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Regulatory T cells (Tregs) play a pivotal role in suppressing immune responses regulating behavior and gene expression in effector T cells, macrophages, and dendritic cells. Tregs infiltrate the infarcted myocardium; however, their role the inflammatory and reparative response after myocardial infarction remains poorly understood. We used FoxP3EGFP reporter mice to study Treg trafficking in the infarcted heart and examined the effects of Treg depletion on postinfarction remodeling using an anti-CD25 antibody. Moreover, we investigated the in vitro effects of Tregs on cardiac fibroblast phenotype and function. Low numbers of Tregs infiltrated the infarcted myocardium after 24-72 h of reperfusion. Treg depletion had no significant effects on cardiac dysfunction and scar size after reperfused myocardial infarction but accelerated ventricular dilation and accentuated apical remodeling. Enhanced myocardial dilation in Treg-depleted animals was associated with increased expression of chemokine (C-C motif) ligand 2 and accentuated macrophage infiltration. In vitro, Tregs modulated the cardiac fibroblast phenotype, reducing expression of a-smooth muscle actin, decreasing expression of matrix metalloproteinase-3, and attenuating contraction of fibroblast-populated collagen pads. Our findings suggest that endogenous Tregs have modest effects on the inflammatory and reparative response after myocardial infarction. However, the anti-inflammatory and matrix-preserving properties of Tregs may suggest a role for Treg-based cell therapy in the attenuation of adverse postinfarction remodeling. [ABSTRACT FROM AUTHOR]

Aims The CXC chemokine CXCL10 is up-regulated in the infarcted myocardium and limits cardiac fibrosis by inhibiting growth factor-mediated fibroblast migration. CXCL10 signals by binding to its receptor CXCR3; however, recently CXCR3-independent CXCL10 actions have been suggested. Our study explores the role of CXCR3 signalling in myocardial infarction and investigates its involvement in mediating the anti-fibrotic effects of CXCL10. Methods and results Wild-type and CXCR3 null mice underwent reperfused infarction protocols. CXCL10 was markedly induced in the infarct; in contrast, expression of the other two CXCR3 ligands, CXCL9 and CXCL11 was extremely low. CXCR3 loss did not affect scar size, geometric ventricular remodelling, collagen deposition, and systolic dysfunction of the infarcted heart. CXCR3 null mice had increased peak neutrophil recruitment and delayed myofibroblast infiltration in the infarcted heart, but exhibited comparable myocardial expression of pro-inflammatory cytokines and chemokines. In vitro, CXCL10 did not modulate Transforming Growth Factor (TGF)-β signalling, but inhibited basic fibroblast growth factor (bFGF)-induced cardiac fibroblast migration in both wild-type and CXCR3 null cells. Treatment of fibroblasts with heparinase and chondroitinase to cleave glycosaminoglycan chains abrogated the inhibitory effects of CXCL10 on cell migration. Conclusion CXCR3 signalling does not critically regulate cardiac remodelling and dysfunction following myocardial infarction. The anti-fibrotic effects of CXCL10 in the healing infarct and in isolated cardiac fibroblasts are CXCR3-independent and may be mediated through proteoglycan signalling. Thus, administration of CXCR3-defective forms of CXCL10 may be an effective anti-fibrotic strategy in the remodelling myocardium without activating a potentially injurious, CXCR3-driven T cell response. [ABSTRACT FROM AUTHOR]

In the infarcted myocardium, activation of the inflammatory cascade clears the wound from dead cells, whereas stimulating matrix degradation and chamber dilation, thus contributing to the development of heart failure. IL-1 is critically involved in the postinfarction inflammatory reaction and mediates adverse dilative remodeling. We hypothesized that IL-1 may regulate postinfarction repair and remodeling through cell-specific actions on leukocytes and fibroblasts. Flow cytometry demonstrated that in mouse infarcts, early recruitment of proinflammatory Ly6Chi cells expressing IL-1R1, the signaling receptor for IL-1, was followed by infiltration with cells expressing the decoy receptor, IL-1R2. Increased expression of IL-1R2 may serve to terminate IL-1-driven inflammation after infarction. Loss of IL-1 signaling in IL-1R1 null mice globally attenuated leukocyte recruitment, reducing the number of infiltrating Ly6Chi and Ly6Clo cells. Nonmyeloid CD11b- cells harvested during the inflammatory phase of cardiac repair exhibited marked upregulation of chemokines and cytokines; their inflammatory activation was IL-1R1 dependent. Moreover, IL-1β attenuated TGF-β-induced contractile activity of fibroblasts populating collagen pads, attenuated a-smooth muscle actin expression, and stimulated matrix metalloproteinase synthesis in an IL-1R1-dependent manner. The effects of IL-1 on TGF-β responses in cardiac fibroblasts were not due to direct effects on Smad activation, but were associated with endoglin suppression and accentuated expression of bone morphogenetic protein and activin membrane-bound inhibitor, a negative regulator of TGF-β signaling. IL-1 may orchestrate fibroblast responses in the infarct; early stimulation of fibroblast IL-1R1 signaling during the inflammatory phase may prevent premature activation of a matrix-synthetic contractile phenotype until the wound is cleared, and the infarct microenvironment can support mesenchymal cell growth. [ABSTRACT FROM AUTHOR]

