Young osteocyte-derived extracellular vesicles facilitate osteogenesis by transferring tropomyosin-1.

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) can undergo inadequate osteogenesis or excessive adipogenesis as they age due to changes in the bone microenvironment, ultimately resulting in decreased bone density and elevated risk of fractures in senile osteoporosis. This study aims to investigate the effects of osteocyte senescence on the bone microenvironment and its influence on BMSCs during aging. RESULTS: Primary osteocytes were isolated from 2-month-old and 16-month-old mice to obtain young osteocyte-derived extracellular vesicles (YO-EVs) and senescent osteocyte-derived EVs (SO-EVs), respectively. YO-EVs were found to significantly increase alkaline phosphatase activity, mineralization deposition, and the expression of osteogenesis-related genes in BMSCs, while SO-EVs promoted BMSC adipogenesis. Neither YO-EVs nor SO-EVs exerted an effect on the osteoclastogenesis of primary macrophages/monocytes. Our constructed transgenic mice, designed to trace osteocyte-derived EV distribution, revealed abundant osteocyte-derived EVs embedded in the bone matrix. Moreover, mature osteoclasts were found to release osteocyte-derived EVs from bone slices, playing a pivotal role in regulating the functions of the surrounding culture medium. Following intravenous injection into young and elderly mouse models, YO-EVs demonstrated a significant enhancement of bone mass and biomechanical strength compared to SO-EVs. Immunostaining of bone sections revealed that YO-EV treatment augmented the number of osteoblasts on the bone surface, while SO-EV treatment promoted adipocyte formation in the bone marrow. Proteomics analysis of YO-EVs and SO-EVs showed that tropomyosin-1 (TPM1) was enriched in YO-EVs, which increased the matrix stiffness of BMSCs, consequently promoting osteogenesis. Specifically, the siRNA-mediated depletion of Tpm1 eliminated pro-osteogenic activity of YO-EVs both in vitro and in vivo. CONCLUSIONS: Our findings suggested that YO-EVs played a crucial role in maintaining the balance between bone resorption and formation, and their pro-osteogenic activity declining with aging. Therefore, YO-EVs and the delivered TPM1 hold potential as therapeutic targets for senile osteoporosis.

Harmine targets inhibitor of DNA binding-2 and activator protein-1 to promote preosteoclast PDGF-BB production.

Osteoporosis is one of the most common metabolic bone diseases affecting millions of people. We previously found that harmine prevents bone loss in ovariectomized mice via increasing preosteoclast platelet-derived growth factor-BB (PDGF-BB) production and type H vessel formation. However, the molecular mechanisms by which harmine promotes preosteoclast PDGF-BB generation are still unclear. In this study, we revealed that inhibitor of DNA binding-2 (Id2) and activator protein-1 (AP-1) were important factors implicated in harmine-enhanced preosteoclast PDGF-BB production. Exposure of RANKL-induced Primary bone marrow macrophages (BMMs), isolated from tibiae and femora of mice, to harmine increased the protein levels of Id2 and AP-1. Knockdown of Id2 by Id2-siRNA reduced the number of preosteoclasts as well as secretion of PDGF-BB in RANKL-stimulated BMMs administrated with harmine. Inhibition of c-Fos or c-Jun (components of AP-1) both reversed the stimulatory effect of harmine on preosteoclast PDGF-BB production. Dual-luciferase reporter assay analyses determined that PDGF-BB was the direct target of AP-1 which was up-regulated by harmine treatment. In conclusion, our data demonstrated a novel mechanism involving in the production of PDGF-BB increased by harmine, which may provide potential therapeutic targets for bone loss diseases.

LncRNA CHRF promotes TGF-ß1 induced EMT in alveolar epithelial cells by inhibiting miR-146a up-regulating L1CAM expression.

