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First published online February 1, 2007
Stem Cells Vol. 25 No. 5 May 2007, pp. 1195 -1203
doi:10.1634/stemcells.2006-0605; www.StemCells.com
© 2007 AlphaMed Press

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THE STEM CELL NICHE

Host Vascular Niche Contributes to Myocardial Repair Induced by Intracoronary Transplantation of Bone Marrow CD34+ Progenitor Cells in Infarcted Swine Heart

Shaoheng Zhanga, Junbo Gea,b, Lan Zhaoa, Juying Qiana, Zheyong Huanga, Li Shena, Aijun Suna, Keqiang Wanga, Yunzeng Zoua,b

aShanghai Institute of Cardiovascular Diseases, Zhongshan Hospital and
bInstitutes of Biomedical Sciences, Fudan University, Shanghai, China

Key Words. Myocardial infarction • Progenitor cells • Transplantation • Collateral vessels • Myocardial repair

Correspondence: Junbo Ge, M.D., Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, 180 Fen Lin Road, Shanghai 200032, China. Telephone: 86-21-64041990, ext. 2152; Fax: 86-21-64223006; e-mail: gejunbo{at}zshospital.net

Received on October 5, 2006; accepted for publication on January 22, 2007.

First published online in STEM CELLS EXPRESS  February 1, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
The effects of bone marrow cell transplantation (BMT) on myocardial infarct might be affected by host intrinsic circumferences. A best vascular niche was shown in the infarcted hearts with collateral vessels at 2 weeks after myocardial infarction (MI). BMT caused the greatest cardiac repairs after MI in the swine with better collateral vessels, which might be relative to richer collateral vessels, greater vessel densities, and higher expressions of basif fibroblast growth factor and stromal cell–derived factor-1 in the hearts before BMT. Our data suggest that existence of intrinsic collateral vessels contributes greatly to the beneficial effects of intracoronary BMT on cardiac repairs after MI.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Basic and clinical research has shown that bone marrow stem cell transplantation (BMT) can improve cardiac function after myocardial infarction (MI), but the mechanisms by which BMT exerts beneficial effects remain largely controversial [1–7]. Moreover, the different effects of BMT on the infarcted heart have been reported in several lines of studies. Although intracoronary administration has been indicated to be advantageous for the tissue repair of infarcted heart muscle in the patients with myocardial infarction [8, 9], Strauer et al. [10] reported that catheter-based intracoronary implantation of autologous bone marrow cells (BMCs) exhibited no statistically significant change in cardiac function. However, Wollert et al. [11] demonstrated that autologous BMC transplantation by the same intracoronary transfer approach promoted left ventricular systolic function after acute myocardial infarction. The differences in these results have caused confusion in the BMT treatment. Because these studies focused mainly on the safety and efficacy of transplanted extrinsic stem cells on cardiac repair after MI, we therefore supposed whether host intrinsic factors might affect the efficacy of BMT.

A variety of growth factors and cytokines, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and stromal cell-derived factor-1 (SDF-1) could be upregulated in myocardium after MI [12]. VEGF and bFGF may regulate neovascularization, and SDF-1 could recruit circulating or resident stem cell populations, such as CD34+ progenitor cells, for myocardial repair and regeneration after MI [13]. However, this autologous cardiac repair is very limited because of a low dividing capacity of terminally differentiated cardiac cells [14]. Use of undifferentiated stem cells may resolve such limitations in the infarcted hearts. The cardiac host environment could determine the fate and the effect of transplanted stem cells [15]. One of the fundamental questions is how the host environment factors, such as the existence of collateral vessels, affect the engraftment and effects of the transplanted cells. Some reports have indicated that transplanted neural stem cells were mainly distributed around blood vessels [16, 17], suggesting that host might provide a vascular niche for neurogenesis.

In this study, we investigated the possible role of host collateral vessels in cardiac repair after intracoronary BMT in a swine MI model. We found that host intrinsic factor, especially the collateral vessels, greatly affected the effects of transplanted CD34+ progenitor cells on the infarcted heart possibly through upregulation of bFGF, which might increase collateral vessels and enhance angiogenesis, and of SDF-1, which could home transplanted CD34+ progenitor cells into the infarcted hearts, leading to improvement of cardiac repair after MI.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Animals
Male mini-swine (4 months old, 20–25 kg) were obtained from Shanghai animal administration center. All animal experiments were approved by the Animal Care and Use Committee of Fudan University and were in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Academy Press (NIH Publication No. 85-23, revised 1996).

