Expression Of Bone Morphogenic Protein Research Articles

Abstract PO4-13-10: The anti-metastatic role of BMP4 through cholesterol biosynthesis inhibition and consequent interaction with statin benefit in breast cancer

Abstract We have previously identified a role for bone morphogenic protein 4 (BMP4) in reducing distant relapse risk after early breast cancer (BrCa) (1). We therefore set out to identify mechanisms underlying the protective effect of BMP4, looking to translate this finding for patient benefit. A highly metastatic variant of the triple negative MDA-MB-231 human BrCa line (231-HM-turboGFP) was transduced with BMP4, leading to sustained BMP4 secretion. These modified tumours were established in NSG mice, resected at a given size and turboGFP-positive cancer cells were recovered from primary tumours for RNA sequencing analysis. Gene set enrichment analysis (GSEA) was completed to identify signalling pathways that were significantly modulated by BMP4. The four most significantly downregulated gene sets were associated with cholesterol synthesis, with the overwhelming majority of genes in this pathway being downregulated. BMP4 downregulation of 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1, involved in cholesterol biosynthesis) was confirmed at the protein level by western blotting on whole tumour lysates. Forced expression of BMP4 also led to a significant reduction in the levels of free and total cholesterol. In a confirmatory analysis of the METABRIC human BrCa dataset, the expression of cholesterol biosynthesis-related genes inversely correlated with the expression of BMP4. Further, for the majority of these genes, expression was elevated in high-grade breast tumours, and predicted worse overall survival of patients. Finally, we tested the effect of a lipophilic statin, lovastatin, on the growth and metastasis of 231-HM-turboGFP tumours. Treatment did not affect the growth of primary tumours but significantly less metastases were observed in the lungs of treated mice, at least partially replicating the anti-metastatic effect of BMP4. Having identified inhibition of cholesterol biosynthesis as a potential mechanism of protective BMP4 action we then asked if BMP4 status interacted with any protective effect of co-administered statins. We therefore returned to the cohort initially employed to demonstrate BMP4 benefit (1) and looked at the interaction between BMP4 protein levels and statin usage with regards to recurrence. BMP4 protein and statin usage data were available on a cohort of 417 patients with early BrCa. Statin use compared to none led to a reduced risk of distant relapse (8 v 22%, 0.0029) at 15 years. BMP4 led to borderline reduction in distant relapse (16 v 24%, p=0.052). In BMP4 negative tumours, statin protected against any relapse (p=0.0025) whereas in BMP4 positive disease no significant risk reduction was not significant (p=0.074). Absence of BMP4 expression may be a biomarker of both higher relapse risk and statin benefit in early BrCa. 1. Eckhardt et al, Cancer Res. 2020;80(6):1304-1315 Citation Format: Lap Hing Chi, Andrew Redfern, Allan Burrows, Suraya Roslan, Leone Oh, Lisa Spalding, Robin Anderson. The anti-metastatic role of BMP4 through cholesterol biosynthesis inhibition and consequent interaction with statin benefit in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-13-10.

Serum Iron Overload Activates the SMAD Pathway and Hepcidin Expression of Hepatocytes via SMURF1.

Liver iron overload can induce hepatic expression of bone morphogenic protein (BMP) 6 and activate the BMP/SMAD pathway. However, serum iron overload can also activate SMAD but does not induce BMP6 expression. Therefore, the mechanisms through which serum iron overload activates the BMP/SMAD pathway remain unclear. This study aimed to clarify the role of SMURF1 in serum iron overload and the BMP/SMAD pathway. A cell model of serum iron overload was established by treating hepatocytes with 2 mg/mL of holo-transferrin (Holo-Tf). A serum iron overload mouse model and a liver iron overload mouse model were established by intraperitoneally injecting 10 mg of Holo-Tf into C57BL/6 mice and administering a high-iron diet for 1 week followed by a low-iron diet for 2 days. Western blotting and real-time PCR were performed to evaluate the activation of the BMP/SMAD pathway and the expression of hepcidin. Holo-Tf augmented the sensitivity and responsiveness of hepatocytes to BMP6. The E3 ubiquitin-protein ligase SMURF1 mediated Holo-Tf-induced SMAD1/5 activation and hepcidin expression; specifically, SMURF1 expression dramatically decreased when the serum iron concentration was increased. Additionally, the expression of SMURF1 substrates, which are important molecules involved in the transduction of BMP/SMAD signaling, was significantly upregulated. Furthermore, in vivo analyses confirmed that SMURF1 specifically regulated the BMP/SMAD pathway during serum iron overload. SMURF1 can specifically regulate the BMP/SMAD pathway by augmenting the responsiveness of hepatocytes to BMPs during serum iron overload.

