BMP-2 Levels Research Articles

Modeling of Healing of Intra-Articular Fractures of Long Tubular Bones in Rats

Background: We present a new original method of modeling an intra-articular fracture of the long tubular bone in laboratory animals.Objective: To create a new experimental model of an intra-articular fracture of the long tubular bone and to approve it for comprehensive evaluation of morphofunctional parameters of cartilage and bone healing.Materials and methods: The experiment was performed on 30 white sexually mature Wistar rats. Modeling involved layer-by-layer dissection of tissues along the anteromedial surface and formation of an incomplete fracture of the femoral medial condyle using a sharp L-shaped working end of an 0.2 × 0.2 сm instrument perpendicular to the medial condyle with a slight single muscle force. The rats were euthanized in groups of 10 animals on days 7, 14, and 30. We performed knee radiography and determined blood osteocalcin and BMP-7 levels. The Mankin score was used in the histological evaluation of the articular cartilage changes. The groups were compared using the Kruskal-Wallis test. P < .05 was considered statistically significant.Results: We noted statistically significant differences (P < .0001) when comparing the groups using the Mankin score. The post hoc test revealed statistically significant differences in the Mankin scores between days 7 and 14 (P = .014) and between days 7 and 30 (P < .0001). Group comparisons showed no statistically significant differences in blood osteocalcin levels (P = .518). However, there were statistically significant differences (P = .0028) between the groups in blood BMP-7 levels. The post hoc test revealed statistically significant differences in blood BMP-7 levels between days 7 and 14 (P = .048) and between days 14 and 30 (P = .0195).Conclusions: The developed experimental model of intra-articular fractures of long tubular bones allows for a comprehensive assessment of reparative processes of cartilage and bone tissue. The model is close to the real mechanism of pathology development.

Circulating BMP-7 Level is Independent of Sarcopenia in Older Asian Adults.

In vitro and animal studies have demonstrated that BMP-7, renowned for its osteogenic properties, also exerts beneficial effects on muscle metabolism by enhancing myogenesis and reversing muscle atrophy. Despite being proposed as a common regulatory factor for both muscle and bone, the impact of BMP-7 on human muscle health has not been thoroughly investigated. This cross-sectional study involved 182 community-dwelling older adults who underwent a comprehensive geriatric assessment in South Korea. Sarcopenia was diagnosed using Asian-specific cutoffs, and serum BMP-7 levels were quantified via enzyme immunoassay. The mean age of the participants was 72.2 ± 7.3 years, with 62.6% being female. After adjustments for confounders, serum BMP-7 levels were not significantly different between individuals with and without sarcopenia, nor were there differences based on skeletal muscle mass, strength, or physical performance levels (P = 0.423 to 0.681). Likewise, no correlations were detected between circulating BMP-7 levels and any sarcopenia assessment metrics such as skeletal muscle index, grip strength, gait speed, or chair stand completion times (P = 0.127 to 0.577). No significant associations were observed between increases in serum BMP-7 concentrations and the risk of sarcopenia or poor muscle phenotypes (P = 0.431 to 0.712). Stratifying participants into quartiles based on serum BMP-7 levels also indicated no differences in sarcopenia-related parameters (P = 0.663 to 0.996). Despite experimental evidence supporting BMP-7's role in muscle metabolism, this study found no significant association between serum BMP-7 levels and clinical indicators of muscle health in older adults. These findings challenge the utility of serum BMP-7 as a biomarker for sarcopenia in this demographic.

The Expression Level of Inflammation-Related Genes in Patients With Bone Nonunion and the Effect of BMP-2 Infected Mesenchymal Stem Cells Combined With nHA/PA66 on the Inflammation Level of Femoral Bone Nonunion Rats

