Stephen D. Farris

The c-myc Insulator Element and Matrix Attachment Regions Define the c-myc Chromosomal Domain

ABSTRACT Insulator elements and matrix attachment regions are essential for the organization of genetic information within the nucleus. By comparing the pattern of histone modifications at the mouse and human c-myc alleles, we identified an evolutionarily conserved boundary at which the c-myc transcription unit is separated from the flanking condensed chromatin enriched in lysine 9-methylated histone H3. This region harbors the c-myc insulator element (MINE), which contains at least two physically separable, functional activities: enhancer-blocking activity and barrier activity. The enhancer-blocking activity is mediated by CTCF. Chromatin immunoprecipitation assays demonstrate that CTCF is constitutively bound at the insulator and at the promoter region independent of the transcriptional status of c-myc. This result supports an architectural role of CTCF rather than a regulatory role in transcription. An additional higher-order nuclear organization of the c-myc locus is provided by matrix attachment regions (MARs) that define a domain larger than 160 kb. The MARs of the c-myc domain do not act to prevent the association of flanking regions with lysine 9-methylated histones, suggesting that they do not function as barrier elements.

Mechanisms of Urokinase Plasminogen Activator (uPA)-mediated Atherosclerosis ROLE OF THE uPA RECEPTOR AND S100A8/A9 PROTEINS

Data from clinical studies, cell culture, and animal models implicate the urokinase plasminogen activator (uPA)/uPA receptor (uPAR)/plasminogen system in the development of atherosclerosis and aneurysms. However, the mechanisms through which uPA/uPAR/plasminogen stimulate these diseases are not yet defined. We used genetically modified, atherosclerosis-prone mice, including mice with macrophage-specific uPA overexpression and mice genetically deficient in uPAR to elucidate mechanisms of uPA/uPAR/plasminogen-accelerated atherosclerosis and aneurysm formation. We found that macrophage-specific uPA overexpression accelerates atherosclerosis and causes aortic root dilation in fat-fed Ldlr−/− mice (as we previously reported in Apoe−/− mice). Macrophage-expressed uPA accelerates atherosclerosis by stimulation of lesion progression rather than initiation and causes disproportionate lipid accumulation in early lesions. uPA-accelerated atherosclerosis and aortic dilation are largely, if not completely, independent of uPAR. In the absence of uPA overexpression, however, uPAR contributes modestly to both atherosclerosis and aortic dilation. Microarray studies identified S100A8 and S100A9 mRNA as the most highly up-regulated transcripts in uPA-overexpressing macrophages; up-regulation of S100A9 protein in uPA-overexpressing macrophages was confirmed by Western blotting. S100A8/A9, which are atherogenic in mice and are expressed in human atherosclerotic plaques, are also up-regulated in the aortae of mice with uPA-overexpressing macrophages, and macrophage S100A9 mRNA is up-regulated by exposure of wild-type macrophages to medium from uPA-overexpressing macrophages. Macrophage microarray data suggest significant effects of uPA overexpression on cell migration and cell-matrix interactions. Our results confirm in a second animal model that macrophage-expressed uPA stimulates atherosclerosis and aortic dilation. They also reveal uPAR independence of these actions and implicate specific pathways in uPA/Plg-accelerated atherosclerosis and aneurysmal disease.

Level of Macrophage uPA Expression Is an Important Determinant of Atherosclerotic Lesion Growth in Apoe −/− Mice

