iMSC-mediated delivery of ACVR2B-Fc fusion protein reduces heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease caused by a gain-of-function mutation in ACVR1, which is a bone morphogenetic protein (BMP) type I receptor. Moreover, it causes progressive heterotopic ossification (HO) in connective tissues. Using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) and mouse models, we elucidated the underlying mechanisms of FOP pathogenesis and identified a candidate drug for FOP. METHODS: In the current study, healthy mesenchymal stem/stromal cells derived from iPSCs (iMSCs) expressing ACVR2B-Fc (iMSCACVR2B-Fc), which is a neutralizing receptobody, were constructed. Furthermore, patient-derived iMSCs and FOP mouse model (ACVR1R206H, female) were used to confirm the inhibitory function of ACVR2B-Fc fusion protein secreted by iMSCACVR2B-Fc on BMP signaling pathways and HO development, respectively. RESULTS: We found that secreted ACVR2B-Fc attenuated BMP signaling initiated by Activin-A and BMP-9 in both iMSCs and FOP-iMSCs in vitro. Transplantation of ACVR2B-Fc-expressing iMSCs reduced primary HO in a transgenic mouse model of FOP. Notably, a local injection of ACVR2B-Fc-expressing iMSCs and not an intraperitoneal injection improved the treadmill performance, suggesting compound effects of ACVR2B-Fc and iMSCs. CONCLUSIONS: These results offer a new perspective for treating FOP through stem cell therapy.

Calcium phosphate ceramics combined with rhBMP6 within autologous blood coagulum promote posterolateral lumbar fusion in sheep.

Posterolateral spinal fusion (PLF) is a procedure used for the treatment of degenerative spine disease. In this study we evaluated Osteogrow-C, a novel osteoinductive device comprised of recombinant human Bone morphogenetic protein 6 (rhBMP6) dispersed in autologous blood coagulum with synthetic ceramic particles, in the sheep PLF model. Osteogrow-C implants containing 74-420 or 1000-1700 µm ceramic particles (TCP/HA 80/20) were implanted between L4-L5 transverse processes in sheep (Ovis Aries, Merinolaandschaf breed). In the first experiment (n = 9 sheep; rhBMP6 dose 800 µg) the follow-up period was 27 weeks while in the second experiment (n = 12 sheep; rhBMP6 dose 500 µg) spinal fusion was assessed by in vivo CT after 9 weeks and at the end of the experiment after 14 (n = 6 sheep) and 40 (n = 6 sheep) weeks. Methods of evaluation included microCT, histological analyses and biomechanical testing. Osteogrow-C implants containing both 74-420 and 1000-1700 µm ceramic particles induced radiographic solid fusion 9 weeks following implantation. Ex-vivo microCT and histological analyses revealed complete osseointegration of newly formed bone with adjacent transverse processes. Biomechanical testing confirmed that fusion between transverse processes was complete and successful. Osteogrow-C implants induced spinal fusion in sheep PLF model and therefore represent a novel therapeutic solution for patients with degenerative disc disease.

Novel Indolyl-Benzimidazole Compounds Promote in vitro Wound Healing and Osteogenic Differentiation of Pluripotent Cells.

BACKGROUND: Increasing or restoring Bone Morphogenetic Protein- (BMP-) signaling through administration of recombinant BMPs (rBMPs) has demonstrated therapeutic efficacy for treating bone fractures or to enhance repair following spinal surgeries. However, direct use of rBMPs has come up against significant obstacles like high cost and incidence of adverse effects. Recently, we reported our findings on the novel indolyl-benzimidazoles, SY-LB-35 and SY-LB-57, that fully activated BMP receptor signaling demonstrating activity profiles that mirrored rBMPs. Here, we explored the potential of these compounds to substitute for rBMPs in processes like wound healing and osteogenesis. METHODS: Cell-based assays including cell viability, short- and long-term phosphorylation, protein expression, wound healing and bone differentiation assays were carried out in the pluripotent myoblast C2C12 cell line with select assays performed in multiple cell lines. Several assays included conditions in the presence of a selective inhibitor of type I BMP receptor, Activin-like kinase 2 (ALK2), or inhibitors of BMP-stimulated downstream signaling. All assays were repeated at least 3 times with replicates per condition where indicated. Statistical tests were carried out using Student's two-tailed, t-test. RESULTS: Sustained activation of non-canonical BMP signaling pathways was observed after 24-hour exposure to SY-LB-35 and SY-LB-57. Moreover, this treatment increased the expression of targets of BMP-mediated transcription such as the Id1 transcription factor. SY-LB-35 and SY-LB-57 promoted substantial increases in cell viability in three distinct cell types and increased the rate of wound closure in scrape-wounded C2C12 cell cultures. Cell viability and wound closure induced by SY-LB compounds required ALK2-, PI3K- and p38-dependent pathways. In contrast, responses to SY-LB compounds were not affected by ERK inhibition. Expression of bone differentiation markers beginning at 4 hours and evidence of calcium deposition detected after 21 days in C2C12 cell cultures exposed to SY-LB-35 and SY-LB-57 demonstrated the osteogenic potential of these compounds. CONCLUSIONS: The functional similarities between these novel compounds and rBMPs indicates that SY-LB-35 or SY-LB-57, acting as potent activators of BMP receptor signaling and inducers of osteogenic processes, could potentially replace rBMPs for treating BMP-related pathologies such as bone fracture repair or other wound healing processes.

