beneficial effects of granulocyte-colony stimulating factor on small-diameter heparin immobilized...

Beneficial effects of granulocyte-colony stimulating factor on small-diameter heparin immobilized decellularized vascular graft

Min Zhou,1 Zhao Liu,1 Kun Li,1 Wei Qiao,1 Xuefeng Jiang,2 Feng Ran,1 Tong Qiao,1 Changjian Liu1

1Department of Vascular Surgery, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School,

Nanjing 210008, People’s Republic of China2College of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, People’s Republic of China

Received 15 January 2010; revised 8 April 2010; accepted 21 April 2010

Published online 19 August 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.32864

Abstract: Autologous recellularization of decellularized scaf-

folds is a promising challenge in the field of tissue-engi-

neered vascular graft and could be boosted by endothelial

progenitor cells (EPCs). The purpose of this study was to

examine the effects of granulocyte-colony stimulating factor

(G-CSF) treatment on this process. Heparin immobilized decel-

lularized grafts were fabricated and implanted into 48 rats, of

which 25 rats received G-CSF (50 ug/kg/day) for 14 days after

operation (G-CSF group) and other 23 received saline serving

as control. Five animals of each group were euthanized at 2

weeks for analysis of early graft endothelialization; whereas

the rest were investigated by Doppler ultrasound to monitor

the graft patency rate up to 6 months. After 14 days of G-CSF

administration, the number of CDþ34/CD

þ133 progenitor cells was

increased by 16.2 folds, and endothelial cell-specific immuno-

staining revealed an enhancement of early endothelialization

in G-CSF group. After 6 months of implantation, the G-CSF

treated grafts exhibited a significantly smaller hyperplastic

neointima area compared with the controls, not only at mid-

portion (0.38 6 0.02 vs. 0.47 6 0.07 mm2, p < 0.0001), but also

at distal anastomosis (0.42 6 0.04 vs. 0.70 6 0.13 mm2, p <

0.0001). Moreover, G-CSF treated grafts had a higher patency

rate compared with the control animals (19/20 vs. 12/18, p ¼0.005). In conclusion, G-CSF-induced mobilization of circulat-

ing EPCs regenerated endothelium and inhibited neointimal

hyperplasia of small-diameter heparinized decellularized vas-

cular graft. This cytokine therapy may be a feasible strategy

for the improvement of patency rate of the novel allogeneic

graft. VC 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 95A:

600–610, 2010.

Key Words: decellularized, granulocyte-colony stimulating

factor, endothelialization, neointimal hyperplasia, small-

diameter

INTRODUCTION

Atherosclerosis and heart diseases are still the leadingcauses of morbidity and mortality worldwide. Therapies forcoronary artery and peripheral vascular diseases oftenrequire replacement of the damaged vessels with vasculargrafts. For reconstruction of large arteries, such as the aortaor iliac artery, the current commercial grafts made fromexpanded polytetrafluoroethylene or Dacron are used satis-factorily. However, commercial grafts are not suitable forreconstruction of smaller diameter arteries (<5 mm diame-ter), because of thrombosis, limited endothelialization, andneointimal hyperplasia.1 Therefore, autologous vessels areroutinely used for such reconstruction procedures. Unfortu-nately, approximately 40% of patients who require vascularbypass may not have suitable vessels for successful sur-gery.2 Although considerable research has focused on thedevelopment of novel small-diameter vascular graft formany years, there is still no adequate alternative to the au-tologous vessels.3

Recently, a promising new approach for the engineeringof small-diameter vascular grafts is the use of allogeneicdecellularized vessels that are totally depleted of cellularantigens. Studies provide evidence that these biological scaf-folds have superior adhesive properties for endothelial cells(ECs), as well as mechanical and hemodynamic propertiesresembling natural transplants.4 Several recent studies havefocused on the creation of living vascular tissue replace-ments prepared by recellularizing cardiovascular allograftswith host cells before implantation.5–8 However, reseedingprocedures may only be appropriate for patients withplanned surgery because suitable implants cannot be proc-essed in a short time. The alternative research pathway isto enhance the process of migration and proliferation ofECs, so that the ‘‘spontaneous’’ endothelialization wouldoccur in vivo which may be a more appropriate concept inthe clinical setting. As we know, ECs are able to preventthrombosis and secrete many antiproliferative factors, suchas nitric oxide and vascular natriuretic peptides, which