Bone nonunion delays fracture end repair and is associated with inflammation. Although bone nonunion can be effectively repaired in clinical practice, many cases of failure. Studies have confirmed that BMP-2 and nHA/PA66 repaired bone defects successfully. There are few studies on the effects of the combined application of BMP-2 and NHA/PA66 on bone nonunion osteogenesis and inflammation. We aimed to investigate the expression level of inflammation-related genes in patients with bone nonunion and the effect of BMP-2-infected mesenchymal stem cells combined with nHA/PA66 on the level of inflammation in femur nonunion rats. We searched for a gene expression profile related to bone nonunion inflammation (GSE93138) in the GEO public database. Bone marrow mesenchymal stem cells (MSCs) of SD rats were cultured and passed through. We infected the third generation of MSCs with lentivirus carrying BMP-2 and induced the infected MSCs to bone orientation. We detected the expression level of BMP-2 by RT-PCR and the cell viability and alkaline phosphatase (ALP) activity by CCK8 and then analyzed the cell adhesion ability. Finally, the levels of related inflammatory factors, including C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and Erythrocyte Sedimentation Rate (ESR), were detected in nonunion rats. Our findings: The patients with nonunion had up-regulated expression of 26 differentially inflammatory genes. These genes are mainly enriched in innate immune response, extracellular region, calcium ion binding, Pantothenate and CoA biosynthesis pathways. The expression level of BMP-2 in the Lenti-BMP-2 group was higher (vs. empty lentivirus vector group: t=5.699; vs. uninfected group t=3.996). The cell activity of the MSCs + BMP-2 + nHA/PA66 group increased gradually. After being combined with nHA/PA66, MSCs transfected with BMP-2 spread all over the surface of nHA/PA66 and grew into the material pores. MSCs + BMP-2 + nHA/PA66 cells showed positive ALP staining, and the OD value of ALP was the highest. The levels of CRP, IL-6, TNF-α, and ESR in the MSCs + BMP-2 + nHA/PA66 group were lower than those in the MSCs and MSCs + nHA/PA66 group but higher than those in MSCs + BMP-2 group. The above comparisons were all P˂0.05. The findings demonstrated that the expression level of inflammation-related genes increased in the patients with bone nonunion. The infection of MSCs by BMP-2 could promote the directed differentiation of MSCs into osteoblasts in the bone marrow of rats, enhance the cell adhesion ability and ALP activity, and reduce inflammation in rats with bone nonunion.

Integrin α2β1 deficiency enhances osteogenesis via BMP-2 signaling for accelerated fracture repair

Previous studies have shown that the absence of the collagen-binding integrin α2β1 confers protection against osteoporosis, primarily by enhancing osteoblast-mediated matrix formation, with a particular increase in collagen type I production. This study aimed to elucidate the mechanism underlying this increased matrix production. Our findings demonstrate that osteoblasts lacking integrin α2 secrete a pro-osteogenic factor that activates both TGF-β and BMP signaling pathways. Among these, BMP-2 was identified as the key signaling protein responsible for this effect, as its expression was significantly upregulated during osteoblast differentiation. Moreover, integrin α2 deficiency led to earlier and elevated BMP-2 secretion at the cell surface during osteogenesis, which promoted accelerated osteoblast differentiation. This phenomenon likely contributes to enhanced matrix production in aging animals, providing a protective effect against osteoporosis.To explore the broader implications of this phenotype, we utilized a fracture healing model. In integrin α2-deficient 12 weeks old female mice, elevated serum levels of BMP-2 were detected during the early stages of fracture repair. This upregulation of BMP signaling within the fracture callus accelerated the healing process, resulting in faster formation and mineralization of the cartilaginous callus. Additionally, the elevated BMP-2 levels facilitated earlier differentiation of chondrocytic cells, evidenced by the premature appearance of collagen type II- and type X-positive cells during endochondral ossification. Despite the accelerated healing, the overall biomechanical integrity of the repaired fractures remained uncompromised.Thus, the modulation of integrin α2β1 presents a promising therapeutic target for enhancing fracture repair by regulating BMP-2 signaling in a physiologically relevant manner.

Assessment of experimental circulating biomarkers in volume and pressure overload: insights from a cohort study

