| Peripheral arterial diseases(PAD) are characterized by stenosis and occlusion of arterial, which occur most commonly in lower extremities. The classic treatments for PAD are medicine and operation, but these treatments are considered to be associated with high mortality, non-reduction of amputation and non-improvement of peak walking time and distance and so on. Therapeutic angiogenesis, which aim to promote the angiogenesis of ischemic limbs and the periphery circulation by partially or systemic intervention with the use of the genes and proteins of vascular growth factors or endothelial progenitor cells, has been emerged to be a potential strategy for PAD. Angiogenesis is the formation of new blood vessels from pre-existing blood vessels and involves the digestion of the extracellular matrix and the activation, proliferation and invasion of vascular endothelial cells(VECs), which develop into new capillary or migrate into new vessel networks. Survivin(SVV), a pleiotropic gene expresses in the embryonic tissues and malignancies, has been found to be involved in the progression, metastasis and angiogenesis of a tumor through several signaling pathways that are related to the apoptosis, cycle and migration of cancer cells. Nowadays, the function of SVV in tumor angiogenesis has been extensively elaborated, while its role in the angiogenesis of normal tissue or cell has not been known. In the present study, adenovirus-mediated SVV gene was transfected into rat aortic ECs(RAECs) to test its effects on the proliferation, migration, anti-apoptosis and angiogenesis of RAECs, which suggests a new potential therapeutic angiogenesis factor for patients with PAD.PART I Transfection and Identification of the Rat Aortic Endothelial Cells Infected with Survivin-encoding AdenovirusObjective: To determine the gene and protein expression of survivin in rat aortic endothelial cells(RAECs) after adenovirus-mediated infection.Methods: RAECs were transfected with adenovirus labeled with green fluorescent protein and SVV genes in different multiply of infection(MOI=0, 10, 20, 50, 100). Fluorescent microscope and flow cytometry were respectively performed to observe RAECs and calculate their infection rate at 12 h, 24 h, 48 h, and 72 h after adenovirus-mediated infection, then the m RNA and protein level of SVV in RAECs was detected by RT-q PCR and western blot respectively.Results: The infection rates of MOI=50 and 100 are(95.67±2.16)% and(94.53±1.76)% at 48 h after transfection, respectively, with no significant difference among three groups(P>0.05). RT-PCR analysis demonstrated that the gene level of SVV in Ad- GFP/SVV group was significantly enhanced compared with the blank and negative control group with significantly differences among three groups(P<0.05), which was further confirmed by western blot analysis of SVV protein level.Conclusions: The gene and protein level of SVV in RAECs are significantly enhanced by adenovirus-mediated transfection at 48 h with MOI=50.PART Ⅱ Effects of Survivin on the Proliferation of Rat Aortic Endothelial Cells.Objective: To address the effects of SVV on proliferative rat aortic endothelial cells.Methods: RAECs were divided into three groups: Ad-GFP/SVV(transfected by Ad-GFP-SVV), Ad-GFP(negative control group, transfected by Ad-GFP) and RAEC(blank control group, untreated) group. The expression of proliferating cell nuclear antigen(PCNA) was measured by immunofluorescence and cell viability was determined by MTT assay in RAECs 48 h after adenovirus-mediated infection. Then cellular cycle related proteins were detected by western blot assay.Results: immunofluorescence analysis demonstrated that the PCNA expression in Ad-GFP/SVV group, Ad-GFP and RAEC group was(85.35±4.93)%,(51.04±6.86)% and(52.57±5.24)% respectively. As shown by MTT assay, at the first day after adenovirus-mediated infection, the OD value of Ad-GFP /SVV group, Ad-GFP and RAEC group was 0.93±0.06, 0.77 ± 0.09 and 0.71 ± 0.14 respectively; at the second day after adenovirus-mediated infection, the OD value was 1.28±0.12; 0.91±0.05 and 0.87±0.06 in each group respectively. The level of PCNA expression and OD value of MTT was significantly higher in Ad-GFP/SVV group compared with those in Ad-GFP and RAEC group(P<0.05). Western blot assay indicated that the expression of cellular cycle related proteins cyclins(cyclin B1, cyclin D1, and cyclin E) and CKDs(CDC2, CDK 4 and CDK2) were significantly increased in Ad-GFP/SVV group compared with those in Ad-GFP and RAEC group(P<0.05).Conclusions: SVV significantly enhances the proliferation of RAECs by promoting cell cycle progression.PART Ⅲ Effects of Survivin on