The association between CD31hiEmcnhi endothelial cells and bone mineral density in Chinese women.

Osteoporosis is the most common bone disease in humans. During bone remodeling, specialized blood vessels influenced by the endothelial cells (CD31hiEmcnhi, also called type H cells) are formatted to supply nutrients. Reductions in vascular supply are associated with bone loss resulting in osteoporosis. Therefore, the objective of the present study was to explore the association between the CD31hiEmcnhi endothelial cells and bone mineral density (BMD). In this prospective study, 134 Chinese women were enrolled and examined. BMD was measured by DEXA method while the percentage of CD31hiEmcnhi endothelial cells in the intertrochanteric part was measured by flow cytometry. The percentage of CD31hiEmcnhi endothelial cells in postmenopausal subjects was significantly lower compared with premenopausal women (8.7 ± 4.0% vs 13.2 ± 5.6%, P < 0.01). Meanwhile, the CD31hiEmcnhi endothelial cell levels in osteopenia and osteoporosis were significantly lower compared with subjects with normal BMD (9.84 ± 4.2% in osteopenia and 7.11 ± 3.2% in osteoporosis vs 12.7 ± 5.6% in subjects with normal T score, P < 0.01). Multiple regression analyses showed that the CD31hiEmcnhi endothelial cells level was positively associated with femur neck and total hip BMD, but not with lumbar BMD. Our study suggests a significantly positive association between CD31hiEmcnhi endothelial cells and local BMD in Chinese women. The proportion of CD31hiEmcnhi endothelial cells is a marker of bone quality and represents a potential target for treatment of bone loss.

PCLAF induces bone marrow adipocyte senescence and contributes to skeletal aging.

Bone marrow adipocytes (BMAds) affect bone homeostasis, but the mechanism remains unclear. Here, we showed that exercise inhibited PCNA clamp-associated factor (PCLAF) secretion from the bone marrow macrophages to inhibit BMAds senescence and thus alleviated skeletal aging. The genetic deletion of PCLAF in macrophages inhibited BMAds senescence and delayed skeletal aging. In contrast, the transplantation of PCLAF-mediated senescent BMAds into the bone marrow of healthy mice suppressed bone turnover. Mechanistically, PCLAF bound to the ADGRL2 receptor to inhibit AKT/mTOR signaling that triggered BMAds senescence and subsequently spread senescence among osteogenic and osteoclastic cells. Of note, we developed a PCLAF-neutralizing antibody and showed its therapeutic effects on skeletal health in old mice. Together, these findings identify PCLAF as an inducer of BMAds senescence and provide a promising way to treat age-related osteoporosis.

Mechanosensitive protein polycystin-1 promotes periosteal stem/progenitor cells osteochondral differentiation in fracture healing.

Background: Mechanical forces are indispensable for bone healing, disruption of which is recognized as a contributing cause to nonunion or delayed union. However, the underlying mechanism of mechanical regulation of fracture healing is elusive. Methods: We used the lineage-tracing mouse model, conditional knockout depletion mouse model, hindlimb unloading model and single-cell RNA sequencing to analyze the crucial roles of mechanosensitive protein polycystin-1 (PC1, Pkd1) promotes periosteal stem/progenitor cells (PSPCs) osteochondral differentiation in fracture healing. Results: Our results showed that cathepsin (Ctsk)-positive PSPCs are fracture-responsive and mechanosensitive and can differentiate into osteoblasts and chondrocytes during fracture repair. We found that polycystin-1 declines markedly in PSPCs with mechanical unloading while increasing in response to mechanical stimulus. Mice with conditional depletion of Pkd1 in Ctsk+ PSPCs show impaired osteochondrogenesis, reduced cortical bone formation, delayed fracture healing, and diminished responsiveness to mechanical unloading. Mechanistically, PC1 facilitates nuclear translocation of transcriptional coactivator TAZ via PC1 C-terminal tail cleavage, enhancing osteochondral differentiation potential of PSPCs. Pharmacological intervention of the PC1-TAZ axis and promotion of TAZ nuclear translocation using Zinc01442821 enhances fracture healing and alleviates delayed union or nonunion induced by mechanical unloading. Conclusion: Our study reveals that Ctsk+ PSPCs within the callus can sense mechanical forces through the PC1-TAZ axis, targeting which represents great therapeutic potential for delayed fracture union or nonunion.

