AMD3100 Accelerates Reendothelialization of Neointima in Rabbit Saccular Aneurysm After Flow Diverter Treatment

Accepted Manuscript AMD3100 accelerates reendothelialization of neointima in rabbit saccular aneurysm after flow diverter treatment Zifu Li, Rui Zhao, Xinggen Fang, Jiahao Zhou, Guoquan Jiang, Qinghai Huang, Jianmin Liu PII:

S1878-8750(17)31228-7

DOI:

10.1016/j.wneu.2017.07.128

Reference:

WNEU 6190

To appear in:

World Neurosurgery

Received Date: 6 June 2017 Revised Date:

20 July 2017

Accepted Date: 22 July 2017

Please cite this article as: Li Z, Zhao R, Fang X, Zhou J, Jiang G, Huang Q, Liu J, AMD3100 accelerates reendothelialization of neointima in rabbit saccular aneurysm after flow diverter treatment, World Neurosurgery (2017), doi: 10.1016/j.wneu.2017.07.128. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT AMD3100 accelerates reendothelialization of neointima in rabbit saccular aneurysm after flow diverter treatment Zifu Li1 a, Rui Zhao1 a, Xinggen Fang2, Jiahao Zhou2, Guoquan Jiang2，Qinghai Huang1 *, Jianmin

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Liu1**

1 Neurosurgery Department, Changhai Hospital, Second Military Medical University, Shanghai

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200433, China;

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2 Neurosurgery Department, Yijishan Hospital, Wuhu 241001, Anhui province, China

These two authors contributed equally to this work.

Corresponding authors:

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* Qinghai Huang, Neurosurgery Department, Changhai Hospital, Second Military Medical University, Shanghai 200433, China, E-mail: [email protected].

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** Jianmin Liu, Neurosurgery Department, Changhai Hospital, Second Military Medical

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University, Shanghai 200433, China, E-mail: [email protected].

Running Title: AMD3100 promotes reendothelialization after FD treatment.

Abstract Objective To inspect the role of AMD3100 that acts as an antagonist of SDF-1/CXCR4 on the formation of neointima in rabbit saccular aneurysm after flow diverter (FD) treatment. Methods Twenty saccular aneurysm models were established by using porcine pancreatic elastase. Three weeks later, a FDs of Tubridge was implanted into the saccular aneurysm. All treated

ACCEPTED MANUSCRIPT models were immediately divided into two groups: the AMD3100 group was subcutaneously injected with AMD3100 (5 mg/kg per day), while control group

with saline. Morphology and

thickness of the neointima were investigated 2 and 4 weeks after FD treatment, using hard tissue section and masson trichrome staining. Scanning electron microscope (SEM) was used to observe

of neointima biomarkers, such as KDR, VE-cadherin, CD34, Tie2.

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endothelial-like cells, and fluorescence quantitative PCR was used to determine mRNA expression

Results Two and four weeks after FD treatment, the AMD3100 group had more endothelial-like cells than the control group in the neointima. Masson trichrome staining showed that the

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neointima in the AMD3100 group was more intact and thicker than that the control group. Furthermore, increased mRNA levels of KDR, VE-cadherin, Tie2 in the neointima were found in

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AMD3100 group compared with control group.

Conclusions Interval use of AMD3100 promotes the formation of neointima in rabbit saccular aneurysm and facilitates the endothelialization of the neointima after FD treatment. Key words: AMD3100, endothelial-like cells, flow diverter treatment, saccular aneurysm.

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Introduction

The flow diverter (FD) has been widely used to treat the intracranial aneurysms (IAs) because it could alter the hemodynamic effect of IAs to form thrombus and promote formation of neointima

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in the neck of IAs at the same time 1. The reconstruction of parent artery mainly provides scaffold for endothelial cells to grow and aneurysm neck for neointima formation to repair the saccular 1-3

.Then, if the neointima presents endothelialization, the repair of saccular aneurysm

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aneurysm

neck would be completed. Our previous studies showed that endothelial progenitor cells (EPCs) could take part in the neointima formation and accelerate endothelialization of the neointima

4,5

.