Inhibition of BMP signaling delays tauopathy and cognitive deficits in P301S mouse model

AbstractBackgroundAging is the highest risk factor for sporadic Alzheimer’s disease (AD). Expression of bone morphogenic protein 4 (BMP4) increases 10‐fold with age, while expression of the BMP antagonist, noggin, decreases. We have observed that activating BMP signaling in induced human neurons increased levels of phosphorylated tau. The increase of p‐tau has been correlated with the activated non‐canonical BMP pathway leading to p38 activity. We hypothesized that modulating BMP signaling in an AD mouse model of tau would rescue disease phenotype.MethodP301S (PS19) mice were injected with lentivirus overexpressing noggin (LV‐noggin) or lentivirus mcherry (LV‐mcherry) control in the dentate gyrus. Cognitive behavioral testing was run at 7 months of age and brain tissue was collected for protein and immunofluorescent imaging.ResultWe observe that levels of BMP4, as well as BMP signaling, are increased in the brains of PS19 mice compared to littermate wildtype mice. Analysis of noggin injected PS19 brains show downregulation of BMP signaling in the DG shown by decreased pSMAD1/5/8, as well as non‐canonical signaling through p38 kinase activity. Inhibition of BMP signaling through overexpression of noggin improves cognitive ability shown through Barnes maze testing. Downregulation of BMP signaling in P301S mice led to a decrease in tau phosphorylation seen both in immunoblotting, as well as immunocytochemical analysis in the DG.ConclusionPS19 model of tauopathy show increased levels of BMP signaling. We have shown that increased levels of BMP signaling lead to increased tau phosphorylation and reduced degradation of p‐tau. We show that by inhibiting BMP signaling in vivo we can delay cognitive behavioral deficits seen in these mice by decreasing tau phosphorylation. This suggests that the age‐related increase in BMP signaling may participate in the initiation and progression of AD pathology and help to explain why age is the largest risk factor for AD.

Bone Morphogenic Proteins and Their Antagonists in the Lower Airways of Stable COPD Patients.

Bone morphogenic proteins (BMPs) and their antagonists are involved in the tissue development and homeostasis of various organs. To determine transcriptomic and protein expression of BMPs and their antagonists in stable COPD. We measured the expression and localization of BMPs and some relevant antagonists in bronchial biopsies of stable mild/moderate COPD (MCOPD) (n = 18), severe/very severe COPD (SCOPD) (n = 16), control smokers (CS) (n = 13), and control non-smokers (CNS) (n = 11), and in lung parenchyma of MCOPD (n = 9), CS (n = 11), and CNS (n = 9) using immunohistochemistry and transcriptome analysis, in vitro after the stimulation of the 16HBE cells. In bronchial biopsies, BMP4 antagonists CRIM1 and chordin were increased in the bronchial epithelium and lamina propria of COPD patients. BMP4 expression was decreased in the bronchial epithelium of SCOPD and MCOPD compared to CNS. Lung transcriptomic data showed non-significant changes between groups. CRIM1 and chordin were significantly decreased in the alveolar macrophages and alveolar septa in COPD patients. External 16HBE treatment with BMP4 protein reduced the bronchial epithelial cell proliferation. These data show an imbalance between BMP proteins and their antagonists in the lungs of stable COPD. This imbalance may play a role in the remodeling of the airways, altering the regenerative-reparative responses of the diseased bronchioles and lung parenchyma.

Hypoxia Treatment of Adipose Mesenchymal Stem Cells Promotes the Growth of Dermal Papilla Cells via HIF-1α and ERK1/2 Signaling Pathways.

Dermal papilla cells (DPCs) cultured in vitro induce hair follicle formation. Using a hypoxic microenvironment to culture adipose mesenchymal stem cells (ADSCs) can promote hair follicle growth. However, the exact molecular mechanisms underlying this process remain unclear. In this study, ADSCs and DPCs from Arbas Cashmere goats were used. A hypoxic microenvironment promoted the proliferation of ADSCs and increased the pluripotency of ADSCs. The growth factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) were upregulated in ADSCs in the hypoxia-conditioned medium (Hypo-cm). Hypo-cm also enhanced the ability of DPCs to induce hair follicle formation. Inhibitors of the ERK1/2 signaling pathway caused the expressions of growth factors that increased in hypoxic microenvironments to decrease; moreover, hypoxia-inducible factor-1α (HIF-1α) increased the expression levels of VEGF, bFGF, and PDGF and inhibited the expression of bone morphogenic protein 7 (BMP7). In conclusion, these findings improve the theoretical basis for the development of gene therapy drugs for the treatment of alopecia areata and hair thinning.

