Mechanism of vascular endothelial growth factor on the prevention of restenosis after angioplasty

Abstract

To evaluate the mechanism of vascular endothelial growth factor (VEGF) on the prevention of restenosis after angioplasty, the recombinant adenovirus vector containing hVEGF₁₆₅ cDNA was constructed and transfected into vascular smooth muscle cells (VSMC) in vitro. The conditioned medium containing VEGF was collected 72 h after the infection. Then, the VSMC and human umbilical vein endothelial cells (HUVEC) were divided into control group, H₂O₂-treated group and H₂O₂+VEGF-treated group to observe the proliferation and apoptosis by water soluble tetrazolium (WST-1) method, in situ nick end labeling (TUNEL) and flow cytometry (FCM). Compared with the control and H₂O₂+VEGF-treated groups, the absorbance (A) value of HUVEC was decreased, and apoptosis of HUVEC was significantly increased in H₂O₂-treated group. The changes of A value and apoptosis of VSMC were contrary to those of HUVEC. H₂O₂ could stimulate the proliferation of VSMC and induce the apoptosis of HUVEC, inhibit the proliferation of HUVEC and the apoptosis of VSMC and induce restenosis. VEGF could inhibit the effect of H₂O₂ on HUVEC and VSMC and prevent restenosis. These results offered further theoretical evidence for VEGF on the prevention of restenosis after angioplasty.

Keywords

vascular endothelial growth factors / restenosis / reactive oxygen species / endothelial cells / vascular smooth muscle cell

Introduction

Restenosis following percutaneous coronary intervention (PCI) is one of the major research subjects in coronary heart disease. The mechanism is quite complicated and not clear yet, but it has been known that the impairment of endothelium is the primary evocative factor on the progress of restenosis, and excessive proliferation and decreasing apoptosis of vascular smooth muscle cells (VSMC) are also important factors on the progress of restenosis. It is supposed to prevent the restenosis after PCI by accelerating restoration of endothelial integrity and function, inhibiting proliferation and inducing apoptosis of VSMC. In some experimental studies, exogenic vascular endothelial growth factor (VEGF) was proven to prevent restenosis following angioplasty, but the mechanism was not yet clear [1-3]. In this study, the effects of H₂O₂ and VEGF on proliferation and apoptosis of human umbilical vein endothelial cells (HUVEC) and rabbit aorta VSMC were observed to offer further theoretical evidence for VEGF on the prevention of restenosis after PCI.

Materials and methods

Plasmid

pSVI21 containing hVEGF₁₆₅ cDNA was offered by Boston University (USA). pACCMV·pLpA and pJM17 were supplied by Vanderbilt University (USA). pJM17 plasmid consisted of a modified Ad5 genome lack of E1 region; pACCMV. pLpA plasmid was derived from the pAC plasmid by the insertion of the CMV promoter element, a PUC19 cloning cassette and the SV40 polyadenylation signal. The Ad5 sequence containing in pACCMV.pLpA was completely identical with the part of pJM17 DNA sequence.

Reagents

The reagents used in this study included: EcoR I, Nco I, Hind III, Taq DNA polymerase, calf intestines alkaline phosphatase (CIP), T₄ DNA ligase, AMV reverse transcriptase, λDNA/Hind III and protein Marker (Promega, USA); fetal bovine serum (FBS), RPMI1640 and DMEM (high-glucose, 4.5 g/L) (Sigma, USA); rabbit polyclonal antibody against hVEGF and goat anti-rabbit IgG with peroxidase-conjugated (Santa Cruz, USA); TRIzol and kollagenase I (Gibco, USA); WST-1 and DIG Labeling DNA Detection Kit (Boehringer Mannheim, Germany).

