Abstract
Aberrant transcriptional regulation contributes to the pathogenesis of both congenital and adult forms of heart disease. While the transcriptional regulator friend of Gata 2 (FOG2) is known to be essential for heart morphogenesis and coronary development, its tissue-specific function has not been previously investigated. Additionally, little is known about the role of FOG2 in the adult heart. Here we used spatiotemporally regulated inactivation of Fog2 to delineate its function in both the embryonic and adult mouse heart. Early cardiomyocyte-restricted loss of Fog2 recapitulated the cardiac and coronary defects of the Fog2 germline murine knockouts. Later cardiomyocyte-restricted loss of Fog2 (Fog2MC) did not result in defects in cardiac structure or coronary vessel formation. However, Fog2MC adult mice had severely depressed ventricular function and died at 8–14 weeks. Fog2MC adult hearts displayed a paucity of coronary vessels, associated with myocardial hypoxia, increased cardiomyocyte apoptosis, and cardiac fibrosis. Induced inactivation of Fog2 in the adult mouse heart resulted in similar phenotypes, as did ablation of the FOG2 interaction with the transcription factor GATA4. Loss of the FOG2 or FOG2-GATA4 interaction altered the expression of a panel of angiogenesis-related genes. Collectively, our data indicate that FOG2 regulates adult heart function and coronary angiogenesis.
Authors
Bin Zhou, Qing Ma, Sek Won Kong, Yongwu Hu, Patrick H. Campbell, Francis X. McGowan, Kate G. Ackerman, Bingruo Wu, Bin Zhou, Sergei G. Tevosian, William T. Pu
Citation Information: J Clin Invest. 2009. https://doi.org/10.1172/JCI36433.
Abstract
Leukocyte and platelet accumulation at sites of cerebral ischemia exacerbate cerebral damage. The ectoenzyme CD39 on the plasmalemma of endothelial cells metabolizes ADP to suppress platelet accumulation in the ischemic brain. However, the role of leukocyte surface CD39 in regulating monocyte and neutrophil trafficking in this setting is not known. Here we have demonstrated in mice what we believe to be a novel mechanism by which CD39 on monocytes and neutrophils regulates their own sequestration into ischemic cerebral tissue, by catabolizing nucleotides released by injured cells, thereby inhibiting their chemotaxis, adhesion, and transmigration. Bone marrow reconstitution and provision of an apyrase, an enzyme that hydrolyzes nucleoside tri- and diphosphates, each normalized ischemic leukosequestration and cerebral infarction in CD39-deficient mice. Leukocytes purified from Cd39–/– mice had a markedly diminished capacity to phosphohydrolyze adenine nucleotides and regulate platelet reactivity, suggesting that leukocyte ectoapyrases modulate the ambient vascular nucleotide milieu. Dissipation of ATP by CD39 reduced P2X7 receptor stimulation and thereby suppressed baseline leukocyte αMβ2-integrin expression. As αMβ2-integrin blockade reversed the postischemic, inflammatory phenotype of Cd39–/– mice, these data suggest that phosphohydrolytic activity on the leukocyte surface suppresses cell-cell interactions that would otherwise promote thrombosis or inflammation. These studies indicate that CD39 on both endothelial cells and leukocytes reduces inflammatory cell trafficking and platelet reactivity, with a consequent reduction in tissue injury following cerebral ischemic challenge.
Authors
Matthew C. Hyman, Danica Petrovic-Djergovic, Scott H. Visovatti, Hui Liao, Sunitha Yanamadala, Diane Bouïs, Enming J. Su, Daniel A. Lawrence, M. Johan Broekman, Aaron J. Marcus, David J. Pinsky
Citation Information: J Clin Invest. 2009. https://doi.org/10.1172/JCI35548.