Intracellular labile iron is a key regulator of hepcidin expression and iron metabolism.

Liver iron loading can induce hepatic expression of hepcidin and regulate iron metabolism. However, the mechanism by which hepatocyte senses iron loading and further regulates iron metabolism remains unclear. Intracellular labile iron is nonferritin-bound and redox active; it is transitory, and it serves as a crossroads of cellular iron metabolism, the effect of intracellular labile iron in iron metabolism regulation is particularly poorly understood. An intracellular labile iron overload cell model was established using ferric ammonium citrate (FAC) and the lipophilic iron chelator 8-hydroxyquinoline (8HQ/FAC). RNA-Seq was performed to screen the genes that were highly expressed exclusively in 8HQ/FAC-treated HepG2 cells. High-iron-diet mice model and Hfe knockout hemochromatosis mice were used to investigate the importance of tumor necrosis factor α (TNFα) in iron metabolism. Intracellular labile iron in hepatocytes had a dual function in iron metabolism: It induced hepatocytes to express hepcidin via endoplasmic reticulum stress-induced transcription factors, and it stimulated expression of bone morphogenic protein 6 (BMP6, regulator of iron metabolism) in liver sinusoidal endothelial cells (LSECs) via promoting the secretion of TNFα by the hepatocytes. Blockade of TNFα dysregulated iron metabolism during iron overload. Furthermore, administration of TNFα could reduce iron burden in Hfe knockout hemochromatosis mice. Our findings reveal the importance of intracellular labile iron in iron metabolism, and propose that TNFα might be a novel therapeutic target for HFE-associated hemochromatosis.

Aberrant activation of KRAS in mouse theca-interstitial cells results in female infertility.

KRAS plays critical roles in regulating a range of normal cellular events as well as pathological processes in many tissues mediated through a variety of signaling pathways, including ERK1/2 and AKT signaling, in a cell-, context- and development-dependent manner. The in vivo function of KRAS and its downstream targets in gonadal steroidogenic cells for the development and homeostasis of reproductive functions remain to be determined. To understand the functions of KRAS signaling in gonadal theca and interstitial cells, we generated a Kras mutant (tKrasMT) mouse line that selectively expressed a constitutively active Kras G12D in these cells. Kras G12D expression in ovarian theca cells did not block follicle development to the preovulatory stage. However, tKrasMT females failed to ovulate and thus were infertile. The phosphorylated ERK1/2 and forkhead box O1 (FOXO1) and total FOXO1 protein levels were markedly reduced in tKrasMT theca cells. Kras G12D expression in theca cells also curtailed the phosphorylation of ERK1/2 and altered the expression of several ovulation-related genes in gonadotropin-primed granulosa cells. To uncover downstream targets of KRAS/FOXO1 signaling in theca cells, we found that the expression of bone morphogenic protein 7 (Bmp7), a theca-specific factor involved in ovulation, was significantly elevated in tKrasMT theca cells. Chromosome immunoprecipitation assays demonstrated that FOXO1 interacted with the Bmp7 promoter containing forkhead response elements and that the binding activity was attenuated in tKrasMT theca cells. Moreover, Foxo1 knockdown caused an elevation, whereas Foxo1 overexpression resulted in an inhibition of Bmp7 expression, suggesting that KRAS signaling regulates FOXO1 protein levels to control Bmp7 expression in theca cells. Thus, the anovulation phenotype observed in tKrasMT mice may be attributed to aberrant KRAS/FOXO1/BMP7 signaling in theca cells. Our work provides the first in vivo evidence that maintaining normal KRAS activity in ovarian theca cells is crucial for ovulation and female fertility.

A mucoadhesive gingival patch with Epigallocatechin-3-gallate green tea (Camellia sinensis) as an alternative adjunct therapy for periodontal disease: A narrative review

