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(Circulation Research. 2006;99:891.)
© 2006 American Heart Association, Inc.

Integrative Physiology

Cardiac Overexpression of Monocyte Chemoattractant Protein-1 in Transgenic Mice Prevents Cardiac Dysfunction and Remodeling After Myocardial Infarction

Hajime Morimoto, Masafumi Takahashi, Atsushi Izawa, Hirohiko Ise, Minoru Hongo, Pappachan E. Kolattukudy, Uichi Ikeda

From the Department of Cardiovascular Medicine and Regeneration (H.M., U.I.) and Department of Cardiovascular Medicine (M.H.), Division of Cardiovascular Sciences (M.T., A.I., H.I., U.I.); Department of Organ Regeneration, Shinshu University Graduate School of Medicine, Matsumoto, Japan; and Burnett College of Biomedical Sciences (P.E.K.), University of Central Florida, Orlando.

Correspondence to Masafumi Takahashi, MD, PhD, Division of Cardiovascular Sciences, Department of Organ Regeneration, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan. E-mail masafumi{at}sch.md.shinshu-u.ac.jp

	Abstract

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Myocardial infarction (MI) is accompanied by inflammatory responses that lead to the recruitment of leukocytes and subsequent myocardial damage, healing, and scar formation. Because monocyte chemoattractant protein-1 (MCP-1) (also known as CCL2) regulates monocytic inflammatory responses, we investigated the effect of cardiac MCP-1 overexpression on left ventricular (LV) dysfunction and remodeling in a murine MI model. Transgenic mice expressing the mouse JE-MCP-1 gene under the control of the -cardiac myosin heavy chain promoter (MHC/MCP-1 mice) were used for this purpose. MHC/MCP-1 mice had reduced infarct area and scar formation and improved LV dysfunction after MI. These mice also showed induction of macrophage infiltration and neovascularization; however, few bone marrow-derived endothelial cells were detected in MHC/MCP-1 mice whose bone marrow was replaced with that of Tie2/LacZ transgenic mice. Flow cytometry analysis showed no increase in endothelial progenitor cells (CD34⁺/Flk-1⁺ cells) in MHC/MCP-1 mice. Marked myocardial interleukin (IL)-6 secretion, STAT3 activation, and LV hypertrophy were observed after MI in MHC/MCP-1 mice. Furthermore, cardiac myofibroblasts accumulated after MI in MHC/MCP-1 mice. In vitro experiments revealed that a combination of IL-6 with MCP-1 synergistically stimulated and sustained STAT3 activation in cardiomyocytes. MCP-1, IL-6, and hypoxia directly promoted the differentiation of cardiac fibroblasts into myofibroblasts. Our results suggest that cardiac overexpression of MCP-1 induced macrophage infiltration, neovascularization, myocardial IL-6 secretion, and accumulation of cardiac myofibroblasts, thereby resulting in the prevention of LV dysfunction and remodeling after MI. They also provide a new insight into the role of cardiac MCP-1 in the pathophysiology of MI.

Key Words: cytokines • heart failure • hypertrophy • inflammation • myocardial infarction

	Introduction

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Myocardial infarction (MI) is accompanied by inflammatory responses that lead to the recruitment of leukocytes and subsequent myocardial damage, healing, and scar formation.¹ Recruitment and activation of monocytes/macrophages in the infarcted myocardium have been shown to contribute importantly to the processes that occur after MI. The activated macrophages lead to the release of cytokines and proteinases, which can induce further inflammation and left ventricular (LV) remodeling. Meanwhile, recent evidence indicates that some endothelial progenitor cells (EPCs) are derived from monocytic lineage cells and participate in neovascularization in ischemic tissues.^2–4 Moreover, monocytic-derived EPCs secrete a large amount of angiogenic factors such as the vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF),⁴ thereby suggesting that monocytes/macrophages could improve LV dysfunction and remodeling after MI.