Animal Model, Study Design, and Biopsy
Mini-swine were sedated with ketamine (15–20 mg/kg), diazepam (1.5–2 mg/kg), and atropine (30–50 ug/kg), and anesthesia was maintained with thiopental (1–2 mg/kg/minutes, i.v.). Animals were intubated and mechanically ventilated with a respirator. A median sternotomy was performed, and the pericardium was opened. A silk suture was placed around the left anterior descending (LAD) coronary artery approximately 1/2 of the distance from the apex to allow ligation of the vessel. Occlusion was confirmed by electrocardiography (ECG), echocardiography, and coronary angiography (CAG) 60 minutes after ligation. A lidocaine bolus (2 mg/kg) was given intravenously before coronary occlusion. Lidocaine was subsequently infused at a rate of 50 µg/kg/minute. Two weeks later, animals were subjected to CAG again. Collateral filling of occluded vessels was graded according to the Rentrop score as described previously [18] (absent = 0, filling of side branches of the target occluded epicardial vessel without visualization of the vessel itself = 1, partial filling of the epicardial segment via collateral circulation = 2, complete filling of the epicardial segment of the occluded vessel = 3). Animals with Rentrop score = 0 (R0) or one (R1) were chosen. To exactly valuate the pre-existing vascular environment at the tissue, cellular, and molecular levels, we took five biopsy specimens from the different peri-infarcted areas per heart both in BMT and vehicle groups before cell therapy according to left ventriculography. These biopsy specimens were used to evaluate bFGF, VEGF, and SDF-1 expression and vessel density at the peri-infarct areas. Thereafter, the animals received either intracoronary vehicle (phosphate-buffered saline [PBS]) or CD34+ progenitor cell infusion.

Preparation, Labeling, and Intracoronary Transplantation of CD34+ Progenitor Cells
Two weeks after MI, bone marrow ({approx}80 ml) was aspirated from the ileum in all swine with R0 or R1. CD34+ progenitor cells were prepared by Ficoll-Hypaque gradient centrifugation (Lymphoprep; Axis-Shield plc, Dundee, Scotland, http://www.axis-shield.com/), incubated with MACS colloidal super-paramagnetic microbeads conjugated with anti-CD34 antibodies (Miltenyi Biotec Inc., Auburn, CA, http://www.miltenyibiotec.com). The CD34+ cells were then collected, and then stained with 4',6-diamidino-2'-phenylindole (DAPI; Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) as described previously [19]. Cells were suspended in ice-cold PBS at a density of 1 x 107cells per milliliter and kept on ice before being infused into swine. A total of 5 x 107 CD34+ progenitor cells in 20 ml of PBS (cells) or 20 ml of PBS were then introduced by intracoronary infusion with the use of a balloon catheter as described previously [7]. After exact positioning of the balloon at the site of the occluded artery, the balloon was inflated with low pressure to completely block blood flow for 3 minutes. Cell suspension (4 ml) or PBS (4 ml) was infused into the ligated artery through the center port of the balloon catheter. This maneuver was repeated five times to accommodate infusion of 5 x 107 cells or 20 ml of PBS.

To label the transfected cells genetically, we constructed a lentivirus vector inserted with an enhanced green fluorescent protein (EGFP) cDNA as described previously [20] and transfected it into some CD34+ progenitor cells before transplantation. More than 50% of CD34+ progenitor cells were EGFP-positive, as determined by flow cytometry.

Coronary Angiography and Echocardiography Examinations
Coronary angiography and echocardiography were performed in sedated animals at baseline (preinfarction), immediately after, and 2 and 6 weeks after infarct. Two-dimensional images were obtained at midpapillary and apical levels. To calculate fractional shortening (FS), the dimension of the internal left ventricle was measured at end systole and end diastole from M-mode recording at the level cord tendon. Left ventricular volumes at end-diastolic and end-systolic phase and ejection fraction (EF) were calculated by the modified Simpson's method.

