feature of cardiovascular disease and VC caused by hyperphosphatemia is one of the hallmarks of chronic kidney disease (CKD). Therefore, elucidating the pathogenesis of VC may have significant clinical benefits for CKD patients. Vascular smooth muscle cells challenged with high-phosphate (Pi) actively participate to VC by trans-differentiating into simil-osteoblastic cells that acquire the capability to deposit hydroxyapatite crystals in the extracellular matrix of tunica media in arteries. The role of endothelial cells (ECs) in high-Pi calcification has been poorly investigated until now. Nonetheless, following particular stimuli ECs can lose their endothelial characteristics and acquire several different phenotypes transdifferentiating with a process known as endothelial-to-mesenchymal transition (EndMT). Given the central role of VC for CKD patients wellbeing we decided to investigate whether ECs may have a role in VC process. Method We set up an in vitro calcification model with human aortic ECs (HAECs) challenged with 2, 2.5, 3, 3.5 mM high-Pi for 7 days. Calcium deposition, cell viability and cell trans-differentiation were evaluated. Results In our model we observed deposition of calcium phosphate crystals induced by high-Pi that resulted significant from 3 up to 3.5 mM Pi (Ctr 0.43 ± 0.01; 3 mM Pi 6.72 ± 1.15; 3.5 mM Pi 17.31 ± 1.88; micromol Ca++/mg protein; P &amp;lt; .01; Fig 1A-B). We found that calcification is mediated by Pi-influx, as demonstrated by the inhibition of calcium deposition by Phosphonoformic acid (PFA), an inhibitor of Na/Pi cotransporter Pit-1 (3.5 mM Pi 15.52 ± 1.97; 1 mM PFA 0.6 ± 0.05; micromol Ca++/mg protein; P &amp;lt; .0001; Fig 1A, C). In addition, in our model high-Pi did not show any significant toxic effect at every concentration tested compared to control cells. In Pi-treated ECs we observed a clear change in cell-morphology from cobblestone to spindle shaped, a recognized sign of EndMT. Moreover, we observed a change in extracellular matrix composition more similar to osteoblastic matrix. In facts, ECs treated with Pi were Alcian Blue positive, a staining that binds glycosaminoglycans contained in bone matrix, with blue granules of calcium-phosphate in the cytoplasm of treated cells. Starting from day 5 up to day 7 the transition was confirmed by an up-regulation of SNAIL and N-cadherin mRNA expressions, one of the master genes and one the markers of EndMT process, respectively. A modification of endothelial phenotype was also supported by a progressive decrease in protein expression of Von Willebrand factor and VE-Cadherin observed at day 9 of calcification (−53.7% and −32.6% respectively). Moreover, from day 5 till day 7 we found some osteoblastic differentiation signs such as a progressive increase of RUNX2 and BMP2 mRNA expression and an increase of protective MGP mRNA levels. Conclusion Our data demonstrate that HAECs are able to calcify. High-Pi induces trans-differentiation in simil-osteoblastic cells and a progressive loss of endothelial characteristics through the EndMT process. More studies are needed to better elucidate the role for ECs in CKD VC process.

Diamond-like carbon (DLC)-coated titanium surface inhibits bacterial growth and modulates human alveolar bone cell responses in vitro

Modifying the titanium alloy (Ti-6Al-4V) surface plays a key role in improving biological responses. The present work aimed to determine the response of human alveolar bone cells to the diamond-like carbon (DLC) film and to evaluate the antibacterial effect of DLC-coated titanium. The alloys were deposited with different compositions, including C2H2 (DLC), carbon tetrafluoride (CF4:C2H2; F-DLC), and tetramethylsilane (Si(CH3)4:C2H2; Si-DLC). The surface morphology and wettability were measured using an atomic force microscopy (AFM) and a contact angle measurement. Titanium coated with DLC, F-DLC, or Si-DLC increased surface roughness and hydrophobicity. The ratio of spreading cells to initial rounded attaching cells at 20 min of all modified surfaces was higher than that of the control surfaces. F-DLC showed better cell spreading at 20 min compared to other conditions, as observed by scanning electron microscopy (SEM). The DLC, F-DLC, and Si-DLC coating significantly promoted cell proliferation compared to the Ti-6Al-4V control surfaces. ALP mRNA expression was significantly reduced on DLC surfaces compared to the control on day 14. Additionally, ALP and BMP2 mRNA expression increased on F-DLC-coated surfaces, but there were no statistically significant differences. However, DLC surfaces promoted higher mineral deposition than the control. The lower Staphylococcus aureus colony was observed on the surface of the DLC and F-DLC. In conclusion, the DLC-modified Ti-4Al-6V surface modulates cell attachment, proliferation, osteogenic marker gene expression, and bacterial growth. Herein, DLC-coated titanium alloys are still needed for continued development as an alternative biomaterial in dental applications.