The effects of endothelial progenitor cells on rat atherosclerosis

Xianyou Wang1,2 † Feng Wang1,3,4 † Nana Li5 Min Hu6 Yun Chen7 ∗ Mengqun Tan1

1 Experimental

Hematology Laboratory, Department of Physiology, Xiang-Ya School of Medicine, Central South University, Changsha, People’s Republic of China

2 Hunan

Normal University, Changsha, People’s Republic of China

3 Department

of Physiology and Neurobiology, Xinxiang Medical University, Xinxiang, People’s Republic of China

4 Biomedical

Research Institute, Shenzhen Peking University–The Hong Kong University of Science and Technology Medical Center, Shenzhen, People’s Republic of China

5 Key

Laboratory for Medical Tissue Regeneration of Henan Province, Xinxiang Medical University, Xinxiang, People’s Republic of China

6 Hunan

Center for Disease Control and Prevention, Changsha, People’s Republic of China

7 Department

of Ultrasound, Peking University Shenzhen Hospital, Shenzhen, People’s Republic of China

Abstract Atherosclerosis (AS) is a progressive disease characterized by endothelial injury and lipid aggregation in the arterial walls. Studies have reported that endothelial progenitor cells (EPCs) derived from the bone marrow (BM) might provide an endogenous repair mechanism by differentiating into endothelial cells to replace the dysfunctional endothelium. Our study aims to investigate the effect of EPCs derived from rat BM on AS. EPCs transduced by recombinant adeno-associated virus-green fluorescent protein (GFP) were transplanted into a rat AS model. After 2 months of transplantation, the localization of GFP-labeled cells, morphology, and lipid content in the aorta were examined. GFP-labeled EPCs were found in the endothelial monolayer of the artery vessel in the GFP/EPC group. Hematoxylin and eosin staining suggested

that the lipid deposits in the aortic endothelium in the EPC/GFP group were less compared with those in the untreated group. Oil Red O staining of liver slices showed that lipid droplets were obviously decreased in the GFP/EPC group. The endothelial nitric oxide synthase and apolipoprotein E mRNA levels in the GFP/EPC group were significantly higher, but the intercellular cell adhesion molecule-1 mRNA level was significantly lower compared with the control group. The results suggest that EPCs derived from the BM can repair the injured endothelium and promote an atherosclerotic lesion regression. Therefore, EPCs may provide a useful tool for the C 2014 International Union of Biochemistry and treatment of AS.  Molecular Biology, Inc. Volume 00, Number 0, Pages 1–7, 2014

Keywords: endothelial progenitor cells, atherosclerosis, adeno-associated virus-2, gene therapy

Abbreviations: AAV, adeno-associated virus; ApoE, apolipoprotein E; AS, atherosclerosis; BM, bone marrow; eNOS, endothelial nitric oxide synthase; EPCs, endothelial progenitor cells; GFP, green fluorescent protein; HDL-C, high-density lipoprotein cholesterol; H&E, hematoxylin and eosin; ICAM-1, intercellular cell adhesion molecule-1; MNCs, mononuclear cells; SD, Sprague–Dawley; TC, total cholesterol; TG, triglyceride.

Xiang-Ya Road, Changsha, Hunan 410078, People’s Republic of China. Tel.: +86-731-82355051; Fax: +86-731-82355056; e-mail: [email protected]. †These authors contributed equally to this work. Received 18 October 2012; accepted 23 May 2014

∗ Address for correspondence: Mengqun Tan, PhD, Department of Physiology, Experimental Hematology Laboratory, Xiang-Ya School of Medicine, Central South University, School Building 2, Room 315, 110

DOI: 10.1002/bab.1254 Published online in Wiley Online Library (wileyonlinelibrary.com)

