By using bone microvascular endothelial cells extracted 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 0.3mg/mL would induce typical proliferation inhibition and apoptosis of BMECs[11]. In this study, the extracted extracellular vesicles exhibited classic morphological and biochemical features of exosomes, and we demonstrated that autocrine activity of exosomes ameliorated glucocorticoid-induced apoptosis of BMECs as well as improved cell viability, migration and angiogenesis. Loads of evidence investigated the clinical application of icariin, in which dosage of icariin generally varied from 5×10−6 M to 5×10−5 M[27]. The present study has shown that exposure to icariin could remarkably enhance BMECs viability in a dose-dependent and dose-saturable manner and based on the results of CCK-8 assay, and 5×10−5 M was identified as the optimal concentration for promoting BMECs viability and reinforcing the protective effect of autocrine exosomes for glucocorticoid-induced injury of BMECs. This effect was partially realized by changing protein and RNA profiling of secreted exosomes of bone microvascular endothelial cells.
BMECs-derived exosomes, defined as exosomes released by BMECs during external activation, might be initiated by chemical (e.g., TNFα and IL-6) and physical (e.g., shear stress) factors[28]. Responses of endothelial exosomes to various medications are demonstrably different. Zhao and his colleagues[29] shown that lipopolysaccharide increased the protein quantity of exosomes released from pulmonary artery ECs, which further enhanced proliferation and prevented apoptosis of pulmonary smooth muscle cells. TNF-α would not influence endothelial exosome concentration, but might alter exosome-carried protein and miRNA composition. For BMECs, icariin treatment significantly changed the size and total protein level of secreted exosomes, but could alter the composition of contents packaged in exosomes, dramatically increasing the amounts of VEGF, TGF-β1, hsa-miR-133a-5p, and has-miR-10b-5p in exosomes while leading to lesser hsa-miR-1469. VEGF and TGF-β1 are often co-expressed in tissues and considered to be prominent promoters of angiogenesis; however, they show opposite effects for ECs[30]. The upregulated expression level of VEGF can be initiated by hypoxia, multiple cytokines and growth factors, including TGF-β1. 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[31]. On the other hand, the possible mechanisms of TGF-β1 for enhancing angiogenesis and differentiation of ECs is manifold[32]. 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-β1 could increase the expression of VEGF and other angiogenic factors in ECs. Third, ECs apoptosis induced by TGF-β1 is a transient process, occurring rapidly and followed by a prolonged refractory period. Last but not least, it has been indicated that TGF-β1 promoted angiogenesis through the regulation of immune cells in vivo.
Moreover, to systematically identify novel inflammatory cytokines related to the protective effect of icariin, we performed a screen among the Model group, Icariin-treated group and the control group by protein array assay. The present study found that 29 proteins were dysregulated in exosomes secreted by BMECs after icariin intervention and most of these proteins were upregulated. Based on the PPI network, 9 proteins (ICAM1, MMP3, BSG, LGALS3, CXCL9, CXCL16, MMP10, IL36RN, and TNFRSF1B) with high connectivity were identified to play essential roles in the protective effect of icariin to glucocorticoid-induced injury of BMECs. ICAM1 is a cell surface glycoprotein typically expressed on endothelial cells[33]. Meanwhile, ICAM1 might exert dual effects on bone homeostasis, especially in osteoclastogenesis[34]. MMP3 and MMP10 belong to the matrix metalloproteinase family and also participate in osteogenic differentiation[35]. LGALS3, which is widely expressed in various tissues including bone tissues, is considered as a marker of bone formation[36]. Important miRNAs (e.g., hsa-miR-330-3p and hsa-miR-17) and transcription factors (e.g., MYC and SP1) may involve in the dysregulation of these proteins. In former studies, these molecules have been demonstrated to be vital for vascular development and angiogenesis [37, 38]. Cytokine-cytokine receptor interaction pathway is predicted to be significantly enriched by identified DEPs, which included CCL14, TNFRSF10C, CXCL9, IL36RN, BMP7, TNFRSF1B, CXCL16, and GH1.
ECs-derived exosome affects recipient cells’ function and modulates key events in the development of multiple disorders; the effect could be either beneficial or detrimental. It has also been shown that capillary endothelial tip cells secrete exosomes incorporating Delta-like 4 ligands, which are passed to tip cells of endothelial sprout, promote cell motility and suppress their proliferation[39]. Senescent human ECs-derived exosomes could knock down Frizled-3 of human MSCs and inhibit osteogenic differentiation[40]. Our study demonstrated that exosome intervention resulted in upregulated production of angiogenetic (e.g., VEGF, pERK) and vasodilating factors (e.g., eNOS and COX-2), while inhibited the activation of pro-apoptotic factors (cleaved caspase-3) in endothelial cells, indicating that endothelium-dependent signaling pathways might involve in the protective effect of autocrine exosomes to glucocorticoid-induced endothelial cellular injury. Apoptosis is a kind of programmed cell death occurring in a multicellular organism to maintain homeostasis, with caspase-dependent apoptosis being demonstrated to be the most classical mechanism of glucocorticoid-induced endothelial cell death[41]. BMECs-derived exosomes intervention significantly inhibited the cleavage of pro-caspases-3 and therefore improved the apoptosis of BMECs. NO is synthesized by eNOS using amino acid L-arginine and is able to regulate vascular tone[18]. Inhibition of the VEGF/Akt/eNOS pathway blocks nitric oxide release and promotes vasoconstriction. As for cyclooxygenase, there are two isoforms discovered currently: COX-1 and COX-2, with the latter one being the rate-limiting key enzyme in the biosynthesis of prostaglandins during inflammatory responses. A complex interplay between eNOS and COX-2 pathways can be observed in the relaxation of vascular smooth muscles[42]. ECs-derived exosome intervention may lead to enhanced NO and PGs production in vivo conditions; although eNOS and COX-2 inhibitors did not impact the viability of endothelial cells in rescue experiments, whether these factors ameliorate apoptosis and migration remains to be confirmed. In our studies, exosomes pretreatment promoted the level of the phosphorylated extracellular regulated protein kinases, the active form of ERK, revealing that BMECs-derived exosomes improve glucocorticoid-induced BMEC injury partially via activation of ERK pathways. Among four classical mitogen-activated protein kinases (MAPK) signaling pathways, MAPK/ERK pathway is reported to be associated with regulation of proliferation, survival, apoptosis and angiogenesis of endothelial cells[23].
This study is not without limitations. First, extrapolation of results from cell models to clinical practice should be performed with caution. It is needed to consider the impact of factors in vivo. Second, although hsa-miR-133a-5p and hsa-miR-10b-5p were enriched after icariin intervention, we did not perform the further experiment to test whether they could take effects in the reversion of glucocorticoid-induced cellular injury. Last but not least, it is well-acknowledged that icariin could affect multiple cell types existing in bones, for example, MSCs and osteocytes; meanwhile, exosomes can modulate inflammation, regulate activation and migration of monocytes as well as affect differentiation of MSCs. Therefore, it is rational to assume that other kinds of cells in the bone or even cells of cardiovascular systems in the distance may also engulf BMECs-derived exosomes may also be engulfed by other kinds of cells. In vivo and animal studies are required to confirm these findings and further illuminate the action mechanism of identified miRNAs and proteins for the protective effects of icariin to glucocorticoid-induced injury of BMECs.