Abstract Introduction Experimental biomarkers including Angiopoietin-2 (ANG2), bone morphogenetic protein 10 (BMP10), fibroblast growth factor 23 (FGF23), and insulin-like growth factor-binding protein 7 (IGFBP7) are hypothesized to reflect cardiac pathophysiological processes that are potentially present in adverse cardiac remodeling in volume or pressure overload. This study aims to elucidate the relationship between these circulating biomarkers and the presence of hemodynamically significant pressure or volume overload and adverse outcomes and compare it to N-terminal pro B-type natriuretic peptide (NT-proBNP). Methods We studied the association between the four experimental circulating biomarkers in patients with pressure or volume overload with reference to none/mild valvular disease in N=1302 of an observational outpatient cardiology cohort. The median age was 63.0 (25th/75th quartile 51.5, 71.4) years, 445 (34.2%) were women. Circulating biomarkers were quantified using a novel antibody-based method. Logistic regression models with cox regression plots were employed to assess the association of each biomarker with the severity of valve disease concerning volume or pressure overload, adjusted for age and sex. Results In the overall population, elevated levels of ANG2, BMP10, FGF23 and IGFB7 were positively associated with the presence of significant volume overload (ANG2: Odds ratio (OR) 1.26 (95% confidence interval (CI) 1.17-1.35), p<0.001; BMP10: OR 2.57 (CI 1.89-3.48), p<0.001; FGF23: OR 1.51 (CI 1.14-1.20), p=0.004; IGFBP7: OR 1.39 (CI 1.14-1.69), p=0.001, NT-proBNP: OR 1.69 (CI 1.47-1.95), p<0.001). No statistically significant association between circulating biomarkers and pressure overload was observed. Over a median follow-up of 4 years, 31 (20.1%) participants died in the group of volume overload and 9 (40.9%) individuals in the group of pressure overload. After multivariable-adjustment, elevated levels of BMP 10 showed a borderline association with an increased risk of all-cause mortality in individuals with volume overload compared to none/mild valvular disease: hazard ratio (HR) 1.51 (CI 0.96- 2.38), p=0.075. Higher concentrations of all biomarkers were predictive of all-cause mortality for both individuals with volume overload and those with none/mild valvular disease (ANG2: HR 3.72 (CI 1.67-8.33), p=0.001; BMP10: HR 1.65 (CI 1.20-2.26), p=0.002; FGF23: HR 2.08 (CI 1.65-2.62), p<0.001; IGFBP7: HR 1.55 (CI 1.25-1.91), p<0.001; NT-proBNP: HR 1.72 (CI 1.47-2.00), p<0.001). Conclusions This study demonstrates that circulating biomarkers involved in distinct pathophysiological pathways of inflammation, fibrosis and calcification are elevated in patients with hemodynamically significant volume overload. Since they are also related to mortality, their clinical role needs to be explored further, also in context with the routine biomarker NT-proBNP.

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.

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.

6421 Imaging Phenotype May Predict Genotype In Human Pheochromocytoma: Relationships Between Genetic Mutation Clusters, Metabolite Transporter Expression, And Radiotracer Uptake

Abstract Disclosure: I. Sakuma: None. M. Fujimoto: None. D.F. Vatner: None. K. Yokote: None. T. Tanaka: None. Context [1]23I-metaiodobenzylguanidine scintigraphy (MIBG) and [1]8F-fluoro-2-doxy-D-glucose positron emission tomography (FDG-PET) are helpful for the localization of pheochromocytoma. The norepinephrine transporter (NET) and the glucose transporter 1 (GLUT1) are thought to be critical for the uptake of these radioisotopes. NET expression is activated by Paired Like Homeobox (PHOX2) and glucocorticoid receptor (GR) in neuroblastoma cells. Around 70% of pheochromocytomas carry well-known mutations. Pheochromocytoma-associated genes are divided into two clusters: the pseudohypoxia-related cluster 1 and the kinase signaling-related cluster 2. Cluster 1 mutation are associated with noradrenergic pheochromocytomas with an elevated metastatic risk. Cluster 2 tumors demonstrate an adrenergic phenotype with a less aggressive course. How mutations of cluster 1 and cluster 2 impact the expression of NET/GLUT1 and on radiotracer uptake remains unclear. Hypothesis Cluster 1 and cluster 2 will have different effects on the expression of transcription factors associated with chromaffin cell differentiation with resultant differential effects on NET/GLUT1 expression. Methods 42 patients with pheochromocytoma were evaluated by MIBG and FDG-PET. Tumor gene expression was evaluated by real-time qPCR, and mutation analyses were performed by Sanger or next-generation sequencing. Results 88% of tumors were MIBG-avid, and 67% were FDG-avid. The expression of NET in MIBG-negative tumors was 70% lower than in MIBG-positive tumors. The expression of GLUT1 in FDG-PET-negative tumors was 75% lower than in FDG-PET-positive tumors. The expression of NET positively correlated with transcription factors involved in chromaffin cell maturation, including PHOX2A, PHOX2B, GATA binding protein 3 (GATA3), GR, catecholamine synthases, and insulinoma associated 1 (INSM1). The JASPAR database for promoter analysis revealed putative responsive regions of the transcription factors PHOX2A, PHOX2B, GATA3, GR, and INSM1 on the NET gene. Seven patients (16.7%) had mutations in cluster 1, and fourteen patients (33.3%) had mutations in cluster 2. Cluster 2 exhibited elevated NET, PHOX2A, PHOX2B, GATA3, GR, and INSM1 expression levels compared to cluster 1. Cluster 1 showed a higher expression level of GLUT1 and BMP4 that is associated with early chromaffin cell differentiation. Conclusions In pheochromocytomas, MIBG and FDG uptake correlated with the expression levels of NET and GLUT1, respectively. NET expression is associated with chromaffin cell differentiation transcription factor expression. We propose that pheochromocytomas that carry Cluster 1 mutations are more likely to be missed in MIBG due to low NET expression, while pheochromocytomas that carry Cluster 2 mutations are more likely to be missed on FDG-PET due to low GLUT1 expression with a more mature chromaffin cell phenotype. Presentation: 6/2/2024