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1. Introduction Atherosclerosis (AS) is responsible for a significant part of morbidity and mortality all over the world. Currently available therapeutics for AS patients is inadequate, so the development of novel therapies is needed. AS is a disorder with endothelial dysfunction. The integrity and functional activity of the endothelial monolayer play an important role in the prevention of AS [1, 2]. Endothelial dysfunction and injury are considered to be the first steps in atherogenesis [3, 4]. Recent studies indicated that endothelial progenitor cells (EPCs) derived from the bone marrow (BM) have a characteristic of homing to the injured endothelium and upon arrival at the injured endothelial layer, EPCs may differentiate into the endothelial cells (ECs), proliferate, and replace the injured ECs [5, 6]. Published data from animal studies have revealed that EPCs effectively contribute to the restoration of endothelial function and in diminishing neointimal formation after arterial injury. Moreover, AS could be inhibited by locally delivered EPCs into the carotid artery with the injured endothelium [6, 7]. In our study, to evaluate the potential roles of EPCs in the restoration of endothelial injury and the prevention of atherosclerotic plaque formation, EPCs transduced by recombinant adeno-associated virus (rAAV)-green fluorescent protein (GFP) were transplanted into a diet-induced AS rat model. AAV is one of the most promising viral vectors for gene delivery [8]. A major attraction of AAV is that it mediates long-term gene expression in numerous dividing or nondividing cells [9]. Over the past several years, recombinant AAV vectors have been extensively used to deliver a variety of transgenes with little or no toxicity and inflammation [10, 11]. In the present study, we attempt to investigate whether EPCs transduced by rAAV-GFP are capable of homing to the injured endothelium, and whether the local AS formation can be prevented by the administration of EPCs.

2. Materials and Methods The Animal Care and Use Committee of the Central South University approved the present animal experiment. A total of 28 Sprague–Dawley (SD) rats (male, 200–300 g) were recruited for the study.

2.1. Preparation of virus rAAV-2 vectors were prepared by using the AAV HelperFree System (Agilent Technologies, Santa Clara, CA, USA). pAAV-hrGFP, along with the pAAV-RC and the pHelper, was cotransfected into 293 cells (human embryonic kidney cells) using a calcium-phosphate-based protocol. Three days later, the 293 transfected cells were collected. The rAAV vectors were purified by single-step column purification of rAAVGFP by gravity flow based on its affinity to heparin [12]. The virus titer of the particle was determined by quantitative DNA dot-blot hybridization.

Plus (Sigma, St. Louis, MO, USA). These isolated MNCs were resuspended in a complete endothelial basal medium (EBM2, Clonetics, San Diego, CA, USA) containing 10 ng/mL of endothelial growth factor and 10% fetal bovine serum, and were then immediately plated on culture dishes coated with fibronectin (Sigma). After culturing at 37 ◦ C with 5% CO2 for 4 days, the nonadherent cells were removed, and a fresh medium was applied to the culture, which was maintained through day 7. To study the characteristics of the ECs, the cultured cells for 7 days were analyzed for morphology with immunohistochemical staining using antibodies against EC markers such as vWF, CD31, and CD144 (Cell Sciences, Canton, MA, USA). In addition, the uptake of red fluorescence labeled as acetylated low-density lipoprotein (Dil-acLDL, Molecular Probes, Eugene, OR, USA) and staining with FITC-conjugated UEA-1-lectin (Sigma, 10 µg/mL) were used to assess the EPC identification. The EC colonies were observed when the cells were plated at a low density.

AAV-mediated GFP transfection to EPCs EPCs at approximately 80% confluence in six-well culture plates were washed three times with phosphate buffered saline (PBS) and exposed to 200 µL of AAV-GFP (multiplicity of infection [MOI], 100). After 2 H of incubation, the cells were washed with PBS and a new medium was applied. After 60 H, the cells were observed under an inverted fluorescence microscope to determine the transduction efficiency.

The establishment of the AS rat model A total of 28 healthy male SD rats (200–300 g) were randomly divided into three groups. For 3 months, the control group (A) rats were fed with basic food, whereas in other groups, the rats were loaded with a high-fat diet. The high-fat diet contained 77.8% normal chow, 10% lard, 1% cholesterol, 10% egg yolk powder, 0.5% multivitamin, 0.2% propylthiouracil, and 0.5% chleolate. Plasma levels of total cholesterol (TC) and triglyceride (TG) were analyzed using enzymatic methods (Thermo Fisher Scientific, Vantaa, Finland). High-density lipoprotein cholesterol (HDL-C) was measured after magnesium chloride dextran sulfate precipitation from the supernatant using the enzymatic method (Thermo Fisher Scientific). Blood samples were collected overnight and were centrifuged at 3,000g for 15 Min at 4 ◦ C on the next day. The pathological changes in the

