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European Journal of Heart Failure 2007 9(10):974-985; doi:10.1016/j.ejheart.2007.06.012
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© 2007 European Society of Cardiology

Combined therapy with human cord blood cell transplantation and basic fibroblast growth factor delivery for treatment of myocardial infarction

Seung-Woo Choa,b, Il-Kwon Kimc,d, Suk Ho Bhanga,b, Boyoung Jounge, Young Jin Kimf, Kyung Jong Yood, Yoon-Sun Yangg, Cha Yong Choib and Byung-Soo Kima,*

a Department of Bioengineering, Hanyang University Seoul 133-791, Republic of Korea
b School of Chemical and Biological Engineering, Seoul National University Seoul 151-742, Republic of Korea
c Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine Seoul 120-752, Republic of Korea
d Division of Cardiovascular Surgery, Cardiovascular Center, Yonsei University College of Medicine Seoul 120-752, Republic of Korea
e Division of Cardiology, Cardiovascular Hospital and Research Institute, Yonsei University College of Medicine Seoul 120-752, Republic of Korea
f Department of Radiology, Yonsei University College of Medicine Seoul 120-752, Republic of Korea
g Department of Research and Development for Cellular Therapy, Medipost Biomedical Research Institute Yongin, 449-795, Republic of Korea

* Corresponding author. Tel.: +82 2 2220 0491; fax: +82 2 2298 4101. E-mail address: bskim{at}hanyang.ac.kr (B.-S. Kim).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Background: Transplanting cord blood-derived cells has been shown to augment neovascularization in ischaemic tissue.

Aim: To test whether sustained delivery of basic fibroblast growth factor (bFGF) enhances the efficacy of angiogenic cord blood mononuclear cell (CBMNC) transplantation therapy in treating myocardial infarction.

Methods: Three weeks after myocardial infarction, Sprague-Dawley rats were randomised to either injection of medium only (control), CBMNC transplantation, sustained bFGF delivery, or combined CBMNC transplantation and sustained bFGF delivery. Six weeks after treatment, tissue formation, neovascularization, and apoptotic activity in the infarct regions were evaluated by histology and immunohistochemistry. Left ventricular (LV) dimensions and function were evaluated by magnetic resonance imaging.

Results: Combined bFGF delivery and CBMNC transplantation significantly enhanced neovascularization in the ischaemic myocardium, as compared with either therapy alone. The enhanced neovascularization was likely due to increased VEGF and bFGF expression. The combined therapy also exhibited a reduced infarct area and apoptosis in the ischaemic myocardium, as compared with either individual therapy. The combined therapy did not attenuate LV dilation or increase ejection fraction significantly over either individual therapy.

Conclusion: This study demonstrates that sustained bFGF delivery enhances the angiogenic efficacy of CBMNC transplantation in rat myocardial infarction models.

Key Words: Neovascularization • Basic fibroblast growth factor • Cord blood mononuclear cell • Myocardial infarction

Received September 16, 2006; Revised April 7, 2007; Accepted June 25, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Therapeutic angiogenesis is an important strategy to salvage ischaemic tissues. Angiogenic stem cell transplantation [1-3] and angiogenic protein administration [4-6] have been used to induce neovascularization in animal models of limb and myocardial ischaemia. Transplanting endothelial progenitor cells, isolated from peripheral blood [1] or bone marrow [2,3], has been shown to induce collateral blood vessel development in both ischaemic limb and myocardium. Administering recombinant angiogenic growth factors, such as vascular endothelial growth factor (VEGF) [4,5] and basic fibroblast growth factor (bFGF) [4,6], also enhances angiogenesis in ischaemic tissue. Preliminary evidence suggests that there may be a clinical benefit to using angiogenic stem cell [7] and protein [8] therapy in treating severe limb or myocardial ischaemia in humans.

Human umbilical cord blood cells (HUCBCs) are a possible cell source for therapeutic angiogenesis. HUCBCs contain endothelial progenitor cells with extensive in vitro proliferative capacity [9]. Cord blood-derived CD34-positive cells can also differentiate into endothelial cells (ECs) [10]. Indeed, HUCBC transplantation has been shown to improve neovascularization and increase blood flow in animal models of hind limb [9,11,12] and myocardial [13,14] ischaemia. Both mononuclear cells [15] and CD34-positive cells [16] isolated from human cord blood have been shown to enhance neovascularization in ischaemic myocardium and improve cardiac function.