Background: Periodontitis is a progressive destructive periodontal disease. The prevalence of periodontal disease in Indonesia reaches 74.1% and mostly occurs in the productive age group. Most of the periodontopathogenic bacteria are gram-negative bacteria and have endotoxin in the form of lipopolysaccharide (LPS), which can penetrate the periodontal tissue and induce an inflammatory response. In inflammatory conditions, osteoclastic activity is higher than osteoblastic activity, which causes bone destruction. This results in an imbalance between osteoclast-induced bone resorption and osteoblast-induced bone formation. The current preferred treatment for periodontitis is scaling root planning (SRP), but this therapy cannot repair the damaged periodontal tissue caused by periodontitis. Purpose: To describe the possibility of using a mucoadhesive gingival patch with Epigallocatechin-3-gallate (EGCG) green tea (Camellia sinensis) as alternative adjunct therapy for periodontal disease. Review: EGCG is the main component of green tea catechins, which have antitumor, antioxidant, anti-inflammatory, anti-fibrotic, and pro-osteogenic effects. However, the weaknesses so far regarding the use of EGCG as an alternative treatment is its low oral bioavailability and the concentration of EGCG absorbed by the body decreasing when accompanied by food. EGCG can be used with a mucoadhesive gingival patch to optimise bioavailability and absorption and increase local concentration and sustained release of EGCG. EGCG encourages bone development and braces mesenchymal stem cells (MSCs) differentiation for osteoblast by enhancing the expression of bone morphogenic protein 2 (BMP2). EGCG also has been proven to increase the expression of RUNX2 and ALP activity that induces osteoblast differentiation and bone mineralisation. Conclusion: A mucoadhesive gingival patch containing EGCG Green Tea (C. sinensis) may potentially induce osteoblastic activity as an adjunct therapy to repair the periodontal tissue damage due to periodontal disease.

Biocompatibility and pro-mineralization effect of tristrontium aluminate cement for endodontic use

Background/purposeTristrontium aluminate (S3A) is a hydraulic cement with setting behavior similar to that of mineral trioxide aggregate (MTA). This study examined the biological effects of S3A on mouse dental papilla cells (MDPs) in vitro and on rat exposed pulps in vivo. Materials and methodsExtracts of S3A and MTA were prepared by immersing each cement in ultrapure water. MDPs were cultured with S3A or MTA extracts, and cell proliferation was evaluated with a tetrazolium-salt assay. Attachment of MDPs on the set cements was examined with scanning electron microscopy (SEM). mRNA expression of bone morphogenic protein (Bmp2), osteocalcin (Oc) and osteopontin (Opn) in MDPs exposed to S3A or MTA extracts was determined with reverse transcription-quantitative polymerase chain reaction. Mineralized nodule formation was evaluated with Alizarin Red S staining. Simulated body fluid (SBF)-dipped S3A was examined with SEM and energy dispersive X-ray analysis (EDX). Exposed molar pulps of male Wistar rats capped with S3A or MTA were histologically examined. ResultsS3A extract did not inhibit proliferation of MDPs. Set S3A and MTA exhibited attachment of MDPs on their surface. S3A extract showed significantly higher mineralized nodule formation and mRNA expression of Bmp2, Oc, and Opn than did MTA extract. SBF-dipped S3A exhibited formation of surface precipitates, which were composed of Ca, P, Sr, and Al. Direct pulp capping with S3A and with MTA induced mineralized tissue repair of the exposed pulp. ConclusionS3A possesses biocompatibility and pro-mineralization effects comparable to those of MTA.

Expression of BMP-4 in dentigerous cyst and ameloblastoma: Is it a differentiation measure?

This study aimed to determine the expression of Bone Morphogenic Protein-4 (BMP-4) in dentigerous cyst (DC), unicystic-ameloblastoma (UA), and Multicysticameloblastoma (MA), and assess whether this marker can be a differentiation measure. This study included 30 DC, 30 UA, and 30 MA blocks if the histopathologic diagnosis of the lesion was definitive, the clinical information and medical records were complete, and the microscopic slides and the paraffin block were available. Age, gender, and location of the lesion were recorded. The samples were analyzed after the immunohistochemical staining (Envision technique). BMP-4 marker was evaluated and reported using Intensity Score (IS), Proportional Score (PS), and Total score (TS). The data were analyzed using SPSS version 21.0. Kruskal-Wallis and Mann-Whitney U tests were applied at the significance level of 0.05. In this study, DCs, UA, and MA had a significant tendency to occur in males compared to females (p<0.001, p<0.001, and p<0.001 respectively), and in the mandible compared to the maxilla (p=0.02, p=0.024, and p=0.02 respectively). The epithelial IS was significantly different among three lesions (p<0.001). IS was higher in MA than UA and DC (p<0.001 and p=0.006, respectively). The IS was not significantly different among the three lesions in connective tissue and around micro-vessels (p=0.3 and p=0.26 respectively). The PS in the epithelium and connective tissue of DC, UA, and MA had no statistical difference (p=0.549 and p=0.540 respectively). The epithelial TS was statistically different among DC, UA, and MA (p<0.001). The TS was higher in UA than MA and DC (p=0.004 and p<0.001 respectively). The expression of BMP-4 in the epithelium was higher in ameloblastoma compared to DCs. BMP-4 is a potential measure to differentiate different types of ameloblastoma and dentigerous cyst. The differentiation of these lesions is important as the right treatment plan changes according to the diagnosis.

