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Am J Physiol Heart Circ Physiol 288: H1444-H1450, 2005. First published November 11, 2004; doi:10.1152/ajpheart.00266.2004
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Adrenomedullin enhances therapeutic potency of bone marrow transplantation for myocardial infarction in rats

Takafumi Fujii,1 Noritoshi Nagaya,2,3 Takashi Iwase,2 Shinsuke Murakami,2 Yoshinori Miyahara,1 Kazuhiro Nishigami,3 Hatsue Ishibashi-Ueda,5 Mikiyasu Shirai,1 Takefumi Itoh,2 Kozo Ishino,6 Shunji Sano,6 Kenji Kangawa,4 and Hidezo Mori1

Departments of 1Cardiac Physiology, 2Regenerative Medicine and Tissue Engineering, 3Internal Medicine, 4Biochemistry, and 5Pathology, National Cardiovascular Center, Osaka; and 6Department of Cardiovascular Surgery, Okayama University Medical School, Okayama, Japan

Submitted 18 March 2004 ; accepted in final form 19 October 2004


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Adrenomedullin (AM), a potent vasodilator, induces angiogenesis and inhibits cell apoptosis through the phosphatidylinositol 3-kinase/Akt pathway. Transplantation of bone marrow-derived mononuclear cells (MNC) induces angiogenesis. We investigated whether infusion of AM enhances the therapeutic potency of MNC transplantation in a rat model of myocardial infarction. Immediately after coronary ligation, bone marrow-derived MNC (5 x 106 cells) were injected into the ischemic myocardium, followed by subcutaneous administration of 0.05 µg·kg–1·min–1 AM (AM-MNC group) or saline (MNC group) for 3 days. Another two groups of rats received subcutaneous administration of AM alone (AM group) or saline (control group). Hemodynamic and histological analyses were performed 4 wk after treatment. Cardiac infarct size was significantly smaller in the MNC and AM groups than in the control group. A combination of AM infusion and MNC transplantation demonstrated a further decrease in infarct size. Left ventricular (LV) maximum change in pressure over time and LV fractional shortening were significantly improved only in the AM-MNC group. AM significantly increased capillary density in ischemic myocardium, suggesting the angiogenic potency of AM. AM infusion plus MNC transplantation demonstrated a further increase in capillary density compared with AM or MNC alone. Although MNC apoptosis was frequently observed 72 h after transplantation, AM markedly decreased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells among the transplanted MNC. In conclusion, AM enhanced the angiogenic potency of MNC transplantation and improved cardiac function in rats with myocardial infarction. This beneficial effect may be mediated partly by the angiogenic property of AM itself and by its antiapoptotic effect on MNC.

angiogenesis; apoptosis; mononuclear cell

DESPITE THE RECENT REMARKABLE progress in medical and surgical treatment for ischemic heart disease, this disease remains a major cause of death worldwide (5). Bone marrow-derived mononuclear cells (MNC) contain various kinds of cell lineages and numerous cytokines that contribute to neovascularization (1, 15). In fact, autologous transplantation of bone marrow cells has been shown to enhance angiogenesis and improve cardiac function in an animal model of cardiac ischemia (6, 9, 10). Recent human studies have demonstrated beneficial effects of transplanted MNC in patients with ischemic heart disease (23, 25). However, some patients fail to respond to this cell therapy. Thus a novel therapeutic strategy to enhance the angiogenic property of MNC is desirable.

Adrenomedullin (AM) is a potent vasodilator peptide that was originally isolated from human pheochromocytoma (8). We have shown that infusion of AM has beneficial hemodynamic and renal effects in patients with heart failure (17). On the other hand, AM has been shown to activate the phosphatidylinositol 3-kinase (PI3-kinase)/Akt-dependent pathway in vascular endothelial cells, which is considered to regulate multiple critical steps in angiogenesis including endothelial cell proliferation, migration, and capillary-like formation (14, 22). In fact, we have shown that AM gene transfer induces therapeutic angiogenesis in a rabbit model of hindlimb ischemia via activation of Akt (24). These findings suggest that AM may play an important role in the regulation of vascular regeneration. In addition, AM has been shown to exert an antiapoptotic effect on a variety of cells including vascular endothelial cells (7, 20). Taking these findings together, combination therapy with MNC transplantation and AM infusion may have additional or synergetic effects on therapeutic angiogenesis for the treatment of ischemic heart disease.