A Runx2/miR-3960/miR-2861 regulatory feedback loop during mouse osteoblast differentiation.

Our recent study showed that miR-2861 promotes osteoblast differentiation by targeting histone deacetylase 5, resulting in increased runt-related transcription factor 2 (Runx2) protein production. Here we identified another new microRNA (miRNA) (miR-3960) that played a regulatory role in osteoblast differentiation through a regulatory feedback loop with miR-2861. miR-3960 and miR-2861 were found clustered at the same loci. miR-3960 was transcribed during bone morphogenic protein 2 (BMP2)-induced osteogenesis of ST2 stromal cells. Overexpression of miR-3960 promoted BMP2-induced osteoblastogenesis. However, the inhibition of miR-3960 expression attenuated the osteoblastogenesis. Homeobox A2 (Hoxa2), a repressor of Runx2 expression, was confirmed to be a target of miR-3960. Electrophoretic mobility shift assay and chromatin immunoprecipitation experiments confirmed that Runx2 bound to the promoter of the miR-3960/miR-2861 cluster. Furthermore, overexpression of Runx2 induced miR-3960/miR-2861 transcription, and block of Runx2 expression attenuated BMP2-induced miR-3960/miR-2861 transcription. Here we report that miR-3960 and miR-2861, transcribed together from the same miRNA polycistron, both function in osteoblast differentiation through a novel Runx2/miR-3960/miR-2861 regulatory feedback loop. Our findings provide new insights into the roles of miRNAs in osteoblast differentiation.

Parathyroid hormone alleviates non-alcoholic liver steatosis via activating the hepatic cAMP/PKA/CREB pathway.

Non-alcoholic fatty liver disease (NAFLD), hallmarked by liver steatosis, is becoming a global concern, but effective and safe drugs for NAFLD are still lacking at present. Parathyroid hormone (PTH), the only FDA-approved anabolic treatment for osteoporosis, is important in calcium-phosphate homeostasis. However, little is known about its potential therapeutic effects on other diseases. Here, we report that intermittent administration of PTH ameliorated non-alcoholic liver steatosis in diet-induced obese (DIO) mice and db/db mice, as well as fasting-induced hepatic steatosis. In vitro, PTH inhibits palmitic acid-induced intracellular lipid accumulation in a parathyroid hormone 1 receptor (PTH1R)-dependent manner. Mechanistically, PTH upregulates the expression of genes involved in lipid ß-oxidation and suppresses the expression of genes related to lipid uptake and de novo lipogenesis by activating the cAMP/PKA/CREB pathway. Taken together, our current finding proposes a new therapeutic role of PTH on NAFLD.

UBE2E3 regulates cellular senescence and osteogenic differentiation of BMSCs during aging.

BACKGROUND: Osteoporosis has gradually become a public health problem in the world. However, the exact molecular mechanism of osteoporosis still remains unclear. Senescence and osteogenic differentiation inhibition of bone marrow mesenchymal stem cells (BMSCs ) are supposed to play an important part in osteoporosis. METHODS: We used two gene expression profiles (GSE35956 and GSE35958) associated with osteoporosis and selected the promising gene Ubiquitin-conjugating enzyme E2 E3 (UBE2E3). We then verified its function and mechanism by in vitro experiments. RESULTS: UBE2E3 was highly expressed in the bone marrow and positively associated with osteogenesis related genes. Besides, UBE2E3 expression reduced in old BMSCs compared with that in young BMSCs. In in vitro experiments, knockdown of UBE2E3 accelerated cellular senescence and inhibited osteogenic differentiation of young BMSCs. On the other hand, overexpression of UBE2E3 attenuated cellular senescence as well as enhanced osteogenic differentiation of old BMSCs. Mechanistically, UBE2E3 might regulate the nuclear factor erythroid 2-related factor (Nrf2) and control its function, thus affecting the senescence and osteogenic differentiation of BMSCs. CONCLUSION: UBE2E3 may be potentially involved in the pathogenesis of osteoporosis by regulating cellular senescence and osteogenic differentiation of BMSCs.

