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Original article
Transplantation of human umbilical cord-derived endothelial progenitor cells promotes re-endothelialization of the injured carotid artery after balloon injury in New Zealand white rabbits
HU Cheng-heng, KE Xiao, CHEN Kui, YANG Da-ya, DU Zhi-min and WU Gui-fu Keywords: endothelial progenitor cell; cell transplantation; neointimal; umbilical cord blood
Background Cell transplantation has great potential for promoting endothelial repair and reducing the complications of percutaneous coronary intervention (PCI). The aim of this study was to investigate the effect of transplantation of human umbilical cord blood endothelial progenitor cells (EPCs) on injured arteries. Methods Umbilical cord blood mononuclear cells were obtained from post-partum lying-in women, and EPCs were isolated, cultured, expanded and identified by immunofluorescence. The carotid arterial endothelium of New Zealand white rabbits was injured by dilatation with a 3F balloon, and the EPCs were injected into the lumen of the injured artery in the transplanted group (n=16), while an equal volume of phosphated buffered saline (PBS) was injected into the control group after balloon injury (n=16). The animals were sacrificed after either 2 or 4 weeks, and the grafted cells were identified by double immunofluorescence staining with human nuclear antigen (HNA) and CD31 antibodies. Arterial cross sections were analyzed by pathology, immunohistochemisty and morphometry to evaluate the reparative effects of EPCs. Proliferating cell nuclear antigen (PCNA) and transforming growth factor (TGF)-1 mRNA expression were detected by reverse transcription-polymerase chain reaction (RT-PCR). Results Fluorescence-labeled EPCs were found in the neointima. The neointimal area and the neointimal/medial area ratio were significantly lower in the transplanted group than in the control group (P <0.05). von Willebrand factor (vWF) immunohistostaining showed more VWF-positive cells in the transplanted animals than in the controls (8.752.92 vs. 4.501.77, P <0.05). Compared with the control group, the transplanted group had lower expression of PCNA mRNA (0.670.11 vs. 1.250.40, P <0.01) and higher expression of TGF-1 mRNA (1.100.21 vs. 0.820.07, P <0.05). Conclusions EPCs derived from human umbilical cord blood were successfully transplanted into injured vessels. The transplanted EPCs inhibited neointimal hyperplasia and promoted vascular re-endothelialization. Chin Med J 2013;126 (8): 1480-1485

ercutaneous coronary intervention (PCI) has become an important treatment of coronary heart disease, but it can also cause endothelial injury. Compared with bare-metal stents (BMSs), drug-eluting stents (DESs) significantly reduce the restenosis rate of target vessels, but at the same time inhibit vascular endothelial repair and delay vascular re-endothelialization, thereby increasing the stent thrombosis rate. In addition, delayed vascular endothelial repair after PCI may further increase the restenosis rate of target vessels. Cell transplantation is a new technology developed in recent years that has great potential for promoting endothelial repair and reducing the complications of PCI. In the present study, we established an acute vascular endothelial injury model using a balloon catheter to injure the endothelium of the rabbit common carotid artery, and then transplanted endothelial progenitor cells (EPCs) derived from human umbilical cord blood to investigate whether EPCs can promote re-endothelialization of injured vessels. METHODS Materials A total of 30 human umbilical cord blood samples were obtained from women with complicated pregnancies who

delivered full-term newborns in the First Affiliated Hospital of Sun Yat-san University (gestational age of 3740 weeks). Each sample contained 80100 ml of blood and was centrifuged within 6 hours. EGM-2 culture medium and endothelial growth supplement kits were purchased from Clonetics Corporation, USA. Other materials included fetal calf serum (FCS) (Invitrogen-Gibco Corporation, USA), human peripheral blood monocyte isolation solution (Tianjin TBD Ltd., China), phosphate buffered saline (PBS) solution (Wuhan Boster, Ltd., China), trypsin (Sigma, USA), mouse anti-human nuclear antigen (HNA) monoclonal antibody (Chemicon, Inc., USA), rabbit anti-human CD31
DOI: 10.3760/cma.j.issn.0366-6999.20122355 Division of Cardiology, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510080, China (Hu CH, Ke X, Chen K, Yang DY and Du ZM) Key Laboratory on Assisted Circulation, Ministry of Health, Guangzhou, Guangdong 510080, China (Wu GF) Peoples Hospital of Futian Shenzhen, Shenzhen, Guangdong 518033, China (Wu GF) Correspondence to: WU Gui-fu, Peoples Hospital of Futian Shenzhen, Shenzhen, Guangdong 518033, China (Tel: 86-20-87330775. Email: eecpchina@yahoo.com.cn) This study was supported by a grant from the National Natural Science Foundation of China (No. 81170272).