Construction and identification of recombinant adenovirus vector containing the hVEGF₁₆₅ gene [4]

The cDNA encoding hVEGF₁₆₅ was obtained by EcoR I from plasmid pSVI21 and was subcloned into pACCMV·pLpA vector. After digestion by EcoR I, Nco I and Hind III, subclone pACCMV·pLpA and pJM17 were purified by the Sambrook method, and then cotransfected into human embryo kidney 293 cells by calcium phosphate co-precipitation to obtain the replication-deficient recombinant adenovirus containing hVEGF₁₆₅ cDNA by homologous recombination. After identification by polymerase chain reaction (PCR) and Dot blot, the recombinant adenovirus was propagated with the titers of 2×10¹² pfu/mL. Forty-eight hours after the infection with recombinant adenoviruses, the expression of VEGF mRNA and protein was confirmed in the rabbit aorta VSMC by reverse transcription-PCR (RT-PCR) and Western blot in vitro.

Culture and identification of rabbit aorta VSMC

VSMC were cultured by using a collagenase-digested method [4]. The second and third passage VSMC were used for experiment.

Effects of H₂O₂ and VEGF on proliferation of HUVEC and VSMC

The HUVEC (American Type Culture Collection, USA) and VSMC at logarithmic growth phase were seeded in a 96-well plate and divided into 8 groups, with 8 wells in each group, and 4×10³ cells in each well. Groups 1-4 were HUVEC and groups 5-8 were VSMC. Seventy-two hours after confluent VSMC were infected with 0, 5 and 20 pfu/cell recombinant adenoviruses containing the hVEGF₁₆₅ gene, conditioned media 1, 2, and 3 (CM 1, 2 and 3) were collected respectively from the supernatants. CM1 into groups 1, 2, 5 and 6, CM2 into groups 3 and 7, and CM3 was added into groups 4 and 8, respectively. H₂O₂ (200 μmol/L) was added into groups 2-4 and groups 6-8, reaching the final 100 μL in each well. After culturing at 37°C in a 5% CO₂ incubator for 48 h, 10 μL water soluble tetrazolium (WST-1) was added into each well and incubation was continued for 4 h. Absorbance (A) value was measured at a wavelength of 450 nm.

Effects of H₂O₂ and VEGF on apoptosis of HUVEC and VSMC

The HUVEC and VSMC at logarithmic growth phase were seeded in 12-well plates and divided into 8 groups, with 6 wells in each group, a cover glass slice of 0.5 cm×0.5 cm size was added into each well, and the cells were incubated at 37°C in a 5% CO₂ incubator. After the HUVEC and VSMC were confluent, the CM and H₂O₂ were added into the well in the same way as above 2.5. After continuous culture of 12 h, the cover glass slices were taken out to make in situ nick end labeling (TUNEL), and the surplus cells were digested and detected by flow cytometry (FCM).

TUNEL

TUNEL kit (Wuhan Boster Biological Technology, Ltd., China) included: dNTP (Promega, USA), DIG-dUTP, Proteinase-K (Boehringer Mannheim, Germany), Biotin-anti-DIG antibody (Sigma, USA) and Streptoaffinitin-peroxidase (Wuhan Boster Biological Technology, Ltd., China). Yellow granules in cell nucleus under microscope indicated apoptosis. The mean of apoptosis under 5 high power visual fields was counted by using computer-based image analysis system.

FCM analysis

The collected cells were washed twice with PBS, and fixed with cold 70% ethanol at 4°C overnight. After centrifugation, the ethanol was discarded, and 200 μL (1 g/L) RNase was added into the Eppendorf (EP) tube and kept at 37°C for 30 min. Then 800 μL PI dye liquor (100 mg/L, PI, Sigma; 1.0% Triton-X 100, Servea; 0.9% NaCl) was added into the EP tube and kept at 4°C for 30 min in darkness. FCM analysis was performed (FACSort, BD, USA), and 8000 cells were counted in each sample. The percentage of apoptosis was calculated.

Statistical analysis

Data were presented as \overline{x}\pm s, and Student t test was applied for statistical analysis.

Results

A value

The A value of HUVEC was lower in the H₂O₂-treated group compared with that in the control and H₂O₂+VEGF-treated groups. The changes of A value in VSMC were contrary to those inHUVEC. These results indicated that H₂O₂ could stimulate the proliferation of VSMC, inhibit the proliferation of HUVEC, and VEGF could inhibit the effect of H₂O₂ on HUVEC and VSMC in a dose-dependent manner (Tables 1, 2).