Histology and Immunohistochemistry
At the end of each study, the heart was removed and sectioned from the ligation location to apex into five transverse slices in a plane parallel to the atrioventricular groove. The left ventricular (LV) sections were divided into three portions: the infarct zone, defined as a myocardial region devoid of myocytes; the peri-infarct region, the region 2 cm away from the infarct zone; and the distant region, the region 5 cm away from the infarction. To observe myocardial expression of bFGF and VEGF, immunofluoroscence staining was performed on the removed hearts. The antibodies were included: rabbit anti-bFGF (1:500; Chemicon, Temecula, CA, http://www.chemicon.com) or VEGF (LabVision, Fremont, CA, http://www.labvision.com/) polyclonal antibodies. For observation of vessel density, immunohistochemistry was performed on a series of the paraffin-embedded sections at the peri-infarct area. The sections were examined using standard immunohistochemical techniques with anti-human factor VIII polyclonal antibody (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and an immunoperoxidase kit (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Vessel density was expressed as the number of factor VIII+ endothelial cells per square millimeter [5]. Under fluorescence microscope, the number of 4,6-diamidino-2-phenylindole (DAPI)-positive cells was evaluated on the cryostat sections. To observe the myocardial distribution, vasculogenesis, and myocardial differentiation of transplanted DAPI+ cells, the cryostat sections were stained with the factor VIII antibody and anti-myosin heavy chain (MHC; Chemicon) antibody. The cell and vessel density counts in each heart were averaged from 25 fields (five slides and five areas in each slide).

To determine the biopsy specimen character, immunofluoroscence was performed on a series of the cryostat sections. The primary antibodies used in this study included: anti-human factor VIII, MHC, or VEGF antibodies, anti-bFGF (Upstate, Charlottesville, VA, http://www.upstate.com) or SDF-1 (Sigma-Aldrich) antibodies. Vessel density was counted as mentioned above.

A pathologist who was blinded to group identity evaluated the capillary density and cell count by counting vessels and cells in the chosen areas. Appropriate immunohistological controls were performed to assess specificity, including exclusion of primary antibody and use of mouse, goat, and rabbit sera isotype in place of the antibodies.

Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from the infarct-related artery area of left ventricular tissue at 2 or 6 weeks after infarct, using TRIzol reagent (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com). The expressions of bFGF, SDF-1, VEGF, and positive control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at the mRNA level were evaluated with reverse transcription-polymerase chain reaction (RT-PCR). Five micrograms of RNA was used to make cDNA. The cDNA (1 µl) was subject to amplification. The amplification conditions were as followed at Table 1. The PCR products were subject to electrophoresis on 1.5% agarose gels, scanned, and semiquantitated using 1D Image Analysis software (Kodak 1D v3.53, 4 Science Park, New Haven, CT).


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  		Table 1. Primer sequences

 
Western Blot Analyses
The expression of bFGF, SDF-1, and VEGF at protein levels was detected by Western blotting in total protein fraction prepared from the peri-infarct area at 2 or 6 weeks after infarct. GAPDH (Upstate) was served as a positive control. Protein extract (100 µg) was subject to electrophoresis on 10% SDS polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were incubated with an anti-bFGF, SDF-1, VEGF, or GAPDH antibody. The levels of these proteins were determined as ratios of the target protein/GAPDH using ImageQuant software.

Statistical Analysis
Data are expressed as mean ± SEM. Comparison of means between groups was performed by one-way analysis of variance followed by analysis with the Bonferroni t test. The two-way analysis of variance with repeated measures was used to compare the EF and FS among various groups and time points. Correlation analysis between the density of transplanted cells and local expression of bFGF protein was performed by bivariate correlations. Differences between groups were considered significant at p < .05.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Thirty-one of the 47 experimental animals survived the MI operation and 20 animals with R0 or R1 (n = 10 each) received PBS injection (R0+PBS or R1+PBS, n = 5 each) or CD34+ progenitor cells implantation (R0+BMT, R1+BMT, n = 5 each). All 20 animals survived to the scheduled study end.