Objective—Enhanced plasminogen activation, mediated by overexpression of urokinase-type plasminogen activator (uPA), accelerates atherosclerosis in apolipoprotein E–null mice. However, the mechanisms through which uPA acts remain unclear. In addition, although elevated uPA expression can accelerate murine atherosclerosis, there is not yet any evidence that decreased uPA expression would retard atherosclerosis. Methods and Results—We used a bone marrow transplant (BMT) approach and apolipoprotein E–deficient (Apoe−/−) mice to investigate cellular mechanisms of uPA-accelerated atherosclerosis, aortic dilation, and sudden death. We also used BMT to determine whether postnatal loss of uPA expression in macrophages retards atherosclerosis. BMT from uPA-overexpressing mice yielded recipients with macrophage-specific uPA overexpression; whereas BMT from uPA knockout mice yielded recipients with macrophage-specific loss of uPA expression. Recipients of uPA-overexpressing BM acquired all the vascular phenotypes (accelerated atherosclerosis, aortic medial destruction and dilation, severe coronary stenoses) as well as the sudden death phenotype of uPA-overexpressing mice. Moreover, fat-fed 37-week-old recipients of uPA-null BM had significantly less atherosclerosis than recipients of uPA wild-type marrow (40% less aortic surface lesion area; P=0.03). Conclusions—The level of uPA expression by macrophages—over a broad range—is an important determinant of atherosclerotic lesion growth in Apoe−/− mice.

Mechanisms of Bone Marrow-Derived Cell Therapy in Ischemic Cardiomyopathy with Left Ventricular Assist Device Bridge to Transplant

BACKGROUND Clinical trials report improvements in function and perfusion with direct injection of bone marrow cells into the hearts of patients with ischemic cardiomyopathy. Preclinical data suggest these cells improve vascular density, which would be expected to decrease fibrosis and inflammation. OBJECTIVES The goal of this study was to test the hypothesis that bone marrow stem cells (CD34+) will improve histological measurements of vascularity, fibrosis, and inflammation in human subjects undergoing left ventricular assist device (LVAD) placement as a bridge to cardiac transplantation. METHODS Subjects with ischemic cardiomyopathy who were scheduled for placement of an LVAD as a bridge to transplantation underwent bone marrow aspiration the day before surgery; the bone marrow was processed into cell fractions (bone marrow mononuclear cells, CD34+, and CD34-). At LVAD implantation, all fractions and a saline control were injected epicardially into predetermined areas and each injection site marked. At the time of transplantation, injected areas were collected. Data were analyzed by paired Student t test comparing the effect of cell fractions injected within each subject. RESULTS Six subjects completed the study. There were no statistically significant differences in complications with the procedure versus control subjects. Histological analysis indicated that myocardium injected with CD34+ cells had decreased density of endothelial cells compared to saline-injected myocardium. There were no significant differences in fibrosis or inflammation between groups; however, density of activated fibroblasts was decreased in both CD34+ and CD34- injected areas. CONCLUSIONS Tissue analysis does not support the hypothesis that bone marrow-derived CD34+ cells promote increased vascular tissue in humans with ischemic cardiomyopathy via direct injection.

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.

Heart failure with preserved ejection fraction and skeletal muscle physiology

Heart failure with preserved ejection fraction (HFpEF) accounts for half of all heart failure in the USA, increases in prevalence with aging, and has no effective therapies. Intriguingly, the pathophysiology of HFpEF has many commonalities with the aged cardiovascular system including reductions in diastolic compliance, chronotropic defects, increased resistance in the peripheral vasculature, and poor energy substrate utilization. Decreased exercise capacity is a cardinal symptom of HFpEF. However, its severity is often out of proportion to changes in cardiac output. This observation has led to studies of muscle function in HFpEF revealing structural, biomechanical, and metabolic changes. These data, while incomplete, support a hypothesis that similar to aging, HFPEF is a systemic process. Understanding the mechanisms leading to exercise intolerance in this condition may lead to strategies to improve morbidity in both HFpEF and aging.