Hydrolyzed Collagen Tripeptide Promotes Longitudinal Bone Growth in Childhood Rats via Increases in Insulin-Like Growth Factor-1 and Bone Morphogenetic Proteins.

Previous studies have reported that collagen tripeptide (CTP) derived from collagen hydrolysate has various beneficial effects on health by protecting against skin aging and improving bone formation and cartilage regeneration. Collagen-Tripep20TM (CTP20), which is a low-molecular-weight CTP derived from fish skin, contains a bioactive CTP, Gly-Pro-Hyp >3.2% with a tripeptide content >20%. Herein, we investigated the osteogenic effects and mechanisms of CTP20 (<500 Da) on MG-63 osteoblast-like cells and SW1353 chondrocytes. And we measured promoting ratio of the longitudinal bone growth in childhood rats. First, CTP20 at 100 µg/mL elevated the proliferation (15.0% and 28.2%), alkaline phosphatase activity (29.3% and 32.0%), collagen synthesis (1.25- and 1.14-fold), and calcium deposition (1.18- and 1.15-fold) in MG-63 cells and SW1353, respectively. In addition, we found that CTP20 could promote the longitudinal growth and height of the growth plate of the tibia in childhood rats. CTP20 enhanced the protein expression of insulin-like growth factor-1 (IGF-1) in MG-63 and SW1353 cells, and in the growth plate of childhood rats, along with Janus Kinase 2, and signal transducer and activator of transcription 5 activation in MG-63 and SW1353 cells. CTP20 also elevated the expression levels of bone morphogenetic proteins (BMPs) in MG-63 and SW1353 cells and in the growth plates of childhood rats. These results indicate that CTP20 may promote the endochondral ossification and longitudinal bone growth, through enhancing of IGF-1 and BMPs. (Clinical Trial Registration number: smecae 19-09-01).

Enhanced Osteogenic Potential of Noggin Knockout C2C12 Cells on BMP-2 Releasing Silk Scaffolds.

The CRISPR/Cas9 mechanism offers promising therapeutic approaches for bone regeneration by stimulating or suppressing critical signaling pathways. In this study, we aimed to increase the activity of BMP-2 signaling through knockout of Noggin, thereby establishing a synergistic effect on the osteogenic activity of cells in the presence of BMP-2. Since Noggin is an antagonist expressed in skeletal tissues and binds to subunits of bone morphogenetic proteins (BMPs) to inhibit osteogenic differentiation, here Noggin expression was knocked out using the CRISPR/Cas9 system. In accordance with this purpose, C2C12 (mouse myoblast) cells were transfected with CRISPR/Cas9 plasmids. Transfection was achieved with Lipofectamine and confirmed with intense fluorescent signals in microscopic images and deletion in target sequence in Sanger sequencing analysis. Thus, Noggin knockout cells were identified as a new cell source for tissue engineering studies. Then, the transfected cells were seeded on highly porous silk scaffolds bearing BMP-2-loaded silk nanoparticles (30 ng BMP-2/mg silk nanoparticle) in the size of 288 ± 62 nm. BMP-2 is released from the scaffolds in a controlled manner for up to 60 days. The knockout of Noggin by CRISPR/Cas9 was found to synergistically promote osteogenic differentiation in the presence of BMP-2 through increased Coll1A1 and Ocn expression and mineralization. Gene editing of Noggin and BMP-2 increased almost 2-fold Col1A1 expression and almost 3-fold Ocn expression compared to the control group. Moreover, transfected cells produced extracellular matrix (ECM) containing collagen fibers on the scaffolds and mineral-like structures were formed on the fibers. In addition, mineralization characterized by intense Alizarin red staining was detected in transfected cells cultured in the presence of BMP-2, while the other groups did not exhibit any mineralized areas. As has been demonstrated in this study, the CRISPR/Cas9 mechanism has great potential for obtaining new cell sources to be used in tissue engineering studies.