Therefore, it might be beneficial of EPCs in blood in the process of FD treatment. Although EPCs could be involved in the formation of neointima, the mechanism of EPCs homing to injury region has been thought correlated with the SDF-1α/CXCR4 axis 6,7. SDF-1 can interact with the CXCR4 receptor, and it has two subtypes, SDF-1α and SDF-1β. It is SDF-1α that promotes EPCs homing through the SDF-1α/CXCR4 axis 8. Based on our previous studies, we found that EPCs in the bone marrow could enter blood when New Zealand White Rabbits were injected with rhSDF-1α and the continuous injection of rhSDF-1α could accelerate the endothelialization of neointima in

ACCEPTED MANUSCRIPT the neck of rabbit saccular aneurysm after FD treatment 4. AMD3100 is a specific antagonist to the SDF-1α/CXCR4 axis 9. It was usually used to suppress the entry of HIV virus, while it can also mobilize hematopoietic stem cells into blood 10,11. Due to this dual function, we inferred that the CXCR4 receptor may be suppressed by AMD3100, and the

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role of SDF-1α on migration of EPCs would be blocked 12, or the AMD3100 could promote the effect of EPCs migration through competition with SDF-1α/CXCR4 axis

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. According to these

inferences, we aimed to inspect whether AMD3100 could influence the EPC homing and chemotaxis via inhibiting the SDF-1α/CXCR4 axis, and ultimately affect the formation and

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endothelialization of neointima in the neck of rabbit saccular aneurysm after FD treatment.

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Material and Methods Materials

AMD3100 and porcine pancreatic elastase were obtained from Sigma-Aldrich (St.Louis, MO, USA). The flow cytometry antibodies were as follow: FITC-conjugated anti-human KDR (vascular endothelial growth factor receptor-2) and APC-conjugated anti-human CD34 were

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obtained from R&D Systems (Minneapolis, MIN, USA)；PE-conjugated anti-human CD133 was purchased from Miltenyi Biotec (Auburn, CA). FD of TubridgeTM was purchased from Shanghai MicroPort Medical Instrument Co., Ltd (Shanghai, China). TaqMan Real-Time PCR Master Mixes

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Kit was purchased from Thermo Fisher Scientific (Massachusetts, MA, USA).

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Rabbit Saccular Aneurysm Models All animals studies were approved by the Ethic Committee of Changhai Hospital, Second Military Medical University. Twenty New Zealand White male rabbits (10-12 months old, 3-3.5 kg) were used to establish the saccular aneurysm models. Under anesthesia state (by intramuscular injection of xylazine in 0.1 mg/kg and intravenous injection of 1% sodium pentobarbital in 1 mg/kg), the right common carotid artery (RCCA) was exposed and the partial wall of subclavian artery was clipped with an arcuate aneurysm clip. Subsequently, the 2 cm proximal RCCA to the origin was ligated after clipping the origin. Afterwards, 75-150 units of porcine pancreatic elastase was injected into the inner lumen of the proximal segment of RCCA by using a 22-gauge catheter, and then the inner lumen was incubated with porcine pancreatic elastase for about 20 min. Finally, the

ACCEPTED MANUSCRIPT clip was removed after the catheter puncture point ligated. After the operation, all rabbits were fed with adequate water and food in cages and intravenously injected with heparin (400 U) for three days.

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Flow Cytometry Analysis Five normal New Zealand White male rabbits were used for this experiment. The peripheral blood sample from median artery of ears of these rabbits was collected before and 4, 8, 12, 24 h after subcutaneously injection of AMD3100 with dosage of 5 mg/kg per day. Then BD FACS Calibur

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(New Jersey, NJ, USA) was performed to detect the number of endothelial progenitor cells (EPCs) in blood sample using antibodies of anti-CD34 ISO IgG, anti-KDR ISO IgG, anti-CD133 ISO IgG,

FD and AMD3100 Treatment

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anti-KDR, anti-CD133 and anti-CD34.