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polyclonal antibody (Abcan, Ltd., Canada), DiI-ac-LDL (Molecular Probes, Ltd., USA), FITC-UEA-I (Vector Inc., Germany), mouse anti- von Willebrand factor (vWF) antibodies (Abcan, Ltd), and 3F Fogarty catheters (Edwards Lifesciences Corporation, USA). Cell separation, culture and identification Human umbilical cord blood was diluted with PBS solution at a 1:1 ratio and then added along with human peripheral blood monocyte isolation solution in a 5:3 ratio to a 50 ml centrifuge tube. The monocytes were isolated with the Ficoll-Hypaque technique and then resuspended in EGM-2 culture medium containing 20% fetal bovine serum (FBS). Cells were pipetted into a 6-well plate that was covered with fibronectin (5 g/cm2), and incubated at 37C under 5% CO2 for 4 days. Nonadherent cells were removed with PBS buffer and adherent cells were continuously cultured. On day 7, the cultured EPCs were digested with 0.25% trypsin and resuspended in 500 l of PBS for transplantation. EPCs were identified by DiI-ac-LDL and FITC-UEA-I double staining as follows: 7-day cultured human EPCs were washed twice with PBS buffer then 0.02 mg/ml of DiI-ac-LDL solution was added and incubation was performed at 37C for 2 hours. The cells were then washed twice with PBS buffer, fixed with 4% paraformaldehyde for 15 minutes and then rinsed with PBS buffer and 0.01 mg/ml of FITC-UEA-I solution was added and incubated for 1 hour. Finally, the cells were observed under a fluorescence microscope. The differentiated EPCs were DiI-ac-LDL (red) and UEA-I (green) double positive. Animal experiments A total of 32 healthy purebred male New Zealand rabbits weighing 2.02.5 kg were provided by the Experimental Animal Center of Sun Yat-sen University, China. The rabbits were caged individually and given a normal diet. They were divided into four groups with 8 rabbits in each group; 2-week and 4-week EPC transplant groups, and 2-week and 4-week control groups (balloon injury only). Rabbits were anesthetized with 3% pentobarbital sodium (30 mg/kg) through ear vein injection. The neck hair was removed. After conventional disinfection, an anterior midline incision was made, and the right common carotid artery and internal and external carotid arteries were isolated. The distal end of the external carotid artery was ligated and the proximal common carotid artery and internal carotid artery were temporarily occluded with artery clips. The external carotid artery near the ligation was punctured using the Seldingers technique. A 3F Fogarty balloon catheter was inserted into the artery for about 3 cm, and 800 l of air was injected into the balloon. The balloon catheter was pulled back, the balloon was deflated and the catheter was advanced. The inflated balloon was pulled back a total of three times in the same arterial segment before the catheter was withdrawn. The cell suspension containing 1106 EPCs cultured in vitro was injected locally into the arterial