PURPOSE: Idiopathic pulmonary fibrosis (IPF) is a type of progressive lung fibrosis disease. The survival time of diagnosed IPF patients is often only 2 years. Currently much evidence showed that the epithelial-mesenchymal transition (EMT) process is the main cause of the occurrence and development of IPF. LncRNA cardiac hypertrophy related factor (CHRF) was reported to be related with IPF development. Here we explored the functions and regulatory mechanisms of CHRF on EMT in IPF. MATERIALS AND METHODS: A549 cells were treated with transforming growth factor-ß1 (TGF-ß1) for 48 h to construct IPF cell model. CHRF and miR-146a expression were quantified using qPCR. The expression of L1 cell adhesion molecule (L1CAM) and EMT related indicators (E-cadherin, Vimentin, Slug and N-cadherin) were detected by qPCR and western blot. Dual luciferase reporter experiment was conducted to prove the molecular interaction of miR-146a and L1CAM, as well as CHRF and miR-146a. RESULTS: CHRF and L1CAM expression were significantly upregulated and promoted the EMT process in A549 after treatment of TGF-ß1. MiR-146a was obviously down-regulated, and knockdown of CHRF inhibited the EMT process by up-regulating miR-146a, in A549 after treatment of TGF-ß1. Meanwhile, overexpression of miR-146a inhibited EMT process via targeting L1CAM. In addition, L1CAM overexpression eliminated the inhibitory effect of sh-CHRF on the EMT process. CONCLUSIONS: These results provided evidence that CHRF promoted EMT process in A549 after treatment of TGF-ß1, which proposed a new insight for depth understanding the pathological mechanisms of IPF.

Rapid and sensitive detection of superoxide dismutase in serum of the cervical cancer by 4-aminothiophenol-functionalized bimetallic Au-Ag nanoboxs array.

Early, efficient and sensitive detection of serum markers in cervical cancer is very important for the treatment and prognosis to cervical cancer patients. In this paper, a SERS platform based on surface enhanced Raman scattering technology was proposed to quantitatively detect superoxide dismutase in serum of cervical cancer patients. Au-Ag nanoboxs array was made by oil-water interface self-assembly method as the trapping substrate. The single-layer Au-AgNBs array was verified by SERS for possessing excellent uniformity, selectivity and reproducibility. 4-aminothiophenol (4-ATP) was used as Raman signal molecule, it will be oxidized to dithiol azobenzene under the surface catalytic reaction with the condition of PH = 9 and laser irradiation. The quantitative detection of SOD could be achieved by calculating the change of characteristic peak ratio. When the concentration was from 10 U mL-1-160 U mL-1, the concentration of SOD could be accurately and quantitatively detected in human serum. The whole test was completed within 20 min and the limit of quantitation was 10 U mL-1. In addition, serum samples from the cervical cancer, the cervical intraepithelial neoplasia and healthy people were tested by the platform and the results were consistent with those of ELISA. The platform has great potential as a tool for early clinical screening of cervical cancer in the future.

Reassessing endothelial-to-mesenchymal transition in mouse bone marrow: insights from lineage tracing models.

Endothelial cells (ECs) and bone marrow stromal cells (BMSCs) play crucial roles in supporting hematopoiesis and hematopoietic regeneration. However, whether ECs are a source of BMSCs remains unclear. Here, we evaluate the contribution of endothelial-to-mesenchymal transition to BMSC generation in postnatal mice. Single-cell RNA sequencing identifies ECs expressing BMSC markers Prrx1 and Lepr; however, this could not be validated using Prrx1-Cre and Lepr-Cre transgenic mice. Additionally, only a minority of BMSCs are marked by EC lineage tracing models using Cdh5-rtTA-tetO-Cre or Tek-CreERT2. Moreover, Cdh5+ BMSCs and Tek+ BMSCs show distinct spatial distributions and characteristic mesenchymal markers, suggestive of their origination from different progenitors rather than CDH5+ TEK+ ECs. Furthermore, myeloablation induced by 5-fluorouracil treatment does not increase Cdh5+ BMSCs. Our findings indicate that ECs hardly convert to BMSCs during homeostasis and myeloablation-induced hematopoietic regeneration, highlighting the importance of using appropriate genetic models and conducting careful data interpretation in studies concerning endothelial-to-mesenchymal transition.

Aged bone matrix-derived extracellular vesicles as a messenger for calcification paradox.

Adipocyte differentiation of bone marrow mesenchymal stem/stromal cells (BMSCs) instead of osteoblast formation contributes to age- and menopause-related marrow adiposity and osteoporosis. Vascular calcification often occurs with osteoporosis, a contradictory association called "calcification paradox". Here we show that extracellular vesicles derived from aged bone matrix (AB-EVs) during bone resorption favor BMSC adipogenesis rather than osteogenesis and augment calcification of vascular smooth muscle cells. Intravenous or intramedullary injection of AB-EVs promotes bone-fat imbalance and exacerbates Vitamin D3 (VD3)-induced vascular calcification in young or old mice. Alendronate (ALE), a bone resorption inhibitor, down-regulates AB-EVs release and attenuates aging- and ovariectomy-induced bone-fat imbalance. In the VD3-treated aged mice, ALE suppresses the ovariectomy-induced aggravation of vascular calcification. MiR-483-5p and miR-2861 are enriched in AB-EVs and essential for the AB-EVs-induced bone-fat imbalance and exacerbation of vascular calcification. Our study uncovers the role of AB-EVs as a messenger for calcification paradox by transferring miR-483-5p and miR-2861.

