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Interact CardioVasc Thorac Surg 2008;7:767-770. doi:10.1510/icvts.2007.169896
© 2008 European Association of Cardio-Thoracic Surgery
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Work in progress report - Experimental

Adenovirus-mediated stromal cell-derived- factor-1{alpha} gene transfer induces cardiac preservation after infarction via angiogenesis of CD133+ stem cells and anti-apoptosis{star}

Junming Tanga,b,*, Jianing Wanga,*, Jianye Yanga and Xia Konga

a Institute of Clinical Medicine, Renmin Hospital, Yunyang Medical College, Shiyan, Hubei 442000, People's Republic of China
b Department of Physiology, Yunyang Medical College, Shiyan, Hubei 442000, People's Republic of China

Received 11 October 2007; received in revised form 25 May 2008; accepted 26 May 2008

{star} This study was supported by grants from National Natural Science Foundation of China (30700306).

*Corresponding authors. Tel.: +86-719/8637170; fax: +86-719/8637011.

E-mail address: tangjm416{at}163.com (J. Tang), rywjn{at}vip.163.com (J. Wang).


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgements
 References
 
In our study, we found cardiocytes expressed CXCR4, and the number of cardiocytes apoptosis with SDF-1 treatment decreased obviously through SDF-1 induced the up-regulation of phosphorylated Akt. On day 7 after myocardial infarction, marked expression of SDF-1{alpha}, and the number of CD133+ cells was the highest in the AdV-SDF-1 injection hearts. On day 28 post-treatment, blood vessel density in the AdV. SDF-1 group was higher in infracted zones. Infarct size and collagen accumulation in the infracted area decreased significantly, thickness of LV wall, vessels and cardiocytes' density increased obviously in the AdV-SDF-1 group than in control or Adv-LacZ group, and hemodynamics showed the improvement of left ventricle heart function in the AdV.SDF-1 group. Therefore, SDF-1{alpha} could improve cardiac structure and function through the combined effects of angiogenesis and anti-apoptosis.

Key Words: Myocardial infarction; SDF-1{alpha}; Stem cell; Angiogenesis; Apoptosis


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgements
 References
 
Bone marrow (BM)-derived stem cells have been shown to home into the periinfarct region of the heart. This observation prompted a search for chemotactic factors that facilitate this ‘homing’ process [1]. Experimental myocardial infarction (MI) in mice increased the expression of stromal derived factor-1 alpha (SDF-1{alpha}) in the infarct and border region [2]. This provides the required stimulus for mobilization of stem cells from BM niches to the damaged site as a part of a natural repair process [3, 4]. SDF-1{alpha} specifically interacts with its receptor CXCR4 and orchestrates the mobilization and homing of stem cells from bone marrow (BM) to the ischemic tissue [1]. However, the effect of intrinsic SDF-1{alpha} upregulated expression is transient and insufficient for cardiac repair [3, 4]. Therefore, forced expression of SDF-1 by adenoviral gene delivery and SDF-1{alpha} protein increased the recruitment of BM-derived stem cells to the infarcted heart. Furthermore, SDF-1{alpha} protein and gene delivery to the ischemic heart promotes endothelial progenitor cells (EPCs, one of characteristic surface markers of EPCs is CD133) recruitment into the ischemic muscles to promote angiogenesis [4–6]. These findings raise the possibility that SDF-1{alpha} have the effects on myocardial structure and function via angiogenesis of stem cells homing. However, little information is available about the role of SDF-1 on angiogenesis and anti-apoptosis after MI.

