Targeting endothelial PDGFR-ß facilitates angiogenesis-associated bone formation through the PAK1/NICD axis.

The intricate orchestration of osteoporosis (OP) pathogenesis remains elusive. Mounting evidence suggests that angiogenesis-driven osteogenesis serves as a crucial foundation for maintaining bone homeostasis. This study aimed to explore the potential of the endothelial platelet-derived growth factor receptor-ß (PDGFR-ß) in mitigating bone loss through its facilitation of H-type vessel formation. Our findings demonstrate that the expression level of endothelial PDGFR-ß is reduced in samples obtained from individuals suffering from OP, as well as in ovariectomy mice. Depletion of PDGFR-ß in endothelial cells ameliorates angiogenesis-mediated bone formation in mice. The regulatory influence of endothelial PDGFR-ß on H-type vessels is mediated through the PDGFRß-P21-activated kinase 1-Notch1 intracellular domain signaling cascade. In particular, the endothelium-specific enhancement of PDGFR-ß facilitates H-type vessels and their associated bone formation in OP. Hence, the strategic targeting of endothelial PDGFR-ß emerges as a promising therapeutic approach for the management of OP in the near future.

SPTBN1 Prevents Primary Osteoporosis by Modulating Osteoblasts Proliferation and Differentiation and Blood Vessels Formation in Bone.

Osteoporosis is a common systemic skeletal disorder that leads to increased bone fragility and increased risk of fracture. Although ßII-Spectrin (SPTBN1) has been reported to be involved in the development of various human cancers, the function and underlying molecular mechanisms of SPTBN1 in primary osteoporosis remain unclear. In this study, we first established a primary osteoporosis mouse model of senile osteoporosis and postmenopausal osteoporosis. The results showed that the expression of SPTBN1 was significantly downregulated in primary osteoporosis mice model compared with the control group. Furthermore, silencing of SPTBN1 led to a decrease in bone density, a small number of trabecular bones, wider gap, decreased blood volume fraction and number of blood vessels, as well as downregulation of runt-related transcription factor 2 (Runx2), Osterix (Osx), Osteocalcin (Ocn), and vascular endothelial growth factor (VEGF) in primary osteoporosis mice model compared with the control group. Besides, the silencing of SPTBN1 inhibited the growth and induced apoptosis of mouse pre-osteoblast MC3T3-E1 cells compared with the negative control group. Moreover, the silencing of SPTBN1 significantly increased the expression of TGF-ß, Cxcl9, and the phosphorylation level STAT1 and Smad3 in MC3T3-E1 cells compared with the control group. As expected, overexpression of SPTBN1 reversed the effect of SPTBN1 silencing in the progression of primary osteoporosis both in vitro and in vivo. Taken together, these results suggested that SPTBN1 suppressed primary osteoporosis by facilitating the proliferation, differentiation, and inhibition of apoptosis in osteoblasts via the TGF-ß/Smad3 and STAT1/Cxcl9 pathways. Besides, overexpression of SPTBN1 promoted the formation of blood vessels in bone by regulating the expression of VEGF. This study, therefore, provided SPTBN1 as a novel therapeutic target for osteoporosis.

NF-κB/TWIST1 Mediates Migration and Phagocytosis of Macrophages in the Mice Model of Implant-Associated Staphylococcus aureus Osteomyelitis.

Staphylococcus aureus (S. aureus) infection-induced osteomyelitis is a great challenge in clinic treatment. Identification of the essential genes and biological processes that are specifically changed in mononuclear cells at an early stage of S. aureus osteomyelitis is of great clinical significance. Based on transcriptional dataset GSE16129 available publicly, a bioinformatic analysis was performed to identify the differentially expressed genes of osteomyelitis caused by S. aureus infection. ERBB2, TWIST1, and NANOG were screened out as the most valuable osteomyelitis-related genes (OMRGs). A mice model of implant-associated S. aureus osteomyelitis was used to verify the above genes. We found significantly up-regulated expression of TWIST1 in macrophages and accumulation of macrophages around the infected implant. Meanwhile, S. aureus infection increased the expression of TWIST1, MMP9, and MMP13, and stimulated the migration and phagocytosis function of Raw 264.7 cells. Additionally, knock-down of the expression of TWIST1 by siRNA could significantly down-regulate MMP9 and MMP13 and suppress the migration and phagocytosis ability of macrophages in response to S. aureus infection. Furthermore, we found that NF-κB signaling was activated in Raw 264.7 cells by S. aureus and that inhibition of NF-κB signaling by Bay11-7082 blocked the expression of TWIST1, MMP9, and MMP13 as well as cell migration and phagocytosis evoked by S. aureus. Our findings demonstrate that NF-κB/TWIST1 is necessary for migration and phagocytosis of macrophages in response to S. aureus infection. Our study highlights the essential role of NF-κB/TWIST1 in early innate immune response to S. aureus infection in bone.

High cholesterol induces apoptosis and autophagy through the ROS-activated AKT/FOXO1 pathway in tendon-derived stem cells.

