Abstract-Heart failure remains a leading cause of morbidity and mortality. The cellular mechanism underlying the development of cardiac dysfunction is a decrease in the number of viable cardiomyocytes. Recent observations have suggested that the adult heart may contain a progenitor cell population. Side population (SP) cells, characterized by a distinct Hoechst dye efflux pattern, have been shown to exist in multiple tissues and are capable of tissue-specific differentiation. In this report, we confirm the existence of a cardiac SP cell population, immunophenotypically distinct from bone marrow SP cells. Moreover, we demonstrate that among cardiac SP cells, the greatest potential for cardiomyogenic differentiation is restricted to cells negative for CD31 expression and positive for stem cell antigen 1 (Sca1) expression (CD31 Ϫ /Sca1 ϩ ). Furthermore, we determine that CD31 Ϫ /Sca1 ϩ cardiac SP cells are capable of both biochemical and functional cardiomyogenic differentiation into mature cardiomyocytes, with expression of cardiomyocyte-specific transcription factors and contractile proteins, as well as stimulated cellular contraction and intracellular calcium transients indistinguishable from adult cardiomyocytes. We also determine the necessity of cell-extrinsic signaling through coupling, although not fusion, with adult cardiomyocytes in regulating cardiomyogenic differentiation of cardiac SP cells. We, therefore, conclude that CD31 Ϫ /Sca1 ϩ cardiac SP cells represent a distinct cardiac progenitor cell population, capable of cardiomyogenic differentiation into mature cardiomyocytes through a process mediated by cellular coupling with adult cardiomyocytes. (Circ Res. 2005;97:52-61.)

It is established that the adipocyte-derived cytokine adiponectin protects against cardiovascular and metabolic diseases, but the effect of this adipokine on macrophage polarization, an important mediator of disease progression, has never been assessed. We hypothesized that adiponectin modulates macrophage polarization from that resembling a classically activated M1 phenotype to that resembling alternatively-activated M2 cells. Peritoneal macrophages and the stromal vascular fraction (SVF) cells of adipose tissue isolated from adiponectin knock-out mice displayed increased M1 markers, including tumor necrosis factor-␣, interleukin-6, and monocyte chemoattractant protein-1 and decreased M2 markers, including arginase-1, macrophage galactose N-acetyl-galactosamine specific lectin-1, and interleukin-10. The systemic delivery of adenovirus expressing adiponectin significantly augmented arginase-1 expression in peritoneal macrophages and SVF cells in both wild-type and adiponectin knock-out mice. In culture, the treatment of macrophages with recombinant adiponectin protein led to an increase in the levels of M2 markers and a reduction of reactive oxygen species and reactive oxygen species-related gene expression. Adiponectin also stimulated the expression of M2 markers and attenuated the expression of M1 markers in human monocyte-derived macrophages and SVF cells isolated from human adipose tissue. These data show that adiponectin functions as a regulator of macrophage polarization, and they indicate that conditions of high adiponectin expression may deter metabolic and cardiovascular disease progression by favoring an anti-inflammatory phenotype in macrophages.Obesity activates low grade inflammation that contributes to the pathogenesis of obesity-linked diseases, such as type 2 diabetes and atherosclerosis (1). Adipose tissue macrophages play an important role in the establishment of the chronic inflammatory state and metabolic dysfunction that is associated with obesity (2, 3). Recent findings show that adipose tissue macrophages from lean organisms express markers of the M2 or "alternatively activated" macrophage, whereas obesity leads to a reduction of these markers and an increase of genes associated with the M1 or "classically activated" macrophage (4). M1 macrophage polarization is associated with inflammation and tissue destruction, whereas the M2 macrophage has an anti-inflammatory phenotype that is associated with wound repair and angiogenesis.Macrophages are polarized to the M1 state by interferon-␥ and inducers of tumor necrosis factor-␣ (TNF-␣) 2 , such as lipopolysaccharide (LPS). M1 macrophages up-regulate proinflammatory cytokines including TNF-␣, interleukin (IL)-6, and IL-12, and they increase the production of reactive oxygen species (ROS) and nitrogen intermediates (5). In contrast, macrophages are polarized to the M2 state by IL-4 and IL-13. M2 macrophages up-regulate scavenger receptors, mannose receptor and IL-1 receptor antagonist. M2 cells also secrete the antiinflammatory cytokine IL-10 and...