The purpose of this study was to investigate whether SDF-1{alpha} directly decreases the cardiocytes apoptosis and, indirectly, induces CD133+ EPCs migration into the injured heart, and thereby changes myocardial histomorphology and improves cardiac function in a rat model of MI.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgements
 References
 
Methods in detail are available as a supplement from the author.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgements
 References
 
3.1. Effect of hSDF-1{alpha} on myocardial conservation

CXCR4 expression in the cultured cadiocytes (Fig. 1a–c) and normal heart (Fig. 1d) is demonstrated (Fig. 1a). Cardiac myocytes in the infarct zone (Fig. 1e) and infarct border zone (Fig. 1f) 72 h after MI expressed CXCR4 and the level of cardiac myocyte CXCR4 expression within the infarct border zone increased greater than in infarct zone and normal heart. SDF-1 within the infarct zone led to a significant decrease in the number of TUNEL-positive cardiac myocyte nuclei (53% decreases) (Fig. 1g–h). The heart that overexpressed SDF-1 had greater phosphorylated Akt than control heart (Fig. 1i). This decrease in cardiac myocyte apoptosis was accompanied by a significant increase in the area of surviving bundles of cardiac myocytes within the infarct zone compared with control group (Fig. 3).


Figure 1
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  		Fig. 1. (a) Representative immunofluorescent staining for CXCR4 (green) and cardiac Troponin T (red) in cultured cadiocytes. (b–d) Merged image of representative immunofluorescent staining for CXCR4 (green) and cardiac Troponin T (red) in the heart of sham group (b) and infarct border zone (c) and infarct zone (d) 72 h after LAD ligation. (e–f) SDF-1 showed the effect of anti-apoptosis on cultured cadiocytes. (e) cadiocytes treated with SDF-1 after oxygen deficit and FCS-starvation administration; (f) cadiocytes treated with oxygen deficit and FCS-starvation. TUNEL (green) and their nucleus stained with DAPI (blue) (400x). (g–h). Merged image from an animal 72 h after LAD ligation in control group (g) and SDF-1 group (h) with cardiac Troponin T (red), TUNEL (green) and their nucleus stained with DAPI (blue) (400x). (i) Western-blotting of rat heart protein extract showed that besides elevated hSDF-1{alpha} expression.

 

Figure 3
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  		Fig. 3. Immunofluorescent staining of myocardium and blood vessel. (a–d) cTnt staining 28 days after MI. cTnt positive cells were observed in the infarction area of the control (a), AdV.LacZ (b), AdV.SDF-1 (c) and sham groups (d). (e–h) von Willebrand factor (vWF) staining after MI. Blood vessel was observed in the infarction area of the control (e), AdV.LacZ (f), AdV.SDF-1 (g) and sham groups (h), respectively. (i) The value of cTnt positive red fluorescence photodensity was analyzed by per high power field (200x). (j) The number of blood vessel was analyzed per high power field (400x) by count method using image pro5.02, respectively. Data are mean±S.D. (n=5 each). *Denotes P=0.002 vs. control or AdV.LacZ group.

 
3.2. Effect of hSDF-1{alpha} on CD133+ stem cells homing into myocardium

CD133 mRNA expression levels increased obviously in the infarcted area of the AdV.SDF-1 group compared with control group or AdV.LacZ group. Simultaneously, the number of cells expressing CD133 surface markers was significantly higher in the infarct areas of heart in the AdV.SDF-1 group, as compared with the other groups (Fig. 2).


Figure 2
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  		Fig. 2. Representative immunofluorescent staining of homing stem cells in the myocardium infarction area. (a–c) CD133 staining 7 days after MI. CD133 positive cells were observed in the infarction area of the AdV.SDF-1 (a, 400x), control groups (b, 400x) and AdV-LacZ group. CD133+, red fluorescence; cellular nucleus marked by DAPI, blue fluorescence. (f) The number of CD133+ cells were analyzed per high power field (400x) by count method using image pro5.02, respectively. Data are mean±S.E.M. (n=5 each). (d–e) RT-PCR analysis of CD133 mRNA expressions in the infarction area and non-infarction area. Rat GAPDH served as an internal control. Data are the means of experiments carried out in duplicate. Values are mean±S.D. (n=5, each). *denotes P=0.005 vs. control or AdV.LacZ group.