Correspondence to: C. Liu; e-mail: [email protected]

Contract grant sponsor: Important Science Project of Jiangsu Health; contract grant number: K200609

Contract grant sponsor: Key Research Program for Scientific and Technological Development of Municipal Medical Sciences of Nanjing City;

contract grant number: ZKX0012

600 VC 2010 WILEY PERIODICALS, INC.

modulate vascular smooth muscle cells (VSMCs) growth.9

The lack of ECs might promote abnormal vascular growth,such as thrombosis, hyperplasia, and obstruction. On thecontrary, the presence of normal ECs might lead to the nor-mal growth of VSMCs, and normal vascular regeneration inthe long run.

More recently, studies evidenced that mature ECs andimmature endothelial-like cells can float in circulating pe-ripheral blood.10–12 Mature ECs are derived from endothe-lial progenitor cells (EPCs), which fall out from the bonemarrow (BM), are expected to have much higher prolifera-tive potential than mature ECs.11,12 The granulocyte-colonystimulating factor (G-CSF), a member of hematopoietic cyto-kines, is clinically used to mobilize progenitor cells from BMand increase them in the circulation. Quite a few reportshave shown that G-CSF stimulates EPCs out from BM to thesites of EC-denuded vessels, and accelerates the recovery ofendothelial integrity, resulting in the inhibition of neointimalhyperplasia and restoration of vasodilatation activity.13–17

Specifically, the possible use of G-CSF in vascular graft hasbeen recently verified experimentally by Bhattacharyaet al.18 and Shi et al.,19 who demonstrated that G-CSFenhanced endothelialization of small-diameter nondegrad-able polymer vascular grafts with no cell seeding in caninemodels. Therefore, we hypothesized that G-CSF-induced mo-bilization of circulating EPCs (CEPCs) may provide a poten-tially effective strategy to enhance early endothelializationand inhibit neointimal hyperplasia of small-diameter decel-lularized graft in vivo.

For that purpose, a novel small-diameter heparin-immo-bilized decellularized vascular graft was fabricated andimplanted into rat infrarenal abdominal aorta. After that, weevaluated the efficacy of postoperative short-term G-CSFtreatment as a strategy for increasing the CEPCs, promotingendothelialization and subsequently inhibiting neointimalhyperplasia. Furthermore, the ability of G-CSF was alsotested through long-term patency rate of the heparinizeddecellularized grafts, which will eventually be needed todemonstrate its clinical applicability.

MATERIALS AND METHODS

Fabrication and characterization of decellularized graftAbdominal aorta’s grafts were harvested from adultSprague–Dawley rats (250–300 g body weight). Meticulousdissection under microscopic visualization was performedto remove the perivascular fat and connective tissue fromthe graft. Then they were immediately stored in Hanks’ bal-anced salt solution (HBSS, Biochrom, Berlin, Germany) at4�C. Decellularization was performed as previouslydescribed.6 Briefly, the vessels were placed into a decellula-rizing solution containing 1% Triton X-100 and 0.1% ammo-nium hydroxide (Sigma, St. Louis, MO) in phosphate buf-fered saline (PBS) and placed on a mechanical shaker (120revolutions per min) at 4�C for 72 h. The solution waschanged every 24 h.

To evaluate the effect of decellularization, a segmentfrom the scaffolds were fixed in neutral buffered 10% for-malin, embedded in paraffin and sliced for hematoxylin-

eosin (H&E) staining, and sliced samples were also incu-bated with horseradish peroxidase (HRP) -conjugated anti-mouse major histocompatibility complex class I (MHC I)antibody (Biomeda), and followed by visualization with 3,30-diaminobenzidine (DAB).