Therapeutic Potential of Plant Metabolites in Bone Apoptosis: A Review.

Osteoporosis is one of the skeletal diseases of major health concern worldwide. Homeostasis of bone occurs with the help of cells, namely, osteoblasts and osteoclasts. Physiological and pathological conditions involve the death of the cells by apoptosis, autophagy, and necrosis. Apoptosis is a key factor in the growth, development, and maintenance of the skeleton. Apoptosis is generated by two pathways: the intrinsic (mitochondria) and extrinsic (death receptor) pathways. Osteoblast apoptosis is governed by the factors like B cell lymphoma 2 (Bcl-2) family proteins, extracellular signal-regulated kinase (ERK), mitogen-activated protein kinases (MAPK), phosphoinositide- 3-kinase/ protein kinase B (PI3-K/Akt), Janus kinase 2 (JAK2), bone morphogenetic protein (BMP), and bone matrix protein. Cytokines interact with osteocytes and induce apoptosis. A pro-inflammatory signal stimulates osteocyte apoptosis and increases osteocyte cytokines production. Current therapies have adverse effects which limit their applications. Various plant metabolites have shown beneficial effects on bone. The present review converses about normal bone metabolism and the mechanism of apoptosis leading to bone deterioration. Furthermore, it discusses the role of plant metabolites on bone apoptosis with related indications of efficacy in various experimental models.

Novel bone Morphogenetic Protein (BMP)-2/4 Consensus Peptide (BCP) for the Osteogenic Differentiation of C2C12 Cells.

BACKGROUND: Despite the promising clinical potential of bone morphogenetic protein (BMP)-related therapies for bone formation, their side effects warrant the need for alternative therapeutic peptides. BMP family members can aid in bone repair; however, peptides derived from BMP2/ 4 have not yet been investigated. METHODS: In this study, three candidates BMP2/4 consensus peptide (BCP) 1, 2, and 3 were identified and their ability to induce osteogenesis in C2C12 cells was analyzed. First, an alkaline phosphatase (ALP) staining assay was performed to evaluate the osteogenic effects of BCPs. Next, the effects of BCPs on RNA expression levels and protein abundances of osteogenic markers were explored. Furthermore, the transcriptional activity of ALP by BCP1 and in silico molecular docking model on BMP type IA receptor (BRIA) were performed. RESULTS: BCP1-3 induced higher RUNX2 expression than BMP2. Interestingly, among them, BCP1 significantly promoted osteoblast differentiation more than BMP2 in ALP staining with no cytotoxicity. BCP1 significantly induced the osteoblast markers, and the highest RUNX2 expression was observed at 100 ng/mL compared to other concentrations. In transfection experiments, BCP1 stimulated osteoblast differentiation via RUNX2 activation and the Smad signaling pathway. Finally, in silico molecular docking suggested the possible binding sites of BCP1 on BRIA. CONCLUSION: These results show that BCP1 promotes osteogenicity in C2C12 cells. This study suggests that BCP1 is the most promising candidate peptide to replace BMP2 for osteoblast differentiation.

BMP-induced non-canonical signaling is upregulated during autophagy-mediated regeneration in inflamed mesothelial cells.