Chemokines are a family of potent chemotactic cytokines that regulate locomotion and trafficking of leukocytes in basal and inflammatory processes; however, it has been recently observed that chemokines are expressed by nonhematopoietic cells such as endothelial cells, smooth muscle cells, and cardiomyocytes, and their function extends far beyond leukocyte activity.¹ Monocyte chemoattractant protein-1 (MCP-1) (also known as CCL2) is a major chemokine that induces the recruitment and activation of monocytes, T cells, and NK cells, and it has been implicated in diseases characterized by monocyte-rich infiltrates. MCP-1 has been shown to be upregulated in experimental MI models and promotes mononuclear cell recruitment into the infarcted heart.⁵ Anti-MCP-1 gene therapy improves survival and attenuates LV dilatation and dysfunction in a murine MI model.⁶ Furthermore, targeted deletion of its receptor CCR2 in mice also improved LV dilatation and dysfunction after MI, suggesting a deleterious role for MCP-1 in postinfarct LV dysfunction and remodeling.⁷ Hence, MCP-1 inhibition is presently regarded to be a target for therapeutic intervention against MI. However, angiogenic and cardioprotective effects of MCP-1 have been reported.^8,9 These studies suggest diverse effects of MCP-1 on myocardial damage and remodeling after MI. Because MCP-1 is produced in the infarcted heart¹⁰ and the effect of MCP-1 is regulated by its topical concentration,^11,12 we hypothesized that MCP-1 that is topically produced in the heart plays a critical role in the pathophysiology of MI. In the present study, we investigated the effect of cardiac MCP-1 overexpression on LV dysfunction and remodeling in a murine MI model. Transgenic mice that express the mouse JE-MCP-1 gene under the control of the -cardiac myosin heavy chain promoter (MHC/MCP-1 mice) were used for this purpose. The findings obtained from this study provide a new insight into the role of cardiac MCP-1 in the pathophysiology of MI.

	Materials and Methods

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MHC/MCP-1 mice (background: FVB) were generated as previously described.¹³ FVB/N mice were purchased from Clea Japan Inc (Tokyo, Japan) and used as the age-matched wild-type controls for MHC/MCP-1 mice. At 12 weeks of age, no differences in the cardiac function and hypertrophy between wild-type and MHC/MCP-1 mice were observed (Figure I of the online data supplement, available at http://circres.ahajournals.org). FVB transgenic mice that express ß-galactosidase under the control of the Tie-2 promoter (Tie2/LacZ mice) were purchased from The Jackson Laboratory (Bar Harbor, Me). Mice aged 8 to 12 weeks (n=191) were used in this study. They were fed a standard diet and water and were maintained on a 12-hour light and dark cycle. All experiments in this study were performed in accordance with the Shinshu University Guide for Laboratory Animals, which conforms to NIH Guidelines.

Cell culture, MI protocols, histology, immunohistochemistry, X-gal staining, bone marrow transplantation, flow cytometry analysis, serum cytokine levels, echocardiography, real-time RT-PCR analysis, and statistical analysis are described in the online data supplement.

	Results

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Infarct Area, Scar Formation, and LV Function
To investigate the role of cardiac overexpression of MCP-1 in MI, infarct area and scar formation were assessed at 14 days after MI. As shown in Figure 1A and 1B, the infarct area was significantly decreased in MHC/MCP-1 mice compared with wild-type mice (P<0.05). Masson’s trichrome staining showed that scar formation was also significantly reduced in MHC/MCP-1 mice compared with wild-type mice (P<0.01) (Figure 1C and 1D). We also assessed the acute infarct size at 24 hours after MI and found that there was no significant difference between wild-type and MHC/MCP-1 mice (27.4%±2.3% versus 23.3%±3.8%, P=0.381, n=6).

View larger version (29K): [in this window] [in a new window]   Figure 1. Infarct area and scar formation. A and C, Heart sections were obtained from wild-type and MHC/MCP-1 mice at 14 days after MI, and these were stained with hematoxylin/eosin (A) and Masson’s trichrome (C). B and D, Bar graphs showing MI area (B) and scar formation (D). Results are expressed as means±SEM (n=10).

We next assessed LV function at baseline, 48 hours, 7 days, 14 days, 21 days, and 28 days after MI by using echocardiography. Table 1 shows that both wild-type and MHC/MCP-1 mice had similar LV dimension and function under baseline conditions. In wild-type mice, a marked decrease in percentage of fractional shortening (FS) was observed at 48 hours after MI, and this decrease was sustained for 28 days. In contrast, percentage of FS was maintained after MI in MHC/MCP-1 mice (14 days: 34.1%±1.0% [wild-type] versus 42.6%±0.7% [MHC/MCP-1]; P<0.001). Furthermore, in MHC/MCP-1 mice, the diastolic and systolic LV wall thicknesses were significantly increased after MI compared with that at baseline (P<0.01 to P<0.001).