Scara3 regulates bone marrow mesenchymal stem cell fate switch between osteoblasts and adipocytes by promoting Foxo1.

OBJECTIVES: Scavenger receptor class A, member 3 (Scara3) was involved in adipogenesis. However, the effect of Scara3 on the switch between osteogenesis and adipogenesis of bone marrow mesenchymal stem cells (BMSCs) remains elusive. MATERIALS AND METHODS: The correlations between SCARA3 with the osteogenic-related were analysed based on the GTEx database. The effects of Scara3 on osteogenic or adipogenic differentiation of BMSCs were evaluated by qPCR, Western blot (WB) and cell staining. The mechanisms of Scara3 regulating Foxo1 and autophagy were validated by co-expression analysis, WB and immunofluorescence. In vivo, Scara3 adeno-associated virus was injected into intra-bone marrow of the aged mice and ovariectomized (OVX) mice whose phenotypes were confirmed by micro-CT, calcein double labelling and immunochemistry (HE and OCN staining). RESULTS: SCARA3 was positively correlated with osteogenic-related genes. Scara3 expression gradually decreased during adipogenesis but increased during osteogenesis. Moreover, the deletion of Scara3 favoured adipogenesis over osteogenesis, whereas overexpression of Scara3 significantly enhanced the osteogenesis at the expense of adipogenesis. Mechanistically, Scara3 controlled the cell fate by promoting Foxo1 expression and autophagy flux. In vivo, Scara3 promoted bone formation and reduced bone marrow fat accumulation in OVX mice. In the aged mice, Scara3 overexpression alleviated bone loss as well. CONCLUSIONS: This study suggested that Scara3 regulated the switch between adipocyte and osteoblast differentiation, which represented a potential therapeutic target for bone loss and osteoporosis.

Dendrobium officinale polysaccharides regulate age-related lineage commitment between osteogenic and adipogenic differentiation.

OBJECTIVES: Excessive oxidative stress and diminished antioxidant defences could contribute to age-related tissue damage and various diseases including age-related osteoporosis. Dendrobium officinale polysaccharides (DOPs), a major ingredient from a traditional Chinese medicine, have a great potential of antioxidative activity. In this study, we explore the role of DOP in age-related osteoporosis that remains elusive. MATERIALS AND METHODS: Oxidative stimulation and DOP were used to treat bone marrow mesenchymal stem cells (BMSCs), whose lineage commitment was measured by adipogenic- and osteoblastic-induced differentiation analysis. The oxidative stress and antioxidant capacity of BMSCs under the treatment of DOP were analysed by the level of MDA, SOD. Related mechanism studies were confirmed by qRT-PCR, Western blotting and siRNA transfection. DOP was orally administrated in aged mice whose phenotype was confirmed by micro-CT, immunofluorescence, immunochemistry and calcein double-labelling analysis. RESULTS: Dendrobium officinale polysaccharide treatment markedly increased osteogenic differentiation of BMSCs, while inhibiting adipogenic differentiation. In vitro, DOP could rescue H2O2-induced switch of BMSCs differentiation fate. However, this effect was abolished in BMSCs when interfered with Nrf2 siRNA. Furthermore, administration of DOP to aged mice significantly increased the bone mass and reduced the marrow adipose tissue (MAT) accompanied with decreased oxidative stress of BMSCs. CONCLUSIONS: Our study reveals that DOP can attenuate bone loss and MAT accumulation through NRF2 antioxidant signalling, which may represent as potential therapeutic agent for age-related osteoporosis.

Preservation of type H vessels and osteoblasts by enhanced preosteoclast platelet-derived growth factor type BB attenuates glucocorticoid-induced osteoporosis in growing mice.