Thus the purposes of this study were 1) to investigate whether infusion of AM enhances the angiogenic potency of MNC transplantation in a rat model of myocardial infarction, and 2) to investigate the effects of AM on survival and differentiation of the transplanted MNC to examine the underlying mechanisms of the effects induced by AM.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Animal model. Myocardial infarction was produced in male Lewis rats weighing 200–220 g by left coronary ligation. In brief, after rats were anesthetized by intraperitoneal injection of pentobarbital sodium (30 mg/kg body wt), they were ventilated artificially. The heart was exposed via left thoracotomy, and the left coronary artery was ligated 2–3 mm from its origin between the pulmonary artery conus and the left atrium using a 6-0 proline suture. Finally, the heart was restored to its normal position, and the chest was closed. The Animal Care Committee of the National Cardiovascular Center approved this experimental protocol.

Preparation of MNC. After Lewis rats were killed, bone marrow from the femur and tibia was collected and put in PBS. Marrow cells were loaded on a 1.077 gradient of Ficoll (Lymphoprep; Nycomed Pharma, Oslo, Norway) and centrifuged at 1,500 rpm for 20 min. The cells were then washed with 10 ml PBS to remove the Ficoll and centrifuged at 2,000 rpm for 10 min. The cells were finally suspended in PBS at a concentration of 5 x 106 cells in 50 µl PBS for transplantation. Fluorescence-activated cell sorting analysis demonstrated that 22 ± 1% of MNC were positive for lectin from ulex europaeus (UEA)-1 lectin (Sigma, St. Louis, MO).

MNC transplantation and AM infusion. Transplantation of bone marrow-derived MNC and/or 3-day infusion of AM was performed immediately after coronary ligation. MNC (5 x 106 cells in 50 µl PBS) were injected into the myocardium at five points in the border zone surrounding the infarct by using a 27-gauge needle. Recombinant human AM (0.05 µg·kg–1·min–1) was subcutaneously administered by using an osmotic minipump (model 2004; Alza, Palo Alto, CA) for 3 days. The pump was positioned in a pocket constructed in the subcutaneous tissue just below the subscapular region. For control, 5% glucose was infused in a similar manner in the rats receiving coronary ligation. This protocol resulted in the creation of four groups: 1) AM infusion plus MNC transplantation (AM-MNC group, n = 15), 2) vehicle infusion plus MNC transplantation (MNC group, n = 14), 3) AM infusion plus PBS injection (AM group, n = 14), and 4) vehicle infusion plus PBS injection (control group, n = 13).

Echocardiographic studies. Echocardiographic studies were performed 4 wk after surgery using a 7.5-MHz phased-array transducer (model HP SONOS 5500; Hewlett-Packard, Andover, MA). Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (30 mg/kg body wt) as a supplement to maintain mild anesthesia. M-mode tracings were obtained at the level of the papillary muscles. Anterior and posterior end-diastolic wall thickness, left ventricular (LV) end-diastolic and end-systolic dimension, and LV fractional shortening were measured from three consecutive cardiac cycles by the American Society for Echocardiology leading-edge method (21).

Cardiac catheterization. Cardiac catheterization was performed 4 wk after surgery. Rats were anesthetized with intraperitoneal pentobarbital and placed on a heating pad to maintain body temperature at 37–38°C throughout the study. A 1.5 Fr micronanometer-tipped catheter was inserted in the right carotid artery for measurement of heart rate and mean arterial pressure. The catheter was then advanced into the LV for measurement of LV end-diastolic pressure and then replaced with a thermomicroprobe for measurements of cardiac output. These hemodynamic variables were measured with a pressure transducer (UFI, Morro Bay, CA) connected to a polygraph and recorded with a thermal recorder (model 7758 B system; Hewlett-Packard).

Infarct size measurement. After completion of hemodynamic measurements, the heart was arrested by an injection of 2 mmol KCl through the carotid artery, and the cardiac ventricles were excised. The size of myocardial infarction was determined by a previously described method (2). In brief, incisions were made in the LV so that the tissue could be pressed flat. The circumference of the entire flat LV and the visualized infarcted area, as judged from both the epicardial and endocardial sides, was outlined on a clear plastic sheet. The difference in weight between the two marked areas on the sheet was used to determine infarction size and was expressed as a percentage of LV surface area.