TUNEL and FCM

To HUVEC, the apoptosis was significantly higher in H₂O₂-treated group compared with control and H₂O₂+VEGF-treated groups. To VSMC, the changes of apoptosis were contrary to HUVEC. These results indicated that H₂O₂ could induce the apoptosis of HUVEC, inhibit the apoptosis of VSMC, and VEGF could inhibit the effect of H₂O₂ on HUVEC and VSMC (Tables 1, 2).

Discussion

PCI has become a successful and widely used method for treatment of coronary heart disease, but restenosis occurs in about 30%-50% of the patients within 6 months after PCI. Up to now, no methods have been proven to effectively prevent clinical restenosis. This problem not only influences the prognosis, but also restricts the application of PCI. Restenosis is a serial reaction of arterial response to injury, and the mechanism is quite involved. The impairment of endothelium is the primary evocative factor on the progress of restenosis. Excessive proliferation and decreasing apoptosis of VSMC are also important factors inducing the progress of restenosis. Based on this recognition, it is supposed to prevent the restenosis after PCI by accelerating re-endothelialization, inhibiting excessive VSMC proliferation and inducing apoptosis of VSMC.

Active oxygen could be produced during ischemia, hypoxia, ischemia-reperfusion and angioplasty, but the effects of active oxygen on vascular endothelial cell and VSMC have been reported by few researchers. In this study, the results demonstrated that H₂O₂ could improve the proliferation of VSMC, induce the apoptosis of HUVEC, inhibit the proliferation of HUVEC and the apoptosis of VSMC, but VEGF could inhibit the effect of H₂O₂ on HUVEC and VSMC partially in a dose-dependent manner. Active oxygen could stimulate the excessive intimal proliferation after arterial injury [5,6], which might have relation with above effect of H₂O₂. Active oxygen could induce vascular endothelial cell peroxidation lipid, protein degeneration and ligation, split and degeneration of DNA, and inhibit the repair of damaged DNA, inhibit the proliferation and promote death of vascular endothelial cell [7]. The contrary effect of H₂O₂ on VSMC is probably achieved through H₂O₂-induced up-regulation on proto-oncogene c-myc and c-fos expression [8]. VEGF could stimulate vascular endothelial cell effective and rapid mitogenesis and growth, inhibit the effect of H₂O₂ on vascular endothelial cells partially. The mechanism on the inhibitory effect of VEGF on H₂O₂ induced VSMC proliferation is not clear yet. H₂O₂ could inhibit the expression of vascular cell adhesion molecule gene and induce the apoptosis of vascular endothelial cell [9], but VEGF could stimulate the expression of vascular cell adhesion molecule gene and inhibit the H₂O₂-induced apoptosis of vascular endothelial cells [10]. The inhibitory effect of H₂O₂ on VSMC apoptosis was probably achieved through an up-regulation of Bcl-2 gene expression [11,12], but the inducing effect of VEGF on VSMC apoptosis was probably achieved by the down-regulation of Bcl-2 gene expression.

VEGF has been shown to be an endothelial cell-specific mitogen in vitro, and could improve effective proliferation of vascular endothelial cells and accelerate the restoration of endothelial integrity and function after PCI. It probably plays an important role in prevention of restenosis. In experimental study and clinical trial, it was confirmed that hVEGF could accelerate restoration of endothelial integrity and function after experimental angioplasty [1-3], but now there are not enough mechanisms to explain these results. It is probably achieved by improving proliferation and regulating secretion of VEGF on vascular endothelial cells [13]. In this study, these results demonstrated that active oxygen produced during angioplasty could stimulate the proliferation of VSMC, induce the apoptosis of vascular endothelial cells, inhibit the proliferation of vascular endothelial cells and the apoptosis of VSMC, and thus, induce the restenosis. VEGF could inhibit the effect of active oxygen on HUVEC and VSMC, and prevent restenosis. These results offered further theoretical evidence for VEGF on the prevention of restenosis after angioplasty.

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