## 4 Discussion  
By using bone microvascular endothelial cells isolated from the femoral head, the experimental conditions are closer to the microvasculature in bone than those using human umbilical vein endothelial cells. The preceding finding has revealed that a hydrocortisone concentration of more than 0.3 mg/mL would induce typical proliferation inhibition and apoptosis of BMECs [11]. In this study, the isolated EVs exhibited classic morphological and biochemical features of EVs, and we demonstrated that autocrine activity of EVs ameliorated glucocorticoid-induced apoptosis of BMECs, as well as improved cell viability, migration, and angiogenesis. A great deal of the evidence investigated the clinical application of Icaririn, in which the dosage of Icaririn generally varied from 5 \(\times\) 10\({}^{-6}\) M to 5 \(\times\) 10\({}^{-5}\) M [20]. The present study has shown that exposure to Icaririn could remarkably enhance BMECs' viability in a dose-dependent and dose-saturable manner and based on the results of CCK-8 assay, and 5 \(\times\) 10\({}^{-5}\) M was identified as the optimal concentration for promoting BMECs' viability and reinforcing the protective effect of autocrine EVs for glucocorticoid-induced injury of BMECs. This effect was partially realized by changing the protein and RNA profiling in secreted EVs of bone microvascular endothelial cells. The BMECs-derived EVs, defined as EVs released by BMECs during external activation, might be initiated by chemical (e.g., TNF\(\alpha\) and IL-6) and physical (e.g., shear stress) factors [21]. The responses of endothelial EVs to various medications are demonstrably different. [PERSON] and his colleagues [22] showed that lipopolysaccharideackmide increased the pro  
Figure 8: Co-culture with EVs improved the viability and decreased apoptosis of BMECs. These effects are more pronounced in Icaririn-BMEC-EVs group than in BMEC-EVs group. (**A**) Detection of OD values at various time points after hydrocortisone intervention; (**B**) cell viability at each time point; (**C**) cell apoptosis detected by flow cytometry. Data were analyzed using one-way analysis of variance and student \(t\)-test, error bars indicate standard deviation, and ** indicates \(p\)-value < 0.01.  
tein quantity of EVs released from pulmonary artery ECs, further enhancing proliferation and preventing apoptosis of pulmonary smooth muscle cells. TNF-\(\alpha\) would not influence endothelial EVs concentration, but might alter EVs-carried protein and miRNA composition. For BMECs, icarini treatment did not significantly change the size and total protein level of secreted EVs. However, it could alter the composition of contents packaged in EVs, dramatically increasing the amounts of VEGF, TGF-\(\beta\)1, hsa-miR-133a-5p, and hsa-miR-10b-5p in EVs, while leading to lesser hsa-miR-1469. VEGF and TGF-\(\beta\)1 are often co-expressed in tissues and considered to be prominent promoters of angiogenesis; however, they show opposite effects for ECs [23]. The upregulated expression level of VEGF can be initiated by hypoxia, multiple cytokines, and growth factors, including TGF-\(\beta\)1. After combining with VEGF receptor 2 (VEGFR2), VEGF further transduces signals to downstream molecules (e.g., ERK1/2, c-Jun N-terminal kinase/JNK, phosphatidylinositol 3 kinase/PI3K, AKT, eNOS, and P70S6K), inhibits the apoptosis of ECs, and modulates a series of other physiological functions [24]. On the other hand, the possible mechanisms of TGF-\(\beta\)1 for enhancing angiogenesis and differentiation of ECs are manifold [25]. First, the optimal level of ECs apoptosis is a precondition for the formation of a functional vascular network, and inhibition of apoptosis may lead to abnormality of angiogenesis. Second, TGF-\(\beta\)1 could increase the expression of VEGF and other angiogenic factors in ECs. Third, ECs' apoptosis induced by TGF-\(\beta\)1 is a transient process, occurring rapidly and followed by a prolonged refractory period.