Collateral Vessels
We first observed the collateral vessels by coronary angiography at various time points after MI. The proximal end of the LAD in all animals was blocked after ligation (Fig. 1A, arrowhead). At 2 weeks after ligation, there were some bridging collaterals in the distal occluded LAD from other blood vessels in R1 groups (e.g., left circumflex artery, right coronary artery, or the diagonal branches of the proximal LAD), by which blood flow bypass the occluded portion. Rentrop score was significantly higher in R1+BMT group compared with R0+BMT and R1+PBS groups 6 weeks after MI (Fig. 1B) and the absolute increase of Rentrop score from 2–6 weeks after MI was also significant higher in R1+BMT group than that of R1+PBS group, whereas Rentrop scores were similar between R0+PBS and R0+BMT groups 6 weeks after MI (Fig. 1C).


Figure 1
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  		Figure 1. Rentrop scores derived from coronary angiography before MI, immediately after, and 2 and 6 weeks after MI. (A): Photographs showing coronary angiography results in individual groups. White arrowheads and black arrow show blocked left anterior descending coronary arteries and collateral circulation, respectively. (B): Quantitative data of Rentrop scores in various groups 6 weeks after MI. (C): Quantitative data of Rentrop score changes in various groups 6 weeks after MI. {dagger}, p < .05 versus R0+BMT; {ddagger}, p < .05 versus R1+PBS (mean ± SD, n = 5 each). Abbreviations: BMT, bone marrow cell transplantation; R0, Rentrop score = 0; R1, Rentrop score = 1; MI, myocardial infarction; PBS, phosphate-buffered saline.

 
Cardiac Function
EF (Fig. 2A) and FS (Fig. 2B) were significantly decreased immediately after MI and at 2 weeks after MI in all groups. EF and FS were significantly higher in the R0+BMT group compared with the R0+PBS group and further increased in the R1+BMT group, whereas EF and FS were similar in both PBS-treated groups at 6 weeks after MI.


Figure 2
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  		Figure 2. EF and FS assessed by echocardiography before MI, immediately after, and 2 and 6 weeks after MI. *, p < .05 versus R0+PBS; {dagger}, p < .05 versus R0+BMT; {ddagger} p < .05 versus R1+PBS; §, p < .05 versus Pre-MI; II, p < .05 versus MI; #, p < .05 versus MI 2 weeks (mean ± SD, n = 5 each). Abbreviations: BMT, bone marrow cell transplantation; EF, ejection fraction; FS, fraction of shortening; MI, myocardial infarction; PBS, phosphate-buffered saline.

 
Host Vascular Niche
To determine the mechanism for the increased Rentrop, biopsy specimens were performed on the peri-infarct myocardial tissue 2 weeks after MI. The expressions of bFGF, VEGF, and SDF-1 were examined with RT-PCR, Western blot, and immunofluorescence. As shown in Figure 3, bFGF and SDF-1 were more abundantly expressed in the R1 group than in the R0 group (Fig. 3A, 3B). bFGF expressed mainly in the cytoplasm of the blood vessels (Fig. 3C-a, arrowhead) and more abundantly in the R1 group than in the R0 group (data not shown). SDF-1 mainly expressed in the cytoplasm of cardiomyocytes around rich blood vessel regions (Fig. 3C-b, arrowhead), and more abundantly in the R1 group than in the R0 group (data not shown). VEGF showed no significant difference between the R0 group and the R1 groups (Fig. 3A, 3B, and 3C-c). We also observed the number of factor VIII+ endothelial cells per square millimeter under light microscope in the peri-infarct regions of hearts from the R0 (Fig. 3D-a) group and R1 group (Fig. 3D-b). The vessel density was greater in the R1 group compared with the R0 group (Fig. 3D-c).