Deficiency of Omentin-1 leads to delayed fracture healing through excessive inflammation and reduced CD31hiEmcnhi vessels.

Fracture healing is a complicated process affected by many factors, such as inflammatory responses and angiogenesis. Omentin-1 is an adipokine with anti-inflammatory properties, but whether omentin-1 affects the fracture healing process is still unknown. Here, by using global omentin-1 knockout (omentin-1-/-) mice, we demonstrated that omentin-1 deficiency resulted in delayed fracture healing in mice, accompanied by increased inflammation and osteoclast formation, and decreased production of platelet-derived growth factor-BB (PDGF-BB) and osteogenesis-promoting vessels that are strongly positive for CD31 and Endomucin (CD31hiEmcnhi) in the fracture area. In vitro, omentin-1 treatment suppressed the ability of the tumor necrosis factor-α (TNF-α)-activated macrophages to stimulate multi-nuclear osteoclast formation, resulting in a significant increase in the generation of mono-nuclear preosteoclasts and PDGF-BB, a pro-angiogenic protein that is abundantly secreted by preosteoclasts. PDGF-BB significantly augmented endothelial cell proliferation, tube formation and migration, whereas direct treatment with omentin-1 did not induce obvious effects on angiogenesis activities of endothelial cells. Our study suggests a positive role of omentin-1 in fracture healing, which may be associated with the inhibition of inflammation and stimulation of preosteoclast PDGF-BB-mediated promotion of CD31hiEmcnhi vessel formation.

Neuronal Induction of Bone-Fat Imbalance through Osteocyte Neuropeptide Y.

A differentiation switch of bone marrow mesenchymal stem/stromal cells (BMSCs) from osteoblasts to adipocytes contributes to age- and menopause-associated bone loss and marrow adiposity. Here it is found that osteocytes, the most abundant bone cells, promote adipogenesis and inhibit osteogenesis of BMSCs by secreting neuropeptide Y (NPY), whose expression increases with aging and osteoporosis. Deletion of NPY in osteocytes generates a high bone mass phenotype, and attenuates aging- and ovariectomy (OVX)-induced bone-fat imbalance in mice. Osteocyte NPY production is under the control of autonomic nervous system (ANS) and osteocyte NPY deletion blocks the ANS-induced regulation of BMSC fate and bone-fat balance. γ-Oryzanol, a clinically used ANS regulator, significantly increases bone formation and reverses aging- and OVX-induced osteocyte NPY overproduction and marrow adiposity in control mice, but not in mice lacking osteocyte NPY. The study suggests a new mode of neuronal control of bone metabolism through the ANS-induced regulation of osteocyte NPY.

Extracellular Vesicles from Child Gut Microbiota Enter into Bone to Preserve Bone Mass and Strength.

Recently, the gut microbiota (GM) has been shown to be a regulator of bone homeostasis and the mechanisms by which GM modulates bone mass are still being investigated. Here, it is found that colonization with GM from children (CGM) but not from the elderly (EGM) prevents decreases in bone mass and bone strength in conventionally raised, ovariectomy (OVX)-induced osteoporotic mice. 16S rRNA gene sequencing reveals that CGM reverses the OVX-induced reduction of Akkermansia muciniphila (Akk). Direct replenishment of Akk is sufficient to correct the OVX-induced imbalanced bone metabolism and protect against osteoporosis. Mechanistic studies show that the secretion of extracellular vesicles (EVs) is required for the CGM- and Akk-induced bone protective effects and these nanovesicles can enter and accumulate into bone tissues to attenuate the OVX-induced osteoporotic phenotypes by augmenting osteogenic activity and inhibiting osteoclast formation. The study identifies that gut bacterium Akk mediates the CGM-induced anti-osteoporotic effects and presents a novel mechanism underlying the exchange of signals between GM and host bone.

Exercise benefits cardiovascular health in hyperlipidemia rats correlating with changes of the cardiac vagus nerve.