Three weeks after establishment of rabbit saccular aneurysm models, all of the rabbit models were implanted with FDs of Tubridge in their saccular aneurysms and the rabbit model would receive

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the second FD of Tubridge if the first one did not satisfy the therapeutic requirements. The details of this procedure have been described elsewhere 4,14. In brief, all the rabbits were fed with aspirin (15 mg/kg.d) and clopidogrel (15 mg/kg.d) by gavage for 5 days before the operation. At the

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moment of the operation, these rabbits were subcutaneously injected with 3 ml 2% lidocaine and heparin in a dose of 300 IU/kg, and their femoral artery was exposed using sterile surgical

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techniques. Then the proximal segment of femoral artery was temporarily blocked with an aneurysm clip and the distal was reserved to ligate. Subsequently, 5F sheath (Terumo, Tokyo, Japan) was inserted into the femoral artery and fixed with ligation. Next, 5F catheter (Cordis Corporation, Miami, USA) was introduced and positioned in the innominate artery. At same time, digital subtraction angiography (DSA) was performed to measure the innominate artery and parameters of the induced saccular aneurysms. After verification of aneurysm location and measurement of aneurysm profiles, a FD of Tubridge was implanted via VASCO microcatheter (Balt, Montmorency, France) to cover the induced aneurysm neck, which was braided with 40% metal coverage. Eventually, these rabbits were again fed with adequate water and food, aspirin (15 mg/kg.d) and clopidogrel (15 mg/kg.d) by gavage in cages until been executed after the operation.

ACCEPTED MANUSCRIPT Furthermore, these rabbit models were immediately divided at random into 2 groups after the FD operation and 10 models in each group, one group was AMD3100 treatment and another was control with saline. The AMD3100 group was subcutaneously injected at vastus lateralis with

Scanning Electron Microscope Analysis

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AMD3100 (5 mg/kg.d) 15,16 .

At 2 and 4 weeks after FD treatment, the FDs of Tubridge in all rabbit saccular aneurysm models were removed and 3 rabbit models of each group were chosen randomly to perform scanning

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electron microscope analysis. After disinfection and infiltration anesthesia, their hearts of the 3 rabbit models were exposed by a sterile technique and the left ventricular of every rabbit was

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infused with 200 ml saline containing heparin (400 U) and 250 ml 4% paraformaldehyde (PFA) for 10 min. Then arcus aortae, bilateral subclavian artery, saccular aneurysm, subclavian stents, left common carotid artery of every rabbit models were fixed with 4% PFA for 24-48 h. At last, these tissues were immersed into 2.5% glutaraldehyde solution for fixation. Endothelial-like cells of three random and independent fields were observed and counted under scanning electron

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microscopy (SU-8010, HITACHI, Tokyo, Japan).

Quantitative realtime PCR

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The sample processing for fluorescence quantitative PCR was similar with the SEM analysis except the 4% PFA. The tissues of 3 rabbit models of each group which were chosen at random

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were infused only with saline containing heparin (400 U) and frozen at -80ºC. Afterwards, TaqMan Real-Time PCR Master Mixes Kit was used to conduct the analysis of fluorescence quantitative PCR. In brief, firstly, we designed the primers of Tie2, CD34, KDR, VE-cadherin and GAPDH as showed in Table 1, where GAPDH was the reference gene. Secondly, Total RNA of all samples of the rabbit saccular aneurysms was extracted as described in the instruction of TaqMan Real-Time PCR Master Mixes Kit. Thirdly, the concentration and purity of the total RNA were determined and then the cDNA were reversely transcribed through the general PCR. Lastly, the cDNA levels of Tie2, CD34, KDR, VE-cadherin and GAPDH were analyzed by fluorescence quantitative PCR and the 2−∆∆CT method was used to circulate the relative mRNA expression levels of Tie2, CD34, KDR and VE-cadherin.

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Hard Tissue Section and Masson Trichrome Staining The sample processing for hard tissue section was same with the SEM analysis. Samples of 4 animals of each group were dehydrated and fixed for 2 weeks in acetone. Afterwards, the samples

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were put into methyl methacrylate solution for 30 min of vacuumizing at 25ºC and dehydrated for one week at 40ºC. Then, samples were cut into 50 µm sections, and these specimens were stuck on glass slides.