Figure 3
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  		Figure 3. Host vascular niche at 2 weeks after MI. (A, B): The electrophoresis and quantitative analysis data of bFGF, SDF-1 and VEGF at mRNA and protein levels measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot in the R0 and R1 groups. (mean ± SD, n = 30, RT-PCR experiments were performed three times. The results were the average of 30 experiments on 10 animals in each group). (C): Immunofluoroscence staining showing double-staining of bFGF (red) and factor VIII (green), SDF-1 (green), and myosin heavy chain (MHC; red), and local expression of VEGF (red) at the peri-infarct area from the R1 (C-a, C-b, and C-c, respectively) group (original magnification, x40). Some of vascular endothelial or myocardial cells were double-stained with bFGF or SDF-1 and factor VIII or MHC (yellow, arrowheads). (D-a, D-b): Representative slides showing factor VIII+ endothelial cells in the peri-infarct regions from the R0 group (A) and the R1 group (B) 2 weeks after MI. The capillaries were stained red with anti-factor VIII staining (original magnification, x10). (D-c): Quantitative data of vessel numbers in various groups. *, p < .05 versus the R0 group. (mean ± SD, n = 50, vessel counts were performed five times. The results were the average of 50 experiments on 10 animals in each group). Abbreviations: bFGF, basic fibroblast growth factor; SDF-1, stromal cell–derived factor-1; VEGF, vascular endothelial growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 
Assessment of Regional Angiogenesis
Figure 4 shows the number of factor VIII+ endothelial cells per square millimeter under light microscope in the peri-infarct regions of hearts from R0+PBS (Fig. 4A, 4E), R0+BMT (Fig. 4B, 4F), R1+PBS (Fig. 4C, 4G), and R1+BMT (Fig. 4D, 4H) groups under lower or higher magnification microscopy. The vessel density was increased in both BMT-treated groups compared with respective PBS-treated groups and was even higher in the R1+BMT group than in the R0+BMT group (Fig. 4I).


Figure 4
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  		Figure 4. Angiogenesis analysis. (A–H): Representative slides showing factor VIII+ endothelial cells in the peri-infarct regions from R0+PBS (A, E), R0+BMT (B, F), R1+PBS (C, G), and R1+BMT (D, H) groups 6 weeks after MI. The capillaries were stained with DAB (original magnification, x 10 [A–D], x 40 [E–H]). (I): Quantitative data of vessel numbers in various groups. *, p < .05 versus R0+PBS; {dagger}, p < .05 versus R0+BMT; {ddagger}, p < .05 versus R1+PBS (mean ± SD, n = 25 each, vessel counts were performed five times; the results were the average of 25 experiments on five animals in each group). Abbreviations: BMT, bone marrow cell transplantation; PBS, phosphate-buffered saline.

 
bFGF and VEGF Expressions in the Ischemic Myocardium
The mRNA and protein expressions of bFGF (Fig. 5A, 5B) in the peri-infarct areas were significantly higher in the R0+BMT and R1+BMT groups compared with respective PBS treated groups and were also higher in the R1+BMT group compared with the R0+BMT group. Similar results were revealed on bFGF immunofluorescence photomicrographs from the R0+PBS group (Fig. 5C), R0+BMT group (Fig. 5D), R1+PBS group (Fig. 5E), and R1+BMT group (Fig. 5F). bFGF was mainly found in endothelial cells of capillary vessels. Moreover, the cell count of CD34+ progenitor cells double-stained with DAPI and bFGF were greater in the R1+BMT group than in the R0+BMT group (Fig. 5D, 5F, arrowheads). However, the mRNA and protein levels of VEGF expression were not significantly different among four groups (data not shown). In addition, the density of transplanted cells was positively correlative with local expression of bFGF protein assessed by western blot (Fig. 5G, r = .965, p < .05).


Figure 5
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  		Figure 5. bFGF expressions in the ischemic myocardium. (A, B): The electrophoresis and quantitative analysis data of bFGF at mRNA and protein levels measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot in various groups. (C-F): Local expressions of bFGF at the peri-infarct area from the R0+PBS (C), R0+BMT (D), R1+PBS (E), and R1+BMT (F) group. (D, F): Double immunostaining results with 4',6-diamidino-2'-phenylindole (DAPI) and bFGF (vascular endothelial cytoplasm were stained red) in the R0+BMT and R1+BMT groups, respectively. Some of the transplanted CD34+ progenitor cells were double-stained with DAPI and bFGF (arrowheads). *, p < .05 versus R0+PBS; {dagger}, p < .05 versus R0+BMT; {ddagger}, p < .05 versus R1+PBS (mean ± SD, n = 15, RT-PCR experiments were performed three times. The results were the average of 15 experiments on five animals in each group). (G): The correlation between the density of transplanted cells and local expression of bFGF protein assessed by Western blot (r = .965, p < .05) in the R1+BMT group. Abbreviations: bFGF, basic fibroblast growth factor; BMT, bone marrow cell transplantation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline.