Dual regulatory actions of LncBMP4 on BMP4 promote chicken primordial germ cell formation.

The unique characteristics of chicken primordial germ cells (PGCs) provide potential strategies for transgenic animal generation; however, insufficient PGC availability has limited their application. Regulation of bone morphogenic protein 4 (BMP4), a crucial factor for PGCs formation, may provide new strategies for PGC generation. We here identify a long noncoding RNA (lncRNA) that targets BMP4 (LncBMP4). LncBMP4 has similar functions as BMP4, in that it facilitates the formation and migration of PGCs. LncBMP4 promotes BMP4 expression by adsorbing the miRNA gga-mir-12211, thus reducing its inhibitory effect on BMP4 expression. In addition, the small peptide EPC5 encoded by LncBMP4 promotes the transcription of BMP4. The competing endogenous RNA (ceRNA) effect of LncBMP4 requires N6-methyladenosine (m6A) modification, in a dose-dependent manner, and high levels of m6A modification hinder EPC5 translation. Understanding the molecular mechanisms through which LncBMP4 promotes BMP4 expression during PGC formation may provide new avenues for efficient PGC generation.

Effects of sclerostin antibody on bone healing.

Promoting bone healing after a fracture has been a frequent subject of research. Recently, sclerostin antibody (Scl-Ab) has been introduced as a new anabolic agent for the treatment of osteoporosis. Scl-Ab activates the canonical Wnt (cWnt)-β-catenin pathway, leading to an increase in bone formation and decrease in bone resorption. Because of its rich osteogenic effects, preclinically, Scl-Ab has shown positive effects on bone healing in rodent models; researchers have reported an increase in bone mass, mechanical strength, histological bone formation, total mineralized callus volume, bone mineral density, neovascularization, proliferating cell nuclear antigen score, and bone morphogenic protein expression at the fracture site after Scl-Ab administration. In addition, in a rat critical-size femoral-defect model, the Scl-Ab-treated group demonstrated a higher bone healing rate. On the other hand, two clinical reports have researched Scl-Ab in bone healing and failed to show positive effects in the femur and tibia. This review discusses why Scl-Ab appears to be effective in animal models of fracture healing and not in clinical cases.

Bone morphogenetic protein-7 inhibits endothelial-to-mesenchymal transition in primary human umbilical vein endothelial cells and mouse model of systemic sclerosis via Akt/mTOR/p70S6K pathway

BackgroundSystemic sclerosis (SSc) is an autoimmune inflammatory and vascular disorder that causes tissue fibrosis of the skin and internal organs. Endothelial-to-mesenchymal transition (EndoMT) has been considered an important mechanism in the pathogenesis of vascular remodeling in SSc. Recent studies suggested that bone morphogenic protein 7 (BMP-7) has anti-fibrotic effects in several fibrotic diseases. ObjectivesTo investigate the mechanism of BMP-7 in inhibiting TGF-β-induced EndoMT in systemic sclerosis (SSc). MethodsSkin tissues of both healthy controls and SSc patients were detected the distribution of BMP-7. TGF-β was applied to induce the EndoMT model of human umbilical vein endothelial cells (HUVECs), and bleomycin was used to established the SSc mouse model. After treatment of BMP-7, the protein levels of endothelial specific markers, mesenchymal cell products, transcription factors and Akt signal pathway were examined by western blotting, immunofluorescence or immunohistochemistry both in vivo and in vitro. ResultsThe expression of BMP-7 was decreased in the basal layer of epidermis and dermis of SSc patients. EndoMT in TGF-β-treated HUVECs and skins of SSc mouse model were markedly attenuated after treatment with rh-BMP-7. Moreover, Akt/mTOR/p70S6K phosphorylation was involved in EndoMT and BMP-7 suppressed TGF-β- or bleomycin-induced theses phosphorylation in HUVECs or SSc mouse model. ConclusionBMP-7 reduced the production of TGF-β-induced EndoMT in HUVECs and SSc mouse model through Akt/mTOR/p70S6K signaling pathway. These findings suggested that BMP-7 could be employed as a promising antifibrotic therapy for SSc.

Efficacy of Photobiomodulation and Vitamin D on Odontogenic Activity of Human Dental Pulp Stem Cells.

