Proliferation of endothelial cell on polytetrafluoroethylene vascular graft materials carried VEGF gene plasmid

Proliferation of endothelial cell on polytetrafluoroethylene vascular graft materials carried VEGF gene plasmid 1 first-author 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:chenli@mail.hz.zj.cn †E-mail:chenli@mail.hz.zj.cn chenli@mail.hz.zj.cn 1 1 1 2 3 Department of Surgery, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Department of Surgery, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Department of Surgery, School of Medicine, University of Miami, Miami, Fla 33101, USA Department of Surgery, School of Medicine, University of Miami, Miami, Fla 33101, USA Vascular Biology Institute, School of Medicine, University of Miami, Miami, Fla 33101, USA Vascular Biology Institute, School of Medicine, University of Miami, Miami, Fla 33101, USA Objective: 2 To investigate whether vascular endothelial growth factor (VEGF) gene plasmid carried by polytetrafluoroethylene (PTFE) vascular graft materials could transfect endothelial cells (ECs) and promote their growth. Methods: 2 PTFE vascular graft materials carried with pCDI-hVEGF 121 , pCDI or pEGFP were incubated in Tris-buffer solution and the values of optical density of 260 nm at different time were plotted, then the DNA controlled release curve was made. ECs derived from human umbilical vein were seeded on the pCDI-hVEGF 121 /pCDI/pEGFP-PTFE materials or tissue culture plates, ECs numbers were counted and VEGF protein concentrations at different time were measured by enzyme-linked immunoadsorbent assay method. Green fluorescent protein (GFP) expression in ECs on pEGFP-PTFE materials was examined with fluorescence microscopy. Results: 2 The controlled release curve showed that the gene released from PTFE materials was rapid within 8 h, then slowed down and that the gene released continuously even after 72 h. At 24, 72 and 120 h, ECs number and proliferation rate of pCDI-hVEGF 121 -PTFE materials were higher than those of pCDI or pEGFP-PTFE materials ( P < 0.05). VEGF protein concentration of pCDI-hVEGF 121 -PTFE materials was higher than that of pCDI or pEGFP-PTFE materials at 6, 24, 72 and 120 h ( P < 0.01). GFP expression in ECs on the pEGFP-PTFE materials could be detected by fluorescence microscopy. Conclusion: 2 PTFE graft can be used as a carrier of VEGF gene plasmid, VEGF gene carried by PTFE can transfect ECs and promote ECs growth. Polytetrafluoroethylene Vascular endothelial growth factor Vascular grafts Gene Endothelial cell INTRODUCTION 1 Synthetic biomaterials have been widely employed as vascular grafts in clinical practice over the past 30 years (Stansby et al., 1994 1994 ), but none of the currently available small-caliber grafts (≤6 mm ID) have acceptable long-term patency (Anderson et al., 1987 1987 ). The major cause of grafts failure is the inherent thrombogenicity of the synthetic graft surface and intima hyperplasia which leads to progressive occlusion and closure of the graft (Callow, 1982 1982 ). It is considered that the lack of endothelium is critical to both thrombosis and intima hyperplasia (Yu et al., 2003 2003 ). The seeding of endothelial cells on synthetic grafts was studied to find ways to reduce surface thrombogenicity and increase the long-term patency of synthetic grafts (Herring et al., 1978 1978 ; Eickhoff et al., 1987 1987 ). Unfortunately, although improved patency had been reported in animal models (Pearce et al., 1987 1987 ; Shindo et al., 1987 1987 ), human clinical studies yielded mixed results (Jensen et al., 1994 1994 ; Herring et al., 1994 1994 ; Deutsch et al., 1999 1999 ). In the recent years, great progress has been made in gene therapy for vascular diseases. Some investigators began to use gene to modify the artificial blood vessel with the purpose of promoting endothelium growth. Several earlier studies demonstrated that the transfection of some growth stimulation and thrombolytic genes into endothelial cells could improve cellular adhesion, proliferation and enhance fibrinolytic capability in culture when cells were seeded onto prosthetic grafts (van Belle et al., 1997 1997 ; Grandas et al., 2001 2001 ; Pels et al., 2003 2003 ; Yu et al., 2005 2005 ). Although these methods