Experimental groups

TABLE 1 Group

Treatment

A

Control

PBS

EPC culture and characterization

B

Untreated

PBS

Mononuclear cells (MNCs) were isolated from a rat femur BM via density gradient centrifugation using Ficoll-Paque

C

GFP/EPC

EPCs transduced with AAV-GFP

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The Effects of EPCs on Atherosclerosis

FIG. 1

Characterization of EPCs derived from rat BM in vitro. (A) Monolayer of EPCs with a cobblestone morphology after 7–10 days in culture. (B) Wright’s Giemsa staining of EPCs. (C) Uptake of acetylated LDL. (D) Binding to FITC-labeled lectin. Immunofluorescence staining with anti-rat vWF, CD31, and CD144 antibody was positive in EPCs (E, F, and G, respectively) and negative in the negative control (H, fibroblast). Scale bar: 50 μm for panels (A–C) and (E–H); 25 μm for panel (D).

aorta were observed after 3 months. All experimental protocols were approved by Health and Anti-Epidemic Station of the Hunan province.

2.2. Experimental groups Three months after being fed with a high-fat diet, each group (n = 9) was given treatment as shown in Table 1 (A: normal diet, B and C: high-fat diet). No rat died during the experimentation. Rat BM-derived EPCs from the same strain (EPCs 2 × 106 cells/1 mL PBS) were injected via the tail vein.

Tissue collection and preparation The animals were sacrificed by intravenous injection of potassium chloride 2 months after cell transplantation. The aorta and liver were harvested and divided into two parts. One part was fixed overnight in 4% paraformaldehyde and embedded in paraffin for hematoxylin and eosin (H&E) staining and immunohistochemistry staining. The other part was snap frozen in liquid nitrogen and stored at –80 ◦ C for reverse transcription polymerase chain reaction (RT-PCR) and cryosectioning analysis.

tocols for the HE staining. The specimens were examined using an AXIOVET-40 inverted microscope (Zeiss, Jena, Germany). To examine the expression of the GFP, the tissues stored at –80 ◦ C were embedded in an optimal cutting temperature compound at –20 ◦ C. Sections (10 µm) were cut on a cryostat and placed on glass slides. GFP expressions were observed using a Zeiss inverted fluorescence microscope. Tissue sections of the liver were incubated in 100% propylene glycol for 5 Min and then stained with 0.5% of Oil Red O in propylene glycol (Sigma) for approximately 72 H at 40 ◦ C. Then, the tissue sections were washed with 85% propylene glycol in bidistilled water for 2 Min followed by bidistilled water for 4–5 Min. Finally, the tissue sections were counterstained with hematoxylin and mounted in Kaiser’s glycerol gelatin (Merck, Darmstadt, Germany).

RT-PCR analysis The mRNA expression of the endothelial nitric oxide synthase (eNOS), intercellular cell adhesion molecule-1 (ICAM1), and apolipoprotein E (ApoE) was assessed by (RT-PCR) using the following primers: eNOS primer 1 (forward) 5 AGAGCATACCCGCACTTCTG-3 and primer 2 (reverse) 5 GGAAGTAAGTG-zAGAGCCTGG-3 ; ICAM-1 primer 3 (forward) 5 -AGGTATCCATCCATCCCACA-3 and primer 4 (reverse) 5 -AGTGTCTCATTCCCACGGA-3 ; ApoE primer 5 (forward) 5 -CCAGGCTTTGAGTGACCG-3 and primer 6 (reverse) 5 -CGTTGCG-GTTTCCTCTTC-3 ; β-actin primer 7 (forward) 5 -CGTTGACATCCGTAAAGA-3 and primer8 (reverse) 5 AGCCACCAATCCACACAG-3 . RT-PCR was performed on a sample of all animals in each group and at least three times for each animal.

Histological analysis The aorta were fixed in a 4% paraformaldehyde solution and embedded in paraffin. Cross-sections of the artery were cut to a thickness of 5 µm and stained according to the standard pro-

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Statistical analysis Results are expressed as mean ± SD. Comparisons between groups were analyzed by one-way analysis of variance and

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FIG. 2

Morphous of rat BM-derived ECs in different culture time primary EPCs were trypsinized and assessed for clonogenic potential capacity by single cells replating assay. (A) Day 4 after replating, (B) day 8 after replating, and (C) day 12 after replating. Scale bar: 50 μm.