lumen in the two transplant groups, and the same amount of PBS buffer was injected into the arterial lumen in the two control groups. The proximal end of the external carotid artery was ligated before the injection of EPCs or PBS. After 30 minutes, the artery clips on the proximal common carotid artery and internal carotid artery were released to restore blood flow. The skin incision was closed with sutures. The animals were given intravenous heparin (0.1 IU/Kg) preoperatively to prevent coagulation and 800 000 units of penicillin postoperatively for three consecutive days to prevent infection. Progenitor cell survival and differentiation identification Four weeks after transplantation of EPCs, rabbits were sacrificed with an overdose of sodium pentobarbital. One cm of target vessel was taken and frozen. Cryosections were immediately double stained with anti-HNA antibody and anti-CD31 antibody to observe double-positive cells and their distribution under a fluorescence microscope. Neointimal measurement Animals transplanted with EPCs and control animals were sacrificed at 2 and 4 weeks after arterial injury. One cm of target vessel was fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin and sectioned at various intervals. Four sections of each vessel were randomly selected for conventional HE staining and analyzed. The following indicators were calculated with Image Proplus software; neointimal area (IA), neointimal/medial area ratio (IA/MA), and the residual lumen area (LA). Immunohistochemistry Some parts of target vessels were stained with anti-VWF antibody according to the manufacturers instructions. The positive granules appeared as brown or brown-black particles distributed in the cytoplasm. The expression of endothelial cells was quantified by the total cellular immunity specific index score (TISS = positive rate positive degree). The re-endothelialization of the arterial wall was expressed as the TISS of VWF antibody staining-positive cells per unit length of intima. RT-PCR for PCNA and TGF-1 mRNA expression Total RNA was extracted from 1 cm of vessel with Trizol, and the expression of PCNA and TGF-1 mRNA was detected by RT-PCR. The primers were designed with PRIMER3 software. For PCNA, the forward primer was 5'-CTGAGGGCTGAAGATAATGC-3', and the reverse primer was 5'-CTGTAGGAGAAAGCGGAGTG-3' (product band, 406 bp). For TGF-1, the forward primer was 5'-CCGCAACAACACCCACA-3', and the reverse primer was 5'-CGGCACCTCATCATCACTT-3' (product band, 112 bp). Rabbit glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as the internal control with a forward primer of 5'-CCTGCGACTTCAACAGTGC-3' and a reverse primer of 5'-GCCTCTCGTCCTCCTCTG-3' (product band, 210 bp). The PCNA RT-PCR reaction conditions were denatured at 94C for 3

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minutes, 94C for 30 seconds, annealing at 66C for 30 seconds, 72C for 1 minute, 35 cycles and extension at 72C for 7 minutes. The TGF-B1 RT-PCR reaction conditions were denatured at 94C for 3 minutes, 94C for 30 seconds, annealing at 60C for 30 seconds, 72C for 1 minute, 40 cycles and extension at 72C for 7 minutes. The GAPDH RT-PCR reaction conditions were denatured at 94C for 3 minutes, 94C for 30 seconds, annealing at 58C for 30 seconds, 72C for 1 minute, 30 cycles and extension at 72C for 7 minutes. Products were separated by agarose gel electrophoresis. The relative amount of each product was calculated as the ratio of the target band to the GAPDH band. Statistical analysis Statistical analysis was performed using SPSS 13.0 software (SPSS Inc., USA). The measurement data were expressed as mean standard deviation (SD) and between-group comparisons were performed using a Students unpaired t-test. A P value <0.05 was considered statistically significant. RESULTS EPC culture and identification EPCs isolated from human umbilical cord blood by the Ficoll-Hypaque technique were round in shape and suspended in EBM-2 medium. The cell viability was more than 99% by trypan blue exclusion. After 48 hours of culture, cells were adherent and had a shape that was spindle, round or irregular. The medium was changed every three to four days. After seven days, the majority of adherent cells were spindle shaped and had positive double staining for DiI-ac-LDL and FITC-UEA-I (Figure 1). The establishment and evaluation of rabbit carotid artery balloon injury model The HE staining of the right common carotid artery showed that each layer of normal common carotid artery was clearly seen. The endovascular surface was covered

by a layer of flat endothelial cells with blue stained nuclei. The intima and adventitia were clearly visible and complete. Vascular smooth muscle cells in the media were arranged neatly. However, in the balloon-injured artery, the intima was stripped and endothelial cells had disappeared (Figure 2). Effect of EPCs on neointimal hyperplasia and re-endothelialization of injured vessels Each layer of the normal common carotid artery was complete without any newly grown intima inside the lumen. At two and four weeks after surgery, the blood vessels from groups with transplantation of EPCs and the control groups showed obvious intimal proliferation, disorderly and dense intimal and medial cells and narrowed lumens. The IA, IA/MA ratio and LA were significantly decreased in the EPC transplant groups compared with the control groups (all P <0.05) (Table 1 and Figure 3). VWF was synthesized mainly by endothelial cells as a glycosylated protein and stored in Weibel-Palade bodies in the cytoplasm of endothelial cells. Therefore, it can be identified as a specific marker of vascular endothelial cells. In the present study, immunohistochemical staining showed that VWF-positive cells were distributed in the neointimal layer in both the EPC transplant group and the control group, but the number of VWF-stained cells was significantly higher in the EPC transplant group than in the control group (8.752.92 vs. 4.501.77, P <0.05) (Figure 4). In addition, there was a complete intima in the injured vessels in the EPC transplant

Figure 2. The evaluation of vascular balloon injury model (HE, original magnification 200). A: Normal blood vessels. B: Injured vessels.