 
The Number and Distribution of Transplanted Cd34+ Progenitor Cells
Figure 6A showed the distribution of transplanted CD34+ progenitor cells prelabeled with DAPI (nuclei stained blue) in infarcted myocardium 6 weeks after MI. Figure 6B showed that the number of engrafted cells into the infarcted hearts was significantly higher in the R1+BMT group than in the R0+BMT group. These CD34+ progenitor cells were not dispersed throughout the left ventricle but were assembled around blood vessels as revealed by double staining these cells with anti-factor VIII monoclonal antibodies (Fig. 6A, arrowhead). Some DAPI-labeled CD34+ progenitor cells expressed the vascular endothelial cell-specific cytoplasmic protein, factor VIII (arrowhead), but not MHC protein. These cells seemed to be incorporated in the neovascularized areas, contributing to new vessel formation but not to myocardial differentiation. Moreover, the count of cells double-stained with DAPI and factor VIII were greater in the R1+BMT group than in the R0+BMT group. To confirm these results, we also labeled CD34+ progenitor cells genetically with EGFP (plasma stained green). The results were consistent with those of transplanted CD34+ progenitor cells prelabeled with DAPI in the R0+BMT and R1+BMT groups (Fig. 6A, 6C).


Figure 6
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  		Figure 6. Cell count and distribution of transplanted CD34+ progenitor cells in ischemic myocardium. (A): Distribution of transplanted CD34+ progenitor cells prelabeled with DAPI (the nuclei were stained blue; left panel) or EGFP (the cytoplasm was stained green; right) in the R0+BMT and R1+BMT groups, respectively. The middle panel in (A) shows the tri-immunostaining results with DAPI, anti-factor VIII (vascular endothelial cytoplasm were stained red), and anti-myosin heavy chain (MHC; myocardial cytoplasm was stained green) in the R0+BMT and R1+BMT groups, respectively. Some of the transplanted CD34+ progenitor cells were double-stained with DAPI and factor VIII (arrowheads) but not with MHC. (B, C): Quantitative data of transplanted CD34+ progenitor cells engrafted into the infarcted hearts, respectively. *, p < .05 versus R0+BMT (mean ± SD, n = 25 each, cell counts were performed five times. The results were the average of 25 experiments on five animals in each group). Abbreviations: DAPI, 4',6-diamidino-2'-phenylindole; EGFP, enhanced green fluorescent protein.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
The important and unique findings of this study were twofold. First, autologous intracoronary transplantation of CD34+ progenitor cells improved cardiac function and induced angiogenesis in animals with coronary collateral vessels more than in those without collateral vessels after MI. Second, the number of CD34+ progenitor cells engrafted into infarcted hearts and the expression of bFGF and SDF-1 after BMT were more increased in animals with coronary collateral vessels than those without collateral vessels. Thus, these results demonstrate that pre-existence of host collateral vessels magnified the beneficial effects of bone marrow CD34+ progenitor cell transplantation on the infarcted hearts through increasing the number of engrafted progenitor cells, expression of bFGF and SDF-1, and angiogenesis.

Self-renewal, activation, and differentiation of stem cells could be regulated by the cells and proteins that constitute the extracellular environment (or "niche") [21]. Common components of stem cell niches are composed of neighboring cells, their differentiating daughters, and growth factors. We observed that bFGF and SDF-1 were more abundantly expressed in the infarcted hearts with collateral circulation 2 weeks after MI. bFGF mainly expressed in the blood vessel. SDF-1 mainly expressed in the cytoplasm of cardiomyocytes around the regions rich in blood vessels. Consistent with the changes in these cytokines, the blood vessel density was greater in the peri-infarct regions of hearts with collateral circulation compared with that in the hearts without collateral circulation. These data suggested that ischemic heart itself might provide a vascular niche, which might increase collateral circulation, enhance angiogenesis, and home the stem cells into the infarcted hearts.

