Cardiac macrophages adopt profibrotic/M2 phenotype in infarcted hearts: Role of urokinase plasminogen activator.

BACKGROUND: Macrophages (mac) that over-express urokinase plasminogen activator (uPA) adopt a profibrotic M2 phenotype in the heart in association with cardiac fibrosis. We tested the hypothesis that cardiac macs are M2 polarized in infarcted mouse and human hearts and that polarization is dependent on mac-derived uPA. METHODS: Studies were performed using uninjured (UI) or infarcted (MI) hearts of uPA overexpressing (SR-uPA), uPA null, or nontransgenic littermate (Ntg) mice. At 7days post-infarction, cardiac mac were isolated, RNA extracted and M2 markers Arg1, YM1, and Fizz1 measured with qrtPCR. Histologic analysis for cardiac fibrosis, mac and myofibroblasts was performed at the same time-point. Cardiac macs were also isolated from Ntg hearts and RNA collected after primary isolation or culture with vehicle, IL-4 or plasmin and M2 marker expression measured. Cardiac tissue and blood was collected from humans with ischemic heart disease. Expression of M2 marker CD206 and M1 marker TNFalpha was measured. RESULTS: Macs from WT mice had increased expression of Arg1 and Ym1 following MI (41.3±6.5 and 70.3±36, fold change vs UI, n=8, P<0.007). There was significant up-regulation of cardiac mac Arg1 and YM1 with MI in both WT and uPA null mice (n=4-9 per genotype and condition). Treatment with plasmin increased expression of Arg1 and YM1 in cultured cardiac macs. Histologic analysis revealed increased density of activated fibroblasts and M2 macs in SR-uPA hearts post-infarction with associated increases in fibrosis. Cardiac macs isolated from human hearts with ischemic heart disease expressed increased levels of the M2 marker CD206 in comparison to blood-derived macs (4.9±1.3). CONCLUSIONS: Cardiac macs in mouse and human hearts adopt a M2 phenotype in association with fibrosis. Plasmin can induce an M2 phenotype in cardiac macs. However, M2 activation can occur in the heart in vivo in the absence of uPA indicating that alternative pathways to activate plasmin are present in the heart. Excess uPA promotes increased fibroblast density potentially via potentiating fibroblast migration or proliferation. Altering macrophage phenotype in the heart is a potential target to modify cardiac fibrosis.

Aberrant differentiation of fibroblast progenitors contributes to fibrosis in the aged murine heart: role of elevated circulating insulin levels.

With age, the collagen content of the heart increases, leading to interstitial fibrosis. We have shown that CD44(pos) fibroblasts derived from aged murine hearts display reduced responsiveness to TGF-ß but, paradoxically, have increased collagen expression in vivo and in vitro. We postulated that this phenomenon was due to the defect in mesenchymal stem cell (MSC) differentiation in a setting of elevated circulating insulin levels and production that we observed in aging mice. We discovered that cultured fibroblasts derived from aged but not young cardiac MSCs of nonhematopoietic lineage displayed increased basal and insulin-induced (1 nM) collagen expression (2-fold), accompanied by increased farnesyltransferase (FTase) and Erk activities. In a quest for a possible mechanism, we found that a chronic pathophysiologic insulin concentration (1 nM) caused abnormal fibroblast differentiation of MSCs isolated from young hearts. Fibroblasts derived from these MSCs responded to insulin by elevating collagen expression as seen in untreated aged fibroblast cultures, suggesting a causal link between increased insulin levels and defective MSC responses. Here we report an insulin-dependent pathway that specifically targets collagen type I transcriptional activation leading to a unique mechanism of fibrosis that is TGF-ß and inflammation-independent in the aged heart.

Cardiac mesenchymal stem cells contribute to scar formation after myocardial infarction.