View this table: [in this window] [in a new window]   Table 1. Echocardiographic Findings After MI in Wild-Type and MHC/MCP-1 Mice

Macrophage Infiltration and Capillary Formation
Because macrophage infiltration in the heart is a prominent feature of MHC/MCP-1 mice,¹³ immunohistochemical analysis of the macrophage marker F4/80 was performed. In baseline conditions, the number of infiltrated macrophages in the heart of MHC/MCP-1 mice was slightly increased compared with that of wild-type mice (Figure 2B). The number of infiltrated macrophages was increased in the border (infarct) area of the heart after MI in wild-type mice, and this macrophage infiltration was further increased in MHC/MCP-1 mice (P<0.05, Figure 2A and 2B). To assess the capillary density in the border area of the heart, immunohistochemical analysis of the endothelial cell marker CD31 was also performed. The capillary density determined by CD31 expression was significantly increased in MHC/MCP-1 mice compared with that in wild-type mice (P<0.01, Figure 2C and 2D), thereby suggesting that MHC/MCP-1 mice promote macrophage infiltration and neovascularization in the border area in MI.

View larger version (56K): [in this window] [in a new window]   Figure 2. Macrophage infiltration and capillary formation. A and C, Heart sections were obtained from wild-type and MHC/MCP-1 mice at 14 days after MI and stained with F4/80 (macrophages) (A) and anti-CD31 antibody (endothelial cells) (C) by immunohistochemical analysis. B and D, Bar graphs showing the number of macrophages (B) and capillary density (D). Results are expressed as means±SEM (n=5).

Contribution of Bone Marrow-Derived Cells
To determine the contribution of bone marrow-derived EPCs to the neovascularization in MI in MHC/MCP-1 mice, we used bone marrow-transplanted mice whose bone marrow was replaced with that of Tie2/LacZ mice.¹⁴ In wild-type mice whose bone marrow was replaced with that of Tie2/LacZ mice (Tie2/LacZwild-type), no LacZ-positive cells were detected in the heart. In contrast, few LacZ-positive cells were observed in the border area of the heart in MHC/MCP-1 mice whose bone marrow was replaced with that of Tie2/LacZ mice (Tie2/LacZMHC/MCP-1) (Figure 3A).

View larger version (50K): [in this window] [in a new window]   Figure 3. Contribution of bone marrow-derived EPCs. A, Heart sections were obtained from bone marrow-transplanted mice (Tie2/LacZwild-type and Tie2/LacZMHC/MCP-1) at 14 days after MI and stained with hematoxylin/eosin (left and middle) and X-gal (right). The arrow indicates a LacZ-positive cell. B through E, Blood samples were collected from the same wild-type and MHC/MCP-1 mice at baseline, 7 days, and 14 days after MI. The percentage of Mac-1+/Gr-1– cells (monocyte marker), CD34+ cells (hematopoietic and endothelial marker), Flk-1+ cells (endothelial marker), and CD34+/Flk-1+ cells (EPC marker) was assessed by flow cytometry. Results are expressed as means±SEM (n=4 to 5). *P<0.05 and ** P<0.01 vs baseline, P<0.05 and P<0.01 vs wild-type, #P<0.05 and ##P<0.01 vs day 7. F, LV function was assessed by echocardiography at baseline, 7 days, and 14 days after MI in bone marrow-transplanted mice (Tie2/LacZwild-type, n=10; Tie2/LacZMHC/MCP-1, n=5). The bar graph shows percentage of FS. Results are expressed as means±SEM.

To further investigate the contribution of EPCs, we checked whether EPCs were mobilized from the bone marrow to the peripheral circulation after MI in wild-type and MHC/MCP-1 mice. Predictably, flow cytometry analysis showed that the percentage of Mac-1⁺/Gr-1^– cells (monocyte marker) was increased at 7 days after MI in wild-type mice, and the increase after MI in MHC/MCP-1 mice was significantly greater than that in wild-type mice (Figure 3B). The percentage of CD34⁺ cells (hematopoietic and endothelial marker) was increased only at 14 days after MI in wild-type mice, and in MHC/MCP-1 mice, the CD34⁺ cell percentage were significantly greater than that in wild-type mice at baseline, 7 days, and 14 days after MI (Figure 3C). However, in MHC/MCP-1 mice, the percentage of Flk-1⁺ cells (endothelial marker) was not increased (Figure 3D) and the percentage of CD34⁺/Flk-1⁺ double-positive cells (EPC marker) was significantly less than that of wild-type mice (Figure 3E). These results indicate that the number of peripheral monocytes, not EPCs, was highly increased after MI in MHC/MCP-1 mice.

