The Mechanism Study Of High Glucose Induced Endothelial-To-Osteoblasts

Objective:Vascular calcification (VC) is one of the common complications in diabetes mellitus (DM). It is important risk factor for cardiovascular morbidity and mortality among patients with DM. Hyperglycemia is a hallmark of diabetes mellitus, and is the single most important risk factor for the cardiovascular disease. However, the exact mechanism of hyperglycemia induced vascular calcification has not been completely elucidated, although emerging evidence indicates that hyperglycemia may play an important role of the onset and progression of VC and be a potent inducer of vascular calcification. Furthermore, multiple studies reported that in vitro, vascular smooth muscle cells (VSMCs) and pericytes can transforme into osteoblast-like cells. Recent studies showed that conversion of endothelial-to-mesenchymal transition (EndMT) could be triggered into osteoblasts. Our preliminary experiment indicated that high glucose could induce the phenotypic change of the human aortic endothelial cells in a dose and time-dependent manner. And diabetic rats could present with myocardial EndMT in very early stage. On the basis of previous research, the human aortic endothelial cells (HAECs) culture will be used to explore the effect and mechanism of high glucose on EndMT by the ways of pathological changes and molecular techniques. Furthermore, whether high glucose-induces EndMT will transform into osteoblasts could contribute to the vascular calcifation in DM will be investigated. And the possible signal pathway will be explored. This study will provide novel insight into the development of VC and finally contribute to the establishment of new strategy of VC prevention in DM.Method:Our experiment included two parts. In the first part, we aimed to explore whether high glucose (HG)-induced endothelial-to-mesenchymal transition (EndMT) could be transformed into mesenchymal stem cells (MSCs) and further differentiated into osteoblasts. Therefore. HAECs were divided into three groups:normal concentration glucose(NG,5.5mM) group, high concentration glucose (30 mM) group, and mannitol (MN,5.5mM glucose+4.5 mM mannitol) group, and were cultured for 48 h. The protein expressions of CD31, FSP1 and a-SMA were detected by Western blotting. And the expressions of the MSCs markers STRO-1 and CD44 were investigated by immunofluorescence staining, RT-PCR and western blotting. Pathological changes were investigated using fluorescence microscopy and electron microscopy. Immunonuorescence staining was peformed to detect the co-expression of CD31 (endothelial markers) and fibroblast-specific protein 1(FSP1, fibroblast markers). Endothelial-derived MSCs were grown in MODS for one week. The expressions of the marker were detected by western blotting. Calcium deposits were analyzed by alizarin red staining and alkaline phosphatase were analyzed by specific alkaline phosphatase staining.In the second part, we aimed to explore the role of IL-1β on high glucose induced endothelial-to-mesenchymal transition, and the mechanism of IL-1β regulation under high glucose conditions. Therefore, HAECs were exposed to normal glucose (NG,5.5 nM), high glucose (HG,30 nM), IL-1β (10 ng/ml), HG+IL-1β (10 ng/ml) and HG+anti-IL-1β antibodies (1000 ng/ml) or HG+IL-1β small interfering RNA (siRNA). Pathological changes were investigated using confocal microscopy and electron microscopy. Confocal microscopy was performed to detect the co-expression of CD31 and fibroblast specific protein 1 (FSP1). To study the effect of protein kinase C-β (PKCP) activation on IL-1β in HAECs, HAECs were stimulated with 30 nM PMA (PKCP activator) and 0.3 μM PKCβ inhibition (LY317615) for 48 h in the NG or HG group. The expressions of PKCβ and IL-1β were detected y RT-PCR and Western blot. And the concentration of IL-1β in the supernatant of HAECs was measured by ELISA. The xpressions of FSP1, a-SMA and CD31 were detected by Western blotting.Results:In the first part:the incubation of HAECs exposed to HG resulted in CD31 protein expression decreased, and the protein expressions of FSP-1 and a-SMA were significantly increased in the HG group (P<0.05). And the mRNA and protein expressions of STRO-1 and CD44 were significantly increased in dose-and time-dependent manners (P<0.05). Immunofluorescence staning showed that STRO-1 and CD44 expression intensity increased. Double staining of the HAECs indicated a co-localization of CD31 and FSP1 and the cells presented the fibroblast-like phenotype, wherein increased microfilamentation and a roughened endoplasmic reticulum structurethe cells. Additionally, Notch protein expression was increased in HG group (P<0.05). Next, endothelial-derived mesenchymal cells in HG group were grown in MODS medium for one week. Cabfal and Osterix protein expressions markly increased compared with the NG group (P<0.05). Consistent with the evaluation of Cabfal and Osterix expressions, alizarin red staining evaluating calcium deposits and alkaline phosphatase staining in the HG group were both positive compared to the NG group.In the second part:It was shown that the HG resulted in significant increase in the expressions of PKCP and DL-1β in dose-and time-dependent manners. The HG or exogenous IL-1β alone inhibited the expression of CD31 and markly increased the expressions of FSP1 and a-SMA. Furthermore, we observed that the HG and IL-1β synergistically increased FSP1 and a-SMA expressions compared with the HG or IL-1β alone group (P<0.05). Confocal microscopy revealed a colocalization of CD31 and FSP1 and that some cells acquired spindle-shaped morphologies and a loss of CD31 staining. Electron microscopy showed that the HG resulted in the increased microfilamentation and a roughened endoplasmic reticulum structure in the cytoplasm. However, the changes above were attenuated by the intervention of anti-IL-1β antibodies or IL-1β siRNA (P< 0.05). In addition, the PMA induced the expressions of PKCP and IL-1β in HAECs. The PKCβ activation may mediate the effect of the HG on IL-1β production, which could be attenuated by the PKCβ selective inhibitor (LY317615)(P<0.05).Conclusion:1) Our study demonstrated that HG could induce endothelial cells transdifferentiation into osteoblast-like cells via EndMT, which is mediated in part by the activation of the Notch signaling pathway。2) Our study demonstrated that HG induced phenotypic transition in HAECs,which was prevented by the blockade of IL-1β production.3) PKCβ activation may be responsible for upregulation of IL-1β by HG. Our findings implied that IL-1β or PKCβ pathway blockade would be a novel and effective strategy to control diabetic vasculopathy.