In the current study, glucocorticoid-induced ANFH via inhibition of mTOR signaling was found, a mechanism not reported previously. Detailed regulatory mechanism included impaired angiogenesis by subsequent HIF-1 inhibition and increased CHOP-dependent DR5-mediated apoptosis. Those findings offered novel perspective on the impact of glucocorticoids on ANFH.
Role of impairment of angiogenesis in ANFH has been emphasized. It is reported that circulating endothelial progenitor cell (EPC) numbers and functions are reduced in ANFH patients, suggesting that risk factors of ANFH may alter EPCs biology in angiogenesis and vascular repair [
11]. Further, recombinant adeno-associated viral vectors co-expressing the hVEGF(165) and hBMP(7) genes showed efficient gene expression ability on angiogenesis. The VEGF(165) and BMP(7) proteins expressed from the vector have efficient biological activity in vitro [
12]. Small intestinal submucosae matrix (SIS) is composed of highly conserved collagens, glycoproteins, proteoglycans, and glycoaminoglycans in their natural configuration and concentrations. Transplantation of PBSCs cultured with SIS effectively improved ischemic femoral head necrosis with significant increase of new blood vessels [
13]. Co-culture of mesenchymal stem cells with umbilical vein endothelial cells also showed increased cell survival, migration, and angiogenesis, with upregulation of expression of SDF-1α, VEGF, and IL-6 expression [
14]. Those pilot studies pushed forward stem cell investigation in human studies. Intravenous transplantation of allogeneic MSCs can promote vascular and bone regeneration in the necrotic region of the femoral head in a rabbit model of ANFH. The results of our study suggest that the intravenous transplantation of MSCs could be a potential and minimally invasive treatment option for ANFH patients [
15]. Interestingly, a novel calcium phosphate (CPC) composite scaffold, which contains BMP-VEGF-loaded poly-lactic-co-glycolic acid (PLGA) microspheres (BMP-VEGF-PLGA-CPC) was generated and implanted into the bone tunnels of core decompression in the femoral head, showing the scaffold may improve the therapeutic effect of core decompression surgery and be used as a treatment for ANFH [
16]. Those studies provide compelling evidence that angiogenesis plays a critical role in ANFH recovery, and our results indicate that enhancement of mTOR could be another treatment modality. One of the critical findings of the current study was inhibition of mTOR signaling also induced apoptosis of BM-EPCs via CHOP-dependent DR5 induction in response to GR stimulation. This finding is in line with the study by Shimizu et al. [
9], in which authors found that mTOR activation inhibits GR transcription function and efficiently counteracts the catabolic processes provoked by glucocorticoids in skeletal muscle tissue. Other scholars find that rapamycin sensitizes glucocorticoid-resistant acute lymphoblastic leukemia CEM-C1 cells to dexamethasone-induced apoptosis through both mTOR suppression and upregulation and activation of glucocorticoid receptor [
17]. The main risk factors are bone fractures, joint dislocations, alcoholism, and the use of high-dose steroids. Other risk factors include radiation therapy, chemotherapy, and organ transplantation. Unlike mechanic factors that directly disrupt the blood supply of the femoral head, etiology for corticosteroid-induced ANFH remains unclear. Most suggest that the drugs may interfere degradation of lipids, which accumulate and clog the vessels, eventually leading to avascularity. This phenotype is more severe as it is more frequently to affect both hips. Given that mTOR signaling is strongly associated with lipid metabolism, the mTOR-GR axis appears to be a promising target for the disease management.
Combating apoptosis of progenitor cells is another target in treating ANFH. It was reported that The Wnt/β-catenin pathway is involved in the pathogenesis of early stage ANFH in rat model, and it may act through the regulation of c-Myc, which affects the cell cycle and cell apoptosis [
18]. Alternatively, cell death was used to trace early evidence of ANFH. It was shown that (99m)Tc-annexin V is superior to (99m)Tc-MDP for the early detection of glucocorticoid-induced femoral head necrosis in the rabbit and may be a better strategy for the early detection of glucocorticoid-induced femoral head necrosis in patients [
19]. One group reported that Chinese herb, Tao-Hong-Si-Wu decoction (THSWD), significantly promoted the expression of HIF-1α and VEGF in the femoral head tissue of rabbits and markedly inhibit the apoptosis of osteocytes, chondrocytes, and bone marrow cells. In addition, THSWD suppressed caspase-3 expression and induced bcl-2 expression in femoral head tissues. Like in our study, they concluded that THSWD can suppress ANFH by regulating the HIF-1α pathway and cell apoptosis [
20]. Another study pointed out that was able to reduce steroid-induced bone cellular apoptosis, reduce the occurrence of necrosis of the femoral head and, through in vivo metabolism, it may promote the synthesis and release of IGF-1 [
21]. More commonly accessible drug, vitamin E was also reported to benefit ANFH patients via combating apoptosis. Report showed that vitamin E is effective in intervening in apoptosis through decreasing caspase-3 expression and upregulating Bcl-2 expression and by alleviating DNA oxidative damage in bone marrow hemopoietic cells at the early stage of steroid-induced femoral head necrosis in rabbit models [
22]. So far, no study focused on apoptosis of BM-EPC, the direct progenitor for angiogenesis. Our results for the first time revealed that the apoptosis induced by excessive steroid also included endothelial progenitors, and combined impairment of osteocytes and angiogenesis contributes to the pathogenesis.