Because lethal irradiation might affect LV function,¹⁵ we measured LV function after MI in bone marrow-transplanted mice by using echocardiography. Similar to the observation with wild-type and MHC/MCP-1 mice, we confirmed that in comparison with Tie2/LacZwild-type mice, Tie2/LacZMHC/MCP-1 mice showed a significant improvement in LV dysfunction after MI (Figure 3F). These findings suggest a small contribution of bone marrow-derived EPCs to neovascularization after MI in MHC/MCP-1 mice.

Involvement of Inflammatory Cytokines
To investigate whether inflammatory cytokines are involved in the reduction of infarct area and scar formation after MI in MHC/MCP-1 mice, we determined the serum levels of MCP-1, interleukin (IL)-6, IL-10, IL-12p70, interferon (IFN)-, and tumor necrosis factor (TNF)-. As expected, the serum level of MCP-1 was strikingly increased in MHC/MCP-1 mice under baseline conditions, but no increase was detected in the levels of other cytokines (Table 2). At 14 days after MI, the serum levels of MCP-1 and IL-12p70 were increased in wild-type as well as MHC/MCP-1 mice (MCP-1, P<0.01; IL-12p70, P<0.01). There was no difference between wild-type and MHC/MCP-1 mice in terms of the increased MCP-1 levels after MI. Notably, the serum IL-6 level was markedly increased after MI in MHC/MCP-1 mice when compared with that in wild-type mice (P<0.05).

View this table: [in this window] [in a new window]   Table 2. Serum Cytokine Levels at Baseline and Fourteen Days After MI in Wild-Type and MHC/MCP-1 Mice

To explore the cellular sources of IL-6 in MHC/MCP-1 mice, real-time RT-PCR analysis was performed to detect IL-6 and TNF- expression in the infarct heart and peripheral blood cells in the early phase after MI. IL-6 mRNA expression in the heart of wild-type mice was increased at 6 hours after MI (Figure 4A). In MHC/MCP-1 mice, IL-6 mRNA expression in the heart was also clearly increased at 6 hours after MI; however, no increased IL-6 mRNA expression was detected in peripheral blood cells isolated from both wild-type and MHC/MCP-1 mice. Moreover, the level of IL-6 mRNA expression in the heart of MHC/MCP-1 mice significantly increased at 24 hours, compared with that in wild-type mice. There was no significant increase in TNF- mRNA expression in the heart and peripheral blood of wild-type and MHC/MCP-1 mice (supplemental Figure II). Immunohistochemical staining also showed that the expression of MCP-1 and IL-6 proteins was increased in the infarct heart of MHC/MCP-1 mice (Figure 4B). These findings suggest that not only MCP-1 but also IL-6 produced by the infarct heart might be involved in the beneficial effects that were observed in MHC/MCP-1 mice.

View larger version (41K): [in this window] [in a new window]   Figure 4. Involvement of inflammatory cytokines. A, Total RNA was extracted from the heart and peripheral blood (PB) in wild-type and MHC/MCP-1 mice at baseline, 6 hours, and 24 hours after MI and analyzed for IL-6 mRNA expression by real-time RT-PCR. Results are expressed as means±SEM (n=5). B, Heart sections were obtained from wild-type and MHC/MCP-1 mice at 14 days after MI and stained with antibodies against MCP-1 and IL-6. The results are representative of 3 independent experiments.

Cardiac Hypertrophy and STAT3 Activation
Because IL-6 has been reported to induce cardiac hypertrophy and exert a cardioprotective effect through the STAT3 signaling pathway,¹⁶ we tested whether cardiac hypertrophy and STAT3 activation were induced in MHC/MCP-1 mice. Representative histological findings of cross-sectional cardiomyocytes are shown in Figure 5A and 5B. There was no difference between wild-type and MHC/MCP-1 mice in terms of the cross-sectional diameters of cardiomyocytes at baseline. However, the cross-sectional diameters of cardiomyocytes at 14 days after MI in MHC/MCP-1 mice were significantly greater than those of wild-type mice (P<0.001). Furthermore, activated STAT3 (phosphorylated form) was visualized in the nuclei of the infarct heart in MHC/MCP-1 mice but not in those of wild-type mice (Figure 5C).