Heparin immobilization and releaseCovalent immobilization of heparin to decellularized vascu-lar graft was performed using 1-ethyl-3-(3-dimethyl amino-propyl) carbodiimide (EDC) and N-hydroxysuccinimide(NHS; both from Pierce Biotechnology, Rockford, IL) accord-ing to Yao et al.20 In Brief, 1 g heparin, 2 g EDC, and 1.2 gNHS were added into 500 mL 2-morpholinoethanesulfonicacid buffer (0.05M, pH 5.6) for 10 min at 37�C to activatecarboxylic acid groups of heparin, then decellularized graftswere immersed into the reagent solution for 4 h at 37�Cunder gentle shaking. After heparin immobilization, vesselswere rinsed in 0.1M Na2HPO4 (2 h), 4M NaCl (4 times for24 h) and distilled water (3 times for 24 h). After beingcleaned, the vessels were lyophilized and cold-gas sterilized.

The stability of heparinized decellularized vessels(HDVs) was assessed via releasing experiment as previouslydescribed.21 Briefly, the lyophilized HDVs (approximately10 mg weight, n ¼ 8) were transferred to 5 mL release me-dium (0.1M HCl, 2 mg/mL NaCl) at 37�C under continuousshaking, and the medium was replaced every 5 to 20 days.The medium was removed and mixed with 5 mL aqueoussolution of toluidine blue (0.1M HCl, 2 mg/mL NaCl, and0.4 mg/mL toluidine blue) in a test tube for 4 h at roomtemperature, resulting in complexation of toluidine bluewith heparin. Thereafter, the test tube was centrifuged andthe precipitate was washed with distilled water (twice for5 min). Subsequently, toluidine blue complexed to heparinwas solubilized in 5 mL of a 1: 4 (v/v) mixture of 0.1MNaOH and ethanol. The extinction of the resulting solutionwas determined at 530 nm using an Uvikon 930 spectro-photometer (Kontron Instruments, Switzerland). The hepa-rin contents released from HDVs were interpolated from acalibration curve obtained as previous reported.22 In theend, a releasing curve of immobilized heparin was made.

Platelet adhesion experimentThe antithrombogenicity of HDVs were assayed by plateletadhesion experiment.23 Briefly, the sample in a disk shape(5 mm in diameter) was placed into a 24-well tissue cultureplate. The PBS was allowed to stand in the wells for a dayto equilibrate the surfaces. Then, the PBS was removed and1 mL of platelet-rich plasma (Blood Center of Nanjing RedCross) was poured into the wells and stored for 1 and 3 hat 37�C. Samples were rinsed with PBS and treated with2.5% glutaraldehyde for 30 min at room temperature thenrinsed with PBS and dehydrated by systemic immersion in aseries of ethanol–water solutions for 30 min each andallowed to evaporate at room temperature. The platelet-attached surfaces were coated with gold before beingobserved by scanning electron microscope (SEM; Leo1530vp scanning electron microscopy, Germany). The

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | NOV 2010 VOL 95A, ISSUE 2 601

heparin unimobilized decellularized graft film was used as areference.

Surgical implantation of HDVs and administration ofG-CSFSprague–Dawley rats (250 to 300 g body weight) wereanesthetized and maintained using intraperitoneal injectionsof sodium pentobarbital (50 mg/kg). Through a right lateralthoracotomy, abdominal aortas were exposed. After heparin(100 unit/kg) had been administered intravenously, theproximal and distal portions of the infrarenal abdominalaortas were clamped. Segments (5–10 mm) of the native ab-dominal aortas were resected and replaced by the HDVs.Standard microsurgical techniques were used. Anastomoseswere performed using 10-0 polypropylene (Ethicon, Somer-ville, NJ) running suture in end-end manner. No anticoagu-lants or antiplatelets were administered postoperatively.The rats were injected subcutaneously with recombinanthuman G-CSF (50 ug/kg/day; NovaTech Biopharmaceuti-cal, China) once a day for 14 days after grafts implantation(n ¼ 25). The grafts implanted into the rats not receiving G-CSF served as controls (n ¼ 23). Postoperatively, theimplanted grafts patency was monitored with a handleDoppler probe (HP Sonos 4500, Philips, Forestville, CA) ev-ery month. All care and handling of the animals were pro-vided according to the Guide for the Care and Use of Labo-ratory Animals approved by the Ethical Committee ofResearches of Nanjing University.