Figure 1. Immunofluorescence after 7 days of culture (original magnification 100). A: Adherent cells after 7 days of culture. B: FITC-UEA-1 positive cells in green fluorescence. C: DiI-ac-LDL positive cells in red fluorescence. D: Confocal fluorescence images showed the double-positive endothelial cells as yellow. Table 1. Comparison of intimal hyperplasia of the injured common carotid artery in the EPC transplant and control groups (meanSD, mm2)
Groups EPC Control 2-week (n=7) 4-week (n=8) IA I/M LA IA I/M 0.178 0.042* 0.2810.020* 0.8060.264* 0.4960.218* 0.8000.245* 0.4150.099 0.8970.413 0.5000.238 0.7520.300 1.4330.470 * P <0.05 compared with the control group. IA: neointimal area; I/M: neointimal / medial area ratio; LA: luminal area. LA 0.8610.815* 0.4460.260

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Figure 3. Injured vessels with HE staining at 2 and 4 weeks after EPC transplantation (HE, original magnification 100). A: After 2 weeks of EPC transplantation. B: The injured vessels of the control group after 2 weeks. C: After 4 weeks of EPC transplantation. D: The injured vessels of the control group after 4 weeks.

Figure 4. The VWF immunohistochemistry of injured vessels. A: After EPCs transplantation, a complete vascular endothelial layer was formed, showing VWF positive. The neointimal hyperplasia underneath was not obvious (Original magnification 200). B: In the control group, neointimal hyperplasia was seen without VWF-positive cells to cover it (Original magnification 200). C: In EPCs transplant group, there were VWF positive cells on the intimal surface and in the neointimal layer of damaged blood vessel (Original magnification 400). D: Compared the EPCs group with the control group, the immunohistochemical scores were significantly different. Figure 5. Immunofluorescent staining (Original magnification 200). A: Green fluorescence represents vascular endothelial human CD31+ positive cells. B: Red fluorescence represents the HNA-positive endothelial cells in the same blood vessels. C: Confocal fluorescent images show double-positive EPCs in yellow, integrated into the intima of damaged blood vessel.

group without obvious neointimal hyperplasia underneath. In the control group, the intima was incomplete with obvious neointimal hyperplasia underneath. This suggested that EPCs transplanted directly into injured blood vessels differentiated into mature endothelial cells to form a fairly complete intima. The EPCs appeared to inhibit neointimal hyperplasia and promote re-endothelialization of the injured vessels. Identification of EPCs in the rabbit common carotid artery Double immunofluorescent staining with the highly sensitive and specific anti-HNA antibody and anti-CD31 antibody showed that many HNA-positive cells (red fluorescence) and CD31-positive cells (green fluorescence) were distributed in the neointima of injured vessels in the EPC group, suggesting that transplanted EPCs survived in the injured vessels. However, there were no HNA-positive cells in injured vessels in the control group. This indicated that EPCs were involved in vascular repair and re-endothelialization (Figure 5). The mRNA expression of relevant inflammatory cytokines To investigate the possible mechanisms of beneficial

EPCs transplantation, we used RT-PCR to detect the expression of relevant inflammatory cytokines. Compared with the control group, the EPC transplant group showed significantly decreased expression of PCNA mRNA in the carotid artery (0.670.11 vs. 1.250.40, P <0.01) and significantly increased expression of TGF-1 mRNA (1.100.21 vs. 0.820.07, P <0.05) (Figure 6). DISCUSSION PCI has created a new era in the treatment of coronary heart disease. However, postoperative sub-acute and late stent thrombosis, and a high restenosis rate of target vessels remain the key issues in stent implantation in interventional cardiology. Studies have shown that endothelial dysfunction is involved in the pathogenesis of coronary artery disease. 1 Therefore, accelerating re-endothelialization of injured vessels to form an intact intima is meaningful for the recovery of vascular function, the prevention of thrombosis, and the inhibition of excessive neointimal hyperplasia. 2,3 Stem cell transplantation has beco me a ma jor focus of cardiovascular disease treatment in recent years. Stem cell transplantation and its secondary effect of