 
3.3. Effect of hSDF-1{alpha} on surviving cardiocytes and angiogenesis in the heart

The data in Fig. 3 demonstrate that SDF-1{alpha}, to a greater extent, increase the area and number of cardiac myocytes within the infarct zone (Fig. 3). The angiogenic effect of SDF-1{alpha} overexpression was measured in terms of blood vessel density in the infarct regions after immunostaining for vWF-VIII (Fig. 3). Blood vessel counts per area were highest in infarct regions with overexpression of SDF-1{alpha}.

3.4. Effect of hSDF-1{alpha} on LV function

Measurement of hemodynamics in vivo showed that LV function was significantly lower in the AdV.SDF-1 group, AdV.LaZ group, and control group than sham operation group. However, improvement of LV function was significantly greater in the AdV.SDF-1 group than in control group. The differences of LVSP (P<0.05), ±dp/dtmax and LVEDP (P<0.05) were statistically significant between the three groups (Fig. 4).


Figure 4
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  		Fig. 4. Measurement of hemodynamics. Left ventricular (LV) function under baseline resting conditions 4 weeks after surgery; LVSP, left ventricle systolic pressure; LVEDP, left ventricle end-diastolic pressure; +dp/dtmax and –dp/dtmax: rate in rise and fall of ventricular pressure, respectively. *P=0.0006 vs. the sham group and control group (each group, n=10~12).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgements
 References
 
In our and Segret's study, cardiac myocytes and cardiac stem/progenitor cells have been shown to express CXCR4 [7]. SDF-1{alpha} to the cell surface of injured cardiac myocytes could lead to inhibition of myocyte apoptosis as it did to stem/progenitor cells cultured under ischemic conditions via SDF-1 inducing up-regulation of prosurvival signaling [8]. We found not only that cardiac myocytes had been shown to express SDF-1{alpha} receptor CXCR4, and SDF-1{alpha} could lead to inhibition of cardiac myocyte apoptosis via SDF-1/CXCR4 axle inducing up-regulation of Akt and Bcl-2. But also, we observed an ~53% decrease in cardiac myocytes apoptosis at the infarct area in those animals that received SDF-1{alpha}. These changes could lead to a significant increase in the survival of cardiac myocytes within the infarct zone of those animals that received AdV.SDF-1 injection.

Hattori et al. reported that plasma elevation of SDF-1 induced mobilization of hematopoietic stem cells into peripheral blood. We have found that myocardial tissue and plasma SDF-1{alpha} concentrations were manifestly increased in AdV.SDF-1 group. Peripheral mononuclear cells (PBMNCs) increased greater than other groups. And there was a prolonged augmented response of PBMNCs count to SDF-1 after MI induction (Supplement 2). Interestingly, much more CD133+ EPCs were localized in the area of overexpressing SDF-1{alpha}. These suggested that SDF-1 could induce mobilization of CD133+ EPCs and migrate into the infarct area. On the one hand, these EPCs could proliferate and differentiate into vascular cells [6, 9]. On the other hand, EPCs produce a wide array of arteriogenic cytokines and improve perfusion and remodeling in mouse models of hindlimb ischemia, and these effects appear to be mediated through paracrine mechanisms associated with local release of the arteriogenic cytokines [10]. Furthermore, SDF-1 could promote the expression of angiogenic cytokines such as VEGF, fibroblast growth factor (FGF) and hepatocyte growth factor (HGF), and so on [2, 5]. Also, increased VEGF could involve in or trigger angiogenic progress of stem cells, and even might contribute to postnatal neovascularization by mobilizing bone marrow-derived EPCs [11]. These data suggest that SDF-1 could improve the blood provide of cardiocytes in the infarct area of hearts through the ischemic vasculogenesis and angiogenesis.

In conclusion, SDF-1 could improve cardiac structure and function through the symphysial effect of anti-apoptosis and angiogenesis.