Mononuclear cells count and FACS analysisOn the 14th day of G-CSF or saline treatment, 1 mL bloodwas harvested from the inferior vena cava (5 rats of eachgroup were randomly selected), and the grafts wereretrieved for the observation of early endothelialization. Themononuclear cells were isolated by density-gradient centrif-ugation and counted with a Z1 Coulter particle counter(Beckman Coulter). Approximately 106 cells from each ani-mal were suspended in 50 lL PBS containing 5 mmol/Lethylenediamine tetraacetic acid (EDTA) and 0.5% bovineserum albumin and incubated for 30 min on ice with 20 g/mL phycoerythrin-conjugated antimouse c-kit, phycoery-thrin-conjugated antimouse CD133, and fluorescein isothiocy-anate-conjugated antimouse CD34 (all primary antibodieswere purchased from BD Pharminge). Unlabeled cells servedas negative controls. The cells were analyzed with a Caliburflow cytometer (fluorescence activated cell sorter (FACS);Beckman Dickinson).

Specimen retrieval and histological examinationMorphometric assessment of endothelialization was per-formed at 2 weeks after implantation. The aforementioned5 rats of each group were exsanguinated under deep anes-thesia, and the grafts were explanted and histologicallyexamined by H&E staining and SEM. Furthermore, to ana-lyze the EC-recovered area, samples were incubated withHRP-conjugated anti-Factor VIII antibody, followed by visu-alization with DAB, and assessed by planar morphometry.For morphometric analysis of neointimal hyperplasia, all the

rest rats (20 rats of G-CSF group and 18 controls) were ex-sanguinated after 6 months of transplantation. The midpor-tion and distal anastomotic sites of the specimen werestained with H&E and Masson’s trichrome staining, thedegrees of neointima formation were evaluated by histo-morphometry with the use of ImagePro software (ImagePro. Plus 3.0.1 software).

Statistical analysisAll results are presented as mean 6 standard deviation(SD). A pearson’s v2 test was used to compare the patencyrate between two groups. And unpaired t test was used forcomparisons between control and treated groups. A proba-bility value <0.05 was considered to indicate statistical sig-nificance. All analyses were performed using SAS software(version 9.1, SAS Institute, Cary, NC).

RESULTS

Characterization of decellularized vascular graftDecellularized vascular grafts were prepared by removingcellular components and leaving the native extracellular ma-trix (ECM) of the arteries. H&E staining did not show anysigns of remaining nuclear material in the vessel walls,whereas the basic extracellular microstructure remainedintact [Fig. 1(A,B)]. The following immuostaining of MHC Ifurther confirmed the efficacy of the decellularization pro-cess in removing the majority of cellular elements, and rela-tive preservation of the ECM. [Fig. 1(C,D)].

Characterization of heparin immobilizationUpon incubation of HDVs within releasing medium, partialrelease of immobilized heparin occurred during the first 5days, 40.40 6 8.78% of heparin released from HDVs(2.71 6 0.59 mg heparin per gram of HDVs), and after 20days, a total of 59.03 6 10.03% heparin released formHDVs (3.98 6 0.67 mg heparin per gram of HDVs). Heparinrelease leveled off after longer incubation time, but a pla-teau value was not observed during 20 days (Fig. 2).

Platelet adhesion was investigated by SEM in this study.Decellularized vascular graft showed highest platelet adhe-sion, most of the adhered platelets were spread anddeformed shapes and they might be activated [Fig. 3(A)].With the longer platelet adhesion time, the number of plate-let adhered was also increased (data not shown). In com-parison, platelets were rarely observed on the surfaces ofheparinized grafts [Fig. 3(B)].