Allogeneic Precursor Cells for Systolic Heart Failure

Congestive heart failure (CHF) is a major cause of cardiac morbidity and mortality. Loss of cardiomyocytes with replacement fibrosis is a common feature of all end-stage heart disease. Although data suggest that there is at least some regeneration of cardiomyocytes throughout life, it is clear that exogenous drug therapy and endogenous sources of regeneration and repair are insufficient to stop the progression of heart failure. The great need for improved therapies has stimulated the rapid translation of studies of stem and precursor cell therapy into clinical trials for heart failure and myocardial infarction.1,2 In this issue, Perin et al describe a phase II, dose escalation study of a mesenchymal stem cell (MSC) product for advanced heart failure. Article, see p 576 MSCs are mesodermal-derived, multipotent, stromal cells found in several organs, most numerous in bone marrow and adipose tissue. They are defined by the ability to grow well in culture, differentiate into adipocytes, osteoblasts, and chondrocytes, and being positive for several surface markers, including CD90, CD105, MHCI, CD73, and CD271, whereas being negative for CD45 and MHCII.3 Mesenchymal progenitor cells (MPCs) are related, but also positive for surface markers CD31, CD105, Stro-1/3.4 Most MSC-like cells can also be differentiated in vitro into endothelial cells, smooth muscle cells, and fibroblasts.5 MSCs were enthusiastically studied in vitro and showed therapeutic promise to (1) increase endothelial proliferation and vessel formation, (2) promote cardiomyocyte survival, and (3) differentiate into cardiomyocytes. In preclinical animal models, MSC/MPCs can increase endothelial and cardiomyocyte generation and preservation, improved infarct healing, and rescue of heart failure.2,6 Similar in vitro data were generated with other stem cells with mesenchymal origins, including cardiac- and bone marrow–derived (CD34+) stem cells. Spring boarding from these preclinical studies, many small human trials have been …

Allogeneic Precursor Cells for Systolic Heart Failure A Need for Mechanisms in Humans

Congestive heart failure (CHF) is a major cause of cardiac morbidity and mortality. Loss of cardiomyocytes with replacement fibrosis is a common feature of all end-stage heart disease. Although data suggest that there is at least some regeneration of cardiomyocytes throughout life, it is clear that exogenous drug therapy and endogenous sources of regeneration and repair are insufficient to stop the progression of heart failure. The great need for improved therapies has stimulated the rapid translation of studies of stem and precursor cell therapy into clinical trials for heart failure and myocardial infarction.1,2 In this issue, Perin et al describe a phase II, dose escalation study of a mesenchymal stem cell (MSC) product for advanced heart failure. Article, see p 576 MSCs are mesodermal-derived, multipotent, stromal cells found in several organs, most numerous in bone marrow and adipose tissue. They are defined by the ability to grow well in culture, differentiate into adipocytes, osteoblasts, and chondrocytes, and being positive for several surface markers, including CD90, CD105, MHCI, CD73, and CD271, whereas being negative for CD45 and MHCII.3 Mesenchymal progenitor cells (MPCs) are related, but also positive for surface markers CD31, CD105, Stro-1/3.4 Most MSC-like cells can also be differentiated in vitro into endothelial cells, smooth muscle cells, and fibroblasts.5 MSCs were enthusiastically studied in vitro and showed therapeutic promise to (1) increase endothelial proliferation and vessel formation, (2) promote cardiomyocyte survival, and (3) differentiate into cardiomyocytes. In preclinical animal models, MSC/MPCs can increase endothelial and cardiomyocyte generation and preservation, improved infarct healing, and rescue of heart failure.2,6 Similar in vitro data were generated with other stem cells with mesenchymal origins, including cardiac- and bone marrow–derived (CD34+) stem cells. Spring boarding from these preclinical studies, many small human trials have been …

Reply : Could the Interplay Between Macrophages and Fibroblasts Drive Extracellular Matrix Dynamics in End-Stage Heart Failure?