The detailed procedure of masson trichrome staining was as follow. Briefly, these slides were

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washed by hydrogen peroxide, and stovinged in dryer of 40ºC for 10 min. After the temperature on the surface of the slides reached to 40ºC, several drops of hematoxylin were dropt on the

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specimens and the hematoxylin should cover the whole specimen. Next, they were put into the dryer of 40ºC to stain for 30-90 min and then the hematoxylin was washed by water. Subsequently, the slides were polarized with 1% hydrochloric acid alcohol for 15 s and then stained with acidic Ponceau S for 10 min. Further, the dye liquor of acidic Ponceau S was washed with deionized water and the slides were treated with phosphomolybdic acid solution and aniline blue in turn for

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10 min. Moreover, these slides were washed with water for 30 min to wash away the hydrochloric acid alcohol and turned blue. At last, slides were covered with coverslips, observed under the

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microscope, and the thickness of saccular aneurysm neointima was measured.

Statistical Analysis

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All data was presented as mean ± Standard Deviation (SD) in this study. The number of endothelial-like cells on the neointima, and mRNA levels of KDR, CD34, VE-cadherin and Tie2 in the neointima between groups were compared using two-way ANOVA. The numbers of EPCs analyzed by flow cytometry were compared with unpaired t-test. A value of P < 0.05 was considered significant in statistics.

Results AMD3100 promoted the increase of KDR+ cells in peripheral blood of rabbit To inspect the role of AMD3100 on the migration of EPCs, New Zealand White male rabbits were injected with AMD3100. Figure 1A and 1B showed that the number of KDR+ cells in peripheral

ACCEPTED MANUSCRIPT blood of rabbit increased obviously and this increase was especially significant at 4 h after the injection (P<0.01 vs the before injection), and then this trend was attenuated immediately. These results indicated that AMD3100 could promote EPCs into peripheral blood of rabbit.

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Establishment of rabbit saccular aneurysm models The animal models of rabbit saccular aneurysm were induced by porcine pancreatic elastase. The width of saccular aneurysm neck, the height and width of saccular aneurysm, the diameter of common carotid artery and the parameter of rabbit saccular aneurysm were shown in Table 2. The

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mean width of saccular aneurysm neck was 4.33 ± 0.83 mm, the mean height of saccular aneurysm was 6.20 ± 1.55 mm, the mean width of saccular aneurysm was 4.36 ± 1.17 mm and the

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mean diameter of common carotid artery was 2.57 ± 0.24 mm. These data suggested that the rabbit saccular aneurysm model was established successfully.

Results of the FD treatment of Tubridge

After FD treatment, the results of DSA showed that it had retention of contrast medium in the

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rabbit saccular aneurysm (Figure 2D) and the FD of Tubridge adhered suitably to the vascular wall of rabbit artery. Before we executed all the rabbits, we performed DSA again to verify that seventeen of the rabbits were cured by the FD treatment of Tubridge and the parent artery kept

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patent. No lumen stenosis appeared at the proximal and distal of the parent artery. Only one rabbit

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had ascending aortic dissecting aneurysm.

AMD3100 increased Tie2, KDR and VE-cadherin expression in the saccular aneurysm neointima

Four weeks after FD treatment of Tubridge , mRNA levels of Tie2, CD34, KDR and VE-cadherin were evaluated. The results in Figure 3 showed that mRNA levels of Tie2, KDR and VE-cadherin were significantly higher in AMD3100 group than that controls (P<0.05), and there were no significant differences of CD34 expression between groups.

AMD3100 increased the number of endothelium-like cells in the surface of neointima SEM analysis was used to observe the formation of neointima in the neck of saccular aneurysm.

ACCEPTED MANUSCRIPT As shown in Figure 4A, in AMD3100 group, the neointima covered more endothelium like cells at 2 weeks after the FD treatment. The surface of neointima in the neck of saccular aneurysm was rough at 2 weeks after the operation and few endothelium-like cells adhered to the surface when compared to the fusiform or cobblestone-like endothelial cells in the neointima of normal arteries.