Soybean Isoflavones Alleviate Osteoarthritis Through Modulation of the TSC1/mTORC1 Signaling Pathway to Reduce Intrachondral Angiogenesis

ABSTRACT Background The incidence of osteoarthritis (OA) is increasing, yet its pathogenesis remains largely unknown. Recent studies suggest that abnormal subchondral bone remodeling plays a crucial role in OA development, highlighting a gap in clinical treatments targeting this aspect. Soybean Isoflavone (SI) has shown potential in treating OA, although its mechanisms are not fully understood. Methods This research investigated the effects of SI on subchondral bone remodeling in an OA rat model, assessing joint damage, OARSI scores, and type H vessel formation (CD31hiEmcnhi expression). Additionally, the expression of ALP, OCN, BMP, and TSC1 was evaluated to determine involvement of the mTORC1 pathway. In vitro studies on IL-1β-induced osteoblasts further examined the impact of SI on TSC1/mTORC1 signaling and related markers. Results SI treatment reduced joint damage and OARSI scores in the rat OA model, significantly decreasing CD31hiEmcnhi expression, indicating a reduction in type H vessel formation. SI also downregulated ALP, OCN, and BMP expression while upregulating TSC1, suggesting inhibition of the mTORC1 signaling pathway and VEGF release. In vitro, SI increased TSC1 expression and decreased mTORC1 signaling, VEGF, ALP, OCN, and BMP levels in IL-1β-induced osteoblasts. Conclusion SI targets the TSC1/mTORC1 signaling pathway to suppress osteoblast activation and VEGF release, inhibiting type H vessel formation and slowing abnormal subchondral bone remodeling. These findings provide a novel therapeutic approach for OA by focusing on subchondral bone remodeling mechanisms.

Cephalomannine reduces radiotherapy resistance in non-small cell lung cancer cells by blocking the β-catenin-BMP2 signaling pathway

BackgroundThe management of non-small cell lung cancer (NSCLC) often includes the use of radiotherapy, with individual outcomes being impacted by the tumor's response to this treatment modality. Cephalomannine (CPM), a taxane diterpenoid found in Taxus spp, has been found to have anti-tumor activity. This study was aim to the explore the role and mechanism by which CPM affects radiotherapy resistance in NSCLC. MethodsH460 cells were pretreated with different doses of CPM. H460 cells were transfected with β-catenin overexpression plasmids. The cell viability, colony-forming ability, migration ability, and sphere-forming ability and apoptosis of the cells were measured by using CCK-8, colony-forming, transwell, and sphere-forming assay and flow cytometry. Western blot assay was employed to detect the expression of β-catenin and BMP2. ResultsThe cell viability, proliferation, migration and sphere-forming ability of cells in the radiotherapy-resistant (RR) group were significantly higher than those in the radiotherapy-sensitivity (RS) group. Conversely, the apoptosis rate of cells in the RR group was lower than that in the RS group. However, after CPM pretreatment of RR group cells, the above phenomena were reversed in a CPM dose-dependent manner. Subsequently, pretreatment with CPM resulted in a decrease in the expression levels of β-catenin and BMP2 in the RR group. In addition, overexpression of β-catenin mitigated the inhibitory effects of CPM on radiotherapy-resistant NSCLC cells. ConclusionCPM has the potential to decrease radiotherapy resistance in NSCLC cells by inhibiting the β-catenin-BMP2 signaling pathway, promoting apoptosis, and ultimately impeding cell growth.

