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

Clinical studies have indicated that increased serum cholesterol levels raised the risk of tendinopathy in hypercholesterolemia, but the effect of cholesterol on tendon‐derived stem cells (TDSCs) and its underlying mechanism have not been studied. The purpose of this study is to investigate the association between cholesterol and tendinopathy in vitro and in vivo, and its underlying molecular mechanism as well. In TDSCs, the effect of cholesterol was assessed by quantitative polymerase chain reaction, western blot analysis, and immunofluorescence staining. Intracellular levels of reactive oxygen species (ROS) was detected, using flow cytometry. The link between nuclear factor (NF)‐κB signaling and the effect of cholesterol was evaluated using a representative IκB kinase (IKK) inhibitor, BAY 11‐7082. In addition, Achilles tendons from apolipoprotein E mice fed with a high‐fat diet were histologically assessed using hematoxylin and eosin staining and immunohistochemistry. We found that high cholesterol apparently lowered the expression of tendon cell markers (collagen 1, scleraxis, tenomodulin), and elevated ROS levels via the NF‐κB pathway both in vitro and in vivo. The ROS scavenger N‐acetylcysteine (NAC) and BAY 11‐7082 reversed the inhibiting effect of cholesterol on the tendon‐related gene expressions of TDSCs. Moreover, NAC blocked cholesterol‐induced phosphorylation of IκBα and p65. Significant histological alternation in vivo was shown in Achilles tendon in the hypercholesterolemic group. These results indicated that high cholesterol may inhibit the tendon‐related gene expressions in TDSCs via ROS‐activated NF‐кB signaling, implying pathogenesis of tendinopathy in hypercholesterolemia and suggesting a new mechanism underlying hypercholesterolemia‐induced tendinopathy.

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. FASEB J. 33, 11137–11147 (2019). http://www.fasebj.org