AIMS: Therapeutic advances in prevention and treatment of myocardial infarction (MI) have decreased patient mortality and increased concern about efficient repair and scar formation, processes that are necessary to attenuate complications such as adverse remodelling and heart failure. Since the rapid accumulation and activity of cardiac fibroblasts is critical for proper scar formation, we hypothesized that infarct fibroblasts are generated by a cardiac-resident progenitor cell population. METHODS AND RESULTS: We found that infarct fibroblasts in C57BL/6 mice are generated by a mesenchymal stem cell (MSC) population that responds robustly to injury by proliferating and accumulating in the infarct. We report that stem cell-derived fibroblasts contribute to the formation of a scar after an infarction by differentiating into matrix-producing fibroblasts closely associated with fibrillar collagen in the infarct. Further characterization of these cells revealed a heterogenous population with expression of both stem cell and canonical cardiac fibroblast markers, suggesting that some have a commitment to the fibroblast phenotype. Our in vitro study of these cells shows that they have extended self-renewal capability and express the primitive marker Nanog. In keeping with these observations, we also report that these cells are multipotent and differentiate readily into fibroblasts as well as other mesenchymal lineages. CONCLUSION: Cells with the properties of MSCs participate in wound healing after MI in the adult heart.

Immune-inflammatory dysregulation modulates the incidence of progressive fibrosis and diastolic stiffness in the aging heart.

Diastolic dysfunction in the aging heart is a grave condition that challenges the life and lifestyle of a growing segment of our population. This report seeks to examine the role and interrelationship of inflammatory dysregulation in interstitial myocardial fibrosis and progressive diastolic dysfunction in aging mice. We studied a population of C57BL/6 mice that developed progressive diastolic dysfunction over 30 months of life. This progressive dysfunction was associated with increasing infiltration of CD45(+) fibroblasts of myeloid origin. In addition, increased rates of collagen expression as measured by cellular procollagen were apparent in the heart as a function of age. These cellular and functional changes were associated with progressive increases in mRNA for MCP-1 and IL-13, which correlated both temporally and quantitatively with changes in fibrosis and cellular procollagen levels. MCP-1 protein was also increased and found to be primarily in the venular endothelium. Protein assays also demonstrated elevation of IL-4 and IL-13 suggesting a shift to a Th2 phenotype in the aging heart. In vitro studies demonstrated that IL-13 markedly enhanced monocyte-fibroblast transformation. Our results indicate that immunoinflammatory dysregulation in the aging heart induces progressive MCP-1 production and an increased shift to a Th2 phenotype paralleled by an associated increase in myocardial interstitial fibrosis, cellular collagen synthesis, and increased numbers of CD45(+) myeloid-derived fibroblasts that contain procollagen. The temporal association and functional correlations suggest a causative relationship between age-dependent immunoinflammatory dysfunction, fibrosis and diastolic dysfunction.

Bone marrow-derived fibroblast precursors mediate ischemic cardiomyopathy in mice.

We previously described a mouse model of fibrotic ischemia/reperfusion cardiomyopathy (I/RC) arising from daily, brief coronary occlusion. One characteristic of I/RC was the prolonged elevation of monocyte chemoattractant protein 1 (MCP-1), which was obligate to its phenotype and may contribute to the uptake of bloodborne cells. Here we describe in I/RC hearts a population of small spindle-shaped fibroblasts that were highly proliferative and expressed collagen I and alpha-smooth muscle actin (myofibroblast markers), CD34 (a precursor marker), and CD45 (a hematopoietic marker). These cells represented 3% of all nonmyocyte live cells. To confirm the cells' bone marrow origin, chimeric mice were created by the rescue of irradiated C57BL/6 mice with marrow from ROSA26, a congenic line expressing lacZ. I/RC resulted in a large population of spindle-shaped fibroblasts containing lacZ. We postulated that the fibroblast precursors represented a developmental path for a subset of monocytes, whose phenotype we have shown to be influenced by serum amyloid P (SAP). Thus, we administered SAP in vivo, which markedly reduced the number of proliferative spindle-shaped fibroblasts and completely prevented I/RC-induced fibrosis and global ventricular dysfunction. By contrast, SAP did not suppress the inflammation or chemokine expression seen in I/RC. SAP, a member of the pentraxin family, binds to Fcgamma receptors and modifies the pathophysiological function of monocytes. Our data suggest that SAP interferes with assumption of a fibroblast phenotype in a subset of monocytes and that SAP may be an important regulator in the linkage between inflammation and nonadaptive fibrosis in the heart.