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4 weeks after the operation, the endothelium-like cells in the surface of neointima increased significantly (Figure 4B, P<0.05), with a ratio of endothelium-like cells number that AMD3100: control = 92: 60.

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AMD3100 increased the thickness of neointima in the neck of saccular aneurysm

To observe the morphology of neointima in the neck of saccular aneurysm at 2 and 4 weeks after

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the FD treatment, the hard tissue section and masson trichrome staining were performed. Results in Figure 5A showed that the neointima of saccular aneurysm neck in both AMD3100 and control group were orbicular and the thickness increased, but the neointima in AMD3100 group was significantly thicker compared to controls (P<0.001, Figure 5B).

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Discussion

Although AMD3100 is the specific receptor blocker of CXCR4, which can block the touch between SDF-1 and CXCR4, it can also mobilize CD34+ cells and promote EPCs into blood.

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Owing to the bidirectional role of AMD3100, it has not been clearly illustrated whether AMD3100 could influence the formation of neointima in saccular aneurysm after FD. This study verified that

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AMD3100 could promote EPCs entering blood and neointima formation at the aneurysm neck after FD treatment.

In our previous study, we found that AMD3100 could block the migration effect of EPCs on SDF-1α and this blocking role mainly depended on inhibition of CXCR4, a specific receptor of EPCs. Therefore, we inferred that AMD3100 injected subcutaneously may inhibit the migration effect of EPCs toward the injury tissue, and then block the formation of neointima. However, in previous pharmacokinetics study, some researchers accidentally noticed that intravenous injection of AMD3100 could quickly increase the number of peripheral white blood cells of healthy volunteers

11

. In this study, we similarly discovered that AMD3100 increased peripheral KDR+

cells. We cannot directly provide the experiment data that CD34+ cells increased, but we believe

ACCEPTED MANUSCRIPT that most of these cells were from endothelial cell line in artery of marrow. However, these findings draw a conclusion that it inhibits EPC migration along the concentration gradient of SDF-1α or promotes EPCs entering blood. Whether AMD3100 could promote or inhibit repair of injury tissue relies on the way of

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medications. It was mutual contradiction that AMD3100 can mobilize EPCs into blood and block CXCR4 receptor. In this experiment, we discontinuously injected New Zealand White Rabbits with AMD3100 and found that the level of endothelium like cells upregulated significantly at 2 and 4 weeks after FD treatment. This result similarly showed that AMD3100 could promote

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formation of neointima after FD treatment in saccular aneurysm induced by porcine pancreatic elastase.

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However, the mechanism of AMD3100 promoting EPC migration was not illuminated clearly. Jujo et al. showed that the mice of ischemia and reperfusion injury injected with 5 mg/kg AMD3100 had smaller infract size than normal saline group after 3 days

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. And the CXCR4+

cells in AMD3100 group can be mobilized for 7 days while just one day in control group. Furthermore, AMD3100 could upregulate the expression level of marrow endothelial nitric oxide

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synthase (eNOS) and its two targets of this signaling pathway—MMP9 and soluble stem cell factor. AMD3100 can also mobilize the bone marrow-derived progenitor cells, which may exert its effect through the relationship with expression of eNOS. In diabetic mice, Nishimura et al. found

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that AMD3100 could accelerate wound healing, promote release of inflammatory factors such as SDF-1α, PDGF-B and MCP-1, mobilize EPCs into blood, and improve cell capacity for action of 17

. Based on these

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fibrosis, monocytes and macrophages to enhance ability of angiogenesis

publications, it is conceivable that AMD3100 had an effect to promote repair of injury tissue, while continuous use of AMD3100 had contrary effect. Conclusions

This study turned out that AMD3100 could promote formation of the neointima in rabbit saccular aneurysm neck after flow diverter treatment and enhance endothelial progenitor cells dissociating into blood and increase their content in peripheral blood. This effect could quickly accelerate the proliferation of endothelium like cells in the neck of rabbit saccular aneurysm and endothelialization of the neointima, which ultimately promotes the repair of saccular aneurysm.