Histological analysis of microvessel density. LV myocardium was fixed in 10% formalin. Three cross sections of the LV, cut from apex to base, were obtained from individual rats for comparison among four groups (n = 5 each). They were embedded in paraffin and stained with Masson's trichrome for measurement of interstitial fibrosis. In other rats (n = 5 each), LV myocardium was embedded in optimum cutting temperature (OCT) compound (Sakura Finetechnical, Tokyo, Japan), snap frozen in liquid nitrogen, and cut into 5-µm-thick sections. Tissue sections were stained for alkaline phosphatase with an indoxyltetrazolium method to detect capillary endothelial cells (n = 5 in each group). The number of capillary vessels was counted in the peri-infarct area (a 1.0-mm band next to the scar) excluding scar region using a light microscope at a magnification of x200. The numbers in five high-power fields in each rat were averaged and expressed as the number of capillary vessels. These morphometric studies were performed by two examiners who were blinded to treatment.

Detection of MNC apoptosis. To examine the antiapoptotic effect of AM on transplanted MNC, red fluorescence-labeled MNC were transplanted into ischemic myocardium in rats with (n = 5) and without (n = 5) AM infusion. Before implantation into the ischemic heart, suspended MNC were labeled with fluorescent dyes with a PKH26 (Red Fluorescent Cell Linker Kit; Sigma), as reported previously (13). AM was subcutaneously administered by using a minipump for 3 days. Rats were killed 72 h after MNC transplantation. The LV was enucleated, and muscle samples were embedded in OCT compound and snap frozen in liquid nitrogen for the detection of apoptosis. Serial sections of the heart were stained by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) for apoptosis using an in situ apoptosis detection kit (model S7111 Apoptag Fluorescein Kit; Intergen). Apoptosis of transplanted MNC was also evaluated by the detection of cleaved caspase-3-positive cells. In brief, the frozen tissue sections were incubated with anticleaved caspase-3 antibody (Cell Signaling), followed by incubation with FITC-conjugated IgG antibody (BD Pharmingen, San Diego, CA). The number of TUNEL/PKH26 double-positive cells and caspase-3/PKH26 double-positive cells was counted in 10 fields of each rat using a confocal microscopy (Fluoview model 500; Olympus, Tokyo, Japan).

The antiapoptotic effect of AM on MNC was also evaluated by in vitro TUNEL assay. MNC were plated on 12-well plates (1 x 106 cells per well) and cultured in serum-free medium for 24 h with control buffer, AM (1 x 10–7 M), or AM plus wortmannin, a PI3-kinase inhibitor (50 nM). Randomly selected microscopic fields (n = 10) were evaluated for calculating the ratio of TUNEL-positive cells to total cells.

Monitoring of implanted MNC in ischemic heart. Additional rats were used to examine whether transplanted MNC differentiate into endothelial cells, cardiomyocytes, vascular smooth muscle cells, or macrophages in the ischemic heart. PKH26 (red fluorescence)-labeled MNC were injected into the ischemic heart in rats with (n = 8) and without (n = 8) AM infusion. These subgroups of rats were killed 4 wk after coronary ligation. To identify vascular endothelial cells in vivo, FITC-labeled UEA-1 lectin was intravenously administered 30 min before the rats were killed (n = 5 in each group). The LV was enucleated, and muscle samples were then embedded in OCT compound, snap frozen in liquid nitrogen, and cut into sections. Sections were counterstained with 4',6'-diamidino-2-phenylindole (DAPI) to detect nuclei. The number of DAPI/PKH26 double-positive cells and lectin-positive cells in the peri-infarct area was counted in 10 fields of each rat using a confocal microscopy. Frozen sections from other rats (n = 3 in each group) were incubated with mouse anticardiac troponin T (Novocastra, Newcastle, UK), anti-{alpha}-smooth muscle actin antibody (Dako, Copenhagen, Denmark), and anti-ED1 antibody (Serotec, Oxford, UK), followed by incubation with FITC-conjugated IgG antibody. In other rats (MNC group, n = 5; AM-MNC group, n = 5), the cardiac muscle from base to apex was transversely cut into 6-µm slices to calculate the number of transplanted MNC present within the heart 4 wk after transplantation. These morphometric studies were performed by two examiners who were blinded to treatment.