We appreciate Dr. Dias Novaes’ comments on our studies of myocardial inflammation and fibrosis and their relationship to myocardial dysfunction in heart failure [(1)][1]. He correctly points out that we did not use normal ventricular myocardium as a control tissue in our studies. Normal human

What Can We Learn From Children

Despite excellent medical therapy, many patients of all ages with idiopathic cardiomyopathy (ICM) progress to endstage heart failure requiring advanced therapies. Progression occurs due to cardiomyocyte loss, progressive defects in cardiomyocyte function, and extracellular matrix remodeling leading to progressive ventricular dilation. Study of this process has been challenging. Mouse models incompletely replicate human cardiac disease and are rarely on the background of standard medical therapies. Human studies are limited by lack of access to pathologic specimens. Unlike cancer in which tissue is collected at every stage of disease allowing for differentiation of mechanisms at different stages, in heart disease tissue is only collected from either normal or severely damaged hearts limiting mechanistic insights on the remodeling process. In this issue of the Journal, Woulfe et al used cardiac tissue from a biobank of pediatric and adult transplant recipients to elucidate unique properties of pediatric cardiomyopathy that could guide therapeutic strategies. What was striking was that on first analysis of the pediatric samples, very little expansion of the extracellular matrix was seen. Indeed, half of the subjects had minimal fibrosis histologically. This is in contradistinction to adult studies that have reported fibrotic replacement of 18–35% in similar samples. Although the methodology used by Woulfe et al for measuring fibrosis was less precise-most groups use collagen specific stains for quantification-these data are consistent with an MRI study showing minimal fibrosis in a pediatric population with cardiomyopathy. At first glance these results seem simplistic: pediatric hearts have less cardiac matrix at baseline and the duration of disease is less. However, some hearts did develop a similar degree of fibrosis as adults and there was no relationship between the degree of fibrosis over time. Thus, although subjected to similar increases in wall stress, as a whole, pediatric and adult hearts remodel differently. Understanding the mechanisms behind those differences could illuminate insights into basic mechanisms driving pathologic fibrosis in the human heart. Current data support a model in which fibrosis is initiated in ICM by increased wall stress due to cardiomyocyte dysfunction. Animal models of pressure overload have shown that in the presence of increased wall stress, resident fibroblasts are activated to the myofibroblast phenotype which has upregulation of migration, proliferation and the secretion of fibrillar collagen. A positive feedback loop is then established in which collagen deposition stiffens the ventricle, further increasing wall stress with resultant neurohormonal activation. As wall stress and neurohormones increase, macrophages migrate into the perivascular space and elaborate pro-fibrotic factors that further drive myofibroblasts to generate collagen. Given the role of myofibroblasts and macrophages in fibrosis, it is unfortunate that Woulfe et al were unable to quantify their density in pediatric versus adult hearts. However, their data provide insights into their differing roles. First, they demonstrated a discordance in expression of the macrophagederived pro-fibrotic marker galectin-3 in pediatric versus adult tissue. As whole heart tissue was examined, this observation may have been due to different numbers of macrophages within the pediatric versus adult hearts or the presence of macrophages of different phenotypes within the hearts. Intriguingly, Woulfe also noted less perivascular fibrosis in children, confirming a previous observation. As perivascular fibrosis is associated with macrophage activation, these data support the hypothesis that pro-fibrotic macrophages may not play a role in pediatric remodeling. Studies of gene expression in isolated cardiac macrophages in combination with histologic analysis could test this hypothesis. Fibrosis can also be modulated via activity of proteinases that regulate collagen degradation in the heart: matrix metalloproteinases (MMP’s) and their inhibitors, TIMP’s. Although MMP’s and TIMPs are frequently presented as two sides of a proteolytic see-saw, they are complicated molecules with a multitude of functions beyond collagen degradation. MMP’s can both activate and inactivate matrix bound growth factors which may themselves be pro-fibrotic. Likewise, TIMPS can function as both activators and From the University of Washington, Seattle, Washington. Manuscript received February 17, 2017; revised manuscript accepted February 17, 2017. Reprint requests: April Stempien-Otero, MD, PhD, Department of Medicine, University of Washington, 850 Republican Street, Box 35850, Seattle, WA 98109, USA. Tel: 206.616.9054. E-mail: april@u.washington.edu See page 326 for disclosure information. 1071-9164/