ACCEPTED MANUSCRIPT Acknowledgements No.

Funding: This study was supported by grants from the National Natural Science Foundation of

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China (Grant Numbers: 81171093，81671137 and 81571126).

Competing Interests: None of the authors have any conflict of interests to declare.

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promoted by enriched environment and suppresses long-term seizure activity in adult rats of temporal lobe epilepsy. Behav Brain Res. 2017;322(Pt A):83-91. 14.

Li Z, Zhao R, Fang X, Huang Q, Liu J. Recombinant human SDF-1α administration accelerates aneurysm neck reendothelialization in rabbit saccular aneurysm after flow

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Figure legends

Figure 1 AMD3100 promoted the increase of KDR+ cells in peripheral blood of rabbits. (A) The results of flow cytometry analysis for KDR+ cells in peripheral blood of New Zealand White Male Rabbits after injected with AMD3100: a, setting of flow cytometry analysis; b, before the AMD3100 injection; c, 4 h after the AMD3100 injection; d, 8 h after the AMD3100 injection; e, 12 h after the AMD3100 injection; f, 24 h after the AMD3100 injection; (B) The statistics

ACCEPTED MANUSCRIPT analysis for change of KDR+ cells in peripheral blood of rabbits, data are presented as mean ± SD, n=5, * indicates P < 0.05, ** indicates P < 0.01, error bars represent standard deviation.

Figure 2 The entirety morphology of the rabbit saccular aneurysm before and after FD

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treatment of Tubridge. (A) The morphology of the saccular aneurysm after the establishment of rabbit saccular aneurysm models at once; (B) The morphology of the saccular aneurysm at 3 weeks after the establishment of rabbit saccular aneurysm models; (C) The morphology of the saccular aneurysm after the

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transplantation of FD of Tubridge at once; (D) The morphology of contrast stasis in the rabbit saccular aneurysm; (E) The morphology of the saccular aneurysm at one month after the

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transplantation of FD; (F) The morphology of the saccular aneurysm and FD after obtained the sample of rabbit saccular aneurysm; (G) The morphology of HE staining on the rabbit saccular aneurysm.

Figure 3 Results of fluorescence quantitative PCR. After FD treatment of Tubridge to rabbit

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saccular aneurysm models, 3 rabbits in experiment group were injected with AMD3100 for 4 weeks and then fluorescence quantitative PCR was used to detect the mRNA expression levels of Tie2, KDR, CD34 and VE-cadherin of the rabbit saccular aneurysm. Data are presented as mean ±

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SD, n=3, ns indicates no statistical significance, * indicates P < 0.05, ** indicates P < 0.01, ***

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indicates P < 0.001, error bars represent standard deviation.

Figure 4 Results of scanning electron microscope analysis. At 2 and 4 weeks after the FD treatment of Tubridge to rabbit saccular aneurysm models, the saccular aneurysms were obtained to conduct scanning electron microscope analysis. (A) The image of scanning electron microscope, endothelium-like cells were indicated with white arrows. (B) The statistical analysis of endothelium-like cells of four groups, Data are presented as mean ± SD, n=3, * indicates P < 0.05, error bars represent standard deviation.

Figure 5 Results of hard tissue section and masson trichrome staining. At 2 and 4 weeks after the FD treatment of Tubridge to rabbit saccular aneurysm models, the

ACCEPTED MANUSCRIPT saccular aneurysms were obtained to perform hard tissue section and masson trichrome staining. (A) The image of masson trichrome staining: a, the control group after 2 weeks; b, the AMD3100 group after 2 weeks; c, the control group after 4 weeks; d, the AMD3100 group after 4 weeks. (B) The statistical analysis of neointima thickness of four groups, Data are presented as mean ± SD,

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n=4, *** indicates P < 0.001, error bars represent standard deviation.

ACCEPTED MANUSCRIPT Table 1 The primers of KDR, CD34, VE-cadherin, Tie2 and GAPDH.