Understanding the role of fibroblasts in pathologic conditions is hampered by the absence of specific markers. Fibroblast-specific protein (FSP)1 has been suggested as a fibroblast-specific marker in normal and fibrotic tissues; FSP1 reporter mice and FSP1-Cre-driven gene deletion are considered reliable strategies to investigate fibroblast biology. Because fibroblasts are abundant in normal and injured mammalian hearts, we studied the identity of FSP1+ cells in the infarcted and remodeling myocardium using mice with green fluorescent protein (GFP) expression driven by the FSP1 promoter. Neonatal and adult mouse hearts had low numbers of FSP1+ cells. Myocardial infarction induced marked infiltration with FSP1-expressing cells that peaked after 72 h of reperfusion. Using flow cytometry, we identified 50% of FSP1+ cells as hematopoietic cells; many endothelial cells were also FSP1+. Increased infiltration with FSP1+ cells was also noted in the pressure-overloaded myocardium. Although some FSP1+ cells had fibroblast morphology, >30% were identified as hematopoietic cells, endothelial cells, or vascular smooth muscle cells. In contrast, periostin did not stain leukocytes or vascular cells but labeled spindle-shaped interstitial cells and, as a typical matricellular protein, was deposited in the matrix. CD11b+ myeloid cells sorted from the infarcted heart had higher FSP1 expression than corresponding CD11b-negative cells, highlighting the predominant expression by hematopoietic cells. FSP1 is not a specific marker for fibroblasts in cardiac remodeling and fibrosis. [ABSTRACT FROM AUTHOR]

Background: Transforming growth factor-βs regulate a wide range of cellular responses by activating Smad-dependent and Smad-independent cascades. In the infarcted heart, Smad3 signaling is activated in both cardiomyocytes and interstitial cells. We hypothesized that cell-specific actions of Smad3 regulate repair and remodeling in the infarcted myocardium.Methods: To dissect cell-specific Smad3 actions in myocardial infarction, we generated mice with Smad3 loss in activated fibroblasts or cardiomyocytes. Cardiac function was assessed after reperfused or nonreperfused infarction using echocardiography. The effects of cell-specific Smad3 loss on the infarcted heart were studied using histological studies, assessment of protein, and gene expression levels. In vitro, we studied Smad-dependent and Smad-independent actions in isolated cardiac fibroblasts.Results: Mice with fibroblast-specific Smad3 loss had accentuated adverse remodeling after reperfused infarction and exhibited an increased incidence of late rupture after nonreperfused infarction. The consequences of fibroblast-specific Smad3 loss were not a result of effects on acute infarct size but were associated with unrestrained fibroblast proliferation, impaired scar remodeling, reduced fibroblast-derived collagen synthesis, and perturbed alignment of myofibroblast arrays in the infarct. Polarized light microscopy in Sirius red-stained sections demonstrated that the changes in fibroblast morphology were associated with perturbed organization of the collagenous matrix in the infarcted area. In contrast, α-smooth muscle actin expression by infarct myofibroblasts was not affected by Smad3 loss. Smad3 critically regulated fibroblast function, activating integrin-mediated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-2 (NOX-2) expression. Smad3 loss in cardiomyocytes attenuated remodeling and dysfunction after infarction. Cardiomyocyte-specific Smad3 loss did not affect acute infarct size but was associated with attenuated cardiomyocyte apoptosis in the remodeling myocardium, accompanied by decreased myocardial NOX-2 levels, reduced nitrosative stress, and lower matrix metalloproteinase-2 expression.Conclusions: In healing myocardial infarction, myofibroblast- and cardiomyocyte-specific activation of Smad3 has contrasting functional outcomes that may involve activation of an integrin/reactive oxygen axis. [ABSTRACT FROM AUTHOR]

Aims Tissue transglutaminase (tTG) is induced in injured and remodelling tissues, and modulates cellular phenotype, while contributing to matrix cross-linking. Our study tested the hypothesis that tTG may be expressed in the pressure-overloaded myocardium, and may regulate cardiac function, myocardial fibrosis and chamber remodelling. Methods and results In order to test the hypothesis, wild-type and tTG null mice were subjected to pressure overload induced through transverse aortic constriction. Moreover, we used isolated cardiac fibroblasts and macrophages to dissect the mechanisms of tTG-mediated actions. tTG expression was upregulated in the pressure-overloaded mouse heart and was localized in cardiomyocytes, interstitial cells, and in the extracellular matrix. In contrast, expression of transglutaminases 1, 3, 4, 5, 6, 7 and FXIII was not induced in the remodelling myocardium. In vitro, transforming growth factor (TGF)-β1 stimulated tTG synthesis in cardiac fibroblasts and in macrophages through distinct signalling pathways. tTG null mice had increased mortality and enhanced ventricular dilation following pressure overload, but were protected from diastolic dysfunction. tTG loss was associated with a hypercellular cardiac interstitium, reduced collagen cross-linking, and with accentuated matrix metalloproteinase (MMP)2 activity in the pressure-overloaded myocardium. In vitro, tTG did not modulate TGF-β-mediated responses in cardiac fibroblasts; however, tTG loss was associated with accentuated proliferative activity. Moreover, when bound to the matrix, recombinant tTG induced synthesis of tissue inhibitor of metalloproteinases (TIMP)-1 through transamidase-independent actions. Conclusions Following pressure overload, endogenous tTG mediates matrix cross-linking, while protecting the remodelling myocardium from dilation by exerting matrix-preserving actions. [ABSTRACT FROM AUTHOR]