were effective in inhibiting thrombosis, restenosis and increasing patency of synthetic grafts, the procedures including cell harvest and culture, gene transfer, cell seeding and graft implantation are quite complicated and expensive. Furthermore, several weeks are needed to implement the procedures and absolute caution is needed. Polytetrafluoroethylene (PTFE) is a porous hydrophobic polymer with 10 μm to 90 μm inter pore mean diameter (Campbell et al., 1979 1979 ; Anderson et al., 1987 1987 ). These space of pores should be suitable for gene plasmid storage. However, there is no data available in the literature regarding PTFE as a gene carrier for improving endothelialization. The use of PTFE vascular grafts as a carrier for vascular endothelial growth factor (VEGF) gene may be effective. The localized delivery of VEGF gene into implanted vascular and muscular tissue could result in predominant superior expression of VEGF protein and improve the endothelialization of the graft. In the present study, VEGF plasmid DNA was impregnated into a porous PTFE material. The kinetics of DNA release from the impregnated PTFE material was evaluated. The transfection efficiency of plasmid DNA carried by PTFE in cultured human umbilical vein endothelial cells (HUVECs) and the growth and proliferation of HUVECs on VEGF gene impregnated PTFE material were further investigated. MATERIALS AND METHODS 1 Plasmid preparation 2 The vector pCDI-hVEGF 121 was kindly provided by Peking University Center for Human Disease Genomics (China), and consisted of human VEGF 121 complementary deoxyribonucleic acid (cDNA) full-length sequence and highly efficient eukaryotic expression plasmid pCDI. The vector pEGFP, coding for enhanced green fluorescence protein, was purchased from Clontech Corporation (Clontech, USA). These plasmids were amplified in E . coli DH5a, and retrieved and purified using QIAGEN Plasmid Maxi Kit (QIAGEN, Germany) according to the manufacturers’ instructions. Preparation of PTFE vascular grafts carried with plasmid DNA 2 pCDI-hVEGF 121 , pCDI and pEGFP were mixed with fibronectin (Sigma, USA) in TBS (10 mmol/L Tris/HCl pH 7.4, 150 mmol/L NaCl) respectively. The final concentration of each plasmid and fibronectin was 1 mg/ml. These three solutions were injected into 4 mm-diameter ePTFE grafts respectively. Two ends of each graft were clipped, then the graft was pressed until the external walls appeared as liquid drops in that plasmids solution impregnated through the wall and entered into the fine pores of the grafts. These grafts were dried and cut into small disks (6.5 mm in diameter). As control, one graft was provided only with fibronectin TBS without any plasmid. The PTFE disks were named pCDI-hVEGF 121 -PTFE disks, pCDI-PTFE disks, pEGFP-PTFE disks, and fibronectin-PTFE disks according to the different substances they carried. Plasmid DNA release in vitro 2 PTFE disks carrying pCDI-hVEGF 121 , pCDI or pEGFP (5 samples per plasmid) were incubated in 1 ml TBS separately on rocking bed at 37 °C. Two microlitres solution was taken out from each sample at 0.5, 1, 2, 4, 8, 24, 48, 72, 120, 168 h. The concentrations of DNA released in the solution at different time were measured spectrophotometrically at 260 nm. The value of optical density was converted into DNA concentration. The controlled release curve was made using those results. HUVEC culture 2 HUVECs were harvested from freshly obtained human umbilical cord using the collagenase isolation technique described by Jaffe et al.( 1973 1973 ) and Marin et al.( 2001 2001 ) with slight modification. Briefly, the umbilical vein was rinsed with phosphate buffer solution (PBS, pH 7.4), filled with 0.1% collagenase I (Sigma, USA) and incubated for 15 min at 37 °C. After harvesting, cells were placed in 25 cm 2 tissue culture flasks (Falcon, USA). The complete growth medium consisted of 80% (v/v) MCDB131 medium and 20% (v/v) fetal bovine serum, supplemented with 100 U/ml penicillin, 100 mg/L streptomycin, 4 mmol/L L-glutamine (above substances were all purchased form GibcoBRL, USA), 50 mg/L endothelial cell growth supplement (ECGS, Sigma, USA) and 100 mg/L heparin (Sigma, USA). The cells were cultured in the medium at 37 °C in an atmosphere with 5% CO 2 . The