BACKGROUND: Hypercholesterolemia increases the risk of tendon pain and tendon rupture. Tendon-derived stem cells (TDSCs) play a vital role in the development of tendinopathy. Our previous research found that high cholesterol inhibits tendon-related gene expression in TDSCs. Whether high cholesterol has other biological effects on TDSCs remains unknown. METHODS: TDSCs isolated from female SD rats were exposed to 10 mg/dL cholesterol for 24 h. Then, cell apoptosis was assessed using flow cytometry and fluorescence microscope. RFP-GFP-LC3 adenovirus transfection was used for measuring autophagy. Signaling transduction was measured by immunofluorescence and immunoblotting. In addition, Achilles tendons from ApoE -/- mice fed with a high-fat diet were histologically assessed using HE staining and immunohistochemistry. RESULTS: In this work, we verified that 10 mg/dL cholesterol suppressed cell proliferation and migration and induced G0/G1 phase arrest. Additionally, cholesterol induced apoptosis and autophagy simultaneously in TDSCs. Apoptosis induction was related to increased expression of cleaved caspase-3 and BAX and decreased expression of Bcl-xL. The occurrence of autophagic flux and accumulation of LC3-II demonstrated the induction of autophagy by cholesterol. Compared with the effects of cholesterol treatment alone, the autophagy inhibitor 3-methyladenine (3-MA) enhanced apoptosis, while the apoptosis inhibitor Z-VAD-FMK diminished cholesterol-induced autophagy. Moreover, cholesterol triggered reactive oxygen species (ROS) generation and activated the AKT/FOXO1 pathway, while the ROS scavenger NAC blocked cholesterol-induced activation of the AKT/FOXO1 pathway. NAC and the FOXO1 inhibitor AS1842856 rescued the apoptosis and autophagy induced by cholesterol. Finally, high cholesterol elevated the expression of cleaved caspase-3, Bax, LC3-II, and FOXO1 in vivo. CONCLUSION: The present study indicated that high cholesterol induced apoptosis and autophagy through ROS-activated AKT/FOXO1 signaling in TDSCs, providing new insights into the mechanism of hypercholesterolemia-induced tendinopathy. High cholesterol induces apoptosis and autophagy through the ROS-activated AKT/FOXO1 pathway in tendon-derived stem cells.

Hydroxytyrosol promotes autophagy by regulating SIRT1 against advanced oxidation protein product­induced NADPH oxidase and inflammatory response.

Advanced oxidation protein products (AOPPs) can trigger NADPH oxidase (NOX) and lead to the production of reactive oxygen species (ROS) in the pathophysiology of rheumatoid arthritis (RA). Hydroxytyrosol (HT) is a phenolic composite in olive oil that has antioxidant and anti­inflammatory effects and enhances autophagy. Early research has revealed that HT can activate the silent information regulator 1 (SIRT1) pathway to induce autophagy and alleviate the cartilage inflammatory response caused by H2O2. However, whether HT can attenuate AOPP­induced NOX and inflammatory responses remains to be elucidated. The present study aimed to investigate how HT can alleviate the damage caused by AOPPs. In cell experiments, chondrocytes were pre­stimulated with HT and then exposed to AOPPs. First, it was found that HT promoted autophagy through the SIRT1 pathway, increased the expression of autophagy­related proteins including microtubule­associated protein 1 light chain 3, autophagy related (ATG)5 and ATG7, and decreased the expression of P62. Furthermore, HT reduced the expression of NOX, which was affected by AOPPs in chondrocytes through the SIRT1 pathway. Finally, the expression of inflammatory cytokines caused by AOPPs was downregulated following HT treatment. In conclusion, it was found that HT reduced the expression of NOX and inhibited the inflammatory response caused by AOPPs in chondrocytes through the SIRT1 pathway.

The role of EGFR signaling in age-related osteoporosis in mouse cortical bone.

So far, there has been no effective cure for osteoporotic cortical bone, the most significant change in long bone structure during aging and the main cause of bone fragility fractures, because its underlying molecular and cellular mechanisms remain largely unknown. We used 3- and 15-mo-old mice as well as 15-mo-old mice treated with vehicle and gefitinib to evaluate structural, cellular, and molecular changes in cortical bone. We found that the senescence of osteoprogenitors was increased, whereas the expression of phosphorylated epidermal growth factor receptor (EGFR) on the endosteal surface of cortical bone down-regulated in middle-aged 15-mo-old mice compared with young 3-mo-old mice. Further decreasing EGFR signaling by gefitinib treatment in middle-aged mice resulted in promoted senescence of osteoprogenitors and accelerated cortical bone degeneration. Moreover, inhibiting EGFR signaling suppressed the expression of enhancer of zeste homolog 2 (Ezh2), the repressor of cell senescence-inducer genes, through ERK1/2 pathway, thereby promoting senescence in osteoprogenitors. Down-regulated EGFR signaling plays a physiologically significant role during aging by reducing Ezh2 expression, leading to the senescence of osteoprogenitors and the decline in bone formation on the endosteal surface of cortical bone.-Liu, G., Xie, Y., Su, J., Qin, H., Wu, H., Li, K., Yu, B., Zhang, X. The role of EGFR signaling in age-related osteoporosis in mouse cortical bone.