The role of exercise training on hemodynamic parameters, blood lipid profiles, inflammatory cytokines, cholinesterase-positive nerves and muscarinic cholinergic (M(2)) receptors expression in the heart was investigated in Sprague-Dawley male rats with hyperlipidemia (HL). The rats were subjected to a high-fat diet and exercise training for 8 weeks, and then the hemodynamic parameters, the profiles of blood lipid and inflammatory cytokines, and the expression of cholinesterase-positive nerves and M(2) receptors were measured. HL rats displayed cardiac dysfunction, dysregulation of inflammatory cytokines, and decreased cholinesterase-positive nerves and M(2) receptors expression. The combination of hyperlipidemia with exercise training (AT) restored the profiles of blood lipids and the levels of inflammatory cytokines. In addition, AT and HL + AT improved cardiac function with increasing cholinesterase-positive nerves and M(2) receptors expression. Overall, these data show that the increased expression of cholinesterase-positive nerves and M(2) receptors in the heart is partially responsible for the benefits of exercise training on cardiac function in hyperlipidemia rats.

Ontogenetic characterization of sporangium and spore of Huperzia serrata: an anti-aging disease fern.

BACKGROUND: Huperzia serrata is a medicinal plant used in Traditional Chinese Medicine, which has been used to prevent against aging diseases. It is mainly propagated by spores and grows extremely slowly. Due to severe harvest, it is a highly endangered species. In this report, we characterize ontogenesis of sporangia and spores that are associated with propagation. A wild population of H. serrata plants is localized in western Hunan province, China and protected by Chinese Government to study its development (e.g. sporangia and spores) and ecology. Both field and microscopic observations were conducted for a few of years. RESULTS: The development of sporangia from their initiation to maturation took nearly 1 year. Microscopic observations showed that the sporangial walls were developed from epidermal cells via initiation, cell division, and maturation. The structure of the mature sporangial wall is composed of one layer of epidermis, two middle layers of cells, and one layer of tapetum. Therefore, the sporangium is the eusporangium type. Spore development is characterized into six stages, initiation from epidermal cell and formation of sporogenous cells, primary sporogenous cell, secondary sporogenous cell, spore mother cell, tetrad, and maturation. CONCLUSION: The sporangial development of H. serrata belongs to the eusporangium type. The development takes approximately 1 year period from the initiation to the maturation. These data are useful for improving propagation of this medicinal plant in the future.

Aerobic exercise inhibits sympathetic nerve sprouting and restores ß-adrenergic receptor balance in rats with myocardial infarction.

BACKGROUND: Cardiac sympathetic nerve sprouting and the dysregulation of ß-adrenergic receptor (ß-AR) play a critical role in the deterioration of cardiac function after myocardial infarction (MI). Growing evidence indicates that exercise provides protection against MI. The aims of this study were to investigate whether aerobic exercise following MI could inhibit sympathetic nerve sprouting and restore the balance of ß3-AR/ß1-AR. METHODS: Male Sprague-Dawley rats were divided into three groups: sham-operated control group (SC), MI group (MI), and MI with aerobic exercise group (ME). The rats in ME group were assigned to 8 weeks of exercise protocol (16 m/min, 50 min/d, 5 d/wk). The expression of nerve growth factor (NGF), the sympathetic nerve marker-tyrosine hydroxylase (TH), the nerve sprouting marker-growth associated protein 43 (GAP43), and ß1- and ß2-AR expression in the peri-infarct area of the left ventricle (LV) were measured by Western blot and immunohistochemistry, while ß3-AR expression was determined by Western blot and immunofluorescence. Endothelial nitric oxide synthase (NOS2), phospho-NOS2 (p-NOS2), and neuronal nitric oxide synthase (NOS1) were measured by Western blot. RESULTS: MI increased LV end-diastolic pressure (LVEDP), and decreased LV systolic pressure (LVSP). Compared with the MI group, aerobic exercise significantly decreased LVEDP and increased LVSP. The protein expression of TH, GAP43 and NGF was significantly increased after MI, which was normalized by exercise. Compared with the SC group, the ratios of ß2-AR/ß1-AR and ß3-AR/ß1-AR were elevated in the MI group, and the protein expression of ß3-AR and NOS1 increased after MI. Compared with the MI group, the ratios of ß2-AR/ß1-AR and ß3-AR/ß1-AR were normalized in the ME group, while the protein expression of ß3-AR and NOS1 significantly increased, and NOS2 was activated by exercise. CONCLUSIONS: Aerobic exercise inhibits cardiac sympathetic nerve sprouting, restores ß3-AR/ß1-AR balance and increases ß3-AR expression through the activation of NOS2 and NOS1 after myocardial infarction.