medium was firstly changed after 8 h and then every 2 d until the cells were confluent. At confluence, cells’ passage referred to traditional trypsin-EDTA method. In this study, passage 1-3 HUVECs were used for experiments. Cell adhesion and proliferation 2 PTFE disks carrying pCDI-hVEGF 121 , pCDI, pEGFP or fibronectin were fixed to the bottom of 96-well tissue culture plates (TCP, Falcon, USA). HUVECs were seeded onto the various PTFE materials and empty 96-well TCP at a density of 25000 cells per well in 200 μl culture medium (without ECGS). The medium was first changed after 6 h resulting the removal of unattached cells, then every 2 d. At 6, 24, 72 and 120 h after seeding, the cells were detached by using 0.25% trypsin-0.02% EDTA in PBS. The number of cells was counted under hemocytometer. The cell adhesion ratio is defined as the ratio of cell number at 6 h and cell number at seeding. Five replicate samples of various PTFE materials or TCP were evaluated at each time. At 6 and 120 h after seeding, cell growth status on the different PTFE materials was observed under scanning electronic microscopy (SEM). Various PTFE disks fixed in 2.5% glutaral solution were postfixed for 1 h in 10 ml/L OsO 4 , dehydrated in a graded series of alcohol, CO 2 critical-point dried, mounted on aluminum tubs and sputter-coated with gold. Samples were examined with an SEM (Stereoscan 260, Cambridge, UK) operated at 25 kV. Determination of pEGFP gene transfection and the transfection efficiency 2 At 6, 24, 72 and 120 h after seeding, cells grown on the pEGFP-PTFE disks were detached by 0.25% trypsin-0.02% EDTA in PBS. Cell GFP expression was detected by fluorescence microscopy. The transfection efficiency was determined by counting cells expressing GFP in three non-overlapping vision fields in relation to the total cell number determined by phase contrast microscopy. Five replicate samples were evaluated at each time. Enzyme-linked immunoadsorbent assay (ELISA) of VEGF expression 2 At 6, 24, 72 and 120 h after cell seeding, secretion of VEGF in the supernatant of the cultured HUVECs on different materials was measured using ELISA kits for human VEGF (R & D, USA) and following the manufacturer’s instructions. Optical density was determined at wavelength of 450 nm, VEGF concentration was determined by comparison with standard curves. Each material had five replicated samples each time. Statistical analyses 2 Data were expressed as means±standard deviation ( SD ) and at the same time the differences between different materials were assessed by two factors analysis of variance or Kruskal-Walllis H test (SPSS V.10.0). P values less than 0.05 were considered to be statistically significant. RESULTS 1 The release rates of various plasmid DNA from PTFE materials were almost identical, the difference of various plasmid DNA concentration at the same time was not statistically significant (Fig. 1a 1a , P > 0.05). Take the cumulative release curve of pCDI-hVEGF 121 for instance (Fig. 1b 1b ), the release of pCDI-hVEGF 121 from PTFE disk in vitro was characterized by an early burst, with more than 50% total pCDI-hVEGF 121 release within the first 8 h of incubation, then the speed of release decreased obviously. Thought DNA released continuously even after 5 d, the increase of DNA release between 3~7 d had no statistical significance ( P > 0.05). Therefore, the total dose of plasmid that could be carried by one 6.5 mm diameter PTFE disk by our method was about 6 μg. The differences of HUVECs adhesion ratios at 6 h on PTFE disks carrying pCDI-hVEGF 121 , pCDI, pEGFP or fibronectin were not statistically significant (0.41±0.1, 0.43±0.1, 0.44±0.09, 0.44±0.09, respectively, P > 0.05, Fig. 2 2 ), these adhesion ratios were far lower than that on TCP (0.91±0.08, P < 0.01). At 24, 72 and 120 h, the number of HUVECs on pCDI-hVEGF 121 -PTFE disks was far more than that on pCDI/pEGFP/fibronectin-PTFE disks ( P < 0.05), but less than that on TCP ( P < 0.01). However, the proliferation rate of HUVECs in pCDI-hVEGF 121 -PTFE disks in 6~120 h was (7.56±0.82), which was not only higher than that of pCDI-PTFE disks (2.39±0.16), pEGFP-PTFE disks (2.31±0.15) and fibronectin-PTFE disks (2.36±0.14) but also higher than that of TCP (4.63±0.19) obviously ( P < 0.01, Fig. 2 2 ). The differences of cell numbers between pCDI/pEGFP/fibronectin-PTFE disks at 6, 24, 72 and 120 h were not statistically significant. # P < 0.01 compared with pCDI-hVEGF 121 -PTFE/pCDI/pEGFP/fibronectin-PTFE disks; * P < 0.05 compared with pCDI/pEGFP/fibronectin-PTFE disks Representative SEM pictures of HUVECs cultured on the pCDI-hVEGF 121 -PTFE disks and fibronectin-PTFE disks are shown in Fig. 3 3 . At 6 h, cell numbers on pCDI-hVEGF 121 -PTFE disk and fibronectin-PTFE disk were almost identical. However, at 120 h, cell number on pCDI-hVEGF 121 -PTFE disk was more than that on fibronectin-PTFE disk. Though cell morphology was not very well, with some cell clusters, and many cells did not have HUVEC’s typical spindle-shape because of absence of flow environment, these pictures generally demonstrated that HUVECs could quickly proliferate to a certain extent on the pCDI-hVEGF 121 -PTFE disk. The proportion of GFP-positive cells on pEGFP-PTFE disks is positively related to the culture time. At 6, 24, 72 and 120 h, the proportions were (1.19%±0.31%), (2.89%±0.42%), (4.78%±0.73%) and (7.14%±0.94%) respectively. Fig. 4 4 shows representative pictures of fluorescence microscopy and phase contrast microscopy at 120 h. VEGF concentrations in supernatant of the cultured HUVECs on each material at different time are shown in Table 1 1 . VEGF concentration of pCDI-hVEGF 121 group was higher than that of TCP, pCDI, pEGFP and fibronectin group ( P < 0.01). The difference of VEGF concentration between TCP, pCDI-PTFE disks, pEGFP-PTFE disks and fibronectin-PTFE disks was not statistically significant ( P > 0.05). For pCDI-hVEGF 121 -PTFE disk group, expression of VEGF by HUVECs increased continuously. The VEGF level reached 539×10 −12 g/ml at 120 h after cell culture, (23.16±1.64) times higher than that at 6 h. P < 0.01 compared with pCDI/pEGFP/fibronectin-PTFE disks and TCP at the same time P > 0.05 compared with pEGFP/fibronectin-PTFE disks and TCP at the same time Group 6 h 24 h 72 h 120 h TCP 10.72±0.74 11.06±0.90 11.44±1.13 10.8±0.70 pCDI-hVEGF 121 23.36±1.51 # # 191.84±17.49 # # 303.24±13.06 # # 539.22±16.78 # # pCDI 11.74±1.34 * * 11.5±1.22 * * 12.52±1.16 * * 11.3±1.21 * * pEGFP 11.28±1.45 11.4±1.34 12.94±1.45 11.7±1.54 Fibronectin 11.56±1.45 11.26±1.55 12.72±1.57 11.9±1.79 DISCUSSION 1 The development of a small diameter artificial vascular graft is limited by rapid thrombus formation upon blood contact and subsequent intima hyperplasia after implantation (Esquivel and Blaisdell, 1986 1986 ). Rapid and controlled endothelialization and neoarterial regeneration via perianastomotic as well as transmural tissue ingrowth play a critical role in the patency of implanted vascular grafts (Clowes et al., 1986 1986 ). Herring et al.( 1978 1978 ) studied endothelial cell seeding in synthetic vascular grafts, but the results of this method used in clinical trials are still controversial. VEGF is a strong mitogenic and migration-promoting factor for vascular endothelial cells and therefore a major initiator of angiogenesis (Ferrara and Henyel, 1989 1989 ; Leung et al., 1989 1989 ). Increased NO and prostacyclin secretion induced by VEGF is known to inhibit smooth muscle cell proliferation and platelet aggregation in animal models as well (Tsurumi et al., 1997 1997 ; Laitinen et al., 1997 1997 ; Dorafshar et al., 2003 2003 ). Some experimental and clinical studies proved that naked VEGF gene plasmids directly injected into myocardium or skeletal muscle could express VEGF protein and promote angiogenesis (Losordo et al., 1998 1998 ; Isner et al., 1996 1996 ; Shyu et al., 2003 2003 ). Recent trials showed that the transfection of VEGF gene could promote vascular endothelial cell regeneration, decrease thrombosis, reduce proliferation of muscle cells and vascular stenosis rate after arterioplasty and stent implantation (Riessen et al., 1993 1993 ; Walter et al., 2004 2004 ). In the present study, VEGF gene was employed to improve the endothelialization and patency of artificial vascular grafts. As for a carrier for VEGF gene plasmid, vascular grafts was investigated in terms of a simple and practical approach for transferring VEGF gene into the cells of the implant bed tissue, in other