Recently, combined angiogenic stem cell transplantation and protein/gene therapy were shown to act synergistically in inducing angiogenesis in ischaemic tissue. In a recent study, intra-myocardial transplantation of bone marrow mesenchymal stem cells transfected with the VEGF gene, significantly enhanced angiogenesis and improved cardiac function in infarcted myocardium compared to untransfected cells [17]. It was subsequently reported that combined bone marrow mononuclear cell transplantation and in vivo angiopoietin-1 gene transfer promoted functional neovascularization in hind limb ischaemic tissue [18]. Our previous study demonstrated that treatment with granulocyte colony-stimulating factor enhanced the efficacy of cellular cardiomyoplasty by embryonic stem cell transplantation in infarcted myocardium [19]. However, to date no study has shown that the HUCBC transplantation angiogenic efficacy is enhanced in combination with angiogenic protein therapy.

The present study was performed to evaluate whether sustained bFGF delivery enhances the efficacy of angiogenic cord blood mononuclear cell (CBMNC) transplantation therapy in treating myocardial infarction. Three weeks after myocardial infarction, Sprague-Dawley rats randomly received either an injection of medium only (control), CBMNC transplantation, sustained bFGF delivery, or combined CBMNC transplantation and sustained bFGF delivery. The angiogenic efficacy of these treatments was determined by the arteriole density and angiogenic growth factor expression in the infarcted myocardium. Whether the combined therapy improved left ventricular (LV) dilatation and LV function more than the individual therapies was also investigated.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1. Rat myocardial infarction model
Induction of myocardial infarction, administration of treatments, and all analyses were performed according to the schedule in Fig. 1. The rat myocardial infarction model was prepared as previously described [20]. Sprague-Dawley rats (200-250 g, Jungang Animal Lab, Seoul, Korea) were anaesthetized by intra-muscular (i.m.) administration of ketamine hydrochloride (90 mg/kg) and xylazine hydrochloride (5 mg/kg). The anaesthetized rats were intubated and placed on a ventilator (Model 683, Harvard Apparatus, South Natick, MA). The heart was exposed through a 2-cm left lateral thoracotomy. Cryoinjury was performed using a metal probe (8 mm diameter) cooled by immersion in liquid nitrogen. The probe was applied to the left ventricle free wall twice for 10 s each, then six times for 60 s each. The muscle and skin were then closed with sutures. This cryoinjury method is known to induce comparable myocardial infarction in all animals, and is therefore appropriate for randomisation [21]. The mortality rate of the infarction procedure using cryoinjury method was approximately 30%. All animal care and handling was performed according to the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (NIH publication 85-23, revised 1996).


Figure 01
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  		Fig. 1 Schematic diagram illustrating the experimental design and the order of analyses. For measurement of LVESD, LVEDD, and EF five animals per experimental group were investigated with MRI, one day before sacrifice (41 days after treatment). We could not obtain MRI data from one animal in the bFGF delivery group. Immediately after sacrifice (6 weeks after treatment), the whole hearts retrieved from five animals in each group were examined with optical mapping. The retrieved hearts (n=5) in each group were divided into two specimens, each of which was used for histology, immunohistochemistry, and apoptosis assay (n=5) and RT-PCR assay (n=4), respectively.

 
2.2. CBMNC isolation
Written consent was obtained from all donor mothers prior to cord blood collection. CBMNC isolation was performed as previously described [22]. In brief, cord blood was mixed with an equal volume of phosphate buffered saline (PBS, Sigma, St. Louis, MO) and centrifuged on a Ficoll-Paque density gradient (Amersham Biosciences, Arlington Heights, IL) for 30 min at 2000 rpm. CBMNCs were isolated from the layer between the Ficoll-Paque reagent and the blood plasma and washed three times in PBS.