the Apoptosis of Rat Aortic Endothelial Cells.Objective: To observe the effects of survivin on the apoptosis of RAECs.Methods: RAECs were divided into four groups: Ad-GFP/SVV (RAECs transfected by Ad-GFP-SVV under conditions of hypoxia for 12h), Ad-GFP(negative control group, RAECs transfected by Ad-GFP and cultured under conditions of hypoxia for 12h), Control(hypoxia group, untransfected RAECs under conditions of hypoxia for 12h) and RAEC(untransfected RAECs under conditions of normoxia for 12h). The apoptosis of RAECs was induced by addition of hypoxia and detected by Td T-mediated d UTP nick end labeling(TUNEL) staining and flow cytometry. DNA chromatin fragments were stained with DAPI. Then cellular apoptosis related proteins(cleaved-caspase-3, 8 and 9) were detected by western blot assay.Results: Cell morphology analyses showed well-contacted RAECs in Ad-GFP/SVV group compared with the retracted RAECs in Ad-GFP group and RAEC group. Phalloidin staining observed by fluorescence microscope showed cell retraction, F-actin reorganization and stress fiber formation in Ad-GFP group and Control group compared with RAEC and Ad-GFP/SVV group. Transfection of SVV reversed the deleterious effects of hypoxia on RAECs, as demonstrated by the well-arranged cortical actin rim. Flow cytometry(FCM) analysis showed that the ratios of apoptotic cells were significantly diminished in Ad-GFP/SVV group compared with those in Ad-GFP and Control group. Western blot showed that the expressions of apoptosis related proteins(cleaved-caspase-3, 8 and 9) were significantly reduced in GFP/SVV group compared with those in Ad-GFP and RAEC group.Conclusions: SVV protects against hypoxia-induced apoptosis of RAECs by down-regulating the expression of cleaved caspase-3, 8 and 9.PART Ⅳ Effects of Survivin on the migration and invasion of Rat Aortic Endothelial Cells.Objective: To explore the effects of Survivin on the migration and invasion of RAECs.Methods: RAECs were divided into three groups: Ad-GFP/SVV(transfected by Ad-GFP-SVV), Ad-GFP(negative control group, transfected by Ad-GFP) and RAEC(blank control group, untreated) group. At 48 h after adenovirus-mediated infection, scratch healing assay and transwell cell migration assay was used to evaluate the migration ability of RAECs; Matrigel chamber assay was preformed to test the invasive ability of RAECs; Western blot was employed to detect matrix metalloproteinase(MMPs) protein expression.Results: Scratch healing assay showed that the distance of cells migration in Ad-GFP/SVV group was significantly increased compared with those in Ad-GFP and RAEC group. Matigel chamber assay showed that the number of RAECs migrated through the membrane was significantly increased in Ad-GFP/SVV group compared with those in Ad-GFP and RAEC group. Moreover, the expressions of MMP-2, 7,8 and 9 was significantly increased in Ad-GFP/SVV group in Ad-GFP and RAEC group(P<0.05)。Conclusions: SVV gene significantly enhance the migration and invasion abilities of RAECs by up-regulating the expressions of MMP-2, 7,8 and 9.PART Ⅴ Effects of Survivin on the Angiogenesis of Rat Aortic Endothelial CellsObjective: To evaluate the Effects of Survivin on the Angiogenesis of RAECs.Methods: RAECs were divided into three groups: Ad-GFP/SVV(transfected by Ad-GFP-SVV), Ad-GFP(negative control group, transfected by Ad-GFP) and RAEC(blank control group, untreated) group. Tube formation assay were used to evaluate angiogenic activity of RAECs in vitro by quantifying tubes and branches. VEGF content in cultured cell supernatant was detected by enzyme-linked immunosorbent assay(ELISA). Matrigel mixed with RAECs were subcutaneously injected in Nude mice for angiogenesis evaluation in vivo. The blood vessel in martigel plugs and capsules was quantified to evaluate the angiogenesis by the H&E, Masson’s trichrome and CD31 immunohistochemistry staining analysis.Results: Tube formation assay revealed that the level of tubes and branches was increased in Ad-GFP/SVV group compared with those in Ad-GFP and RAEC group. Moreover, compared with Ad-GFP and RAEC group, the VEGF content in cultured cell supernatant in Ad-GFP/SVV group was significantly higher(P<0.05). In vivo, the orderly laterigraded capillary in caspase was observed in Ad-GFP/SVV group, and the number of neovessels in the capsules and plugs was significantly increased in Ad-GFP/SVV group compared with Ad-GFP and RAEC group(P<0.05).Conclusions: SVV involves in angiogenesis in vivo and contributes to an angiogenic environment, which enhancing the angiogenic capacity of cells in pre-existing capillaries. |
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