View larger version (51K): [in this window] [in a new window]   Figure 5. Cardiac hypertrophy and STAT3 activation. A, Heart sections were obtained from wild-type and MHC/MCP-1 mice at baseline and 14 days after MI and stained with hematoxylin/eosin. B, Bar graph showing the diameters of LV cardiomyocytes. Results are expressed as means±SEM (n=5). C, Heart sections were obtained from wild-type and MHC/MCP-1 mice at 14 days after MI and stained with anti-phospho-STAT3 (pSTAT3) antibody using immunohistochemical analysis. Arrows indicate positive cells. D, Cardiomyocytes were treated with IL-6 (10 ng/mL), MCP-1 (10 ng/mL), or IL-6 combined with MCP-1 for the indicated periods. Cell lysates were prepared and analyzed by Western blotting with antibodies against phospho-STAT3, STAT3, or ß-actin. The results are representative of 3 independent experiments. E, Representative photographs of cardiomyocytes treated with or without IL-6 (50 ng/mL) for 48 hours. F, Bar graph showing the size of cardiomyocytes treated with or without IL-6 (10 and 50 ng/mL) for 48 hours. Results are expressed as means±SEM (n=25). G, Bar graphs showing the body weights, heart weights, and the ratio of heart weights/body weights (H.W./B.W. ratio). Results are expressed as means±SEM (wild-type, n=5; MHC/MCP-1, n=7).

To confirm the activation of STAT3 by IL-6 and investigate the effect of MCP-1 on STAT3 activation in cardiomyocytes, we used mouse neonatal cultured cardiomyocytes in vitro and tested for STAT3 activation by Western blotting.¹⁷ Treatment with IL-6 clearly activated STAT3 within 5 minutes, peaked at 30 minutes, and then declined to the basal level (Figure 5D), whereas treatment with MCP-1 showed only slight STAT3 activation. It is noteworthy that combined treatment of IL-6 with MCP-1 synergistically activated STAT3, and this activation was sustained at least during the 120 minutes observation period. To assess cellular hypertrophy, the 2D cell area was quantified in cardiomyocytes treated with IL-6. As shown in Figure 5E and 5F, treatment with IL-6 for 48 hours increased the size of cardiomyocytes in vitro. Furthermore, we measured the heart weights and body weights and calculated the ratio of heart weights to body weights. The ratio of heart weights to body weights after MI in MHC/MCP-1 mice was significantly increased compared with that in wild-type mice (Figure 5G). These results suggest that IL-6 induced cardiac hypertrophy through STAT3 activation after MI in MHC/MCP-1 mice.

Accumulation of Cardiac Myofibroblasts
Cardiac fibroblasts have been shown to differentiate into myofibroblasts during the process of myocardial repair and remodeling after MI.^10,18 Because myofibroblasts are characterized by the presence of -smooth muscle actin (-SMA), immunohistochemical analysis of -SMA was performed. The number of -SMA-positive myofibroblasts was notably increased at 14 days after MI in MHC/MCP-1 mice compared with that in wild-type mice (Figure 6A and 6B).

View larger version (69K): [in this window] [in a new window]   Figure 6. Accumulation of cardiac myofibroblasts. A, Heart sections were obtained from wild-type and MHC/MCP-1 mice at 14 days after MI and stained with anti--SMA antibody by immunohistochemical analysis. B, Bar graph showing myofibroblast counts. Results are expressed as means±SEM (n=5). C, Cardiac fibroblasts were treated with or without MCP-1 (10 ng/mL), IL-6 (10 ng/mL), or MCP-1 combined with IL-6 for 24 hours under normoxia (top panels) or hypoxia (bottom panels) and then stained with -SMA. Red indicates -SMA; blue, 4',6-diamidino-2-phenylindole (DAPI). The results are representative of 3 independent experiments.