2.3. Myocardial infarction treatments
Three weeks after inducing the myocardial infarction, the rats were randomly assigned to one of four treatment groups (n=5 per group). Intra-myocardial cell and/or growth factor injection was performed under general anaesthesia. Rats were anaesthetized by an i.m. administration of ketamine hydrochloride (90 mg/kg) and xylazine hydrochloride (5 mg/kg). The anaesthetized rats were intubated and placed on a ventilator, prior to administration of the intra-myocardial injection of study treatment. The control group received an injection of medium only. A fibrin matrix was used as the vehicle to provide sustained bFGF delivery. Fibrin has previously been used as a reservoir for growth factor proteins [22-24], genes [25], and cells [20,22-24]. CBMNCs (2.0x107 cells per rat [20,26]) were suspended in 100 µl of fibrin matrix (Greenplast®, Greencross Co., Yongin, Korea) containing heparin (20 µg/ml, Sigma) and injected i.m. at two sites bordering the infarcted myocardium (CBMNC group). A fibrin matrix containing 100 µg of bFGF [27] and heparin (20 µg/ml) was injected i.m. at two sites bordering the infarcted myocardium (bFGF group). A fibrin matrix containing bFGF (100 µg), CBMNCs (2.0x107 cells per rat), and heparin (20 µg/ml) was injected i.m. at two sites bordering the infarcted myocardium (CBMNC+bFGF group). Cyclosporine-A (10 mg/kg body weight, CIPOL®, Chong Kun Dang, Seoul, Korea) was administered two days before and then daily after treatment, for the duration of the experiment to immunosuppress the rats. Approximately 30% of animals died during the injection procedure.

2.4. Histology and immunohistochemistry
Six weeks after treatment, all rats were sacrificed and the hearts were collected. Heart specimens were fixed in 10% (v/v) buffered formaldehyde, dehydrated with a graded ethanol series, embedded in paraffin, and cut into 4-µm sections. Collagen in the tissue sections was stained using Masson's trichrome method. The infarct area was the total infarction area divided by the total LV area. To detect angiogenic growth factors (VEGF and bFGF) in the infarcted regions, tissue sections were immunohistochemically stained with antibodies raised against VEGF and bFGF (Chemicon, Temecula, CA). To detect human cells, tissue sections were immunohistochemically stained with antibodies against human nuclear antigen (HNA, Chemicon). The staining signal was visualized with avidin-biotin complex immunoperoxidase (Vectastain® ABC kit, Vector Laboratories, Burlingame, CA) and a 3,3'-diaminobenzidine (DAB) substrate solution kit (Vector Laboratories).

2.5. Arteriole density estimation
Tissue sections were immunostained for smooth muscle (SM) {alpha}-actin (DAKO, Carpenteria, CA) to visualize arterioles in the infarcted region. The signal was visualized with an avidin-biotin complex immunoperoxidase (Vectastain® ABC kit) and a DAB substrate solution kit. The arterioles in the infarct regions were counted under a light microscope. Ten fields in two heart samples from each rat were randomly selected for these counts.

2.6. Determination of apoptotic activity
Apoptotic activity in the infarcted regions 6 weeks after treatment was determined by the terminal deoxynucleotidetransferase-mediated deoxyuridine triphosphate nick end-labelling (TUNEL) method using a commercially available kit (ApopTaq®, Chemicon). TUNEL-positive cells in regions bordering the infarcted myocardium were visualized with a DAB substrate solution kit. The tissue sections were counterstained with eosin.