The importance of the vascular niche in clinical cases of cellular cardiomyoplasty has been shown in previous articles where vascularized infarct scars seem to be better regenerated than complete ischemic fibrotic areas [22]. However, in our present study, we compared the effects of vascular niche on cellular cardiomyoplasty in swine not only by echocardiographic and ventricle angiographic methods but also by examinations at tissue and molecular levels. In addition, because we found, in the animal MI model without cell infusion, that the ventricular function and myocardial preservation were increased a bit in those with a good collateral circulation (R1+PBS) than in others without collateral circulation (R0+PBS) (although these benefits were limited), it is possible that the spontaneous development of collateral circulation after MI is related to the improvement of ventricular function and viability. Pre-existed coronary collateral vessels might magnify the beneficial effects on cardiac repair induced by intracoronary BMT after MI.

We did not exactly define the collateral source and pattern in the present study, which must involve some bridging collaterals between the occluded artery and other blood vessels (e.g., left circumflex artery, right coronary artery, or the diagonal branches of the proximal LAD). The injected cells might go into the infarcted area through these bridging collaterals. Indeed, we have observed these bridging collateral vessels in the distal occluded LAD in R1 groups, through which blood flow bypassed the occluded portion, helping the cells from distal end of the catheter go into the infarct area. Although the exact roles of bridging collateral vessels needs to be further defined, we were the first to observe that the pre-existing host vascular niche enhances the beneficial effects of intracoronary BMT on cardiac function recovery.

In this study, we found that intracoronary transplanted bone marrow CD34+ progenitor cells were distributed centrally around blood vessels rather than randomly throughout the myocardium. Moreover, the number of engrafted stem cells into the host hearts around the blood vessels was significantly greater in animals with collateral vessels than in animals without collateral vessels. Compared with that in R0 group, SDF-1 expression was more abundantly expressed in the biopsy specimen of the heart from R1 group 2 weeks after MI, which was mainly observed in the cytoplasm of both blood vascular endothelial cells and cardiomyocytes around the regions rich in blood vessels, indicating that existence of vascular niche in the host heart might be helpful to homing of the transplanted CD34+ progenitor cells in the infarcted heart. Our study also showed that both expression of bFGF and angiogenesis in the peri-infarct area were significantly increased in the R1+BMT group compared with R0+BMT group, which was parallel to the density of transplanted cells. The evidence collectively suggests that host SDF-1 and bFGF play a significant role in the recruitment of transplanted cells independent of collateral flow-mediated distribution. This finding is in line with previous studies showing that neural stem cells were positioned predominantly in a vascular niche [17]. Our data also suggest that pre-existing vascular niche might increase the release of growth factors from vascular endothelial cells and angiogenesis in the ischemic myocardium, resulting in more cardiac repair in the R1+BMT group. Shen et al. [23] show that endothelial cells that are enriched in the neural stem cell niche could regulate neural stem cell proliferation by secreting bFGF and induce these stem cells to become neurons in vitro. However, in this study, both the mRNA and protein levels of VEGF expression were not significantly different among individual groups 2 and 6 weeks after MI, consisting of data from the pig study by Heba et al. [24] showing that the expression of VEGF was upregulated within 4 days after MI. We also observed that the expression of VEGF was increased at 2 days, peaked at 7 days, and decreased thereafter in the infarcted hearts of rats (data not shown). These results suggest that the increase in VEGF might be an early response to hypoxia, which serves as a signal for physiologically compensatory angiogenesis and amelioration of endothelial cell apoptosis but not as the main regulator for the effect of the transplanted cells after 2 weeks of MI.

After myocardial infarction, induction of bFGF may also serve as an early signal for physiologically compensatory angiogenesis and amelioration of endothelial cell apoptosis. At the onset of MI (within 4 days), the cytokine was detected extracellularly in the border zone of myocardial infarction and locally in inflammatory infiltrates of infarcted myocardium [24], indicating that the early expression levels of bFGF were related to the degree of hypoxia and inflammatory reaction. At a later stage, however, cytokine was found mainly in vascular cells situated among fibrous tissue of the border zone [24], suggesting that the expression levels of bFGF were related mainly to the amount of surviving vascular cells. In addition, we observed in another experiment that the increase of bFGF appeared as early as 2 days, and the increase persisted for more than 2 weeks after MI in rats (data not shown), suggesting that bFGF might play an important role in the cardiac repair even at a later stage after MI. These results might be used to explain why the expression level of VEGF is same between R0 groups and R1 groups, but bFGF is expressed much more in the myocardium from R1 groups compared with that from R0 groups in the present study.