FIG. 4

FIG. 3

EPCs transfected with rAAV-GFP. At 60 h posttransfection, the expression of GFP was detected by fluorescence microscopy. Scale bar: 100 μm.

Effects of high-fat diet on rat plasma lipids

TABLE 2 Group

P values < 0.05 were considered statistically significant. All analyses were performed with the SPSS13.0 software.

H&E staining of the rat artery of each group 3 months after high-fat diet. (A) Normal diet group and (B) high-fat diet group. Atherosclerotic lesions are shown in the high-fat diet group (B). Scale bar: 50 μm.

TG

TC

HDL-C

Normal diet

0.78 ± 0.29

1.87 ± 0.30

1.77 ± 0.37

High-fat diet

1.82 ± 0.43*

6.64 ± 0.58**

2.43 ± 0.77*

Date are expressed as means ± SD. *P < 0.05,**P < 0.01 versus the normal diet group.

3. Results 3.1. Characterization of EPCs Under the culture conditions used in the present study, the adherent cells with cobblestone morphology emerged approximately 7–10 days after plating (Figs. 1A and 1B). After 2 weeks of culture, almost all of the cells had the ability to take up Dil-acLDL and bind to lectin (Figs. 1C and 1D). More than 90% of the cells expressed vWF, CD31, and CD144 (Figs. 1E– 1G), which are generally accepted as the markers of EPCs. The cells could be passaged several times without discernible alterations in morphology and growth characteristics. In addition, EC colonies were formed when the cells were plated at a low density and the colonies became larger with time (Fig. 2).

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3.2. AAV-mediated GFP transfected EPC We performed transduction of EPCs using AAV-GFP in vitro. EPCs were transduced with 200 µL of AAV-GFP (100 MOI). After 60 H, the expression of the transgenes was detected under the inverted fluorescence microscope (Fig. 3).

3.3. High-fat-diet-induced rat AS model The rat AS model was established by feeding with a high-fat diet for 3 months. The AS plaque from the aorta could be observed in high-fat diet rats (Fig. 4). The TC, TG, and HDL-C levels of high-fat diet rats were significantly higher than those of normal diet rats (P < 0.05) (Table 2).

The Effects of EPCs on Atherosclerosis

FIG. 5

Histological assessment of the arteries 2 months after the transplantation. (A–C) H&E staining of rat artery of each group. (A) Control group, (B) untreated group, and (C) GFP/EPC group. Atherosclerotic lesions are shown in the untreated group (B). (D and E) Fluorescence photomicrograph of rat carotid artery frozen sections; EGFP-expressing ECs are shown on the endothelium of aorta in the GFP/EPC group (D). No EGFP was detected in the control group (E). Scale bar: 100 μm for (D) and (E); 50 μm for (A–C).

3.4. EPCs reduced AS lesion formation induced by high-fat diet To determine whether the transplanted EPCs were localized at the athero lesion, direct fluorescence microscopy was used to detect the presence of GFP in the cryosections of the arteries. Two months after the transplantation, GFP-labeled EPCs were detectable on the endothelium of aorta in the GFP/EPC group (Fig. 5D), but undetectable in the control group (Fig. 5E). The rats under a high-fat diet for 5 months developed noticeable AS lesion (Fig. 5B). Transplantation of EPCs after 2 months of high-fat diet dramatically reduced lesion formation (Fig. 5C).

transplantation (Figs. 7A–7C). The values were expressed with the corresponding optical density (OD) value. Both the eNOS and ApoE mRNA expressions in the untreated group were significantly lower than those of the control group and GFP/EPC group indicating that eNOS and ApoE expressions were downregulated in high-fat-diet-induced AS lesion (Figs. 7A and 7B, Table 3). Transplantation of EPCs significantly elevated both the eNOS and ApoE expressions. The ICAM-1 mRNA expression was increased in high-fat-diet-induced AS lesion. However, the transplantation of EPCs significantly reduced the ICAM-1 expression at the lesion (Fig. 7C, Table 3).

