| Objective: The injury and apoptosis of endothelium is the early pathological process, including hypertension, diabetes, atherosclerosis and restenosis of PCI. This cardiovascular research has focus on the intervetion with endothelium dysfunction. Numerous studies have confirmed that many exogenous factors such as hemodynamic changes, oxidative stress, inflammation and immune responses, lead to the apoptosis of endothelium. These exogenous factors contribute to apoptosis of vascular endothelial cells (VECs) through breaking the endothelium homeostasis, exacerbating its dysfunction, accelerating the vascular disease. In recent years, the intervention with these exogenous facrors that lead to endothelium injury has enhanced and improved the vascular function and delayed the progression of vascular disease. But up to now, the report on the regulation of homestatic disorders about apoptosis of endothelial celsl (ECs) still keep unknown.The cellular repressor of E1A stimulated genes (CREG) was a novel transcriptional factor that maintain differentiaton of tissue and cell. It has been identified that CREG can inhibit the apoptosis of vascular smooth muscle cells (VSMC) and mesenchymal stem cells (MSC). Furthermore, CREG is found to be abuntant in mature vascular endothelium, and is downredgulated significantly in the vascular media after ballon injury in the rat or rabbit carotid artery or in atherosclerotic vessel. These findings revealed that CREG might play an important role in pathological injury and dysfunction of ECs. However, the role of CREG in ECs apoptosis and the underling signaling mechanisms is unknown. Methods: (1) Phoenix 293 cells were used to package and produce the pLNCX-empty, pLNCX-CREG and pLXSN-shRNA-CREG retrovirus. Three kinds of retrovirus were infected VE cells respectively. Stable cell clone with overexpressing CREG or CREG silencing expression was obtained by G418 or puromycin selection. (2) Immunofluorescence stain of CD31 was used to identify the character of ECs. Expression of CREG was detected by Western blot. (3) The effect of CREG on staurosporine (STS) or etoposide (VP-16)-induced cell apoptosis was detected by Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining and Flourescence-Activated Cell Sorting (FACS) analysis. Moreover, caspase-3 activity was detected by Western blot analysis. (4) ELISA assay detected the secretion of vascular endothelial growth factor (VEGF) in 3 kinds of VECs. The expressing of phosphoinositide 3-kinase (PI3K), p-Akt, t-Akt and VEGF were detected by Western blotting. The neutralizing antibody for VEGF and the specific inhibitor of PI3K, LY294002 were added into the ECs to evalute their effect on CREG-regulated EC apoptosis by TUNEL and FACS analysis. (5) Immunofluorescence staining and Western blotting were used to analysis the express of CREG in atherosclerotic blood vessle of apolipoprotein E-null (apoE-/-) mice compared with that in wild-type littermates blood vessle. STS-induced endothelium apoptosis were evaluated by the cleaved caspase-3 and TUNEL staining in apoE-/- mice compared with the wild-type littermates ex vivo. Artery tissues from apoE-/- mouse overexpressing CREG were generated by infection with adenovirus vectors containing CREG, the effect of CREG on endothelium apoptosis were detected by cleaved caspase-3 and TUNEL immunfluorescence staining.Results: (1) The CREG-overexpressing cell (CREG-UP) was obtained by retroviral infection with pLNCX-CREG, while the CREG silenced clone (CREG-DW) was produced by retroviral transfer of shRNAs targeting the open reading frames of human CREG gene. (2)Western blot analysis showed that pLNCX-CREG transduction in CREG-UP cells resulted in an approximately 1.9-fold increase in CREG protein expression compared with the HUVECs infected by retrovirus carrying pLNCX empty vector (CREG-NR). In contrast, stable expression of shRNAs in HUVECs reduced CREG expression by 93% compared with CREG-NR cells. (3) Gain- and loss-of-function analyses revealed that silencing CREG expression significantly enhanced ECs apoptosis, whereas CREG overexpression abrogated apoptosis stimulated by STS or VP-16. (4) ELISA assays identified that the secretion of VEGF was significantly elevated in CREG-UP cells, compared with control or CREG-DW cells. Meanwhile, PI3K and pAKT were also detected to elevate in CREG-UP cells in response to STS or VP-16 stimulation. Furthermore, blocking assays using the neutralizing antibody for VEGF and LY294002 demonstrated that the protective effect of CREG on ECs apoptosis was mainly mediated by activation of the VEGF/PI3K/AKT signaling pathway. (5) CREG expression was decreased in atherogenesis-prone endothelium in apoE-/- mice compared with their wild-type littermates using in situ immunofluorescent staining. TUNEL staining and caspase-3 activity assays determined that treatment of apoE-/- mice arteries with STS (600μmol/L) for 24 h significantly induced endothelial apoptosis associated with a reduction in CREG expression. Moreover, pretreatment the apoE-/- mice arteries with adenovirus-CREG infection, the number of apoptotic- positive ECs was obviously decreased in artery compared with that of uninfected group. While, TUNEL-positive cells also significantly abolished in CREG-infected endothelium compared with that of uninfected endothelium.Conclusions: These data demonstrate that CREG plays a critical role in protecting the vascular endothelium from apoptosis, and the protective effort of CREG against ECs apoptosis is through the activation of the VEGF/PI3K/AKT signaling pathway. |
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