Previously, we showed that after Freund's adjuvant-induced peritonitis, rat mesothelial cells regain their epithelial phenotype through mesenchymal-epithelial transition (MET) accompanied by autophagy. Since bone morphogenetic proteins (BMPs) are well-known MET-inducers, we were interested in the potential expression of BMPs and BMP-induced pathways. Although mesothelial cells expressed lower amounts of BMP7, its level in the peritoneal cavity and mesothelial synthesis of BMP4 were significantly increased during inflammation. BMPR1A and BMPR2 were also significantly expressed. Expression of transforming growth factor beta-activated kinase (TAK1) and c-Jun NH2-terminal kinases (JNK1-JNK2) were more intense than that of phosphorylated Mothers Against Decapentaplegic homolog 1/5 (p-SMAD1/5), confirming that the non-canonical pathway of BMPs prevailed in our model. JNK signaling through B-cell lymphoma-2 (Bcl-2) can contribute to Beclin-1 activation. We demonstrated that TAK1-JNK-Bcl-2 signaling was upregulated simultaneously with the autophagy-mediated regeneration. A further goal of our study was to prove the regenerative role of autophagy after inflammation. We used a specific inhibitor, bafilomycin A1 (BafA1), and found that BafA1 treatment decreased the expression of microtubule-associated protein 1A/1B-light chain 3 (LC3B) and resulted in morphological signs of cell death in inflamed mesothelial cells indicating that if autophagy is arrested, regeneration turns into cell death and consequently, mesothelial cells die.

GDF11 as a friend or an enemy in the cancer biology?

The Growth and Differential Factor 11 (GDF11) is a recently discovered representative of Transforming Growth Factor ß superfamily. The highest expression of GDF11 is detected in the nervous system, bladder, seminal vesicles and muscles whereas the lowest in the testis, liver or breast. GDF11 role in physiology is still not clear. GDF11 is a crucial factor in embryogenesis, cell cycle control and apoptosis, inasmuch it mainly targets cell retain stemness features, managing to the cell differentiation and the maturation. GDF11 is entangled in lipid metabolism, inflammatory processes and aging. GDF11 is strongly related to carcinogenesis and its expression in tumors is intruded. GDF11 can promote cancer growth in the colon or inhibit the cell proliferation in breast cancer. The aberrated expression is probably allied with the impaired maturation. In this article we summarized an impact of GDF11 on the tumor cells and review the all attitudes connecting GDF11 with carcinogenesis.

The method of sinus node-like pacemaker cells from human induced pluripotent stem cells by BMP and Wnt signaling.

The embryonic development of sinus nodes (SAN) is co-regulated by multiple signaling pathways. Among these, the bone morphogenetic protein (BMP) and Wnt signaling pathways are involved in the development of SAN. In this study, the effects of BMP and Wnt signaling on the differentiation of SAN-like pacemaker cells (SANLPCs) were investigated. Human induced pluripotent stem cells (hiPSCs) were divided into four groups: control, BMP4, CHIR-3, and BMP4 + CHIR (CHIR: a Wnt signaling activator). The samples were tested at day (D) 15 of differentiation. The final protocol for the activation of BMP signaling at D0-D3 and reactivation of Wnt signaling at D5-D7 in the differentiation of hiPSCs were determined. The results showed that the mRNA levels of pacemaker markers (TBX18, SHOX2, TBX3, HCN4, and HCN1) were higher in the BMP4 + CHIR group than in the control group, and working myocardial genes were downregulated. The immunofluorescence assay revealed that the expression of SHOX2 and HCN4 increased in the BMP4 + CHIR group compared to that in the other groups. In addition, the results of patch clamps revealed that a funny current of higher density and typical SAN action potentials were recorded, except in the control group, in which the L-type calcium current was higher in the BMP4 + CHIR group than in the other groups. Finally, the proportion of SANLPCs (cTnT+ NKX2.5-) was further enhanced by the combination of BMP4 and CHIR treatment. In summary, the combination of BMP and Wnt signaling promotes the differentiation of SANLPCs from hiPSCs.

Bioactivity, Molecular Mechanism, and Targeted Delivery of Flavonoids for Bone Loss.

Skeletal disabilities are a prominent burden on the present population with an increasing life span. Advances in osteopathy have provided various medical support for bone-related diseases, including pharmacological and prosthesis interventions. However, therapeutics and post-surgery complications are often reported due to side effects associated with modern-day therapies. Thus, therapies utilizing natural means with fewer toxic or other side effects are the key to acceptable interventions. Flavonoids constitute a class of bioactive compounds found in dietary supplements, and their pharmacological attributes have been well appreciated. Recently, flavonoids' role is gaining renowned interest for its effect on bone remodeling. A wide range of flavonoids has been found to play a pivotal role in the major bone signaling pathways, such as wingless-related integration site (Wnt)/ß-catenin, bone morphogenetic protein (BMP)/transforming growth factor (TGF)-ß, mitogen-activated protein kinase (MAPK), etc. However, the reduced bioavailability and the absorption of flavonoids are the major limitations inhibiting their use against bone-related complications. Recent utilization of nanotechnological approaches and other delivery methods (biomaterial scaffolds, micelles) to target and control release can enhance the absorption and bioavailability of flavonoids. Thus, we have tried to recapitulate the understanding of the role of flavonoids in regulating signaling mechanisms affecting bone remodeling and various delivery methods utilized to enhance their therapeutical potential in treating bone loss.