In the present study, we found that the number of cells stained with bFGF or factor VIII counted in DAPI-labeled CD34+ progenitor cells was greater in the R1+BMT group than in the R0+BMT group, suggesting that pre-existing vascular niche is helpful for transplanted CD34+ progenitor cells to form more new vessels. The result of experiment using EGFP-expressing CD34+ progenitor cells was similar to that using DAPI-labeled CD34+ progenitor cells. The increase in vessel density induced by stem cell therapy includes vasculogenesis and angiogenesis [2]. In our study, both the number of capillaries and Rentrop score were greater in the R1+BMT group than in the R0+BMT group, suggesting that the transplantation of CD34+ progenitor cells in animals with pre-existing host vascular niche increased angiogenesis as well as vasculogenesis. The expression of bFGF and angiogenesis were found more in the region rich in gathered transplanted cells than other areas, suggesting that the increase in vessel density was due, at least in part, to the injected cells.

It is well known that niche is composed of stem/progenitor cells and niche-supportive cells, such as mesenchymal cells and extracellular matrix, and functions as regulator of proliferation, differentiation, and quiescence. In the present study, we did not examine the interaction between transplanted cells and niche cells, nor did we assess exactly the role of the niche. These will be addressed in a future study. However, we indeed observed that transplanted CD34+ progenitor cells concentrated around blood vessels rather than being randomly distributed throughout the myocardium. This location places CD34+ progenitor cells near the endothelial cells that line blood vessels, facilitating communication between these two cell types. We also observed that bFGF and SDF-1 proteins were detected mainly at vascular endothelial and myocardial cells. These data suggest that these two cells, especially endothelial cells, were a key regulator for the fate of transplanted cells. The density of transplanted cells was positively correlative with local expression of bFGF protein, indicating that bFGF might play a key role in regulation of the proliferation and differentiation of transplanted progenitor cells.

We found no evidence showing myocardial differentiation from transplanted cells, which is in agreement with other studies demonstrating that hematopoietic stem cells do not transdifferentiate into cardiac myocytes in MI heart of mice [25, 26]. However, infusion of CD34+ progenitor cells induced increases in angiogenesis and vasculogenesis and improved cardiac function, indicating that CD34+ progenitor cells may be an available source for cell therapy in patients with MI or chronic heart failure. In the present study, we used a sustained coronary artery ligation model, which is similar to a completely occluded coronary artery disease in clinic, and infused cells by an intracoronary delivery method. An epicardial or transendocardial injection of the cells into the heart with a completely occluded (ligated) coronary artery is usually considered the optimal approach for the treatment of infarcted areas because epicardial injection needs a surgical approach, which is sometimes associated with the well known perioperative risks. Because it might be difficult to transplant cells by intraventricular injection into a beating heart, we therefore preferred an intracoronary infusion method, and all cells delivered via the balloon catheter in the present study seemed to flow into the infarcted and peri-infarcted tissue through host collateral vessels.

In summary, the beneficial effects of intracoronary delivery of CD34+ progenitor cells after MI on cardiac repair were magnified in the heart with pre-existing coronary collateral vessels, suggesting that host intrinsic factor, especially the vascular niche, plays a critical role in the cell therapy for the infarcted heart.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Mr. Renmin Yao, Mrs. Yang Wang and Mr. Jianguo Jia for technical assistance. This study was supported by the "10.5" Key Technologies R&D Programme (2004BA714B05-2), Shanghai Medical Development Research Fund (2000I-2D002), Shanghai Scientific & Technological Committee Research Fund (03XD14010), Chinese postdoctoral scientific fund (KLF101004), and National Keystone Basic Research Programme (2006CB943704). S.Z., J.G. and Y.Z. contributed equally to this work.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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