Survival of chronic diseases in childhood is often achieved utilizing glucocorticoids, but comes with significant side effects, including glucocorticoid-induced osteoporosis (GIO). Knowledge of the mechanism of GIO is limited to the adult skeleton. We explored the effect of genetic loss and inhibition of cathepsin K (Ctsk) as a potential treatment target in a young GIO mouse model as genetic loss of cathepsin K results in a mild form of osteopetrosis secondary to impaired osteoclast bone resorption with maintenance of bone formation. We first characterized the temporal osteoclast and osteoblast progenitor populations in Ctsk-/- and wild type (WT) mice in the primary and secondary spongiosa, as sites representative of trabecular bone modeling and remodeling, respectively. In the primary spongiosa, Ctsk-/- mice had decreased numbers of osteoclasts at young ages (2 and 4 weeks) and increased osteoblast lineage cells at later age (8 weeks) relative to WT littermates. In the secondary spongiosa, Ctsk-/- mice had greater numbers of osteoclasts and osteoblast lineage cells relative to WT littermates. We next developed a young GIO mouse model with prednisolone 10 mg/m2/day injected intraperitoneally daily from 2 through 6 weeks of age. Overall, WT-prednisolone mice had lower bone volume per tissue volume, whereas Ctsk-/--prednisolone mice maintained a similar bone volume relative to Ctsk-/--vehicle controls. WT-prednisolone mice exhibited a decreased number of osteoclasts, tartrate-resistant acid phosphatase and platelet-derived growth factor type BB (PDGF-BB) co-positive cells, type H endothelial cells, and osteoblasts relative to WT-vehicle mice in both the primary and secondary spongiosa. Interestingly, Ctsk-/--prednisolone mice demonstrated a paradoxical response with increased numbers of all parameters in primary spongiosa and no change in secondary spongiosa. Finally, treatment with a cathepsin K inhibitor prevented WT-prednisolone decline in osteoclasts, osteoblasts, type H vessels, and bone volume. These data demonstrate that cells in the primary and secondary spongiosa respond differently to glucocorticoids and genetic manipulation. Inhibition of osteoclast resorption that preserves osteoclast coupling factors, such as through inhibition of cathepsin K, may be a potential preventive treatment strategy against GIO in the growing skeleton.

Low plasma PDGF-BB levels are associated with estradiol in postmenopausal osteoporosis: PDGF-BB mediated by estradiol in women.

Objective This study aimed to investigate the association between low plasma Platelet-derived growth factor-BB (PDGF-BB) levels and oestradiol in Postmenopausal osteoporosis (PMOP). Methods This prospective study measured plasma PDGF-BB and oestradiol levels in outpatients who were admitted to our hospital. Participants were screened and then allocated to three groups: normal young women, postmenopausal control, and PMOP. Additionally, Sprague-Dawley rats underwent either sham surgery or bilateral ovariectomy (OVX), and were divided into the following groups: sham, OVX, OVX + oestradiol, and OVX + PDGF-BB. Plasma oestradiol and PDGF-BB levels were measured using commercially available ELISA kits. Results A total of 121 participants, including 69 normal young women, 28 patients with primary PMOP, and 24 age-matched postmenopausal women were enrolled. Plasma oestradiol and PDGF-BB levels were lower in postmenopausal women, especially in PMOP ( P < 0.01). Pearson correlations analysis showed that PDGF-BB levels were positively correlated with oestradiol levels and inversely correlated with age ( P < 0.01). The OVX rat model showed that oestradiol replacement increased plasma PDGF-BB levels, while PDGF-BB systematic treatment had no effect on plasma oestradiol levels. Conclusions Plasma PDGF-BB levels are maintained by oestrogen in normal young women and play a major role in PMOP.

Runx2/miR-3960/miR-2861 Positive Feedback Loop Is Responsible for Osteogenic Transdifferentiation of Vascular Smooth Muscle Cells.

We previously reported that Runx2/miR-3960/miR-2861 regulatory feedback loop stimulates osteoblast differentiation. However, the effect of this feedback loop on the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) remains unclear. Our recent study showed that miR-2861 and miR-3960 expression increases significantly during ß-glycerophosphate-induced osteogenic transdifferentiation of VSMCs. Overexpression of miR-2861 or miR-3960 in VSMCs enhances ß-glycerophosphate-induced osteoblastogenesis, whereas inhibition of miR-2861 or miR-3960 expression attenuates it. MiR-2861 or miR-3960 promotes osteogenic transdifferentiation of VSMCs by targeting histone deacetylase 5 or Homeobox A2, respectively, resulting in increased runt-related transcription factor 2 (Runx2) protein production. Furthermore, overexpression of Runx2 induces miR-2861 and miR-3960 transcription, and knockdown of Runx2 attenuates ß-glycerophosphate-induced miR-2861 and miR-3960 transcription in VSMCs. Thus, our data show that Runx2/miR-3960/miR-2861 positive feedback loop plays an important role in osteogenic transdifferentiation of VSMCs and contributes to vascular calcification.