2.7. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from the ischaemic myocardium 6 weeks after treatment. The tissues were homogenized and lysed in 1 ml TRIzol reagent (Invitrogen, Carlsbad, CA). Total RNA was extracted with 200 µl chloroform and precipitated with 500 µl 80% (v/v) isopropanol. After removing the supernatant, the RNA pellet was washed with 75% (v/v) ethanol, air-dried, and dissolved in 0.1% (v/v) diethyl pyrocarbonate-treated water. The RNA concentration was determined by measuring absorbance at 260 nm using a spectrophotometer. The reverse transcription reaction was performed with 5 µg pure total RNA using SuperScriptTM II reverse transcriptase (Invitrogen). The resulting cDNA was amplified by PCR using primers for VEGF (sense 5'-CCA TGA ACT TTC TGC TGT CTT-3' and antisense 5'-ATC GCA TCA GGG GCA CAC AG-3' [28]), bFGF (sense 5'-GAA GAG CGA CCC TCA CAT CAA G-3' and antisense 5'-CTG CCC AGT TCG TTT CAG TG-3' [29]), and β-actin (sense 5'-CCT TCC TGG GCA TGG AGT CCT G-3' and antisense 5'-GGA GCA ATG ATC TTG ATC TTC-3' [30]). The PCR was carried out in 35 cycles consisting of denaturing (94 °C for 30 s), annealing (60 °C for 30 s), and extension (72 °C for 45 s) followed by a final extension at 72 °C for 7 min. The expected RT-PCR products for VEGF, bFGF, and β-actin are 249, 236, and 208 bp, respectively. The PCR products were visualized by electrophoresis on a 2% (w/v) agarose gel containing 0.5 µg/ml ethidium bromide and photographed. The images were scanned and saved using Adobe Photoshop software (Adobe Systems Inc., Mountain View, CA). Densitometric analysis of the images was carried out on the Scion Image program (NIH Image, Scion Corporation, Frederick, MD). The VEGF and bFGF cDNA band intensities were normalized against the corresponding β-actin band.

2.8. Magnetic resonance imaging (MRI)
Non-invasive MRI was used to monitor hypertrophy and LV dilation in the treated rats, as previously described [31]. Briefly, 41 days after treatment, MRI was performed with a 7.05-Tesla BIOSPEC 70/21 scanner (Bruker, Germany) under inhalation anaesthesia (1.5% (v/v) isoflurane supplemented with 0.5 l oxygen per minute) applied with a nose cone. An echocardiogram-triggered fast gradient echo sequence with the following parameters (flip angle; 30 to 40°, echo time; 1.1 ms, repetition time; 3.2 ms, and 12 frames per heart cycle) was used. The total acquisition time for one sequence was 40 to 50 s, depending on the heart rate, with an acquisition window per frame of 6.2 ms (2xrepetition time). Data from each slice was averaged four times to increase the signal to noise ratio. 15 to 18 contiguous ventricular short axis 1-mm slices were acquired across the entire heart to give a quantitative determination of morphology and function. Hence, total scan time was in the range of 15 min. The field of view was 30 to 40 mm with an image matrix of 128 by 128 and an in-plane resolution of 230 to 310 m. Data analysis was performed by a trained observer using an operator-interactive threshold technique. The LV end-systolic diameter (LVESD) and LV end-diastolic diameter (LVEDD) were determined from end-systolic and end-diastolic images by multiplying the compartment area and slice thickness (1 mm). The ejection fraction (EF) was calculated as follows; EF (%)=[(LVEDD3–LVESD3)/LVEDD3]x100.

2.9. Optical mapping
Optical mapping was performed ex vivo on perfused rat hearts, as previously described [32,33]. Image capture and collection using charge-coupled device camera were optimized for rat hearts. Immediately after sacrifice and before sampling for histology, immunohistochemistry, apoptosis assay, and RT-PCR, optical mapping was performed twice for the retrieved whole hearts (n=5) in each group. The retrieved hearts were stained with di-4-ANEPPS (Molecular Probes, Eugene, OR) and then excited with quasi-monochromatic light (500±30 nm) from two green LED lamps (LL-50R30-G25, Optronix, Korea). Fluorescent and scattered light was collected by an image-intensified charge-coupled device camera (Dalsa Inc., Waterloo, Canada). The data were gathered at 3.75 ms sampling intervals, acquiring from 100x100 sites simultaneously over a 35x35 mm2 area with pixel size of 0.27 mm. The mapped area included parts of the right and left ventricular free walls. For detailed mapping of the anterior wall of LV including the transplanted site of the CBMNCs, we magnified the field to a 10x10 mm2 area. To decrease motion related artefacts, diacetyl monoxime was used with an infusion rate of 8 mmol/l [32].