Why Some Mice Are Smarter than Others: The Impact of Bone Morphogenetic Protein Signaling on Cognition.

Inbred mice (C57Bl/6) display wide variability in performance on hippocampal-dependent cognitive tasks. Examination of microdissected dentate gyrus (DG) after cognitive testing showed a highly significant negative correlation between levels of bone morphogenetic protein (BMP) signaling and recognition memory. Cognitive performance decline during the aging process, and the degree of cognitive decline is strongly correlated with aging-related increases in BMP signaling. Further, cognitive performance was impaired when the BMP inhibitor, noggin, was knocked down in the DG. Infusion of noggin into the lateral ventricles enhanced DG-dependent cognition while BMP4 infusion led to significant impairments. Embryonic overexpression of noggin resulted in lifelong enhancement of recognition and spatial memory while overexpression of BMP4 resulted in lifelong impairment, substantiating the importance of differences in BMP signaling in wild-type mice. These findings indicate that performance in DG-dependent cognitive tasks is largely determined by differences in levels BMP signaling in the dentate gyrus.

Bone morphogenetic protein pathway responses and alterations of osteogenesis in metastatic prostate cancers.

BACKGROUND: Prostate cancer is a common cancer in men that annually results in more than 33 000 US deaths. Mortality from prostate cancer is largely from metastatic disease, reflecting on the great strides in the last century of treatments in care for the localized disease. Metastatic castrate resistant prostate cancer (mCRPC) will commonly travel to the bone, creating unique bone pathology that requires nuanced treatments in those sites with surgical, radio and chemotherapeutic interventions. The bone morphogenetic protein (BMP) pathway has been historically studied in the capacity to regulate the osteogenic nature of new bone. New mineralized bone generation is a frequent and common observation in mCRPC and referred to as blastic bone lesions. Less common are bone destructive lesions that are termed lytic. METHODS: We queried the cancer genome atlas (TCGA) prostate cancer databases for the expression of the BMP pathway and found that distinct gene expression of the ligands, soluble antagonists, receptors, and intracellular mediators were altered in localized versus metastatic disease. Human prostate cancer cell lines have an innate ability to promote blastic- or lytic-like bone lesions and we hypothesized that inhibiting BMP signaling in these cell lines would result in a distinct change in osteogenesis gene expression with BMP inhibition. RESULTS: We found unique and common changes by comparing these cell lines response and unique BMP pathway alterations. We treated human PCa cell lines with distinct bone pathologic phenotypes with the BMP inhibitor DMH1 and found distinct osteogenesis responses. We analyzed distinct sites of metastatic PCa in the TCGA and found that BMP signaling was selectively altered in commons sites such as lymph node, bone and liver compared to primary tumors. CONCLUSIONS: Overall we conclude that BMPs in metastatic prostate cancer are important signals and functional mediators of diverse processes that have potential for individualized precision oncology in mCRPC.

CircNUFIP2 overexpression induces GDF11 to ameliorate oxygen-glucose deprivation-induced hippocampal neuron cell apoptosis and oxidative stress after cerebral ischemia.