To explore the factors responsible for the differentiation of fibroblasts into myofibroblasts, we used murine neonatal cardiac fibroblasts in vitro. Treatment with MCP-1 remarkably increased the differentiation of fibroblasts into myofibroblasts under the normoxic condition (Figure 6C). Similarly, treatment with IL-6 alone or combined treatment of MCP-1 with IL-6 also increased its differentiation. Furthermore, hypoxic condition increased its differentiation, and its differentiation was further enhanced in the presence of MCP-1.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major findings of this study are as follows. (1) Cardiac overexpression of MCP-1 reduced infarct area and scar formation and improved LV dysfunction and remodeling after MI. (2) Cardiac overexpression of MCP-1 also induced macrophage infiltration and capillary formation in the border area of MI. However, a small contribution of bone marrow-derived EPCs to the neovascularization was observed. (3) Cardiac overexpression of MCP-1 induced marked myocardial IL-6 secretion, STAT3 activation, and LV hypertrophy in the MI heart. (4) Combined treatment of IL-6 with MCP-1 synergistically stimulated and sustained STAT3 activation in cardiomyocytes in vitro. (5) Cardiac overexpression of MCP-1 increased the accumulation of cardiac myofibroblasts, and an in vitro study demonstrated that MCP-1 and hypoxia synergistically induced the differentiation of cardiac fibroblasts into myofibroblasts. These findings suggest that cardiac overexpression of MCP-1 induced macrophage infiltration, neovascularization, cardiac IL-6 secretion, and accumulation of cardiac myofibroblasts, thereby resulting in the prevention of LV dysfunction and remodeling after MI.

The beneficial role of MCP-1 in MI is controversial. Inhibition of MCP-1 or its signaling has been shown to attenuate LV damage and remodeling after MI.^6,7 Conversely, MCP-1 has direct angiogenic effects, and human endothelial cells express CCR2.⁸ In addition, a recent study demonstrated that MCP-1 has a cardioprotective effect against hypoxia in cardiomyocytes in vitro.⁹ Dewald et al¹⁰ recently demonstrated that MCP-1 gene disruption led to decreased and delayed macrophage infiltration in the healing infarct and delayed replacement of injured cardiomyocytes with granulation tissue. They also showed that MCP-1 deficiency had decreased the expression of inflammatory cytokines such as TNF-, IL-1ß, IL-10, and transforming growth factor (TGF)-ß and had diminished myofibroblast accumulation, thereby suggesting the beneficial role of MCP-1 in myocardial healing after MI. The reason for the discrepancy in the role of MCP-1 in MI remains unclear. We postulate that the topical MCP-1 concentration may be important because the chemotactic function of MCP-1 depends on the MCP-1 concentration gradient.^11,12 Therefore, we used MHC/MCP-1 mice that topically overexpress MCP-1 in hearts and demonstrated that these mice exhibited a reduction in the infarct area and scar formation and prevention of LV dysfunction and remodeling after MI. Martire et al¹⁹ recently reported that cardiac overexpression of MCP-1 could prevent myocardial damage against short-term ischemia/reperfusion injury; their observations supported our data. Taken together, these findings suggest the beneficial effects of topical cardiac MCP-1 production in the heart after MI.

We demonstrated that macrophage infiltration and capillary formation were significantly increased after MI in MHC/MCP-1 mice. In the noninfarct area, the similar number of macrophages in MHC/MCP-1 mice was observed, compared with that of wild-type mice. A recent study reported that Mac-1– but not Mac-3–positive cells were increased in the heart of 2-month-old MHC/MCP-1 mice.²⁰ In this regard, Mac-1 is expressed on both granulocytes and monocytes/macrophages, whereas Mac-3 is specifically expressed by activated macrophages. Similarly, F4/80 antibody used in this study specifically recognizes activated macrophages, suggesting that the number of activated macrophages in the noninfarct area was similar between wild-type and MHC/MCP-1 mice. We also showed the similar acute infarct sizes after MI between wild-type and MHC/MCP-1 mice; this suggests that neovascularization rather than cytoprotective effects might be responsible for the beneficial effects of MCP-1. Recent evidence indicates that MCP-1 promotes neovascularization in ischemic tissues through several possible mechanisms. First, MCP-1 directly stimulates VEGF induction by monocytes/macrophages²¹ and endothelial cells,²² leading to neovascularization. Second, recent evidence indicates that monocytes/macrophages produce matrix metalloproteinase-dependent tunnels and promote neovascularization.²³ In fact, Moldovan et al²⁴ demonstrated that this phenomenon was observed in the heart of MHC/MCP-1 mice, suggesting that this mechanism might contribute to neovascularization in this study. Third, bone marrow-derived monocytic lineage cells are suggested to function as EPCs and participate in neovascularization in the ischemic tissues.^2–4 Flow cytometric analysis showed that there was no increase in the EPCs (CD34⁺/Flk-1⁺ cells) in peripheral circulation after MI in MHC/MCP-1 mice. In this regard, Harraz et al³ reported that CD34-negative angioblasts are a subset of CD14-positive monocytic cells and that these monocytes have the potential to transdifferentiate into endothelial cells, thereby suggesting that monocytic cell-derived EPCs might have other surface markers. We detected few bone marrow-derived endothelial cells after MI in MHC/MCP-1 mice. Interestingly, EPCs have shown to induce the proliferation of the neighboring resident endothelial cells.²⁵ Thus, further investigations are required to elucidate the role of EPCs in the heart of MHC/MCP-1 mice after MI.