2.10. Statistical analysis
Quantitative data are expressed as the mean±standard deviation. Statistical analysis was performed with a one-way analysis of variance (ANOVA) method followed by Bonferroni's procedure to determine a statistical difference between experimental groups. The assumptions of ANOVA were found to satisfy Levene's test for homogeneity of variance and pass tests for normality. An ANOVA p value <0.05 was considered to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1. Transplanted CBMNCs in ischaemic myocardium
Immunohistochemical HNA staining confirmed that some transplanted human CBMNCs were incorporated into the ischaemic region vascular networks (Fig. 2A, arrowheads), and many transplanted human CBMNCs were located in the vicinity of microvessels in the ischaemic region (Fig. 2B, arrows). The HNA-positive cell density was significantly higher (p<0.05) in the CBMNC+bFGF group than in the CBMNC group (Fig. 2C).


Figure 02
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  		Fig. 2 Immunohistochemical staining for HNA in infarcted myocardium, six weeks after CBMNC transplantation. (A) Transplanted human CBMNCs were incorporated into vascular networks (arrowheads) in the ischaemic regions. The scale bar indicates 50 µm. (B) Transplanted human CBMNCs were localized in the vicinity of microvessels (arrows) in ischaemic regions. The scale bar indicates 100 µm. (C) The HNA-positive cell density in the infarct regions (*, p<0.05).

 
3.2. Enhancement of neovascularization by combination of CBMNC transplantation and bFGF delivery
Combined CBMNC transplantation and bFGF delivery resulted in more extensive angiogenesis in the infarcted myocardium than either therapy alone. Immunohistochemical staining for SM {alpha}-actin revealed more arterioles present in the combined therapy group (Fig. 3D) than in the control (Fig. 3A) or single therapy (Fig. 3B and C) groups. The arteriole density in the combined therapy group infarct regions (208±39/mm2) was significantly higher (p<0.05) than in the single therapy (CBMNC; 120±30/mm2 and bFGF; 145±17/mm2) or control (65±14/mm2) groups (Fig. 3E). Immunohistochemical staining for angiogenic growth factors (VEGF and bFGF) showed that angiogenic growth factor expression in the infarct regions was more extensive in the combined therapy group than the control or single therapy groups (data not shown). VEGF and bFGF mRNA expression was up-regulated in the combined therapy group as compared with the other groups (Fig. 4A). In addition, the ratio of VEGF (Fig. 4B) to β-actin was significantly higher (p<0.05) in the combined therapy group than the control or single therapy groups. The ratio of bFGF (Fig. 4C) to β-actin was significantly higher (p<0.05) in the combined therapy group than the control or CBMNC transplantation groups.


Figure 03
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  		Fig. 3 Arteriole density in the infarct region six weeks after therapy. Immunohistochemical staining for SM {alpha}-actin (400x) in (A) control group, (B) CBMNC transplantation using fibrin matrix (CBMNC) group, (C) sustained bFGF delivery (bFGF) group, and (D) combined CBMNC transplantation and sustained bFGF delivery (CBMNC+bFGF) group. The scale bar indicates 100 µm. All photographs were taken at the same magnification. (E) The arteriole density in the infarct regions (**, p>0.05; otherwise p<0.05 between any two groups).

 


Figure 04
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  		Fig. 4 Angiogenic growth factor expression in the infarct region six weeks after therapy. (A) Semi-quantitative RT-PCR analysis using angiogenic growth factor (VEGF and bFGF)-specific primers for the ischaemic myocardium (n=4 per group) 6 weeks after treatment (G1, control; G2, CBMNC; G3, bFGF; G4, CBMNC+bFGF). The ratio of (B) VEGF (**, p>0.05; otherwise p<0.05 between any two groups) and (C) bFGF (*, p<0.05; otherwise p>0.05 between any two groups) to β-actin mRNA expression in each group.

 
3.3. Reduction of tissue apoptosis in ischaemic myocardium by combined therapy
Apoptotic activity in the infarcted myocardium six weeks after treatment was determined by TUNEL staining. The combined therapy significantly reduced apoptosis in the region bordering the infarcted myocardium, as compared with the other groups (Fig. 5A-D). The number of TUNEL-positive cells in the region bordering the infarcted myocardium was significantly smaller (p<0.05) in the combined therapy group (18±10/mm2) than in the control (68±12/mm2) or CBMNC transplantation (40±7/mm2) groups (Fig. 5E). The number of TUNEL-positive cells of the combined therapy group was not significantly different (p>0.05) from that of bFGF group (34±15/mm2).