Statistical analysis. Numerical values were expressed as means ± SE. Comparisons of parameters among the four groups were performed by one-way ANOVA, followed by Newman-Keuls test for unpaired data. Comparisons of parameters between two groups were made by unpaired Student's t-test. A value of P < 0.05 was considered significant.


   RESULTS
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 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Infarct size and ventricular weight. Moderate-to-large infarcts were observed in the control group after coronary ligation (Fig. 1). However, infarct size was smaller in the MNC, AM, and AM-MNC groups than in the control group. In particular, it was very small in the AM-MNC group. Quantitative analysis also demonstrated that cardiac infarct size in the AM-MNC group was smallest among the four groups. Right ventricular weight was significantly lower in the AM and AM-MNC groups than that in the control group (Table 1). LV weight did not significantly differ among the four groups.



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  		Fig. 1. A: representative examples of Masson trichrome-staining of transverse sections of left ventricular (LV) myocardium 4 wk after coronary ligation. B: quantitative analysis of infarct size and LV chamber size. Infarcted area and LV end-diastolic diameter (LVDd) of the adrenomedullin-mononuclear cell (AM-MNC) group were significantly smaller than those of the other groups. Values are means ± SE. *P < 0.05 vs. control; {dagger}P < 0.05 vs. MNC; {ddagger}P < 0.05 vs. AM.

 

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  		Table 1. Physiological profiles of four experimental groups

 
Echocardiographic findings. LV diastolic dimension was smallest in the AM-MNC group, followed by the AM, MNC, and control groups (Fig. 1). LV fractional shortening in the AM-MNC group was also higher than that in the control, MNC, and AM groups (Table 2). Diastolic thickness of the anterior wall was significantly attenuated in the MNC, AM, and AM-MNC groups compared with the control group.


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  		Table 2. Echocardiographic findings

 
Hemodynamics. Cardiac output in the AM-MNC group was significantly higher than that in the control, MNC, and AM groups (Fig. 2). LV end-diastolic pressure in the MNC, AM, and AM-MNC groups was significantly lower than that in the control group. LV maximum change in pressure over time (dP/dt) in the MNC and AM-MNC group were significantly higher than that in the control group. Similarly, LV minimum dP/dt was significantly decreased only in the AM-MNC group.



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  		Fig. 2. Effects of AM infusion and MNC transplantation on hemodynamic parameters. CO, cardiac output; LVEDP, LV end-diastolic pressure; Max dP/dt, LV maximum change in pressure over time; Min dP/dt, LV minimum dP/dt. Values are means ± SE. *P < 0.05 vs. control; {dagger}P < 0.05 vs. MNC; {ddagger}P < 0.05 vs. AM.

 
Capillary density. Alkaline phosphatase staining of ischemic myocardium showed marked augmentation of neovascularization in the MNC, AM, and AM-MNC groups compared with the control group (Fig. 3A). Quantitative analysis demonstrated that capillary density was significantly higher in the AM-MNC group than in the MNC and AM groups (Fig. 3B). Cartilage, bone, or fat was not observed in the transplanted area. No tumor-like cells were seen.



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  		Fig. 3. A: representative examples of alkaline phosphatase staining in peri-infarct area. A combination of AM infusion and MNC transplantation markedly induced myocardial neovascularization. Magnification, x200. B: quantitative analysis of capillary density in peri-infarct area. Capillary density in the AM-MNC group was significantly higher than that in the MNC and AM groups. Values are means ± SE. *P < 0.05 vs. control; {dagger}P < 0.05 vs. MNC; {ddagger}P < 0.05 vs. AM.

 
Antiapoptotic effect of AM on MNC. Red fluorescence-labeled MNC were detected in each recipient heart 72 h after transplantation (Fig. 4). TUNEL-positive cells were frequently observed in the MNC group. In contrast, these apoptotic cells were hardly detected in the AM-MNC group. Semiquantitative analysis demonstrated that the number of TUNEL-positive MNC was significantly lower in the AM-MNC group than in the MNC group. Similarly, the number of caspase-3-positive MNC was significantly lower in the AM-MNC group than in the MNC group. These results suggest that infusion of AM inhibits apoptosis of transplanted MNC.