IL-6 is a pleiotropic cytokine with varying effects on cells of the immune system and other tissues, including cardiomyocytes. Interestingly, we found that a substantial amount of IL-6 was secreted after MI in MHC/MCP-1 mice, although there was no significant increase at baseline. The latter findings were supported by Kolattukudy et al,¹³ who reported that cardiac overexpression of MCP-1 did not induce the production of proinflammatory or morphogenic cytokines, including IL-6, up to 100 days of age. In the present study, cardiomyocytes were defined as the main cellular source of IL-6 production after MI, and IL-6 promoted the cellular hypertrophy of cardiomyocytes. MCP-1 has previously been reported to stimulate IL-6 production in isolated adult cardiomyocytes.²⁶ Additionally, cardiomyocytes respond to hypoxic conditions to augment the production of IL-6.²⁷ Although the role of IL-6 in cardiac tissues has not been fully understood, it is now recognized that the cytokines of the IL-6 family might prevent heart failure through antiapoptotic and hypertrophic effects on cardiomyocytes. Furthermore, recent investigations indicate that the STAT3-mediated signaling pathway is responsible for these beneficial effects.¹⁶ Indeed, Negoro et al²⁸ demonstrated that MI activates the JAK/STAT pathway mainly in the border area of MI and that pharmacological inhibition of this pathway results in the deterioration of myocardial viability. Interestingly, combined treatment of IL-6 with MCP-1 synergistically stimulated and sustained STAT3 activation in cardiomyocytes, thereby suggesting the presence of crosstalk signaling pathways between IL-6 and MCP-1. Taken together, these findings suggest that IL-6 contributes to the prevention of LV damage and subsequent remodeling after MI in MHC/MCP-1 mice.

The present study demonstrated that accumulation of cardiac myofibroblasts was increased in MHC/MCP-1 mice after MI. Previous studies demonstrated that proliferation of myofibroblasts is observed during the process of MI repair,¹⁸ and these cells disappear by an apoptotic mechanism in association with necrotic tissue during fibrotic scar formation,²⁹ thereby suggesting a role for myofibroblasts in scar formation and remodeling after MI. Consistent with our findings, Dewald et al¹⁰ recently reported that MCP-1 deficiency diminished myofibroblast accumulation in a murine MI model. Our in vitro results also suggest that MCP-1 directly stimulated the differentiation of fibroblasts into myofibroblasts, and this differentiation was enhanced by hypoxic conditions. Taken together, these findings suggest a critical role for MCP-1 in cardiac scar formation and remodeling after MI.

In conclusion, we clearly demonstrated that cardiac overexpression of MCP-1 induced macrophage infiltration, neovascularization, cardiac IL-6 secretion, and accumulation of cardiac myofibroblasts, thereby resulting in the prevention of LV dysfunction and remodeling after MI. Although the MCP-1 levels in MHC/MCP-1 mice are not physiological, the current results may provide insights into gene-based drug delivery of MCP-1. Furthermore, several previous studies suggest the inhibition of MCP-1 as a potential target for therapeutic intervention^6,7; however, our study indicates that further investigations are necessary to elucidate the precise role of MCP-1 in ischemic heart diseases before its clinical application.

	Acknowledgments

 
We thank Junko Nakayama, Tomoko Hamaji, and Kazuko Misawa for excellent technical assistance.

Sources of Funding

This study was supported by research grants from the Ministry of Health, Labor and Welfare of Japan (Research on Measures for Intractable Diseases) (to M.T. and U.I.); the Ministry of Education, Science, Sports and Culture (to M.T. and U.I.); the Mitsubishi Pharma Research Foundation (to M.T.); and the NIH (HL69458 to P.K.).

Disclosures

None.

	Footnotes

 
Original received April 23, 2006; resubmission received August 15, 2006; revised resubmission received September 4, 2006; accepted September 8, 2006.

	References

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