Obesity and type 2 diabetes are associated with chronic inflammation. Adiponectin is an adipocyte-derived hormone with antidiabetic and antiinflammatory actions. Here, we demonstrate what we believe to be a previously undocumented activity of adiponectin, facilitating the uptake of early apoptotic cells by macrophages, an essential feature of immune system function. Adiponectin-deficient (APN-KO) mice were impaired in their ability to clear apoptotic thymocytes in response to dexamethasone treatment, and these animals displayed a reduced ability to clear early apoptotic cells that were injected into their intraperitoneal cavities. Conversely, adiponectin administration promoted the clearance of apoptotic cells by macrophages in both APN-KO and wild-type mice. Adiponectin overexpression also promoted apoptotic cell clearance and reduced features of autoimmunity in lpr mice whereas adiponectin deficiency in lpr mice led to a further reduction in apoptotic cell clearance, which was accompanied by exacerbated systemic inflammation. Adiponectin was capable of opsonizing apoptotic cells, and phagocytosis of cell corpses was mediated by the binding of adiponectin to calreticulin on the macrophage cell surface. We propose that adiponectin protects the organism from systemic inflammation by promoting the clearance of early apoptotic cells by macrophages through a receptor-dependent pathway involving calreticulin.

Side population (SP) cells are a rare subset of cells found in various tissues that are highly enriched for stem cell activity. SP cells can be isolated by dual-wavelength flow cytometry because of their capacity to efflux Hoechst dye, a process mediated by the ATP-binding cassette transporter breast cancer resistance protein (Bcrp) 1. By performing flow cytometry of enzymedigested mouse lung stained with Hoechst dye, we found that SP cells comprise 0.03–0.07% of total lung cells and are evenly distributed in proximal and distal lung regions. By RT-PCR, we found that lung SP cells express hepatocyte nuclear factor-3β, but not thyroid transcription factor-1. Surface marker analysis revealed lung SP cells to be stem cell antigen 1 positive, Bcrp1 positive, lineage marker negative, and heterogeneous at the CD45 locus. As expected, we did not detect lung SP cells in Bcrp1-deficient animals. We, therefore, employed nonisotopic in situ hybridization and immunostaining for Bcrp1 as a strategy to localize these cells in vivo. Expression was observed in distinct lung cell types: bronchial and vascular smooth muscle cells and round cells within the distal air space. We confirmed the expression of Bcrp1 in primary bronchial smooth muscle cell cultures (BSMC) and in lavaged distal airway cells, but neither possessed the capacity to efflux Hoechst dye. In BSMC, Bcrp1 was localized to an intracellular compartment, suggesting that the molecular site of Bcrp1 expression regulates SP phenotype.

Tissue-specific progenitor cells contribute to local cellular regeneration and maintain organ function. Recently, we have determined that cardiac side-population (CSP) cells represent a distinct cardiac progenitor cell population, capable of in vitro differentiation into functional cardiomyocytes. The response of endogenous CSP to myocardial injury, however, and the cellular mechanisms that maintain this cardiac progenitor cell pool in vivo remain unknown. In this report we demonstrate that local progenitor cell proliferation maintains CSP under physiologic conditions, with little contribution from extracardiac stem cell sources. Following myocardial infarction in adult mice, however, CSP cells are acutely depleted, both within the infarct and noninfarct areas. CSP pools are subsequently reconstituted to baseline levels within 7 days after myocardial infarction, through both proliferation of resident CSP cells, as well as through homing of bone marrow-derived stem cells (BMC) to specific areas of myocardial injury and immunophenotypic conversion of BMC to adopt a CSP phenotype. We, therefore, conclude that following myocardial injury, cardiac progenitor cell populations are acutely depleted and are reconstituted to normal levels by both self-proliferation and selective homing of BMC. Understanding and enhancing such processes hold enormous potential for therapeutic myocardial regeneration.T issue-specific progenitor cell populations maintain the regenerative capacity of terminally differentiated organs, both under basal conditions and following local tissue injury. Side population (SP) cells, characterized by their intrinsic capacity to efflux Hoechst dye through ATP-binding cassette transporters, contribute to the long-term regenerative potential of hematopoietic and extrahematopoietic tissues. 1 Recently, we have demonstrated that cardiac SP (CSP) cells, immunophenotypically distinct from bone marrow (BM)-derived stem cells (BMC), are present in the adult heart and are capable of both biochemical and functional cardiomyogenic differentiation into mature cardiomyocytes, thereby identifying CSP as a distinct cardiac progenitor cell population. 2 Supplementation of cardiac progenitor cell pools after myocardial infarction (MI) with exogenous cells has been shown to improve ventricular function by regenerating myocardium and cardiac vasculature. [3][4][5] The response of endogenous CSP cells to myocardial injury, however, and the cellular mechanisms that maintain this endogenous cardiac progenitor cell pool under basal and after injury conditions remain unknown. We, therefore, serially assessed CSP pools in hearts following MI and determined the role of selfproliferation and BMC in reconstituting cardiac progenitor pools. Methods and MaterialsCSP were isolated from mouse hearts as described previously. 2 MI was performed in mice via permanent coronary ligation. 6 BM transplantation was performed in lethally irradiated mice using marrow isolated from C57bl/6-Tg(ACTbEGFP) mice. 7 All animals were obtained from The ...