Figure 05
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  		Fig. 5 Apoptotic activity in the infarcted myocardium six weeks after therapy. Apoptotic nuclei (black) in the region bordering the infarcted hearts were stained with DAB by the TUNEL staining method (100x). (A) Control (medium injection). (B) CBMNC transplantation (CBMNC). (C) Sustained bFGF delivery (bFGF). (D) Combined CBMNC transplantation and sustained bFGF delivery (CBMNC+bFGF). The scale bar indicates 200 µm. All photographs were taken at the same magnification. (E) The density of TUNEL-positive cells in the region bordering the infarcted myocardium (**, p>0.05; otherwise p<0.05 between any two groups).

 
3.4. Tissue regeneration in ischaemic myocardium by combined therapy
Six weeks after treatment, combined CBMNC transplantation and bFGF delivery resulted in more extensive tissue regeneration in the infarcted myocardium, than either single therapy alone. Masson's trichrome staining showed a smaller area of fibrous tissue present in the combined therapy group (Fig. 6A) than in the control and single therapy groups. The injured myocardium infarct area in the combined therapy group (16.1±3.7%) was significantly smaller (p<0.05) than in the other groups (control; 35.3±3.3%, CBMNC; 25.0±2.8%, and bFGF; 22.6±3.7%). The newly formed tissues in ischaemic regions of the combined therapy group were stained positively with SM {alpha}-actin, (Fig. 6B), indicating that these tissues might be composed of myofibroblasts not cardiomyocytes.


Figure 06
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  		Fig. 6 Tissue regeneration in the infarct region six weeks after combined CBMNC transplantation and sustained bFGF delivery. (A) Masson's trichrome staining (100x). (B) Immunohistochemical staining for SM {alpha}-actin (100x). The scale bars indicate 200 µm.

 
3.5. LV dimensions and function in ischaemic hearts
LV dimensions and heart function were measured by MRI. It was notable that LVEDD and LVESD in the combined therapy group were significantly lower (p<0.05) than in the control group (Table 1). The LVEDD and LVESD in the combined therapy group, however, were not significantly different (p>0.05) from the single therapy groups (Table 1). The EF of combined therapy group was slightly higher than those of the other three groups, but was only significantly different from that of the control group (Table 1).


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  		Table 1 Data from MRI examination of infarcted hearts 41 days after single or combined therapy

 
3.6. Electrical potential in ischaemic hearts
Optical mapping of the ischaemic hearts 6 weeks after treatment showed that the myocardial region electrically connected to normal myocardium was larger in the angiogenic therapy (CBMNC+bFGF) group (Fig. 7D-F) than in the control group (Fig. 7A-C). Electrical potentials obtained from each site (numbered 1 to 3) in the CBMNC+bFGF group showed synchronization (Fig. 7F), suggesting that each site had established an electrical connection. This result indicates that additional apoptosis in the area bordering the ischaemic myocardium and the resultant expansion of fibrosis was prevented by angiogenic therapy. Thereby, myocardium was preserved after infarction.