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transplantation without the need for immunosuppressive agents. The transplanted cells can survive in the host and mediate angiogenesis.7,8 This has been confirmed by our previous studies by the transplantation of human EPCs to treat acute myocardial infarction in rats.9,10 Therefore, the EPCs cultured in vitro in this study were derived from human umbilical cord blood stem cells. We used an established progenitor cell culture method11,12 and identified DiI-ac-LDL+/FITC-UEA+ progenitor cells after seven days, which showed a successful induction of differentiation into endothelial progenitor cells.13 We established a model of intimal exfoliation and vascular endothelial injury using balloon angioplasty to damage the intima and smooth muscle layer of the rabbit common carotid artery. Our model of endothelial injury was very reproducible, since the contiguous pathological examination showed that the entire intima was stripped, and the subendothelial elastic membrane and medial smooth muscle layer were exposed. We used IA, I/M and LA along with other indicators to evaluate neointimal hyperplasia. Our results indicated that two weeks after transplantation, IA and I/M were significantly lower in the EPC group than in the control group (P <0.01), whereas LA was greater in the EPC group than in the control group. At four weeks after transplantation, IA was increased but LA was significantly reduced in the EPC and control groups compared with after two weeks of injury. These two indicators at four weeks were significantly different (P <0.01) between the EPC group and the control group, suggesting that transplantation of EPCs reduced neointimal hyperplasia and inhibited vascular remodeling. To evaluate the differentiation and survival of EPCs after transplantation, we used highly sensitive anti-HNA and anti-CD31 antibodies to stain the cells. We found HNA and CD31 double-positive cells in the damaged blood vessels of rabbits transplanted with EPCs, indicating that there was colonization of EPCs into the damaged blood vessels. Moreover, the results of immunohistochemical staining showed that the VWF-positive staining was significantly higher in the EPC group than in the control group. VWF-positive cells not only covered the intimal surface of the injured blood vessel to form a complete intima, but were also expressed in the neointimal layer. In the damaged blood vessels with a complete neointimal layer in the two EPC groups, the degree of neointimal hyperplasia was significantly lower than in the control groups, suggesting that transplanted EPCs could differentiate into endothelial cells in damaged vessels, form a fairly complete intima and contribute to re-endothelialization. Our results are consistent with the results of previous studies.14,15 At the present time, the mechanisms by which EPCs participate in vascular endothelial repair are unknown. Most studies have shown that the inflammatory response plays an important role in vascular remodeling after the vascular damage. Vascular inflammation is initiated after endothelial damage. The release of a large number of cytokines and the chemotaxis of various inflammatory

Figure 6. RT-PCR analysis of PCNA and TGF-1 expression. M: Marker DL500; There were a total of 7 bands from top to bottom as 500 bp, 400 bp, 300 bp, 200 bp (the brightest), 150 bp, 100 bp and 50 bp. 1: control group, 2: EPC group. A: PCNA expression. B: TGF-1 expression. The relative expression rate (RER) of PCNA to the control gene was 1:1.87 (P <0.01); the RER of TGF-1 to control gene was 1.34:1 (P <0.05), suggesting decreased PCNA mRNA expression and increased TGF-1 mRNA expression in damaged blood vessels after EPC transplantation.

re-endothelialization may become a new method to suppress atherosclerosis and to prevent restenosis after PCI.4 In recent years, it has been discovered that EPCs can directly proliferate and differentiate into the precursor cells of vascular endothelial cells that participate in the repair of injured vessels and angiogenesis. The role of these cells in the treatment of cardiovascular disease has become increasingly important.5 A number of previous studies have shown that vascular endothelium is necessary for maintaining normal homeostasis of vascular physiological function. DES implantation can lead to inflammatory responses mediated by nuclear eosinophil CD45+ cells. Furthermore, it can inevitably lead to intimal exfoliation, with the release of prothrombotic substances from the endothelium into circulating blood that could result in incomplete re-endothelialization and delayed vascular repair. Although drugs released from DESs inhibit smooth muscle cell proliferation and migration, the drugs are nonspecific. They can also inhibit endothelial cell proliferation and migration, further delaying endothelial repair. In addition, most patients with coronary heart disease undergoing PCI have multiple cardiovascular risk factors such as older age, hypertension, high blood glucose levels, and high cholesterol levels. The circulating levels of EPCs, EPC mobilization, adhesion, and vascular regeneration are significantly impaired in these patients compared to those without coronary heart disease. Therefore, based on this information, we wanted to explore whether in vitro cultured EPCs can colonize damaged endothelium and help the re-endothelialization of an arterial segment to inhibit excessive neointimal hyperplasia and restore vascular function. Asahara et al6 separated EPCs from bone marrow, but umbilical cord blood contains more cells and more primitive pluripotent stem cells and it is easy to obtain and less expensive. They also have a unique immunogenicity that can be used in xenograft