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  		Fig. 4. Detection of transplanted cell apoptosis. A: representative photographs of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Red fluorescence (PKH26) marks transplanted MNC; green fluorescence indicates TUNEL-positive cells. TUNEL-positive cells were frequently observed in the MNC group, whereas they were hardly detected in the AM-MNC group. Magnification, x400. B: semiquantitative analysis of TUNEL-positive cells in the PKH26-positive (transplanted) cells. C: representative photographs of caspase-3 staining. Red fluorescence (PKH26) marks transplanted MNC; green fluorescence indicates caspase-3-positive cells. D: semiquantitative analysis of caspase-3-positive cells in the PKH26-positive cells. Values are means ± SE. *P < 0.05 vs. control.

 
In vitro, serum starvation induced MNC apoptosis. When incubated in the presence of AM (1 x 10–7 M), the percentage of TUNEL-positive cells decreased significantly (19 ± 1 to 9 ± 1%, P < 0.05). However, pretreatment with wortmannin, a PI3-kinase inhibitor, diminished the antiapoptotic effect of AM (17 ± 1%).

Differentiation of MNC into endothelial lineage. Four weeks after transplantation, fluorescence-labeled transplanted cells were more frequently observed in the AM-MNC group than in the MNC group (6.4 ± 0.4 to 3.1 ± 0.2%, P < 0.05). Moreover, some of the transplanted cells were positive for UEA-1 lectin in the AM-MNC group (Fig. 5A), suggesting differentiation of MNC into vascular endothelial cells. Semiquantitative analysis demonstrated that the number of DAPI/PKH26 double-positive cells (viable transplanted cells) was significantly higher in the AM-MNC group than in the MNC group (Fig. 5B). Moreover, the ratio of lectin-positive cells to DAPI/PKH26 double-positive cells was significantly higher in the AM-MNC group than in the MNC group. The ratio of DAPI/PKH26 double-positive cells to lectin-positive cells was small, but significantly higher in the AM-MNC group than in the MNC group (23.9 ± 0.9 to 17.2 ± 0.6%, P < 0.01). Transplanted MNC were negative for troponin T or {alpha}-smooth muscle actin-positive cells. Some of the transplanted MNC were positive for ED1, a marker of macrophage (data not shown).



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  		Fig. 5. A: representative examples of MNC differentiation into endothelial lineage. Red fluorescence (PKH26) marks transplanted cells; green fluorescence indicates ulex europaeus (UEA)-1 lectin, a marker for vascular endothelial cells. Most of the transplanted cells differentiated into endothelial cells in the AM-MNC group. Magnification, x400. B: quantitative analysis of living transplanted cells and endothelial differentiation. The number of living cells after transplantation was significantly higher in the AM-MNC group than in the MNC group. The ratio of lectin-positive cells to living transplanted cells was significantly higher in the AM-MNC group than in the MNC group. Values are means ± SE. *P < 0.05 vs. control. DAPI, 4',6'-diamidino-2-phenylindole.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we demonstrated that 1) infusion of AM enhanced the angiogenic potency of MNC in a rat model of acute myocardial infarction, resulting in decreased infarct size and improved cardiac function. We also demonstrated that 2) AM induced angiogenesis and inhibited apoptosis of the transplanted MNC. Thus a combination of AM and MNC may have beneficial effects in rats with myocardial infarction, partly through the angiogenic potency of AM itself and through its antiapoptotic effect on MNC.

Bone marrow-derived MNC include a variety of stem and progenitor cells (1, 15, 19), some of which can differentiate into endothelial cells and secrete numerous cytokines and chemokines (6, 9, 10). Earlier studies (6, 9, 10, 23, 25) have shown that autologous bone marrow transplantation induces angiogenesis and improves LV function in animals and humans. However, some patients are refractory to this cell therapy. Thus an approach to augment the angiogenic potency of MNC transplantation is required.

The present study showed that MNC transplantation or AM infusion alone reduced infarct size. A combination of AM infusion and MNC transplantation resulted in further decreases in infarct size and LV chamber size. MNC transplantation or AM administration modestly improved LV function. On the other hand, a combination of MNC and AM significantly improved cardiac performance compared with MNC or AM alone, as indicated by increases in cardiac output, fractional shortening, and LV maximum dP/dt. Earlier studies (6, 9, 10) have reported that MNC transplantation induces therapeutic angiogenesis and preserves LV function through inhibition of cardiomyocyte apoptosis in animal models of myocardial infarction. We have shown that AM infusion during the acute phase of ischemia-reperfusion inhibits apoptosis of cardiomyocytes and produces hemodynamic improvement in an animal study (18). These findings suggest that the reduction of infarct size induced by this combination therapy may be attributable to additive cardioprotective effects of MNC and AM.