4. Discussion Endothelial dysfunction is an early indication of atherosclerotic disease. Several studies have shown that EPCs contribute to the reendothelialization of the injured vessels and have a potentially protective role in endothelial dysfunction and in the early stages of AS formation [13, 14]. Evidence suggests that EPCs are mobilized from the BM into the peripheral blood in response to tissue ischemia or injury [15]; these cells migrate

3.5. EPCs alleviate high-fat-diet-induced hepatosteatosis Lipid accumulation of the liver was also examined microscopically following the staining of the tissues with Oil Red O, which detects deposits of neutral lipids. Oil Red O positive droplets were clearly detected within the untreated group and obviously decreased in the GFP/EPC group (Fig. 6).

3.6. EPCs increased mRNA expression of eNOS and ApoE and decreased expression of ICAM The expression of eNOS, ICAM-1, and ApoE genes at the mRNA level was evaluated by RT-PCR 2 months after the

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TABLE 3

Expression of eNOS, ApoE, and ICAM-1 mRNA in artery wall of each group

Group

eNOS/β-actin

APOE/β-actin

ICAM-1/β-actin

Control

1.73 ± 0.18

0.78 ± 0.03

0.65 ± 0.05

Untreated

0.54 ± 0.10

0.51 ± 0.04

0.73 ± 0.03*

GFP/EPC

1.03 ± 0.02#

1.39 ± 0.02*#

0.31 ± 0.02*#

*

*

Date are expressed as means ± SD. *P < 0.05 versus the control group, # P < 0.05 versus the untreated group.

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FIG. 6

FIG. 7

Oil Red O staining of liver slices. (A) Control group, (B) untreated group, and (C) GFP/EPC group. Oil Red O positive droplets were clearly detected within the untreated group (B), and were undetected in the control group and GFP/EPC group (A and C ). Scale bar: 100 μm for (A–C).

The effects on the aortic gene expression 2 months after the transplantation. (A) The expression of eNOS mRNA by RT-PCR. The size of the PCR products for eNOS and β-actin was 625 and 173 bp, respectively. (B) The expression of ApoE mRNA by RT-PCR. The size of the PCR products for ApoE and β-actin was 594 and 173 bp, respectively. (C) The expression of ICAM-1 mRNA by RT-PCR. The size of the PCR products for ICAM-1 and β-actin was 388 and 173 bp, respectively. Lane 1: 100 bp marker; Lane 2: control group (group A); Lane 3: untreated group (group B); Lane 4: GFP/EPC group (group C).

to sites of damaged endothelium and differentiate into ECs [16, 17], thereby improving blood flow and tissue repair [18]. EPCs can find their way to the injured endothelium via a complex signaling network for reendothelialization [19]. A crucial goal in the treatment and prevention of cardiovascular diseases is the promotion of reendothelialization [20, 21]. Since EPCs play a critical role in maintaining an intact and functional endothelium [22], regeneration of ECs either by endogenous or transfused progenitor cells may be an important and novel therapeutic approach for the management of vascular injury in the early stages of AS [23]. However, George et al. demonstrated that transfer of BM cells or EPCs may result in an increase in atherosclerotic lesion size and might also potentially influence plaque stability [24]. Possible reasons for such

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discrepancy are the different time points and different models found in these studies. These results suggest that EPCs may play an important role in the early-stage AS formation. Therefore, it would be interesting to study the function of EPCs on the formation of atherosclerotic plaques with different methods. EPCs can be localized in the BM and peripheral blood or can reside in the arterial wall. EPCs show cobblestone morphology and exhibit high capacity of proliferation. In addition, they can be passaged several times without discernible alterations in cell morphology and growth characteristics. In our study, EPCs were cultured from rat BM. The cultured cells showed cobblestone morphology and displayed a high proliferative potential when we observed the formation of the colonies. Their phenotype was further confirmed by the expression of EC markers (vWF, CD31, and CD144), ac-LDL uptake, and binding to UEA-1-lectin. To evaluate the role of EPC in preventing atherosclerotic lesion formation in vivo, a high-fat-diet-induced rat AS model was used. Using a high-fat diet to feed SD rats for 3 months induced athero lesion formation and hyperlipidemia. In this experiment, EPCs were transfected with GFP by AAV. The expression of GFP is used to track the viability and behavior of the transplanted cells in a host organ in vivo. Two months after the transplantation, EPCs localized in the athero lesion and GFP-expressing ECs were detectable on the endothelium of the aorta in the GFP/EPC group. Transplantation of EPCs