MicroRNA-188 regulates age-related switch between osteoblast and adipocyte differentiation.

Bone marrow mesenchymal stem cells (BMSCs) exhibit an age-dependent reduction in osteogenesis that is accompanied by an increased propensity toward adipocyte differentiation. This switch increases adipocyte numbers and decreases the number of osteoblasts, contributing to age-related bone loss. Here, we found that the level of microRNA-188 (miR-188) is markedly higher in BMSCs from aged compared with young mice and humans. Compared with control mice, animals lacking miR-188 showed a substantial reduction of age-associated bone loss and fat accumulation in bone marrow. Conversely, mice with transgenic overexpression of miR-188 in osterix+ osteoprogenitors had greater age-associated bone loss and fat accumulation in bone marrow relative to WT mice. Moreover, using an aptamer delivery system, we found that BMSC-specific overexpression of miR-188 in mice reduced bone formation and increased bone marrow fat accumulation. We identified histone deacetylase 9 (HDAC9) and RPTOR-independent companion of MTOR complex 2 (RICTOR) as the direct targets of miR-188. Notably, BMSC-specific inhibition of miR-188 by intra-bone marrow injection of aptamer-antagomiR-188 increased bone formation and decreased bone marrow fat accumulation in aged mice. Together, our results indicate that miR-188 is a key regulator of the age-related switch between osteogenesis and adipogenesis of BMSCs and may represent a potential therapeutic target for age-related bone loss.

PDGF-BB secreted by preosteoclasts induces angiogenesis during coupling with osteogenesis.

Osteogenesis during bone modeling and remodeling is coupled with angiogenesis. A recent study showed that a specific vessel subtype, strongly positive for CD31 and endomucin (CD31(hi)Emcn(hi)), couples angiogenesis and osteogenesis. Here, we found that platelet-derived growth factor-BB (PDGF-BB) secreted by preosteoclasts induces CD31(hi)Emcn(hi) vessel formation during bone modeling and remodeling. Mice with depletion of PDGF-BB in the tartrate-resistant acid phosphatase-positive cell lineage show significantly lower trabecular and cortical bone mass, serum and bone marrow PDGF-BB concentrations, and fewer CD31(hi)Emcn(hi) vessels compared to wild-type mice. In the ovariectomy (OVX)-induced osteoporotic mouse model, serum and bone marrow levels of PDGF-BB and numbers of CD31(hi)Emcn(hi) vessels are significantly lower compared to sham-operated controls. Treatment with exogenous PDGF-BB or inhibition of cathepsin K to increase the number of preosteoclasts, and thus the endogenous levels of PDGF-BB, increases CD31(hi)Emcn(hi) vessel number and stimulates bone formation in OVX mice. Thus, pharmacotherapies that increase PDGF-BB secretion from preosteoclasts offer a new therapeutic target for treating osteoporosis by promoting angiogenesis and thus bone formation.

The role of microRNAs in adipocyte differentiation.

Adipocytes differentiate from mesenchymal stem cells (MSCs) in a process known as adipogenesis. The programme of adipogenesis is regulated by the sequential activation of transcription factors and several signaling pathways. There is growing evidence indicating that a class of small non-coding single-stranded RNAs known as "microRNAs (miRNAs)" also are involved in this process. In this review, we summarize the biology and functional mechanisms of miRNAs in adipocyte differentiation. In addition, we further discuss the miRNAs profiling, the miRNAs function and miRNAs target prediction in the adipogenesis.

MicroRNAs and their roles in osteoclast differentiation.

Osteoclasts, which are derived from monocyte-macrophage precursors, are exclusive bone resorption cells. Limited evidence indicates that a class of small non-coding single-stranded RNAs known as "microRNAs" (miRNAs) is also involved in bone resorption. Although various miRNAs regulate cell proliferation and differentiation, few miRNAs have been reported to play a key role in the regulation of osteoclast differentiation. In this short review, the biology and functional mechanisms of miRNAs in osteoclastogenesis are summarized. The profiling, function, and target prediction of miRNAs are discussed as well.