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1485 blood-derived endothelial precursor cells augment postnatal neovascularization. Clin Invest 2000; 105: 1527-1536. Goodwin HS, Bicknese AR, Chien SN. Multilineage differentiation activity by cells isolated from umbilical cord blood: expression of bone, fat, and neural markers. Biol Blood Marrow Transplant 2001; 7: 581-588. Hu CH, Li ZM, Du ZM, Wu GF, Wang XQ. Transplanted human umbilical cord blood mononuclear cells improve left ventricular function through angiogenesis in myocardial infarction. Chin Med J 2006; 119: 1499-1506. Hu CH, Li ZM, Du ZM. Human umbilical cord-derived endothelial progenitor cells promote growth cytokines-mediated neorevascularization in rat myocardial infarction. Chin Med J 2009; 122: 548-555. Gehling UM, Ergun S, Schurnaeher U. In vitro differentiation of endothelial cells from AC133-positive progenitor cells. Blood 2000; 5: 3106-3112. Murohara T, Ikeda H, Duan J. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest 2000; 105: 1527-1536. Vasa M, Fichtlscherer S, Aicher A. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Cir Res 2001; 89: E1-E7. Kauahal S, Amierl GE, Guleserian KJ. Functional small-diameter neovessels created using endothelial progenitor cell expanded ex vivo. Nat Med 2005; 7: 1035-1040. Kong D, Melo LG, Mangi AA. Enhanced inhibition of neointimal hyperplasia by genetically engineered endothelial progenitor cells. Circulation 2004; 109: 1765-1775. Saleh N, Svane B, Jensen J. Stent implantation, but not pathogen burden, is associated with plasma C-reactive protein and interleukin-6 levels after percutaneous coronary intervention in patients with stable angina pectoris. Am Heart J 2005; 149: 876-872. Ruiz-Ortega M, Rodrguez-Vita J, Sanchez Lopez E. TGF-beta signaling in vascular fibrosis. Cardiovasc Res 2007; 74: 196-206 . Flynn AW, Chen X, OConnell E, OBrien T. A comparison of the efficacy of transplantation of bone marrow derived mesenchymal stem cells and unrestricted somatic stem cells on outcome after acute myocardial infarction. Stem Cell Res Ther 2012; 3: 36. Kuraitis D, Ebadi D, Zhang P, Rizzuto E, Vulesevic B, Padavan DT, et al. Injected matrix stimulates myogenesis and regeneration of mouse skeletal muscle after ischaemic injury. Eur Cell Mater 2012; 24: 175-196. Reis LA, Borges FT, Simes MJ, Borges AA, Sinigaglia-Coimbra R, Schor N. Bone marrow-derived mesenchymal stem cells repaired but did not prevent gentamicin-induced acute kidney injury through paracrine effects in rats. PLoS One 2012; 7: e44092.

cells stimulate the over-activation, proliferation and migration of medial smooth muscle cells, leading to negative vascular remodeling and subsequent stenosis.16 PCNA is an auxiliary protein of DNA polymerase , playing an important role in the initiation of cell proliferation. Its expression is positively correlated with the proliferative activity of cells and tissues. TGF-1 is a multifunctional growth factor, widely present in various animal tissues and cells. It has a dual effect in the formation of connective tissue and smooth muscle cell proliferation.17,18 The present study showed PCNA mRNA expression was decreased but TGF-1 mRNA expression was increased in damaged vascular tissue in the EPC transplant group compared with the control group. In summary, the present study demonstrated several beneficial effects of transplantation of EPCs into rabbit carotid arteries that were damaged by balloon angioplasty. The EPCs derived from human umbilical cord blood could reduce the endothelial hyperplasia of the damaged blood vessels via acceleration of re-endothelialization and reducing the local inflammatory response in order to promote vascular repair. Combined with our previous use of EPCs to treat acute myocardial infarction in rats,6,7 we confirmed EPCs have an enormous potential in angiogenesis and vascular repair. The results of the present study provide an important experimental basis for the clinical application of EPCs in the treatment of restenosis after PCI. We anticipate transplantation of EPCs after PCI may become a new treatment option for coronary artery disease as an ideal way to improve vascular remodeling and prevent restenosis.
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(Received August 12, 2012) Edited by WANG Mou-yue