The present study showed that AM infusion significantly increased capillary density in ischemic myocardium. Furthermore, AM infusion plus MNC transplantation demonstrated a further increase in capillary density compared with AM or MNC alone. Contribution of transplanted MNC to neovascularization (the ratio of DAPI/PKH26 double-positive cells to lectin-positive cells) was significantly greater in the AM-MNC group than in the MNC group. A recent study (14) has reported that AM promotes proliferation and migration of human umbilical vein endothelial cells and enhances angiogenesis in a murine gel plug assay through the PI3-kinase/Akt pathway. We have also shown that intramuscular administration of AM DNA induces therapeutic angiogenesis in a rabbit model of chronic hindlimb ischemia via activation of Akt (24). These findings suggest that the beneficial effects of combination therapy using AM and MNC may be attributable, in part, to the angiogenic properties of AM itself. Thus it is possible that AM infusion and MNC transplantation induce additive effects on myocardial damage after myocardial infarction. However, it still remains unknown whether AM infusion plus MNC transplantation induces synergetic effects.

An earlier study has demonstrated that ischemia and mechanical stress induce apoptosis of transplanted cells in the early stage after MNC transplantation (9). These results raise the possibility that the angiogenic potency of MNC transplantation is attenuated by MNC apoptosis. Kim et al. (7) have demonstrated that AM inhibits apoptosis of endothelial cells through the PI3-kinase/Akt pathway in vitro. Activation of the PI3-kinase/Akt pathway has been shown to inhibit apoptosis of endothelial progenitor cells and enhance neovascularization (11). In the present study, AM infusion significantly inhibited MNC apoptosis in ischemic tissue. In vitro, we showed that the antiapoptotic effect of AM on MNC was mediated by activation of the PI3-kinase/Akt pathway. Thus AM may enhance the therapeutic potency of MNC transplantation through a direct action of AM on MNC survival. Moreover, immunohistological examination demonstrated that infusion of AM increased the number of lectin-positive (endothelial) cells in transplanted MNC. These findings raise the possibility that AM may enhance differentiation of MNC into the endothelial lineage. Thus AM may directly act on transplanted MNC, which may result in synergetic effects on the ischemic myocardium.

This study includes some study limitations. Although the labeling efficacy of PKH26 has been shown to persist for >8 wk without cell toxicity (3, 4), the used vital marker PKH26 may have some cell toxic effects and cell or membrane fusion can lead to labeling of neighboring cells in the target tissue. Second, the present study demonstrated that AM prolongs MNC survival through the PI3-kinase/Akt pathway and enhances neovascularization in a peri-infarcted area. However, further studies are necessary to examine the effect of AM on MNC differentiation into endothelial cells.

Autologous cell transplantation may be an alternative treatment for ischemic heart disease in the clinical setting. Because their use does not require immunosuppression, the clinical use of MNC for cellular cardiomyoplasty appears to be most advantageous. Administration of AM peptide is simple and relatively noninvasive. We and others (12, 16, 17) have reported the safety of AM infusion in humans. Thus combination therapy using AM infusion and MNC transplantation may be a new therapeutic strategy for the treatment of ischemic heart disease.

In conclusion, infusion of AM enhanced the angiogenic potency of MNC transplantation and improved cardiac function in rats with myocardial infarction. This beneficial effect may be mediated partly by the angiogenic property of AM itself and by its antiapoptotic effect on MNC. Thus combination therapy using AM infusion and MNC transplantation may be a new therapeutic strategy for the treatment of ischemic heart disease.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research 13470154; Health and Labor Sciences Research Grants nano 001 and genome 005; Ministry of Health, Labor and Welfare Research Grant for Cardiovascular Disease H13C-1 and 16C-6; and grants from New Energy and Industrial Technology Development Organization and the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research of Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. Nagaya, Dept. of Regenerative Medicine and Tissue Engineering, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan (E-mail: nnagaya{at}ri.ncvc.go.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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