   Acknowledgements
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgements
References
 
We thank Professor Yongsheng Ren for his helpful instruction in heart function evaluation. We are grateful to Long Chen for help with immunofluorescence. We are grateful to Yongzhang Huang and Linyun Guo for help with Construction of Adenoviral Vector expressing hSDF-1{alpha}. Thanks also to Fei Zheng for his western blot analysis.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgements
 References
 

  1. Hofmann M, Wollert KC, Meyer GP, Menke A, Arseniev L, Hertenstein B, Ganser A, Knapp WH, Drexler H. Monitoring of bone marrow cell homing into the infarcted human myocardium. Circulation 2005;111:2198–2202.[Abstract/Free Full Text]
  2. Abbott JD, Huang Y, Liu D, Hickey R, Krause DS, Giordano FJ. Stromal cell-derived factor-1alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury. Circulation 2004;110:3300–3305.[Abstract/Free Full Text]
  3. Hattori K, Heissig B, Tashiro K, Honjo T, Tateno M, Shieh JH, Hackett NR, Quitoriano MS, Crystal RG, Rafii S, Moore MA. Plasma elevation of stromal cell-derived factor-1 induces mobilization of mature and immature hematopoietic progenitor and stem cells. Blood 2001;97:3354–3360.[Abstract/Free Full Text]
  4. De Falco E, Porcelli D, Torella AR, Straino S, Iachininoto MG, Orlandi A, Truffa S, Biglioli P, Napolitano M, Capogrossi MC, Pesce M. SDF-1 involvement in endothelial phenotype and ischemia-induced recruitment of BM progenitor cells. Blood 2004;104:3472–3482.[Abstract/Free Full Text]
  5. Hiasa K, Ishibashi M, Ohtani K, Inoue S, Zhao Q, Kitamoto S, Sata M, Ichiki T, Takeshita A, Egashira K. Gene transfer of stromal cell-derived factor-1alpha enhances ischemic vasculogenesis and angiogenesis via vascular endothelial growth factor/endothelial nitric oxide synthase-related pathway: next-generation chemokine therapy for therapeutic neovascularization. Circulation 2004;109:2454–2461.[Abstract/Free Full Text]
  6. Yamaguchi J, Kusano KF, Masuo O, Kawamoto A, Silver M, Murasawa S, Bosch-Marce M, Masuda H, Losordo DW, Isner JM, Asahara. Stromal cell-derived factor-1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization. Circulation 2003;107:1322–1328.[Abstract/Free Full Text]
  7. Segret A, Rucker-Martin C, Pavoine C, Flavigny J, Deroubaix E, Chatel MA, Lombet A, Renaud JF. Structural localization and expression of CXCL12 and CXCR4 in rat heart and isolated cardiac myocytes. J Histochem Cytochem 2007;55:141–150.[Abstract/Free Full Text]
  8. Kayali AG, Van Gunst K, Campbell IL, Stotland A, Kritzik M, Liu G, Flodstrom-Tullberg M, Zhang YQ, Sarvetnick N. The stromal cell-derived factor-1alpha/CXCR4 ligand-receptor axis is critical for progenitor survival and migration in the pancreas. J Cell Biol 2003;163:859–869.[Abstract/Free Full Text]
  9. Kuhlmann CR, Schaefer CA, Reinhold L, Tillmanns H, Erdogan A. Signalling mechanisms of SDF-induced endothelial cell proliferation and migration. Biochem Biophys Res Commun 2006;335:1107–1114.
  10. Miyamoto Y, Suyama T, Yashita T, Akimaru H, Kurata H. Bone marrow subpopulations contain distinct types of endothelial progenitor cells and angiogenic cytokine-producing cells. J Mol Cell Cardiol 2007;43:627–635.[CrossRef][Medline]
  11. Asahara T, Takahashi T, Masuda H, Kalka C, Chen D, Iwaguro H, Inai Y, Silver M, Isner JM. VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J 1999;18:3964–3972.[CrossRef][Medline]



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 Eur. J. Cardiothorac. Surg.Home page
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