Background-Inflammation plays a key role in the pathophysiology of myocardial ischemia/reperfusion (I/R) injury; however, the mechanism by which myocardial I/R induces inflammation remains unclear. Recent evidence indicates that a sterile inflammatory response triggered by tissue damage is mediated through a multiple-protein complex called the inflammasome. Therefore, we hypothesized that the inflammasome is an initial sensor for danger signal(s) in myocardial I/R injury. Methods and Results-We demonstrate that inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes, is crucially involved in the initial inflammatory response after myocardial I/R injury. We found that inflammasomes are formed by I/R and that its subsequent activation of inflammasomes leads to interleukin-1␤ production, resulting in inflammatory responses such as inflammatory cell infiltration and cytokine expression in the heart. In mice deficient for apoptosis-associated speck-like adaptor protein and caspase-1, these inflammatory responses and subsequent injuries, including infarct development and myocardial fibrosis and dysfunction, were markedly diminished. Bone marrow transplantation experiments with apoptosis-associated speck-like adaptor protein-deficient mice revealed that inflammasome activation in bone marrow cells and myocardial resident cells such as cardiomyocytes or cardiac fibroblasts plays an important role in myocardial I/R injury. In vitro experiments revealed that hypoxia/reoxygenation stimulated inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes, and that hypoxia/reoxygenation-induced activation was mediated through reactive oxygen species production and potassium efflux. Conclusions-Our results demonstrate the molecular basis for the initial inflammatory response after I/R and suggest that the inflammasome is a potential novel therapeutic target for preventing myocardial I/R injury. (Circulation. 2011;123:594-604.)Key Words: cytokine Ⅲ heart Ⅲ hypoxia Ⅲ inflammation Ⅲ leukocyte I ncreasing evidence indicates that inflammation is involved in the pathophysiology of myocardial ischemia/reperfusion (I/R) injury. 1 One prominent and early mediator for inflammation in I/R injury is interleukin-1␤ (IL-1␤). 2,3 I/R induces IL-1␤ expression in the heart, and the inhibition of IL-1␤ prevents myocardial injury after I/R, 3 suggesting that the deleterious effects of myocardial I/R are mediated, at least in part, by IL-1␤. In the generation of IL-1␤, pro-IL-1␤, an inactive precursor, undergoes proteolysis by the converting enzyme caspase-1. Caspase-1 is activated within a cytosolic multiprotein complex, the inflammasome. The inflammasome contains cytoplasmic receptors of the NACHT leucine-rich-repeat protein family that are associated with the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which in turn recruits and activates caspase-1. 4,5 Increasing evidence indicates that several sterile inflammatory responses triggered by tissue damage are mediated by th...

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Abstract-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 ϩ /Flk-1 ϩ 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 Ⅲ heart failure Ⅲ hypertrophy Ⅲ inflammation Ⅲ myocardial infarction M yocardial 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][3][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 rece...

Inducible costimulatory molecule (ICOS) plays a pivotal role in T cell activation and Th1/Th2 differentiation. ICOS blockade has disparate effects on immune responses depending on the timing of blockade. Its role in transplantation immunity, however, remains incompletely defined. We used a vascularized mouse cardiac allograft model to explore the role of ICOS signaling at different time points after transplantation, targeting immune initiation (early blockade) or the immune effector phase (delayed blockade). In major histocompatibility-mismatched recipients, ICOS blockade prolonged allograft survival using both protocols but did so more effectively in the delayed-treatment group. By contrast, in minor histocompatibility-mismatched recipients, early blockade accelerated rejection and delayed blockade prolonged graft survival. Alloreactive CD4 + T cell expansion and alloantibody production were suppressed in both treatment groups, whereas only delayed blockade resulted in suppression of effector CD8 + T cell generation. After delayed ICOS blockade, there was a diminished frequency of allospecific IL-10-producing cells and an increased frequency of both IFN-γ-and IL-4-producing cells. The beneficial effects of ICOS blockade in regulating allograft rejection were seen in the absence of CD28 costimulation but required CD8 + cells, cytotoxic T lymphocyte antigen-4, and an intact signal transducer and activator of transcription-6 pathway. These data define the complex functions of the ICOS-B7h pathway in regulating alloimmune responses in vivo.

Vascular endothelial growth factor (VEGF), an established angiogenesis factor, is expressed in allografts undergoing rejection, but its function in the rejection process has not been defined. Here, we initially determined that VEGF is functional in the trafficking of human T cells into skin allografts in vivo in the humanized SCID mouse. In vitro, we found that VEGF enhanced endothelial cell expression of the chemokines monocyte chemoattractant protein 1 and IL-8, and in combination with IFN-γ synergistically induced endothelial cell production of the potent T cell chemoattractant IFN-inducible protein-10 (IP-10). Treatment of BALB/c (H-2d) recipients of fully MHC-mismatched C57BL/6 (H-2b) donor hearts with anti-VEGF markedly inhibited T cell infiltration of allografts and acute rejection. Anti-VEGF failed to inhibit T cell activation responses in vivo, but inhibited intragraft expression of several endothelial cell adhesion molecules and chemokines, including IP-10. In addition, whereas VEGF expression was increased, neovascularization was not associated with acute rejection, and treatment of allograft recipients with the angiogenesis inhibitor endostatin failed to inhibit leukocyte infiltration of the grafts. Thus, VEGF appears to be functional in acute allograft rejection via its effects on leukocyte trafficking. Together, these observations provide mechanistic insight into the proinflammatory function of VEGF in immunity

Inducible costimulatory molecule (ICOS) plays a pivotal role in T cell activation and Th1/Th2 differentiation. ICOS blockade has disparate effects on immune responses depending on the timing of blockade. Its role in transplantation immunity, however, remains incompletely defined. We used a vascularized mouse cardiac allograft model to explore the role of ICOS signaling at different time points after transplantation, targeting immune initiation (early blockade) or the immune effector phase (delayed blockade). In major histocompatibility-mismatched recipients, ICOS blockade prolonged allograft survival using both protocols but did so more effectively in the delayed-treatment group. By contrast, in minor histocompatibility-mismatched recipients, early blockade accelerated rejection and delayed blockade prolonged graft survival. Alloreactive CD4 + T cell expansion and alloantibody production were suppressed in both treatment groups, whereas only delayed blockade resulted in suppression of effector CD8 + T cell generation. After delayed ICOS blockade, there was a diminished frequency of allospecific IL-10-producing cells and an increased frequency of both IFN-γ-and IL-4-producing cells. The beneficial effects of ICOS blockade in regulating allograft rejection were seen in the absence of CD28 costimulation but required CD8 + cells, cytotoxic T lymphocyte antigen-4, and an intact signal transducer and activator of transcription-6 pathway. These data define the complex functions of the ICOS-B7h pathway in regulating alloimmune responses in vivo.

G-CSF treatment accelerated reendothelialization and decreased neointimal formation following vascular injury, although there was little contribution of bone marrow-derived EPCs to the reendothelialization of the artery. These results suggest that G-CSF pretreatment has a therapeutic potential for prevention of restenosis following PCI.

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