Figure 07
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  		Fig. 7 Optical mapping of ischaemic hearts 6 weeks after treatment. (A-C) Control (medium injection). (D-F) Combined CBMNC transplantation and sustained bFGF delivery (CBMNC+bFGF). (A) Schematic diagram of the LV in the control group. Optical mapping was recorded at the anterior LV (box). The numbers 1 to 3 represent the sites where optical signals were taken. The numbers 1, 2, and 3 indicate normal myocardium, the border zone, and the infarcted region, respectively. (B) Pseudo colour membrane voltage maps of the control group. During sinus rhythm, depolarization was initiated on mid-LV (site 1), but delayed at border zone (site 2). Depolarization was not observed on infarcted region (site 3). (C) The optical signals of control group. The action potentials along the excitation wave fronts were observed at normal (site 1) and border zone (site 2) but not the infarcted region (site 3). (D) Schematic diagram of the LV in the CBMNC+bFGF group. (E) Pseudo colour membrane voltage maps of the CBMNC+bFGF group. During sinus rhythm, depolarization was initiated on mid-LV (site 1) and spread to border zone (site 2). Depolarization propagated up to the infarcted region (site 3). (F) The optical signals of the CBMNC+bFGF group. The action potentials along the excitation wave fronts were observed at normal (site 1), border zone (site 2), and infarcted region (site 3).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
HUCBCs have many advantages as a cell source for therapeutic angiogenesis. Compared with adult peripheral blood and bone marrow progenitor cells, HUCBCs have distinct proliferative characteristics. These include the capacity to form more colonies, a faster cell cycle, and longer telomeres [34]. In addition, cord blood can be obtained non-invasively, whereas bone marrow isolation is invasive. Furthermore, over 60 to 90% of nucleated cells can be recovered from cord blood that has been frozen for 15 or more years [35]. HUCBCs for the repair of infarcted myocardium may be particularly important in elderly people, in whom the availability of autologous, functional stem cells from bone marrow or peripheral blood is limited.

Recent studies have demonstrated that the angiogenic potential of transplanted HUCBCs arises from both their differentiation into ECs and their ability to produce various angiogenic growth factors, including VEGF, bFGF, and angiopoietin-1 [13,16,36]. Even though HUCBCs may not directly enter the vasculature in ischaemic sites, they may have beneficial effects on ischaemic tissues [13,16,37], due to the secretion of angiogenic growth factors, cytokines, nitric oxide, or other factors that limit tissue damage and remodelling after ischaemia [15]. Other recent studies have reported enhanced neovascularization and reduced infarct size in ischaemic myocardium following HUCBC transplantation [13,16]. In these studies, however, most HUCBCs were located in the perivascular sites, and were not incorporated in neovessels. This finding suggests that transplanted HUCBCs contribute to neovascularization chiefly by secreting angiogenic factors [13,16]. In the present study, tissues regenerated in the infarcted myocardium were not stained positively with cardiac markers (cardiac troponin I and myosin heavy chain) (data not shown). However, the fact that the tissues were stained positively with SM {alpha}-actin (Fig. 6B), suggests that they may be mainly composed of myofibroblasts. HNA-positive cells were not co-localized with SM {alpha}-actin-positive cells, indicating that SM {alpha}-actin-positive cells were not differentiated from transplanted human CBMNCs. This result is consistent with the finding of a previous study demonstrating that human cord blood-derived CD133-positive cells prevented scar thinning by inducing autologous myofibroblast accumulation into ischaemic myocardium [37]. The autologous myogenic cell accumulation might be due to factors secreted by the transplanted cord blood cells acting in a paracrine fashion rather than trans-differentiation of transplanted human cord blood cells [37].

The present study shows that angiogenesis induced by CBMNC transplantation may occur, in part, due to transplanted cells secreting angiogenic factors in a paracrine manner. Immunostaining for HNA revealed that many HNA-positive cells were located in the perivascular regions of the ischaemic myocardium (Fig. 2B). VEGF and bFGF expression was more extensive in the CBMNC and CBMNC+bFGF groups than in the control group (Fig. 4). In the latter, VEGF and bFGF expression was likely induced by endogenous ischaemia. Since bFGF immobilized in the fibrin matrix is completely released within 1 week [38], any bFGF present 6 weeks after treatment in the CBMNC+bFGF group is not exogenous and must have been secreted by cells in the ischaemic myocardium. Thus, the beneficial effects (enhanced neovascularization, decreased infarct area, and attenuated ischaemic myocardium apoptosis) are partially due to angiogenic factors secreted by transplanted CBMNCs.