Characterization of cytokine-mobilizedmononuclear cellsThroughout this study, there was no acute myocardial in-farction (AMI) or death in both groups of Sprague–Dawleyrats. The number of circulating mononuclear cells at 14days after treatment was significantly higher in G-CSF groupthan in control group (control, 5.98 6 0.29�106 cells; G-CSF, 31.42 6 2.49�106 cells; p < 0.001, n ¼ 5 for eachgroup). FACS analysis of the whole mononuclear fraction atthis time showed a 2.6-fold increase in the percentage ofmononuclear cells expressing the stem cell marker c-kit

602 ZHOU ET AL. VASCULAR TISSUE ENGINEERING FROM DECELLULARIZED SCAFFOLD

(control, 0.71 6 0.04�106; G-CSF, 9.69 6 0.55�106 cells/mL blood) [Fig. 4(A,B,E)]. The early EPCs markers CD34 andCD133 were double-positive in 0.82% of the mononuclearcells from G-CSF treated animals compared with 0.26% ofthe controls, corresponding to an 16.2-fold increase in thenumber of cells expressing both markers [Fig. 4(C,D,F)].These results suggest that G-CSF treatment significantlyincreases the number of circulating cells expressing an en-dothelial lineage phenotype (EPCs).

Gross specimens and morphometric analysisSegments of the rat infrarenal abdominal aorta werereplaced by HDVs without matrix rupture or deformation(Fig. 5). We assessed the effect of G-CSF treatment on re-en-dothelialization in sections stained with H&E, and by SEMat 2 weeks after implantation. Sections from control animalsshowed patchy and interrupted endothelium-like cells stain-ing [Fig. 6(A)], and topographic SEM view of the controlvessels showed incomplete and sparse endothelium [Fig.6(C)]. In contrast, a continuous monolayer of endothelium-like cells was found lining the lumen of G-CSF-treated grafts[Fig. 6(B)], and the SEM of the luminal surface revealed thepresence of densely packed, continuous cobblestone-likecells covering the luminal area [Fig. 6(D)]. Immunohisto-

chemical and following planar morphometric analysisrevealed that the ratio of Factor VIII positive endotheliallayer relative to the total luminal surface was significantlygreater in G-CSF group than in the control (67.4 6 7.9% vs.15.2 6 3.7%; n ¼ 5, p < 0.01) [Fig. 6(E,F)].

FIGURE 2. Release of heparin from heparinized decellularized vascular

graft in culture medium; (n ¼ 8, mean 6 SD); 46 � 35 mm (600 � 600

DPI).

FIGURE 1. Histological examination of decellularized vascular grafts. H&E staining of (A) native rat aorta shows the vascular cells are intact

(original magnification, �100) and (B) decellularized graft does not show any signs of remaining cellular segments in the vessel walls, whereas

the basic extracellular microstructure remained intact (original magnification, �100). Immunohistochemical staining shows that (C) the intact

vascular cells nuclear of native rat aorta are positive of MHC I (original magnification, �100), (D) but not show any signs of MHC I positive nu-

clear material in the decellularized vessels walls (original magnification, �100); 82 � 73 mm (500 � 500 DPI). [Color figure can be viewed in the

online issue, which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE


To monitor the long-term patency of implanted grafts,the animals were periodically investigated by handle Dopp-ler ultrasound (Fig. 7). Of 20 G-CSF-treated grafts, 19 main-tained patent for up to 6 months, one occluded within thefirst month due to thrombus formation, however, only 12control grafts were still patent when explanted at 6 months,4 were observed thrombotic occlusion within first 2months, and other 2 occluded in the 6th month. The pat-ency rate between G-CSF group and control group differedsignificantly (19/20 vs. 12/18, p ¼ 0.005; Fig. 8).

The patent grafts were all retrieved at 6 months afterimplantation to determine the ability of G-CSF treatment toinhibit neointimal hyperplasia of HDVs. By 6 months, Mas-son trichrome staining displayed amount of collagen andelastin reconstituted in both grafts [Fig. 9(A–D)]. And thesubsequent morphometric analysis revealed that the G-CSF-treated grafts (n ¼ 19) exhibited significantly smaller hyper-plastic neointima area compared with the control grafts(n ¼ 12), not only at midportion (0.38 6 0.02 vs. 0.47 60.07 mm2, p < 0.0001), but also at distal anastomosis(0.42 6 0.04 vs. 0.70 6 0.13 mm2, p < 0.0001) [Fig. 9(E)].Comparable changes were also seen in neointima-mediaratios in the G-CSF-treated group compared with the control(0.92 6 0.05 vs. 1.06 6 0.05, p < 0.0001, at midportion;0.90 6 0.09 vs. 1.41 6 0.29, p < 0.0001, at distal anasto-mosis) [Fig. 9(F)].