Background Previous data have indicated the regulation of circular RNA (circRNA) toward cerebral ischemia. This study aims to reveal the effects of circNUFIP2 on cerebral ischemia and the underlying mechanism. Methods Oxygen-glucose deprivation (OGD) hippocampal neuron (HT22) cell model and middle cerebral artery occlusion (MCAO) mouse model were used for this study. The expression of circRNA nuclear FMR1 interacting protein 2 (circNUFIP2), microRNA-1224-5p (miR-1224-5p) and growth differentiation factor 11 (GDF11) was detected by quantitative real-time polymerase-chain reaction. Protein expression was checked by Western blotting. The binding relationships among circNUFIP2, miR-1224-5p and GDF11 were identified by dual-luciferase reporter assay, RNA pull-down assay, and RNA immunoprecipitation assay. Cell proliferation and apoptosis were investigated by 5-Ethynyl-29-deoxyuridine and flow cytometry analysis, respectively. Results CircNUFIP2 and GDF11 expression were decreased, but miR-1224-5p was increased in OGD-treated HT22 cells when compared with their expression in control groups. OGD treatment inhibited HT22 cell proliferation but induced cell apoptosis and oxidative stress; however, these effects were attenuated after circNUFIP2 overexpression. Also, circNUFIP2 upregulation assuaged the cerebral infarction of MCAO mice. Besides, circNUFIP2 bound to miR-1224-5p and mediated OGD-induced HT22 cell damage through miR-1224-5p. Meanwhile, knockdown of GDF11, a target gene of miR-1224-5p, relieved miR-1224-5p depletion-caused effects in OGD-treated HT22 cells. Furthermore, circNUFIP2 regulated GDF11 expression by interacting with miR-1224-5p. Conclusion CircNUFIP2 overexpression protected neuron cells against cerebral ischemia-induced damage, at least in part, by the miR-1224-5p/GDF11 pathway, providing a possible target for the therapy of cerebral ischemic stroke.

Spermidine inhibits high glucose-induced endoplasmic reticulum stress in HT22 cells by upregulation of growth differentiation factor 11.

Hyperglycemia-induced neuronal endoplasmic reticulum (ER) stress is particularly important for the pathogenesis of diabetic encephalopathy. Spermidine (Spd) has neuroprotection in several nervous system diseases. Our current study to explore the potential protective role of Spd in hyperglycemia-induced neuronal ER stress and the underlying mechanisms. HT22 cells were treated with high glucose (HG) to establish an in-vitro model of hyperglycemia toxicity. The HT22 cells' activity was tested by cell counting kit-8 assay. RNA interference technology was used to silence the expression of growth differentiation factor 11 (GDF11) in HT22 cells. The GDF11 expression levels of mRNA were assessed using reverse transcription-PCR (RT-PCR). Western blotting analysis was applied to evaluate the expressions of GRP78 and cleaved caspase-12. Spd markedly abolished HG-exerted decline in cell viability as well as upregulations of GRP78 and cleaved caspase-12 in HT22 cells, indicating the protection of Spd against HG-induced neurotoxicity and ER stress. Furthermore, we showed that Spd upregulated the expression of GDF11 in HG-exposed HT22 cells. While, silenced GDF11 expression by RNA interference reversed the protective effects of Spd on HG-elicited neurotoxicity and ER stress in HT22 cells. These results indicated that Spd prevents HG-induced neurotoxicity and ER stress through upregulation of GDF11. Our findings identify Spd as a potential treatment for diabetic encephalopathy as well as ER stress-related neurologic diseases.

Effects of Soy Milk in Conjunction With Resistance Training on Physical Performance and Skeletal Muscle Regulatory Markers in Older Men.

Purpose: We aimed to determine the effects of 12 weeks of soy milk consumption combined with resistance training (RT) on body composition, physical performance, and skeletal muscle regulatory markers in older men. Methods: In this randomized clinical trial study, 60 healthy elderly men (age = 65.63 ± 3.16 years) were randomly assigned to four groups: resistance training (RT; n = 15), soy milk consumption (SMC; n = 15), resistance training + soy milk (RSM; n = 15), and control (CON; n = 15) groups. The study was double-blind for the soy milk/placebo. Participants in RT and RSM groups performed resistance training (3 times/week) for 12 weeks. Participants in the SMC and RSM groups consumed 240 mL of soy milk daily. Body composition [body mass (BM), body fat percent (BFP), waist-hip ratio (WHR), and fat mass (FM)], physical performance [upper body strength (UBS), lower body strength (LBS), VO2max, upper anaerobic power, lower anaerobic power, and handgrip strength], and serum markers [follistatin, myostatin, myostatin-follistatin ratio (MFR), and growth and differentiation factor 11 (GDF11)] were evaluated before and after interventions. Results: All 3 interventions significantly (p < 0.05) increased serum follistatin concentrations (RT = 1.7%, SMC = 2.9%, RSM = 7.8%) and decreased serum myostatin (RT = -1.3% SMC = -5.4%, RSM = -0.5%) and GDF11 concentrations (RT = -1.4%, SMC = -1.4%, RSM = -9.0%), and MFR (RT = -2.6%, SMC = -3.2%, RSM = -12%). In addition, we observed significant reduction in all 3 intervention groups in BFP (RT = -3.6%, SMC = -1.4%, RSM = -6.0%), WHR (RT = -2.2%, SMC = -2.1%, RSM = -4.3%), and FM (RT = -9.6%, SMC = -3.8%, RSM = -11.0%). Moreover, results found significant increase only in RT and RSM groups for muscle mass (RT = 3.8% and RSM = 11.8%), UBS (RT = 10.9% and RSM = 21.8%), LBS (RT = 4.3% and RSM = 7.8%), upper anaerobic power (RT = 7.8% and RSM = 10.3%), and lower anaerobic power (RT = 4.6% and RSM = 8.9%). Handgrip strength were significantly increased in all 3 intervention groups (RT = 7.0%, SMC = 6.9%, RSM = 43.0%). VO2max significantly increased only in RSM (1.7%) after 12 weeks of intervention. Additionally, significant differences were observed between the changes for all variables in the RSM group compared to RT, SMC, and CON groups (p < 0.05). Conclusions: There were synergistic effects of soy milk and RT for skeletal muscle regulatory markers, body composition, and physical performance. Results of the present study support the importance of soy milk in conjunction with RT for older men.