Overexpression of Bmp4 induces microphthalmia by disrupting embryonic neural retina

Microphthalmia, mostly an autosomal dominant disorder, is a worldwide severe congenital ocular malformation that causes visual impairment. Our investigation unveiled a total of 30 genes associated with microphthalmia. Employing the CytoHubba and PPI network, we identified Bmp4 as the most pivotal hub gene. Subsequently, the conditional overexpression of Bmp4 in the retina caused highly distinctive microphthalmia, manifested by retinal disorganization with ganglion cell misalignment. Significant reduction in the number and abnormal distribution location of retinal cells in microphthalmia model mice. Elevated Bmp4 was associated with an increase in retinal apoptosis and a decrease in proliferating cells, which exacerbates the development of microphthalmia. Here we identify Bmp4 as an extremely important gene responsible for microphthalmia and the involved mechanisms. Overexpression of Bmp4 induces retinal cell ectopic expression and developmental defects, highlighting the importance of a well-balanced Bmp4 level in shaping the embryonic retina during early development.

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.

BMP4-Induced Suppression of Breast Cancer Metastasis Is Associated with Inhibition of Cholesterol Biosynthesis.

We reported previously that in preclinical models, BMP4 is a potent inhibitor of breast cancer metastasis and that high BMP4 protein levels predict favourable patient outcomes. Here, we analysed a breast cancer xenograft with or without enforced expression of BMP4 to gain insight into the mechanisms by which BMP4 suppresses metastasis. Transcriptomic analysis of cancer cells recovered from primary tumours and phosphoproteomic analyses of cancer cells exposed to recombinant BMP4 revealed that BMP4 inhibits cholesterol biosynthesis, with many genes in this biosynthetic pathway being downregulated by BMP4. The treatment of mice bearing low-BMP4 xenografts with a cholesterol-lowering statin partially mimicked the anti-metastatic activity of BMP4. Analysis of a cohort of primary breast cancers revealed a reduced relapse rate for patients on statin therapy if their tumours exhibited low BMP4 levels. These findings indicate that BMP4 may represent a predictive biomarker for the benefit of additional statin therapy in breast cancer patients.

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.

Macrophage-to-osteocyte communication: Impact in a 3D in vitro implant-associated infection model

Macrophages and osteocytes are important regulators of inflammation, osteogenesis and osteoclastogenesis. However, their interactions under adverse conditions, such as biomaterial-associated infection (BAI) are not fully understood. We aimed to elucidate how factors released from macrophages modulate osteocyte responses in an in vitro indirect 3D co-culture model. Human monocyte-derived macrophages were cultured on etched titanium disks and activated with either IL-4 cytokine (anti-inflammatory M2 phenotype) or Staphylococcus aureus secreted virulence factors to simulate BAI (pro-inflammatory M1 phenotype). Primary osteocytes in collagen gels were then stimulated with conditioned media (CM) from these macrophages. The osteocyte response was analyzed by gene expression, protein secretion, and immunostaining. M1 phenotype macrophages were confirmed by IL-1β and TNF-α secretion, and M2 macrophages by ARG-1 and MRC-1.Osteocytes receiving M1 CM revealed bone inhibitory effects, denoted by reduced secretion of bone formation osteocalcin (BGLAP) and increased secretion of the bone inhibitory sclerostin (SOST). These osteocytes also downregulated the pro-mineralization gene PHEX and upregulated the anti-mineralization gene MEPE. Additionally, exhibited pro-osteoclastic potential by upregulating pro-osteoclastic gene RANKL expression. Nonetheless, M1-stimulated osteocytes expressed a higher level of the potent pro-osteogenic factor BMP-2 in parallel with the downregulation of the bone inhibitor genes DKK1 and SOST, suggesting a compensatory feedback mechanisms. Conversely, M2-stimulated osteocytes mainly upregulated anti-osteoclastic gene OPG expression, suggesting an anti-catabolic effect. Altogether, our findings demonstrate a strong communication between M1 macrophages and osteocytes under M1 (BAI)-simulated conditions, suggesting that the BAI adverse effects on osteoblastic and osteoclastic processes in vitro are partly mediated via this communication. Statement of significanceBiomaterial-associated infections are major challenges and the underlying mechanisms in the cellular interactions are missing, especially among the major cells from the inflammatory side (macrophages as the key cell in bacterial clearance) and the regenerative side (osteocyte as main regulator of bone). We evaluated the effect of macrophage polarization driven by the stimulation with bacterial virulence factors on the osteocyte function using an indirect co-culture model, hence mimicking the scenario of a biomaterial-associated infection. The results suggest that at least part of the adverse effects of biomaterial associated infection on osteoblastic and osteoclastic processes in vitro are mediated via macrophage-to-osteocyte communication.

