Research Article

Cell Biology International 10.1002/cbin.10471

Stromal cell-derived factor-1alpha prevents endothelial progenitor cells senescence and enhances reendothelialization of injured arteries via human telomerase reverse transcriptase Xiaohua Shen 1, Yucheng Zhou 2, Xukun Bi1, Jiefang Zhang1, Guosheng Fu1, Hao Zheng 3 Department of Cardiology1 and General Surgery2, Biomedical Research (Therapy) Center, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, P. R. China. 310058. 3

Department of Cardiology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang

University, Hangzhou 310058, China. Department of Cardiology, Hangzhou Xiasha Hospital, Hangzhou, Zhejiang Province, P. R. China. 310058. Co-correspond ing author: Hao Zheng, Ph.D, Department of Cardiology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University. Department of Cardiology, Hangzhou Xiasha Hospital, Hangzhou, Zhejiang Province, P. R. China. 310058. Tel.: +86 571 86006242; Fax: +86 571 86006242 ; E-mail address: [email protected] Guosheng Fu, PhD, Department of Cardiology, Biomedical Research (Therapy) Center, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, P. R. China. 310058. Tel.: +86 571 86006242; Fax: +86 571 86006242; E-mail address: [email protected]

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/cbin.10471]

This article is protected by copyright. All rights reserved Received 4 August 2014; Revised 15 March 2015; Accepted 18 March 2015

Abstract Recent studies have suggested that endothelial progenitor subpopulation (EPCs) number and activity were associated with EPCs senescence. Our previous study had shown that stromal cell-derived factor-1alpha (SDF-1a) could prevent EPCs senescence which maybe via telomerase. In this study, we further investigated the role of human telomerase reverse transcriptase (h-TERT) on the protective effect of SDF-1a against senescence. Knockdown hTERT abrogated the protective effect of SDF -1a, and abolished the effects of SDF -1a on migration and proliferation. Moreover, it inhibited EPCs recruitment. In conclusion, h-TERT served a critical role in the progress that SDF-1a prevented EPCs senescence and enhanced reendothelialization of the injured arteries.

Hightlights: Knockdown of h-TERT inhibits SDF-1a prevent EPCs from senescence which maybe via Akt pathway. h-TERT inhibition abrogate the effects of SDF -1a on EPCs proliferation and migration . Knockdown of h-TERT delayed the re-endothelialization of the injured arteries, and inhibited EPCs recruitment. Short title: SDF-1a enhanced re-endothelialization via h-TERT.

Short main title: SDF -1a prevents EPCs senescence and enhanced re-endothelialization. Keywords: Stromal cell-derived factor-1alpha; senescence; endothelial progenitor cell; telomerase; human telomerase reverse transcriptase

Abbreviations: EPC: endothelial progenitor cell; h-TERT: human telomerase reverse transcriptase; SDF-1a: Stromal cell-derived factor-1 alpha; SA-b-gal: senescence-associated beta-galactosidase; PI3K: phosphatidylinositol 3-kinase.

1. Introduction Since the concept of a circulating human cell that could serve as a progenitor for the endothelial lineage was first proposed in 1997 (Asahara et al., 1997), emerging evidence demonstrated the vascular-promoting and/or vascular-healing capacity of endothelial progenitor cells (EPCs) (Shi et al., 1998; Asahara et al., 1999; Werner et al., 2006). These remarkable properties make EPCs ideal cell therapy for the endothelial regeneration and reendothelialization of ischemic tissue (Thum et al., 2005). Normal diploid cells of humans and other mammals undergo a limited number of cell divisions , eventually entering a state referred as senescence (Hayflick, 1965). Reduction of telomerase activity and shortening of telomere length, which occur through cellular division, have been suggested to be responsible for cellular senescence (Murasawa et al., 2002). Stromal cell-derived factor-1alpha (SDF-1a/CXCL12) is a member of the CXC chemokine family. Recent studies have shown that SDF-1a/CXCR4 interaction plays an influential role in regulating several cellular functions, such as cell proliferation, migration, survival, and senescence (Thum et al., 2005; Zheng et al., 2007, 2008). Our previous study indicated that ex vivo co-incubation with SDF-1a delayed the onset of EPCs senescence, which may via the activation of telomerase (Zheng et al, 2010). Murakami demonstrated that the neutralizing antibody directed against CXCR4 which transient inhibited the SDF-1/CXCR4 pathway blocked the recruitment of EPCs to the tumor tissue (Murakami et al., 2009; Chen et al., 2010). The aim of this study was to further investigate whether telomerase might play a major role in the protective effect of SDF-1a to prevent EPCs from senescence and enhance proliferation and migration, and then also to investigate the effects of SDF -1a and h-TERT on the potential recruitment and reendothelialization properties of EPCs to injury vessels.