DISCUSSION

Small-diameter vascular synthetic grafts exhibit a high rateof failures due to the lack of an antithrombogenic mono-layer in direct contact with circulating blood.1 Seeding andsodding with autologous vascular cells on the luminal sur-face of graft before implantation have provided a muchhigher patency rate than non-cell-seeded grafts.5–7 However,this technology requires time-consuming and labor-intensiveprocedures, so that such a tissue-engineered approach can-not be used in the emergency cases. Therefore, in vivo rapidspontaneous endothelialization technology has been long

awaited. Regarding spontaneous endothelialization of vascu-lar grafts in vivo, currently there are three major mecha-nisms proposed: (1) the migration of ECs inward across theanastomosis from the native vessel; (2) ECs coveragederived from the ingrowth of capillaries through porousgrafts; and (3) the deposition of circulating EPCs onto theluminal surface of the graft.9

In this study, accordingly, we incorporated porous decel-lularized arterial scaffolds (to enable cell transmuralingrowth and adhesion) and G-CSF (to mobilize EPCs fromBM and increase the number of CEPCs) into a promising solu-tion for rapid self-endothelialization of vascular graft in vivo.

The decellularized scaffolds would be an ideal candi-date for a vascular graft. Our H&E staining showed thatthe cellular components were almost removed, leavingthe porous biological three-dimensional architecture. Andimmunohistochemical analysis of MHC I, which wasinvolved in immune responses against transplanted tissueand express in all blood vessel cells, showed the completeremoval of the vascular cells, and relative preservation ofthe ECM. The previous studies have shown that decellu-larized matrices presented porous and multilayer struc-tures, the pore size was mainly below 20 um in diameter,and porosity of the matrices was 66.8%,8 and the micro-porous structures could enable ECs coverage derivedfrom the ingrowing capillaries.24 Additionally, the mechan-ical properties such as burst strength, compliance, andsuture retention strength are not significantly differentbetween decellularized tissue matrices and native vascu-lar tissues.25

After that, we immobilized heparin to the decellularizedscaffolds using EDC and NHS as crosslink agents. This modi-fication leads to covalent linkage of heparin to the decellu-larized scaffolds, which unlike the ionic linkage of heparinused by other group, seems to be more stable and long last-ing.21 Actually, our results showed that approximately 40%of heparin released from HDVs during the first 5 days, anda total of approximately 60% heparin released after 20 days

FIGURE 3. Characterization of HDVs. Scanning electron micrograph after platelet adhesion test exhibited much more platelets adhering on the

(A) nonheparin immobilized decellularized graft compared with (B) heparinized decellularized graft (original magnification, �1500, the scale bar

indicates 10 lm); 82 � 29 mm (500 � 500 DPI).


of incubation. The slow and decreasing release rates indi-cated that a substantial amount of immobilized heparinwould be present on or in the HDVs for prolonged periodsof time. A continuous release of heparin from a matrixresults in a microenvironment of heparinized blood nearthe biomaterial interface, thus inhibits thrombus formation.

In this study, we demonstrated the excellent antithrom-bogenicity of the HDVs via platelet adhesion test in vitro. Aswe know, when a foreign surface comes in contact withblood, the initial response is the adsorption of blood pro-

teins, followed by platelet adhesion and activation of thecoagulation pathways, leading to thrombus formation. Plate-let adhesion on the membranes from human plasma is animportant test for the evaluation of the blood compatibilityof the grafts. Our results showed that platelets were rarelyobserved on the surfaces of HDVs, however, the unimmobi-lized decellularized graft showed higher platelet adhesion,most of the adhered platelets were spread and deformedshapes and they might be activated. This test revealed thatHDVs possessed excellent ability of antiplatelet adhesion.