Follistatin dysregulation impaired trophoblast biological functions by GDF11-Smad2/3 axis in preeclampsia placentas.

INTRODUCTION: Preeclampsia (PE) is one of the main causes of maternal, fetal, and neonatal mortality. So far, the underlying mechanism of this pregnancy-specific syndrome remains unelucidated. The expression of Follistatin (FST) decreased in maternal serum (especially early onset severe PE) and placental trophoblasts of PE patients. However, whether FST-deficiency in preeclamptic placentas alters trophoblast function remains to be determined. METHODS: Trophoblast cell lines were cultured in vitro and LV3 short hairpin RNA (shRNA) was used to silence FST. Growth and differentiation factor 11 (GDF11) expression level in placentas and serum were detected by immunohistochemistry and enzyme-linked immune-sorbent assay, respectively. To verify the effect of reduced FST expression on trophoblasts, microRNA-24-3p, which was predicted to target the 3'-untranslated region (3'-UTR) of FST, was screened out, and miR-24-3p mimic, inhibitor was used to regulate FST expression in trophoblasts. RESULTS: Downregulation of FST significantly enhanced the apoptosis and impaired migration and invasion of trophoblast. Reduced FST caused the upregulation of GDF11 in trophoblasts. Interestingly, GDF11 reduced in preeclamptic placental microvascular endothelial cells. Dysregulation of FST-GDF11-Smad2/3 signaling pathway, leading to increased apoptosis of trophoblast. Expression levels of miR-24-3p, was significantly elevated in preeclamptic placentas. Trophoblast cells transfected with miR-24-3p mimics displayed impaired migration and invasion and increased apoptosis. Treated by miR-24-3p inhibitor, trophoblast cells exhibited rescued function. DISCUSSION: FST-deficiency impaired trophoblast function by upregulating GDF11 levels in trophoblasts. The regulation of FST-GDF11-Smad2/3 axis by microRNAs mimic or inhibitor may be critical to trophoblast function regulation and helps to deepen our understanding of the molecular mechanism of PE.

Bone morphogenetic protein 10 alleviates doxorubicin-induced cardiac injury via signal transducer and activator of transcription 3 signaling pathway.

Doxorubicin (DOX) has limited antitumor applications owing to its association with life-threatening cardiac injury. Oxidative damage and cardiac apoptosis are crucial in DOX-induced cardiac injury. Bone morphogenetic protein 10 (BMP10) is predominantly distributed in the heart and acts as a cardioprotective factor that preserves cardiac function. However, the role of BMP10 in DOX-induced cardiac injury has not yet been explored. The current study aimed to examine the function and mechanism of action of BMP10 in DOX-induced cardiac injury. An adeno-associated viral system was used for the overexpression or silencing of cardiac-specific BMP10, and subsequently, a single dose of DOX was intraperitoneally injected to induce cardiac injury. Results showed that DOX exposure decreased BMP10 expression in the heart. Cardiac-specific overexpression of BMP10 alleviated the oxidative stress and apoptosis and improved cardiac function. Conversely, cardiac-specific silencing of BMP10 aggravated the redox disorder and apoptosis and worsened the cardiac dysfunction caused by DOX. Exogenous BMP10 supplementation amelioratesd the DOX-induced cardiac contractile dysfunction. Mechanistically, we found that phosphorylation of signal transducer and activator of transcription 3 (STAT3) is reduced in DOX-induced cardiotoxicity, and, BMP10 activated impaired STAT3 via a non-canonical pathway. BMP10 lost its cardioprotective function in cardiomyocyte-specific STAT3 knockout (STAT3-cKO) mice. Based on our findings, we suggested that BMP10 is a potential therapeutic agent against DOX-induced cardiac injury and that the cardioprotective effects of BMP10 are dependent on the activation of STAT3.