The Effects of EPCs on Atherosclerosis

and differentiation of EPCs to ECs in the lesion increased the expression of eNOS and ApoE, and decreased the expression of intercellular adhesion molecule (ICAM-1), resulting in the reduced atherosclerotic plaque formation. The first stage of AS is considered to be endothelial dysfunction accompanied with the changes of gene expression including eNOS, ICAM-1, and ApoE. ECs are able to produce both vasoconstrictive and vasodilating substances. One of the most important regulatory and vasoactive substances produced by the ECs is nitric oxide (NO). NO is produced in the endothelium by eNOS [25]. The development of AS is, however, accompanied by an early deficit of NO and a decrease in the expression of eNOS [26]. Adhesion molecules such as ICAM-1 play a significant role in the process of AS [27]. While ICAM-1 is constitutively expressed on resting ECs, it is upregulated by proatherogene factors. Evidences showed that the EC dysfunctional/activation changes also include upregulation of leukocyte adhesion molecules, including ICAM-1 and vascular-cell adhesion molecule 1 [28]. In our study, the reversal of the ICAM-1 and eNOS genes’ expression after EPC transplantation demonstrates the EC function being restored. Human ApoE is a 34 kDa polymorphic plasma protein, which plays a key role in clearing remnant lipoproteins and delivering the intracellular cholesterol from peripheral tissues to the liver [29]. ApoE is not only synthesized by the liver, but also in the brain and by resident macrophages in the atherosclerotic wall, where it exerts atheroprotective actions independent of its role in lipid metabolism [30]. Hyperlipidemia due to ApoE deficiency results in a lower number of EPCs in the blood, which is consequently correlated with enhanced AS. The wild-type BM transplantation results in a restoration of ApoE expression, normalization of plasma cholesterol, and substantial reduction of atherosclerotic lesions in ApoE in mice with irradiated BM [31]. Although the mechanisms involved are still unclear, EPCs seem to contribute to the restoration of the endothelial monolayer [32–34]. Our results indicate that the change of the expressions of eNOS, ApoE, and ICAM1 in the lesion is due to the localization of EPCs and differentiation of EPCs to ECs at the lesion. These changes of gene expression resulted in reduction of atherolesion formation. Thus, EPCs transplantation may represent a very promising atheroprotective treatment option.

5. Acknowledgement This work was supported by grants from the National Natural Science Foundation (grant numbers 81341097, U1204810, and 81101048) and Shenzhen Basic Research Program (grant number JCYJ20120616144352139). The funding sources had no further role in the study design, data collection, analysis, interpretation, writing of the report, and decision to submit the paper for publication. The authors state that they do not have any conflict of interest (financial or otherwise) related to the data presented in this article.