Microbiological and molecular profile of furcation defects in a population with untreated periodontitis.

To describe the microbiological composition of subgingival dental plaque and molecular profile of gingival crevicular fluid (GCF) of periodontal furcation-involved defects. Fifty-seven participants with periodontitis contributed with a degree II-III furcation involvement (FI), a non-furcation (NF) periodontal defect and a periodontally healthy site (HS). Subgingival plaque was analysed by sequencing the V3-V4 region of the 16S rRNA gene, and a multiplex bead immunoassay was carried out to estimate the GCF levels of 18 GCF biomarkers. Aiming to explore inherent patterns and the intrinsic structure of data, an AI-clustering method was also applied. In total, 171 subgingival plaque and 84 GCF samples were analysed. Four microbiome clusters were identified and associated with FI, NF and HS. A reduced aerobic microbiota (p = .01) was detected in FI compared with NF; IL-6, MMP-3, MMP-8, BMP-2, SOST, EGF and TIMP-1 levels were increased in the GCF of FI compared with NF. This is the first study to profile periodontal furcation defects from a microbiological and inflammatory standpoint using conventional and AI-based analyses. A reduced aerobic microbial biofilm and an increase of several inflammatory, connective tissue degradation and repair markers were detected compared with other periodontal defects.

TRPML1 activation ameliorates lysosomal phenotypes in CLN3 deficient retinal pigment epithelial cells

Mutations in the lysosomal membrane protein CLN3 cause Juvenile Neuronal Ceroid Lipofuscinosis (JNCL). Activation of the lysosomal ion channel TRPML1 has previously been shown to be beneficial in several neurodegenerative disease models. Here, we tested whether TRPML1 activation rescues disease-associated phenotypes in CLN3-deficient retinal pigment epithelial (ARPE-19 CLN3-KO) cells. ARPE-19 CLN3-KO cells accumulate LAMP1 positive organelles and show lysosomal storage of mitochondrial ATPase subunit C (SubC), globotriaosylceramide (Gb3), and glycerophosphodiesters (GPDs), whereas lysosomal bis(monoacylglycero)phosphate (BMP/LBPA) lipid levels were significantly decreased. Activation of TRPML1 reduced lysosomal storage of Gb3 and SubC but failed to restore BMP levels in CLN3-KO cells. TRPML1-mediated decrease of storage was TFEB-independent, and we identified TRPML1-mediated enhanced lysosomal exocytosis as a likely mechanism for clearing storage including GPDs. Therefore, ARPE-19 CLN3-KO cells represent a human cell model for CLN3 disease showing many of the described core lysosomal deficits, some of which can be improved using TRPML1 agonists.

BMP Signaling Is a Prognostic Marker in Patients With Colorectal Cancer and Associates With Frailty.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β superfamily of ligands and have been shown to promote or suppress colorectal cancer (CRC) growth. Developing treatments that target BMPs is challenging due to their multiple roles, including involvement in the inflammatory response and nutritional status. The present study evaluated the prognostic value of BMP-4, which is believed to be highly expressed in CRC, and its correlation with inflammatory and nutrition statuses in patients with CRC. We analyzed BMP-4 expression in tumor tissues from 144 patients who underwent CRC surgery using immunohistochemistry and evaluated the relationship between BMP-4 levels and clinical outcomes. Kaplan-Meier analysis revealed that patients with high expression levels of BMP-4 exhibited a shorter overall survival rate than those with low levels of expression. Multivariate analysis revealed that BMP-4 expression was an independent prognostic factor for overall survival and death from other diseases in CRC patients. Furthermore, high BMP-4 expression was significantly correlated with high C-reactive protein/Albumin ratio, sarcopenia, and osteopenia. BMP-4 is a significant prognostic factor in CRC, particularly in predicting death from other diseases, while also showing associations with inflammatory and nutritional statuses.