Introduction: The regeneration of dental pulp tissue using human dental pulp stem cells (HDPSCs) has attracted increasing attention in recent years. Recent studies have suggested that several factors such as photobiomodulation (PBM) and vitamin D affect the proliferation and differentiation of HDPSCs. Therefore, the present study evaluated the effects of PBM and vitamin D on odontogenic differentiation of HDPSCs for dentin -like tissue formation. Methods: HDPSCs were collected, isolated, and characterized and then divided into six groups: group I, control; group II, vitamin D (10-7 Mol); group III, irradiation at 1 J/cm2 of 810 nm diode laser; group IV, irradiation at 1 J/cm2 and culture with vitamin D; group V, irradiation at 2 J/cm2, and group VI, irradiation at 2 J/cm2 and culture with vitamin D, cell viability assay was measured through MTT. Alkaline phosphatase (ALP) enzyme activity and mRNA levels of vascular endothelial growth factor (VEGF), bone morphogenic protein-2 (BMP-2), and dentin sialophosphoprotein (DSPP) were also assessed. Results: PBM at 1 and 2 J/cm2 combined with vitamin D significantly promoted HDPSCs proliferation through MTT assay and odontogenic differentiation through gene expression of VEGF, BMP-2, and DSPP levels (P < 0.0001). Conclusion: PBM at 2 J/cm2 combined with vitamin D enhanced the HDPSCs proliferation and odontogenic differentiation and thus could be a novel strategy for dentin regeneration in dentistry.

1194-P: Promotive Effects of Recombinant Human Adiponectin on Bone Regeneration by Paracrine

Nonunion and delayed fracture healing remains a great challenge in nowadays clinical practice. Bone and fat hold a close relationship to remain balanced through hormones and cytokines. Herein, we provided a facile and efficient method to obtain high purity and high yield recombinant human adiponectin (ADPN). The biocompatibility and the pharmaceutical behaviors were evaluated in the Sprague Dawley rats. In rat tibia fracture model, significantly accelerated bone healing was observed; the indices of osteogenesis enhanced at the fracture site, but not in blood. The underlying mechanism was primarily assessed and the expression of bone morphogenic protein 2 (BMP-2) and the adiponectin receptor 1 (AdipoR1) showed markedly increase at the fracture site with linear relationship. Cell experiments confirmed that the expression of BMP-2 and osteogenesis markers increased obviously in ADPN treatment groups; but reduced significantly when the expression of AdipoR1 was inhibited by SiRNA; and the differentiation of osteoblasts and calcification were significantly delayed after AdipoR1 SiRNA. Our study provided a feasible and efficacious way for bone fracture treatment with local administration of ADPN. Disclosure Y. Gong: None. Funding Military Logistics Research Projects of China (CWS13J054); Translational Medicine Projects of the Chinese People’s Liberation Army General Hospital (2017TM-021); Youth Training Program of Military Medical Science and Technology (20QNPY103)

Positive Association of Leptin and Artery Calcification of Lower Extremity in Patients With Type 2 Diabetes Mellitus: A Pilot Study.

ObjectiveWe aimed to explore the role and possible mechanism of leptin in lower-extremity artery calcification in patients with type 2 diabetes mellitus (T2DM).MethodsWe recruited 59 male patients with T2DM and 39 non-diabetic male participants. All participants underwent computed tomography scan of lower-extremity arteries. The calcification scores (CSs) were analyzed by standardized software. Plasma leptin level was determined by radioimmunoassay kits. Human vascular smooth muscle cells (VSMCs) calcification model was established by beta-glycerophosphate and calcium chlorideinduction. Calcium deposition and mineralization were measured by the o-cresolphthalein complexone method and Alizarin Red staining. The mRNA expression of bone morphogenic protein 2 (BMP2), runt-related transcription factor 2 (Runx2), osteocalcin (OCN) and osteopontin (OPN) was determined by quantitative RT-PCR. The protein levels of BMP2, Runx2, α-smooth muscle actin (α-SMA) and (p)-Akt was determined by Western-blot analysis, and α-SMA was also measured by immunofluorescence analysis.ResultsCompared with controls, patients with T2DM showed higher median calcification score in lower-extremity artery [286.50 (IQR 83.41, 1082.00) vs 68.66 (3.41, 141.30), p<0.01]. Plasma leptin level was higher in patients with calcification score ≥300 than ≥100 (252.67 ± 98.57 vs 189.38 ± 44.19 pg/ml, p<0.05). Compared with calcification medium, intracellular calcium content was significantly increased in VSMCs treated by leptin (200, 400 and 800 ng/ml) combined with calcification medium [11.99 ± 3.63, 15.18 ± 4.55, and 24.14 ± 5.85 mg/ml, respectively, vs 7.27 ± 1.54 mg/ml, all p<0.01]. Compared with calcification medium, Alizarin Red staining showed calcium disposition was more obvious, and the mRNA level of BMP2, Runx2 and OCN was significantly increased, and immunofluorescence and Western blot analysis showed that the expression of α-SMA was downregulated in VSMCs treated by leptin (400 ng/ml) combined with calcification medium, respectively. Compared with calcification medium, the protein level of BMP2 and Runx2 was upregulated in VSMCs treated by leptin (400 ng/ml) combined with calcification medium. Moreover, blocking PI3K/Akt signaling pathway can decrease the protein expression of BMP2 and Runx2 in VSMCs treated by leptin (400 ng/ml) combined with calcification medium.ConclusionsLeptin promoted lower-extremity artery calcification of T2DM by upregulating the expression of BMP2 and Runx2, and regulating phenotypic switch of VSMCs via PI3K/Akt signaling pathway.