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6. References [1] Fadini, G. P., Agostini, C., Sartore, S., and Avogaro, A. (2007) Atherosclerosis 194, 46–54. [2] Hamed, S., and Roguin, A. (2006) Harefuah 145, 358–361, 397. [3] Liu, J. T., Chen, Y. L., Chen, W. C., Chen, H. Y., Lin, Y. W., Wang, S. H., Man, K. M., Wan, H. M., Yin, W. H., Liu, P. L., and Chen, Y. H. (2012) J. Biomed. Biotechnol. 2012, 871272. [4] Zaragoza, C., Gomez-Guerrero, C., Martin-Ventura, J. L., Blanco-Colio, L., Lavin B., Mallavia B., Tarin C., Mas S., Ortiz, A., and Egido, J. (2011) J. Biomed. Biotechnol. 2011, 497841. [5] Lin, C. P., Lin, F. Y., Huang, P. H., Chen, Y. L., Chen, W. C., Chen, H. Y., Huang, Y. C., Liao, W. L., Huang, H. C., Liu, P. L., and Chen, Y. H. (2013) Biomed. Res. Int. 2013, 845037. [6] Kirton, J. P., and Xu, Q. (2010) Microvasc. Res. 79, 193–199. [7] Du, F., Zhou, J., Gong, R., Huang, X., Pansuria, M., Virtue, A., Li, X., Wang, H., and Yang, X. F. (2012) Front. Biosci. 17, 2327–2349. [8] Mount, J. D., Herzog, R. W., Tillson, D. M., Goodman, S. A., Robinson, N., McCleland, M. L., Bellinger, D., Nichols, T. C., Arruda, V. R., Lothrop, C. D., and High, K. A. (2002) Blood 99, 2670–2676. [9] Ellis, B. L., Hirsch, M. L., Barker, J. C., Connelly, J. P., Steininger, R. J., 3rd., and Porteus, M. H. (2013) Virol. J. 10, 74. [10] Chen, C., Akerstrom, V., Baus, J., Lan, M. S., and Breslin, M. B. (2013) Virol. J. 10, 86. [11] Manfredsson, F. P., Okun, M. S., and Mandel, R. J. (2009) Curr. Gene. Ther. 9, 375–388. [12] Auricchio, A., Hildinger, M., O’Connor, E., Gao, G. P., and Wilson, J. M. (2001) Hum. Gene. Ther. 12, 71–76. [13] Dzau, V. J., Gnecchi, M., Pachori, A. S., Morello, F., and Melo, L. G. (2005) Hypertension 46, 7–18. [14] Werner, N., and Nickenig, G. (2006) J. Cell. Mol. Med. 10, 318–332. [15] Aicher, A., Zeiher, A. M., and Dimmeler, S. (2005) Hypertension 45, 321– 325. [16] Kirton, J. P., and Xu, Q. (2010) Microvasc. Res. 79, 193–199. [17] Motwani, M. S., Rafiei, Y., Tzifa, A., and Seifalian, A. M. (2011) Biotechnol. Appl. Biochem. 58, 2–13. [18] Reinders, M. E. J., Rabelink, T. J., and Briscoe, D. M. (2006) J. Am. Soc. Nephrol. 17, 932–942. [19] Lapidot, T., Dar A., and Kollet, O. (2005) Blood 106, 1901–1910. [20] Drachman, D. E., and Simon, D. I. (2005) Curr. Atheroscler. Rep. 7, 44–49. [21] Di Napoli, M., and Papa, F. (2005) Curr. Hypertens. Rep. 7, 44–51. [22] Urbich, C., and Dimmeler, S. (2004) Circ. Res. 95, 343–353. ¨ [23] Werner, N., Priller, J., Laufs, U., Endres, M., Bohm, M., Dirnagl, U., and Nickenig, G. (2004) Arterioscler. Thromb. Vasc. Biol. 22, 1567–1572. [24] George, J., Afek, A., Abashidze, A., Shmilovich, H., Deutsch, V., Kopolovich, J., Miller, H., and Keren, G. (2005) Arterioscler. Thromb. Vasc. Biol. 25, 2636–2641. [25] Govers, R., and Rabelink, T. J. (2001) Am. J. Physiol. Renal. Physiol. 280, F193–F206. [26] Ignarro, L. J., and Napoli, C. (2005) Curr. Diab. Rep. 5, 17–23. [27] Nakashima, Y., Raines, E. W., Plump, A. S., Breslow, J. L., and Ross, R. (1998) Arterioscler. Thromb. Vasc. Biol. 18, 842–851. [28] Du, F., Zhou, J., Gong, R., Huang, X., Pansuria, M., Virtue, A., Li, X., Wang, H., and Yang, X. F. (2012) Front. Biosci. 17, 2327–2349. [29] Greenow, K., Pearce, N. J., and Ramji, D. P. (2005) J. Mol. Med. 83, 329– 342. [30] Yamada, N., Inoue, I., Kawamura, M., Harada, K., Watanabe, Y., Shimano, H., Gotoda, T., Shimada, M., Kohzaki, K., Tsukada, T., Shiomi, M., Watanabe, Y., and Yazaki, Y. (1992) J. Clin. Invest. 89, 706–711. [31] Sakai, Y., Kim, D. K., Iwasa, S., Liang, J., Watanabe, T., Onodera, M., and Nakauchi, H. (2002) Atherosclerosis 161, 27–34. [32] Torsney, E., Mandal, K., Halliday, A., Jahangiri, M., and Xu, Q.. (2007) Atherosclerosis 191, 259–264 [33] Lesnik, P., and Chapman, M. J. (2006) Arterioscler. Thromb. Vasc. Biol. 26, 965–967. [34] Werner, N., and Nickenig, G. (2006) J. Cell Mol. Med. 10, 318–332.

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The effects of endothelial progenitor cells on rat atherosclerosis.

Atherosclerosis (AS) is a progressive disease characterized by endothelial injury and lipid aggregation in the arterial walls. Studies have reported t...
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