The primer

The sequence Forward: 5’-CAACACAGCAGGAATCAGTCAGG -3’

KDR

Reverse: 5’-TGGTTGTCATCTGGGACTACTTT-3’ Forward: 5’- CCTGGAAGTATTTCTAATCACACAC -3’

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CD34

Reverse: 5’- GTCCTTCTTAAAGTCCTCACAGC -3’

Forward: 5’-CAGGTCAACGCAGACACAGGGG -3’

VE-cadherin

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Reverse: 5’-ACGATGAGGGCAACGAGGTGGT-3’

Forward: 5’-TTGAAGTGGAGAGGAGATCTGTG -3’

Tie2

Reverse: 5’-TGATGTTTTCTGGTTGAGGAGG-3’

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Forward: 5’-CGC CTG GAG AAA GCT GCT A -3’

GAPDH

Reverse: 5’-ACG ACC TGG TCC TCG GTG TA -3’

KDR:

Kinase

insert

domain

receptor;

VE-cadherin:

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Endothelial-cadherin; GAPDH: glyceraldehydes phosphate dehydrogenase

Vascular

ACCEPTED MANUSCRIPT Table 2 3 weeks after establishment of rabbit saccular aneurysm models and before the FD treatment of Tubridge, the width of saccular aneurysm neck, the height and width of saccular aneurysm and the diameter of common carotid artery were

The width of

The height of

The diameter of

saccular

saccular

saccular

common carotid artery

aneurysm

aneurysm

aneurysm (mm)

(mm)

neck (mm)

(mm)

51

3.6

3.99

3.99

52

3.6

3.02

2.67

54

5.48

3.28

6.48

57

2.81

2.81

5.29

58

5.02

3.14

60

3.1

2.95

64

4.22

5.7

66

4.3

2.98

67

3.07

3.72

71

3.85

3.85

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94

4.74

99

5.35

101

5.43

102

4.38

3.05

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2.95 2.09 2.44

7.00

2.81

5.96

2.2

9.03

2.82

4.99

2.78

5.63

2.5

6.6

2.45

5.78

6.76

2.43

5.55

6.07

2.50

4.83

6.89

2.68

3.89

6.78

2.56

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Number

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The width of

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measured.

5.37

6.73

7.86

2.67

4.39

4.58

4.56

2.39

115

3.76

5.32

5.73

2.45

116

4.78

5.65

6.88

2.47

120

5.02

4.56

9.23

2.68

122

4.33

4.87

5.58

2.56

105

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109

FD: flow diverter.

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ACCEPTED MANUSCRIPT

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ACCEPTED MANUSCRIPT Highlights 1.

AMD3100 could increase the number of KDR+ cells in peripheral blood of rabbit.

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AMD3100 could increase the mRNA expression levels of Tie2, KDR and VE-cadherin of the

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saccular aneurysm neointima and the number of endothelium-like cells in the surface of neointima after FD treatment.

AMD3100 could increase the thickness of neointima in the saccular aneurysm neck and

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accelerate endothelialization of the neointima to promote repair of saccular aneurysm after

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FD treatment.

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ACCEPTED MANUSCRIPT Abbreviations and Acronyms FD: flow diverter SEM: scanning electron microscope IAs: intracranial aneurysms

CXCR4: CXC Chemokine Receptor 4 KDR: Kinase insert domain receptor VEGFR-2: vascular endothelial growth factor receptor-2 GAPDH: glyceraldehydes phosphate dehydrogenase

DSA: digital subtraction angiography HIV: human immunodeficiency virus RCCA: right common carotid artery PFA: paraformaldehyde

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PCR: Polymerase Chain Reaction

SD: standard deviation

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VE-cadherin: Vascular Endothelial-cadherin UV: ultraviolet

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ANOVA: analysis of variance

VEGF: vascular endothelial growth factor MMP9: matrix metallo proteinase 9

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SDF-1: stromal cell derived factor-1

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EPCs: endothelial progenitor cells

eNOS: endothelial nitric oxide synthase PDGF-B: platelet derived growth factor-B MCP-1: monocyte chemoattractant protein-1