words, pCDI-hVEGF 121 was impregnated into the wall of vascular grafts that were used not only as reconstruction material but also as gene carrier. PTFE is a fine porous hydrophobic polymer with a highly electronegative surface for vascular graft (Esquivel and Blaisdell, 1986 1986 ). We hypothesized that VEGF gene can permeate into and be stored in the pores of PTFE with controlled release. Fibronectin was used besides a substrate matrix for cell attachment as cross-linking agent of PTFE and gene plasmid to increase the retention of the gene on the grafts. The release curve showed gene released rapidly at the beginning because of rapid dissolution of gene attached on the surface and then released slowly when gene hidden in the pores of PTFE began to release after 8 h. These results suggested PTFE vascular graft carrying plasmid DNA has controlled release effect to some extent. The growth of endothelial cells seeded on PTFE is usually very slow. However, in our experiment, HUVECs cultured on pCDI-hVEGF 121 -PTFE disks grew very fast, with a (7.56±0.82)-fold increase in cell number after 120 h, this speed of cell proliferation was not only significantly higher than that on the pCDI-PTFE disks, pEGFP-PTFE disks and fibronectin-PTFE disks but also higher than that on the TCP. ELISA verified that HUVECs cultured on pCDI-hVEGF 121 -PTFE disks produced a great quantity of VEGF protein. These results proved VEGF gene plasmid carried by PTFE materials could transfect HUVECs successfully and result in VEGF protein expression which consequently improve the proliferation of HUVECs. The GFP expression in the HUVECs cultured on pEGFP-PTFE disks also certified that plasmid DNA carried by PTFE material could transfect cells cultured on them. It had been reported that direct contact of cells with pure plasmid DNA added in the culture does not result in gene transfection in the cell (Klugherz et al., 2000 2000 ). However, in the present study, DNA transfection occurred without the use of any enhancing agent such as lipofectamine or calcium phosphate precipitation. The possible mechanisms are supposed to be: (1) PTFE materials impregnated with DNA, when in intimate contact with cell membranes, will lead directly to transfection (Klugherz et al., 2000 2000 ); (2) When DNA is mixed with fibronectin which may interact with some adhesive molecule receptors (such as integrin α ν β 3 ) on cell membranes, internalization of fibronectin-DNA complex can be realized via receptor-mediated endocytosis (Hood et al., 2002 2002 ), although the transfection efficiency of this method was much lower than that when enhancing agent was used. However, if a desired gene product is a secreted protein, the transfection of only a small percentage of target cells may result in production of a biologically relevant concentration of protein since the transfected cells can act as secretory “factories” for the protein of interest (Losordo et al., 1994 1994 ; Nabel et al., 1993 1993 ; Takeshita et al., 1996 1996 ; Pels et al., 2003 2003 ). This hypothesis is well supported by our study result (fast proliferation of HUVECs cultured on pCDI-hVEGF 121 -PTFE disks). In conclusion, we found that the PTFE graft can be used as a carrier of VEGF gene plasmid. VEGF gene releasing from the graft can transfect HUVECs and promote HUVECs proliferation in vitro successfully. In further research, we will investigate whether the VEGF gene carried by PTFE graft could transfect vascular beds tissue and promote endothelialization after graft transplantation. References JS Anderson , TM Price , SR Hanson , LA Harker . 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Neointimal hyperplasia on a cell-seeded polytetrafluoroethylene graft is promoted by transfer of tissue plasminogen activator gene and inhibited by transfer of nitric oxide synthase gene 10.1631/jzus.2006.B0421 Project supported by the Science and Technology Research Foundation of Zhejiang Province, China (No. 991110052) and the Research and Development Funds of the Second Affiliated Hospital, School of Medicine, Zhejiang University, China 2005-10-18 2006-01-12 2006-05-12 2022-01-05 Copyright © 2006, Journal of Zhejiang University Science 2006 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 06 07