Epigallocatechin-3-Gallate Promotes Osteo-/Odontogenic Differentiation of Stem Cells from the Apical Papilla through Activating the BMP-Smad Signaling Pathway.

Stem cells from apical papilla (SCAPs) are desirable sources of dentin regeneration. Epigallocatechin-3-gallate (EGCG), a natural component of green tea, shows potential in promoting the osteogenic differentiation of bone mesenchymal stem cells. However, whether EGCG regulates the odontogenic differentiation of SCAPs and how this occurs remain unknown. SCAPs from immature human third molars (16–20 years, n = 5) were treated with a medium containing different concentrations of EGCG or bone morphogenic protein 2 (BMP2), with or without LDN193189 (an inhibitor of the canonical BMP pathway). Cell proliferation and migration were analyzed using a CCK-8 assay and wound-healing assay, respectively. Osteo-/odontogenic differentiation was evaluated via alkaline phosphatase staining, alizarin red S staining, and the expression of osteo-/odontogenic markers using qPCR and Western blotting. We found that EGCG (1 or 10 μM) promoted the proliferation of SCAPs, increased alkaline phosphatase activity and mineral deposition, and upregulated the expression of osteo-/odontogenic markers including dentin sialophosphoprotein (Dspp), dentin matrix protein-1 (Dmp-1), bone sialoprotein (Bsp), and Type I collagen (Col1), along with the elevated expression of BMP2 and phosphorylation level of Smad1/5/9 (p < 0.01). EGCG at concentrations below 10 μM had no significant influence on cell migration. Moreover, EGCG-induced osteo-/odontogenic differentiation was significantly attenuated via LDN193189 treatment (p < 0.01). Furthermore, EGCG showed the ability to promote mineralization comparable with that of recombinant BMP2. Our study demonstrated that EGCG promotes the osteo-/odontogenic differentiation of SCAPs through the BMP–Smad signaling pathway.

Small Molecule–Mediated Enhanced Osteogenesis of Human Mesenchymal Stem Cells: a Probable Alternate for BMP-2

The use of bone morphogenic protein-2 (BMP-2) to accelerate the healing of bone defects has resulted in side effects such as ectopic bone formation, soft tissue swelling, and cyst-like bone void formation. An equipotential and a cost-effective replacement for BMP-2 is therefore required. In this study, different combinations of small molecules like FK506 (F), an immunosuppressant; purmorphamine (P), a hedgehog agonist; and Ku-0063794 (K), an mTOR inhibitor, were evaluated on human mesenchymal stem cells (hMSCs) to assess the osteoinduction behavior. Dose optimization and various combinations of small molecules were evaluated using MTT and ALP assay. The gene and protein expression of osteogenic markers RunX2, Osteopontin (OPN), and collagen type 1 on treatment with small molecules was evaluated and compared with BMP-2 expression. The confirmation of the osteogenic potential of small molecules was calcium deposits formed in hMSCs after staining with Alizarin Red S. Optimum concentration of individual small molecule (F 5 nM, K 10 nM, and P 2 μM) was determined based on the MTT and ALP activity. Small molecules (F + K + P) with osteogenic media (OS) showed 1.5-fold higher ALP activity with respect to OS. The synergistic effect of F + K + P and BMP-2/OS demonstrated an upregulation of RUNX2, OPN, and collagen 1 proteins and genes. Small molecules alone (F- 5 nM, K- 10 nM, and P- 2 μM) expressed the formation of calcium ions stained by Alizarin Red S which confirmed their osteoinductive effect. Thus, small molecules may have the potential to substitute or enable the use of reduced dose of BMP-2 for enhanced osteogenesis of progenitor cells. The use of Bone Morphogenic Protein-2 (BMP-2) to accelerate the healing of bone defects has resulted in side effects such as ectopic bone formation, soft tissue swelling, and cyst-like bone void formation. We show that cost-effective small molecules, in combination with osteogenic differentiation cocktail, have the potential to substitute BMP-2 for enhanced osteogenesis of progenitor cells. Graphical abstract