The angiogenic efficacy of CBMNC transplantation to treat myocardial infarction was enhanced by sustained bFGF delivery. Our results show that arteriole density and angiogenic growth factor expression, such as VEGF and bFGF, were significantly enhanced by combined therapy compared with either therapy alone. The enhanced angiogenic efficacy may be due to an additive effect of various known and unknown angiogenic growth factors, cytokines, and vasoactive factors secreted by transplanted CBMNCs and exogenously provided bFGF. Several clinical reports have shown that administering a single angiogenic gene or protein is not sufficient to induce significant therapeutic effects in patients with ischaemic disease [39,40]. Combining angiogenic cell transplantation and protein or gene therapies could facilitate a reduction in the number of cells required for significant therapeutic angiogenesis [18].

Another possible explanation for the enhanced angiogenic efficacy may be enhanced transplanted CBMNC survival in the presence of bFGF. The cells transplanted into the ischaemic region were exposed to hypoxia after transplantation due to a lack of initial vasculature. Transplanted cells are prone to undergo apoptosis in hypoxic conditions [41]. It has been reported that transfecting the bFGF gene into bone marrow mesenchymal stem cells improved cell viability under hypoxic conditions [42]. Thus, exogenously administered bFGF might inhibit apoptosis of the transplanted CBMNCs, thereby promoting neovascularization in ischaemic myocardium. Indeed, the HNA-positive cell density (Fig. 2C) at 6 weeks after CBMNC transplantation was significantly higher in the CBMNC+bFGF group than in the CBMNC group, indicating enhanced transplanted CBMNC survival in the presence of bFGF.

Increased neovascularization led to enhanced therapeutic efficacy in treating myocardial infarction. We have shown that combined CBMNC transplantation and sustained bFGF delivery significantly reduced fibrosis and infarct area in injured myocardium, as compared with either therapy alone. The combined therapy also reduced apoptosis in the region bordering the infarcted myocardium, as compared with either therapy alone (Fig. 5). Electrical disconnection between normal and infarcted myocardium can increase the risk of ventricular arrhythmias [43]. Optical mapping (Fig. 7) showed that the combined therapy enhanced electrical viability and impulse propagation in the border zone between normal and infarcted myocardium. Thus, the infarcted hearts in the combined therapy group might be less prone to develop ventricular arrhythmias than those in the control group.

A major limitation of the present study is the small number of animals used in the experiments. Our study demonstrates that the combined therapy did not attenuate LV dilation or increase EF significantly compared with either individual therapy. This might be due to the small number (n=5) of animals used, particularly in view of the variability (for example in LV dimensions) that can exist irrespective of treatment. Therefore, the efficacy of the combined therapy should be further evaluated in additional studies with larger numbers of animals. Study design should include basal analysis of LV dimensions and function using a non-invasive method (MRI) before treatment (3 weeks after induction of myocardial infarction) followed by randomisation. We only performed a final analysis with MRI 6 weeks after treatment. This made it difficult to assess whether the treatments had any beneficial effects on LV remodelling and function. However, we compensated for these limitations by using cryoinjury (standardized infarct size) for preparation of the myocardial infarction model and MRI (the best in vivo imaging modality for small animals) for assessment of cardiac function and morphology.

The functionality of neovessels should be investigated in additional studies with blood perfusion measurements at a longer time point. Although our study suggests that the combined therapy of CBMNC transplantation and bFGF delivery enhanced neovascularization in infarcted regions, we did not perform angiography to verify the improvement in blood perfusion. Enhanced neovascularization may not lead to improved perfusion in ischaemic myocardium. In addition, we only observed the effect of combined therapy on neovascularization at a relatively short time point (6 weeks). Previous studies have shown that the newly formed vessels are too fragile to persist in vivo over a long period, and thus may be non-functional and have no effect on perfusion [44,45]. The long-term maintenance of neovessels and their contribution to improvement in perfusion needs to be addressed in further studies.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
In summary, the angiogenic efficacy of CBMNC transplantation for treating myocardial infarction was enhanced by combining the therapy with sustained bFGF delivery. The combined therapy significantly enhanced neovascularization and reduced the infarct area in a rat model of myocardial infarction, compared to either therapy alone. Additional studies, to optimize the rate and duration of bFGF release and to determine in vivo bFGF release kinetics, are required to maximize angiogenic efficacy. The type and number of HUCBCs used for transplantation should also be optimized in order to maximize angiogenic efficacy.


   Acknowledgement
 
This study was supported by a grant (A050082) from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

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