2. Method and materials: 2.1 Cell isolation and culture EPC subpopulations were cultured on the basis of the technique that was previously described (Zheng et al., 2007, 2008). Briefly, peripheral blood mononuclear cells (PBMNCs) were isolated from healthy volunteers who were all informed with written consent (n = 18, aged 27

±4 years, 10 male, 8 female) by density-gradient centrifugation with Ficoll separating solution (Cedarlane Laboratories Ltd., Ontario, Canada). All blood samples were obtained, processed, and analyzed individually for independent experiments. The cells were cultured in EGM2MV medium (Lonza, Basel, Switzerland). The colonies of late outgrownth EPCs (ECFCs) that were derived from adherent mononuclear cells cultured for 7–21 days displayed a cobblestone morphology.

2.2 Knockdown of h-TERT by small interfering RNA (siRNA) The small interfering RNA (siRNA) was used to inhibit the expression of h-TERT in these cells. siRNA-TERT and nonsilencing siRNA were purchased from Genepharma (shanghai, China).

The

sequence

of

siRNA-TERT

GGAGCAAGUUGCAAAGCAUTT-3’

was

and

positive-sense

antisense

strand

strand

5’5’-

AUGCUUUGCAACUUGCUCCTT -3’. Proliferating cells were transfected with 200 pmol/L siRNA for h-TERT and negative control SiRNA (5’-UUCUCCGAACGUGUCACGUTT-3’ and

5’ACGUGACACGUUCGGAGAATT-3’)

using

Hiperfect

transfection

reagent

(GIAGEN, California, USA) as delivery media for 4-6 hours, according to the manufacturer’s protocol.

2.3 Senescence-associated b-galactosidase activity assay After several concentrations of SDF-1a (Peprotech, Inc., Rocky Hill, USA) were added into EPC subpopulation for 48 h, EPC subpopulation were harvested and senescence-associated beta-galactosidase (SA-ß-gal) activity was measured by Senescence ß-Galactosidase Staining Kit which was purchased from Cell Signaling Technology (Beverly, MA, USA) (Assmus et al., 2003; Imanishi et al., 2004).

2.4 Proliferation assay EPC subpopulation proliferation was assessed from the cell counting kit-8 (CCK-8) (Dojindo laboratory, Japan). After treatment, Cells (1×104 cells/well) were re-incubated in 96-well plastic plates for another 24h, 10% CCK-8 solution (highly waster-soluble tetrazolium salt

WST-8), which allows sensitive colorimetric assays for the determination of the number of viable

cells

in

cell

proliferation,

The amount of the formazan dye

was

added

to

the

plates

for

4

hours.

generated by the activity of dehydrogenases in cells is

directly proportional to the number of living cells. The absorbance at 450 nm was detected with a microplate reader (ELx800, BioTek, USA).

2.5 Transwell chamber assay a 24-well transwell chamber (Millipore, MA, USA) was used to determine the EPCs migration. After the addition of different concentration of SDF-1a, ten thousand cells of each group were seeded on the inner chamber of a transwell plate, with an 8-µm size pore membrane. After incubation for 24 hours, the migrated cells on the lower surface of the filter were fixed with 4 % paraformaldehyde at room temperature for 30 minutes, and stained by DAPI for 5 minutes. Then the migrated EPCs were photographed with a Zeiss fluorescence microscope (BX51, Olympus , Japan) with 10× objective. Cell numbers were counted in the immunofluorescence micrograph.