Peri-Implantitis Risk in Patients Taking Statins and Antihypertensives

Managing modifiable risk factors, such as cholesterol and blood pressure, is the conventional strategy to combat heart disease, the number 1 cause of death in the United States. Medications for lowering cholesterol and blood pressure have also been shown to play a role in bone metabolism. As such, these medications are of potential interest to the osseointegration and health of dental implants. Given the widespread use of these medications, it is imperative to understand their role in enhancing or deteriorating the health of dental implants. The most common class of cholesterol-lowering medications (known as statins) lower serum cholesterol by inhibiting the rate-limiting enzyme in the hepatic cholesterol biosynthesis pathway. Animal studies have shown statins to increase osteogenesis around dental implants, which has been explained by increased expression of bone morphogenic protein-2 and osteoblast differentiation. The applicability of these results to dental implant health in humans, however, remains unclear.1 Many pharmacologic mechanisms exist for lowering blood pressure, and as such, antihypertensives can affect bone physiology in different ways. For example, thiazide diuretics increase renal calcium absorption, beta adrenergic blockers inhibit osteoclasts, and drugs targeting the renin-angiotensin-aldosterone system block the bone resorptive activity of angiotensin II. These mechanisms have been used to explain an association between decreased implant failure and antihypertensive medications.2 This study aims to elucidate how cholesterol-lowering and antihypertensive medications affect long-term peri-implant health. A retrospective cohort study was performed to evaluate implants placed between 2006 and 2013. Medical and dental encounter notes were used to determine which medications a patient had been prescribed. Peri-implantitis was defined as radiographic evidence of bone loss of osseointegrated implants in conjunction with inflammatory signs, such as bleeding on probing, peri-implant probing depth of 5 or more millimeters, with or without suppuration. A minimum follow-up period of 5 years after placement was necessary to be included in the analysis. Of the 1362 implants placed in 398 patients, 881 implants in 284 unique patients met the inclusion criteria. Descriptive statistics were computed using the SAS system (SAS Institute Version 9.4, 2002-2012, Cary, NC). The predictive variables were prescriptions for cholesterol-lowering and antihypertensive medications. The primary outcome variable was peri-implantitis, with P < .05 used to define statistical significance. Of the implants that met the inclusion criteria, 456 (52%) were placed in patients’ prescribed statins. Risk of developing peri-implantitis was 77% higher in patients’ prescribed statins (OR: 1.772; 95% CL 1.285-2.445) compared to non-users (P = .0005). Peri-implantitis risk was not associated with overall antihypertensive medications or specific classes of antihypertensives, including angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, beta adrenergic blockers, calcium channel blockers, diuretics, or clonidine. Peri-implantitis risk was also not associated with other cholesterol-lowering medications, fibrates, or ezetimibe. The results from this retrospective cohort study reveal an increased risk of peri-implantitis in patients taking statins. This finding is clinically significant because there is substantial overlap between patients in need of dental implants and patients taking statins for cardiovascular protection, as evidenced by the numbers in our study. Further studies are needed to expound this association and establish recommendations for increasing long-term implant success in patients taking statins.

Bone morphogenic protein signaling in spinal cord injury.

Spinal cord injury (SCI) is a debilitating injury that results from traumatic or non-traumatic insults to the spinal cord, causing significant impairment of the patient’s activity and quality of life. Bone morphogenic proteins (BMPs) are a group of polyfunctional cytokines belonging to the transforming growth factor beta superfamily that regulates a wide variety of cellular functions in healthy and disease states. Recent studies suggest that dysregulation of BMP signaling is involved in neuronal demyelination and death after traumatic SCI. The focus of this article is to describe our current understanding of the role of BMP signaling in the regulation of cell fate, proliferation, apoptosis, autophagy, and inflammation in traumatic SCI. First, we will describe the expression of BMPs and pattern of BMP signaling before and after traumatic SCI in rodent models and in vitro. Next, we will discuss the role of BMP in the regulation of neuronal and glial cell differentiation, survival, functional recovery from traumatic SCI, and the gap in knowledge in this area that requires further investigation to improve SCI prognosis.