MiR-20a: a key regulator of orthodontic tooth movement via BMP2 signaling pathway modulation

ABSTRACT Aim In this study, we aimed to establish a rat tooth movement model to assess miR-20’s ability in enhancing the BMP2 signaling pathway and facilitate alveolar bone remodeling. Method 60 male SD rats had nickel titanium spring devices placed between their left upper first molars and incisors, with the right side serving as the control. Forces were applied at varying durations (18h, 24h, 30h, 36h, 42h, 1d, 3d, 5d, 7d, 14d), and their bilateral maxillary molars and surrounding alveolar bones were retrieved for analysis. Fluorescent quantitative PCR was conducted to assess miR-20a, BMP2, Runx2, Bambi and Smad6 gene expression in alveolar bone, and western blot was performed to determine the protein levels of BMP2, Runx2, Bambi, and Smad6 after mechanical loading. Result We successfully established an orthodontic tooth movement model in SD rats and revealed upregulated miR-20a expression and significantly increased BMP2 and Runx2 gene expression and protein synthesis in alveolar bone during molar tooth movement. Although Bambi and Smad6 gene expression did not significantly increase, their protein synthesis was found to decrease significantly. Conclusion MiR-20a was found to be involved in rat tooth movement model alveolar bone remodeling, wherein it promoted remodeling by reducing Bambi and Smad6 protein synthesis through the BMP2 signaling pathway.

ATF3 affects osteogenic differentiation in inflammatory hPDLSCs by mediating ferroptosis via regulating the Nrf2/HO-1 signaling pathway

Activating transcription factor 3 (ATF3) has been identified as a regulator associated with osteoblast differentiation. However, the effects of ATF3 on the osteogenic differentiation and proliferation of human periodontal stem cells (hPDLSCs) in periodontitis have not been reported. With the purpose of establishing an in vitro model of periodontitis, hPDLSCs were challenged with lipopolysaccharide (LPS). The Cell Counting Kit-8 assay was applied to assess cell viability, while reverse transcription-quantitative PCR and western blotting were employed to detect ATF3 expression. Inflammatory release was assessed using ELISA, together with western blotting. Lipid peroxidation was explored using the C11 BODIPY 581/591 probe, biochemical kits, thiobarbituric acid reactive substances (TBARS) assay and DCFH-DA staining. Iron and Fe2+ levels, and the expression levels of ferroptosis-related proteins were measured using corresponding kits and western blotting. Osteogenic differentiative capability was evaluated using alkaline phosphatase staining, Alizarin red staining and western blotting. The expression levels of proteins associated with Nrf2/HO-1 signaling were identified using western blotting. The results indicated that ATF3 expression was upregulated in LPS-induced hPDLSCs. The knockdown of ATF3 alleviated the LPS-induced inflammatory response in hPDLSCs, together with increased levels of TNF-α, IL-6, IL-1β, Cox-2 and iNOS, and decreased levels of IL-10. ATF3 silencing also led to lower TBARS production rate, and reduced levels of reactive oxygen species, iron, Fe2+, ACSL4 and TFR1, whereas it elevated the levels of SLC7A11 and GPX4. In addition, ATF3 silencing promoted hPDLSC mineralization and cell differentiation, and elevated the levels of OCN2, RUNX2 and BMP2. Additionally, ATF3 depletion upregulated the expression levels of proteins related with Nrf2/HO-1 signaling. The Nrf2 inhibitor ML385 partially counteracted the effects of ATF3 interference on the LPS-challenged inflammatory response, lipid peroxidation, ferroptosis as well as osteogenic differentiative capability in hPDLSCs. In summary, the results revealed that ATF3 silencing suppressed inflammation and ferroptosis, while it improved osteogenic differentiation in LPS-induced hPDLSCs by regulating Nrf2/HO-1 signaling, which may provide promising therapeutic targets for the treatment of periodontitis.