2.6 Western blot analysis After treatment, cellular proteins were harvested and then separated by 10% SDS polyacrylamide gel and electrotransferred to a polyvinylidene difluoride membrane. The membranes were blocked in blocking solution (5% milk) for 1 h and then incubated overnight with rabbit polyclonal anti-phospho-Akt-Ser 473, anti-Akt, anti-phospho-MAPK/Erk1,2, antiMAPK/Erk1,2,

anti-phospho-P38(Thr180/Tyr182),

anti-P38/MAPK,

anti-phoshpo-NF-

?B(Ser536), anti-NF-?B, anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-SAPK/JNK, antip21, anti-p16, anti-p53, anti-Rb, anti-cyclin D1 antibodies(Cell Signaling Technology, Beverly, MA) or anti-TERT(Bioworld, Georgia, USA), or anti-GAPDH polyclonal antibody (1:1,000). The membranes were washed thoroughly in Tris-buffered saline with 0.1% (v/v) Tween-20 before incubation for 1 h with a secondary anti-rabbit antibody conjugated to horseradish

peroxidase

(1:5,000).

Protein

was

then

chemiluminescence solution (Amersham, Uppsala, Sweden).

visualized

using

enhanced

2.7 Animal study All animal studies were reviewed and approved by the institutional Animal Care and Use Committee of Zhejiang University. The peripheral blood of New Zealand rabbits was collected for EPCs isolation and culture. A total of 25 rabbits (13 male, 12 female) were distributed into 5 groups: (1) carotid injury group without transplantation; (2) transplantation with EPCs; (3) transplantation with EPCs treated with SDF-1a for 48 hour; (4) transplantation with EPCs treated with SDF-1a and SiRNA. (5) control group with sham procedure. A balloon catheter was used to induce vascular injury in rabbits (2.2-2.8kg). Rabbits were anesthetized with 8% chloralhydrate (4ml/kg), an anterior midline incision was made, and the left common carotid artery, internal and external carotid arteries were exposed. The distal end of the external carotid artery was ligated and the proximal common carotid artery and internal carotid artery were temporarily ligated. A 3.0mm×12mm balloon catheter (Quantum Maverick, Boston, USA) was inserted into the common carotid artery via the external carotid artery for about 3cm and the balloon was inflated with the pressure of 10 atmosphere. The balloon catheter was pulled back and advanced for 3 times to injury the vessels. The PBS suspension containing 1×106 EPCs marked with CellTracker™ CM-DiI dye (Invitrogen, USA) was injected locally into the arterial lumen for about 5 min. After that, the distal end of the external carotid artery was closed and the blood flow to the common carotid was restored by release of the ligatures, and the wound was then closed. No adverse neurological or vascular effects were observed in any animal undergoing this procedure. At 1 week after injury and transplantation, rabbits were sacrificed and the target carotid artery were taken and frozen for the measurement of reendothelialization study. Re-endothelialization of artery was assessed by staining with Evans Blue dye (Sigma, USA) according to the method described previously (Lindner et al. , 1993). A fluorescent microscope (Olympus BX51, Japan) was performed to detect homing of transplanted EPCs to the site of vascular injury in separate experiments with the use of CM-DiI-labelled EPCs.

2.8 Statistical Analysis Values are expressed as mean ± standard deviation in the text and figures. Independent t-test was used to compare the differences between two groups. Comparisons among multiple

groups were performed using One-Way analysis of var iance (ANOVA). P < 0.05 was considered statistical significance.

3. Result 3.1 SDF -1a inhibits premature senescence in endothelial progenitor cell with the increased expression of h-TERT To assess the effect of SDF-1a on h-TERT expression, EPCs were stimulated with SDF -1a at the concentration of 100ng/ml. After 48 hours treatment, the senescent phenotype was inhibited judged by SA-ß-gal assay, which has also been illustrated in our previous study (Zheng et al., 2008). As shown in figure 1A, SDF-1a significantly increased expression of hTERT protein in a concentration-dependent manner.

3.2 Inhibition of h-TERT abrogates the protective effect of SDF-1a against premature senescence. To further determine the effect of h-TERT in SDF-1a against premature senescence, h-TERT was knockdown by siRNA. As shown in figure 1C and 1D, SDF -1a induced a significant decrease in SA-ß-gal-positive cells (42.2% ± 5.0 to 19.1% ± 3.1, P