FIGURE 4. The effects of G-CSF treatment on the mononuclear cells by FACS analysis. The percentage of circulating c-kitþ mononuclear cells in

(A) the control group is 11.80%, and (B) the G-CSF treated group is 31.32%. The percentage of circulating CDþ133/CD

þ34 mononuclear cells in (C)

the control group is 0.26%, and (D) the G-CSF treated group is 0.82%. Statistical analysis show the number of (E) c-Kitþ cells and (F) CDþ133/CD

þ34

cells per mL PB are both significantly higher in G-CSF-treated group. *p < 0.001 versus saline-injected group (n ¼ 5); 80 � 119 mm (500 � 500

DPI). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE


FIGURE 6. Histological analyses of intimal surface of the grafts retrieved 2 weeks after implantation. H&E staining of midportion of the retrieved

(A) control graft (original magnification, �100) and (B) G-CSF treated graft, arrows show presence of endothelial monolayer (original magnifica-

tion, �100); SEM of intimal surface of (C) control graft (original magnification, �1000, the scale bar indicates 10 lm) and (D) G-CSF treated graft

(original magnification, �1000, the scale bar indicates 10 lm). And Factor VIII immunostaining of the retrieved (E) control graft (original magnifi-

cation, �400) and (F) G-CSF treated graft, arrows show presence of endothelial monolayer (original magnification, �400); 80 � 90 mm (500 �500 DPI). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 5. Surgical implantation of the vascular grafts. (A) Before implantation, the black arrow indicates heparinized decellularized graft and

white arrow indicates rat abdominal aorta. (B) After implantation, the grafts were interposed to infrarenal abdominal aorta by the end-to-end

anastomosis in rat models, and black arrows indicate the anastomotic sites; 80 � 30 mm (500 � 500 DPI). [Color figure can be viewed in the

online issue, which is available at wileyonlinelibrary.com.]

Additionally, Conklin25 also reported that HDVs eliminatedblood clot formation in vitro, and subsequent animal studiesindicated it reduced the thrombogenicity and improve earlypatency rate in vivo as well.26,27

Although the HDVs may process endothelialization bythemselves after implantation, the time required for thisnative process to restore endothelial function seems to betoo long to prevent the early critical events leading to acti-vation of VSMCs and neointimal hyperplasia.26,27 Thereby, anovel approach that promotes endothelialization is requiredto accelerate this process. In this study, we provided a non-invasive approach based on a well-established protocol ofcytokine-induced mobilization of circulating putative EPCsto enhance rapid endothelialization of HDVs. Many studieshave well evidenced that G-CSF could mobilize EPCs fromBM and increase them in the circulation.13–19 Our resultswere consistent with previous reports, and showed that G-CSF stimulation increased the abundance of c-kitþ stemcells and CDþ

34/CDþ133 circulating EPCs.

As expected, our animal experiments demonstrated thatthe G-CSF treated grafts had a high degree of early endothe-lialization. The histological analysis of the grafts retrieved at2 weeks revealed a complete and continuous endotheliumin G-CSF-treated grafts, and conversely, only a few disor-derly endothelium and even thrombosis deposited on theluminal surface of the control grafts. Morphometric analysisof the endothelium showed >60% ECs coverage of thelumen in G-CSF-treated animals compared with <20% inthe control animals. Moreover, we observed dramatic inhibi-tion of neointimal hyperplasia in G-CSF group after long-term implantation. Figure 9 demonstrated a remarkabledecrease of intimal hyperplasia at midportion and distalanastomotic sites of G-CSF-treated group compared withcontrol (p < 0.0001 for both sites), and the neointima/media ratios (p < 0.0001 for both sites).

The most important finding, in our opinion, is that theanimal experiments demonstrated G-CSF treated grafts hada significantly higher patency rate than control grafts (19/20 vs. 12/18, p ¼ 0.005) after 6 months of transplantation.All of the occluded grafts were found within either the first2 months after implantation or the last month beforeexplantation, due to thrombus formation or intimal hyper-plasia at distal anastomosis, respectively. We hypothesizedthat both early enhanced endothelialization and late inhib-ited intimal hyperplasia induced by G-CSF treatment con-tributed to improving the patency rate of grafts.