Circ_0008365 Suppresses Apoptosis, Inflammation and Extracellular Matrix Degradation of IL-1ß-treated Chondrocytes in Osteoarthritis by Regulating miR-324-5p/BMPR2/NF-κB Signaling Axis.

BACKGROUND: Recent studies have revealed that circular RNAs (circRNAs) play crucial roles in the progression of osteoarthritis (OA). This study aimed to investigate the biological function and regulatory mechanism of circ_0008365 in OA. METHODS: OA cell model in vitro was established in chondrocytes by treatment with Interleukin-1ß (IL-1ß). The levels of inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The expression levels of circ_0008365, microRNA-324-5p (miR-324-5p) and bone morphogenetic protein type 2 receptor (BMPR2) were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was assessed using flow cytometry and caspase3 activity assays. The protein expression was determined via a western blot assay. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP) and RNA pull-down assays were used to analyze the correlation between targets. RESULTS: IL-1ß level and miR-324-5p expression were increased, while circ_0008365 was downregulated in OA patients. IL-1ß treatment-induced cell apoptosis, inflammation and extracellular matrix (ECM) degradation in chondrocytes. Besides, circ_0008365 overexpression partly relieved IL-1ß-induced cell damage in chondrocytes. Circ_0008365 could interact with miR-324-5p, and BMPR2 was a downstream target of miR-324-5p. Overexpression of miR-324-5p or BMPR2 knockdown partly overturned the inhibiting effect of circ_0008365 on cell damage in IL-1ß-induced chondrocytes. In addition, circ_0008365 inactivated NF-κB pathway via regulating miR-324-5p/BMPR2 axis. CONCLUSION: Circ_0008365 reduced IL-1ß-induced cell damage in chondrocytes via inactivating NF-κB signaling pathway and regulating miR-324-5p/BMPR2 axis.Abbreviations OA: osteoarthritis; BMPR2: bone morphogenetic protein type 2 receptor.

Repurposing MDZ as a tool for tissue regeneration in dental cells.

BACKGROUND: Several recent studies have focused on the utility of drug repurposing to expand clinical application of approved therapeutics. Here, we investigate the efficacy of midazolam (MDZ) and cytokines for regenerating calcified tissue, using immortalized porcine dental pulp (PPU7) and mouse skeletal muscle derived myoblast (C2C12) cells, with the goal of repurposing MDZ as a new treatment to facilitate calcified tissue regeneration. HIGHLIGHTS: We noted that PPU7 and C2C12 cells cultured with various MDZ regimens displayed increased bone morphogenic protein (BMP-2), transforming growth factor beta (TGF-ß), and alkaline phosphatase activity. These increases were highest in PPU7 cells cultured with MDZ alone, and in C2C12 cells cultured with MDZ and BMP-2. PPU7 cells cultured under these conditions demonstrated markedly elevated expression of odontoblastic gene markers, indicating their likely differentiation into odontoblasts. Expression levels of osteoblastic gene markers also increased in C2C12 cells, suggesting that MDZ potentiates the effect of BMP-2, inducing osteoblast differentiation in these cells. Newly formed calcified deposits in both PPU7 and C2C12 cells were identified as hydroxyapatite via crystallographic and crystal engineering analyses. CONCLUSION: MDZ increases ALP activity, inducing expression of specific marker genes for both odontoblasts and osteoblasts while promoting hydroxyapatite production in both PPU7 and C2C12 cells. These responses were cell type specific. MDZ treatment alone could induce these changes in PPU7 cells, but C2C12 cell differentiation required BMP-2 addition.