Bcl-xl Overexpression Protects HUVEC From Oxidative Stress Induced Apoptosis And Restores Endothelial Cells' Function

Background and ObjectiveIntimal hyperplasia (IH) resulting from vascular injury initiated by procedures such as bypass graft surgery or percutaneous transluminal angioplasty with or without stent continues to limit the success of these therapeutic interventions. Vascular endothelial cell (EC) apoptosis induced by oxidative stress after injury is often regarded as the triggering event for the development of IH. Many atherosclerosis risk factors, such as diabetes, hyperlipidemia and smoking can accelerate EC damage and IH owing to heightened oxidative stress. Therefore, inhibiting EC apoptosis and protect vascular tissue against oxidative stress injury could be an effective treatment strategy to limit IH. Previous studies suggested administration of antioxidant agents such as N-acetylcysteine (NAC) and probucol improves EC coverage and decreases IH by inhibiting reactive oxygen species (ROS) formation, scavenging ROS, and interfering with ROS pathogenic signaling pathways. But the antioxidant effects can be hardly obtained because of the lower drug concentration in the local region of vascular wall. With the development of gene therapy, locally transferring target gene into vascular wall is gradually becoming a common method for preventing restenosis. In the present study, we transferred Bcl-xl gene, one of anti-apoptotic genes, into the cultured human umbilical vein endothelial cells (HUVECs) in vitro and sought to determine the efficacy of Bcl-xl overexpression protecting EC from ROS mediated insult, and then improving endothelial regeneration, accelerating the recovery of endothelium-dependent biological functions and providing an effective therapeutic adjunct to facilitate transfer of experimental treatments for vascular injury to the clinic.MethodsThe 702-bp human Bcl-xl gene, originally cloned from human testis cDNA library, was firstly subcloned into adenovirus shuttle vector pShuttle-GFP-CMV. And then, the target gene was cloned into the I-Ceu I and I-Sce I sites of adenovirus backbone vector pAdxsi to generate the recombinant adenovirus vectors pAdxsi-GFP-Bcl-xl. We used pAdxsi-GFP empty vector to be control. HUVECs were infected with the recombinant adenovirus at different multiplicities of infection (MOI) in the range 50 to 200. Infection efficiency was determined by flow cytometry. The expression of Bcl-xl in HUVECs after infection was assessed by Western blot.HUVECs were incubated with hydrogen peroxide (H2O2) for 12 hours at different concentrations from 0.2mM to 2.5mM. We assessed the anti-apoptotic effect of Bcl-xl overexpression to HUVECs exposed to 0.2mM H2O2 from aspects of morphology, cell cycles and apoptosis associated protein. After incubated with H2O2, the growth conditions and morphological changes of the HUVECs were observed with an inverted microscope, the nuclear morphology of cells was observed by Hoechst 33258 staining. The percentage of G0/G1 phase HUVECs were measured through PI staining and flow cytometry, and the expression level of apoptosis associated protein Caspase-7 and PARP were detected by Western blot.Furthermore, we explored if Bcl-xl overexpression could protect the biological functions of HUVEC in the condition of oxidant stress. The viability of HUVEC was detected by CCK-8 assay and anti-PCNA cytoimmunohistochemisty staining. In vitro scratching assay was used to assess the immigration function of HUVEC in different groups. The expressions of eNOS mRNA were investigated by Real-time PCR and the concentration of VEGF in the culture medium in different times (0h,24h,36h,48h) were measured using ELISA kit against human VEGF.ResultsOverexpression of Bcl-xl gene in HUVEC after recombinant adenovirus infection was confirmed by fluorescence microscope and Western blot. We observed the HUVECs of the H2O2 and Adv-GFP group were deformed and contracted, had increased intracellular granular material and showed a roughened profile. But the HUVECs of Adv-GFP-Bcl-xl group didn't show these morphologic characteristics. At meantime the proportion of apoptotic cells labeled with Hoechst 33258 was obviously decreased in Adv-GFP-Bcl-xl group. PI/FITC results showed that a lower percentage of HUVECs was accumulated in G0/G1 phase of the cell cycle in Adv-GFP-Bcl-xl group after exposing to H2O2 for 12 h compared to H2O2 and Adv-GFP group. The results of Western Blot assays showed exposing to H2O2 could induced apoptosis associated protein Caspase-7 and PARP activated and produce cleaved fragments, while Bcl-xl overexpression could inhibit these procedures.As for the endothelial cells' functions, the measurement results of cells' viability showed the proliferation speed of Adv-GFP-Bcl-xl group was higher than H2O2 and Adv-GFP group after 3 days exposed to H2O2. The proportion of PCNA positive cells in Adv-GFP-Bcl-xl group was also higher than other two groups. These results suggested Bcl-xl overexpression could accelerate the regeneration of HUVEC in the circumstances of ROS. The results of scratching assay revealed the immigration function was restored after Bcl-xl gene transferring. After 12h and 24h exposing to H2O2, eNOS expression in the level of mRNA in HUVECs were significantly higher in Adv-GFP-Bcl-xl group than the other two groups. H2O2 can increase the expression of VEGF in HUVECs, in the mean time; Adv-GFP-Bcl-xl group had higher VEGF expression than the other two groups.Conclusion1. The optimal MOI for gene transferring to HUVEC by adenovirus vectors is 100.2. Bcl-xl overexpression can attenuate the oxidative stress damage for HUVEC in vitro through exerting anti-apoptotic effect.3. Bcl-xl overexpression protects the biological function of HUVEC against oxidative stress and remains the cell viability and functions of immigration, synthesis and secretion.