However, our observations are consistent with earlierwork indicating that cytokine treatment increased the num-ber of CEPCs and subsequently inhibited the neointimal for-mation in injured vessels models. Kong et al.14 reportedthat mobilization of CEPCs by exogenous G-CSF facilitatesre-endothelialization and inhibits neointimal hyperplasia inthe injured vascular models. Furthermore, Takamiya et al.15

performed BM replacement experiments and showed thatG-CSF treatment increased the number of BM-derived EPCsthat actually contributed to re-endothelialization of the bal-loon-injured arteries, leading to marked inhibition of neoin-timal formation. Recently, Shoji et al.17 demonstrated that

FIGURE 7. Duplex Doppler ultrasound examination of (A) occlusive control graft and (B) patent G-CSF treated graft at 6 months after implanta-

tion; 65 � 22 mm (500 � 500 DPI). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 8. Long-term patency rate of G-CSF treated grafts (n ¼ 20)

and control grafts (n ¼ 18); 59 � 45 mm (600 � 600 DPI).

ORIGINAL ARTICLE


high-dose G-CSF promoted neointimal hyperplasia in theearly phase and inhibited neointimal hyperplasia in the latephase after vascular injury. Also, using G-CSF treatment, Bhat-tacharya et al.18 and Shi et al.19 showed enhanced endotheli-

alization of small-diameter prosthetic grafts in associationwith an elevation in CEPCs. Our present findings provide fur-ther evidence of the therapeutic potential of cytokine-inducedmobilization of progenitor cells in vessel repair. Specifically,

FIGURE 9. Masson trichrome staining comparing degree of neointimal hyperplasia at 6 months after implantation. (A) Midportion of G-CSF

treated graft (original magnification, �50); (B) midportion of control graft (original magnification, �50); (C) distal anastomotic site of G-CSF

treated graft (original magnification, �50); (D) distal anastomotic site of control graft (original magnification, �50). White arrows indicate neointi-

mal formation. The comparison of (E) neointima area and (F) neointima-media ratios at midportion and distal anastomotic sites of G-CSF treated

(n ¼ 19) and control (n ¼ 12) grafts at 6 months after implantation (*p < 0.0001 when compared with controls at the same site); 80 � 129 mm

(500 � 500 DPI). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]


we demonstrate for the first time the therapeutic benefit ofG-CSF treatment as a strategy for prevention of neointimalhyperplasia of small-diameter heparinized decellularized vas-cular graft. We hypothesized that the mechanism of neointi-mal hyperplasia inhibition was principally due to the increas-ing circulating progenitor cells and inducing rapid re-endothelialization, and subsequently timely restoration of en-dothelial function and vascular homeostasis, logically result-ing in inhibition of neointimal hyperplasia. Together, from atherapeutic standpoint, this evidence leads us to consider amore aggressive clinical use of G-CSF to mobilize CEPCs andtreat vasculoproliferative disease. However, the safety andfeasibility of G-CSF treatment focusing on the induction ofvascular occlusion in atherosclerotic lesions appear not yet tohave been established. There are articles reporting the induc-tion of AMI and cerebral infarction in G-CSF-treated BM trans-plantation patients.15 The potential stimulation of inflamma-tory cells by G-CSF was also reported.28 Although a veryrecent clinical study has reported that administration of G-CSF to patients with AMI improves cardiac function withoutany adverse events during 6-month observation, further basicand clinical studies focusing on these issues will berequired.29

In conclusion, this study exhibited a novel heparin im-mobilized small-caliber decellularized vascular graft, whichshowed excellent antithrombogenicity and biocompatibility,especially combined with short-term postoperative adminis-tration of G-CSF, the re-endothelialization of the graft wasaccelerated, and the neointima hyperplasia was markedlyinhibited, moreover, the 6-month patency rate was signifi-cantly improved. These findings suggest that G-CSF treat-ment may be a feasible and efficient therapeutic strategy forimproving graft patency rate after vascular bypass. If anoptimal schedule for administration and the long-term out-come of this strategy can be determined, it has the potentialto become a useful clinical approach for the improvement ofsmall-diameter vascular graft.

ACKNOWLEDGMENTS

We are grateful to Dr. WeiWang from the Department of Vascu-lar, Division of Vascular Ultrasound for performing the profes-sional duplex ultrasound examination of the rat aorta.

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beneficial effects of granulocyte-colony stimulating factor on small-diameter heparin immobilized...

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