Abstract
The uptake of lipoproteins by macrophages is a critical step in the development of atherosclerotic lesions. Cultured monocyte-derived macrophages take up large amounts of native LDL by receptor-independent fluid-phase pinocytosis, either constitutively or in response to specific activating stimuli, depending on the macrophage phenotype. We therefore sought to determine whether fluid-phase pinocytosis occurs in vivo in macrophages in atherosclerotic lesions. We demonstrated that fluorescent pegylated nanoparticles similar in size to LDL (specifically nontargeted Qtracker quantum dot and AngioSPARK nanoparticles) can serve as models of LDL uptake by fluid-phase pinocytosis in cultured human monocyte–derived macrophages and mouse bone marrow–derived macrophages. Using fluorescence microscopy, we showed that atherosclerosis-prone Apoe-knockout mice injected with these nanoparticles displayed massive accumulation of the nanoparticles within CD68+ macrophages, including lipid-containing foam cells, in atherosclerotic lesions in the aortic arch. Similar results were obtained when atherosclerotic mouse aortas were cultured with nanoparticles in vitro. These results show that macrophages within atherosclerotic lesions can take up LDL-sized nanoparticles by fluid-phase pinocytosis and indicate that fluid-phase pinocytosis of LDL is a mechanism for macrophage foam cell formation in vivo.
Authors
Chiara Buono, Joshua J. Anzinger, Marcelo Amar, Howard S. Kruth
Citation Information: J Clin Invest. 2009. https://doi.org/10.1172/JCI36800.
Abstract
Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of oxidized lipoproteins and apoptotic cells. Adaptive immune responses to various oxidation-specific epitopes play an important role in atherogenesis. However, accumulating evidence suggests that these epitopes are also recognized by innate receptors, such as scavenger receptors on macrophages, and plasma proteins, such as C-reactive protein (CRP). Here, we provide multiple lines of evidence that oxidation-specific epitopes constitute a dominant, previously unrecognized target of natural Abs (NAbs) in both mice and humans. Using reconstituted mice expressing solely IgM NAbs, we have shown that approximately 30% of all NAbs bound to model oxidation-specific epitopes, as well as to atherosclerotic lesions and apoptotic cells. Because oxidative processes are ubiquitous, we hypothesized that these epitopes exert selective pressure to expand NAbs, which in turn play an important role in mediating homeostatic functions consequent to inflammation and cell death, as demonstrated by their ability to facilitate apoptotic cell clearance. These findings provide novel insights into the functions of NAbs in mediating host homeostasis and into their roles in health and diseases, such as chronic inflammatory diseases and atherosclerosis.
Authors
Meng-Yun Chou, Linda Fogelstrand, Karsten Hartvigsen, Lotte F. Hansen, Douglas Woelkers, Peter X. Shaw, Jeomil Choi, Thomas Perkmann, Fredrik Bäckhed, Yury I. Miller, Sohvi Hörkkö, Maripat Corr, Joseph L. Witztum, Christoph J. Binder
Citation Information: J Clin Invest. 2009. https://doi.org/10.1172/JCI36806.
Abstract
Nicotinic acid is one of the most effective agents for both lowering triglycerides and raising HDL. However, the side effect of cutaneous flushing severely limits patient compliance. As nicotinic acid stimulates the GPCR GPR109A and Gi/Go proteins, here we dissected the roles of G proteins and the adaptor proteins, β-arrestins, in nicotinic acid–induced signaling and physiological responses. In a human cell line–based signaling assay, nicotinic acid stimulation led to pertussis toxin–sensitive lowering of cAMP, recruitment of β-arrestins to the cell membrane, an activating conformational change in β-arrestin, and β-arrestin–dependent signaling to ERK MAPK. In addition, we found that nicotinic acid promoted the binding of β-arrestin1 to activated cytosolic phospholipase A2 as well as β-arrestin1–dependent activation of cytosolic phospholipase A2 and release of arachidonate, the precursor of prostaglandin D2 and the vasodilator responsible for the flushing response. Moreover, β-arrestin1–null mice displayed reduced cutaneous flushing in response to nicotinic acid, although the improvement in serum free fatty acid levels was similar to that observed in wild-type mice. These data suggest that the adverse side effect of cutaneous flushing is mediated by β-arrestin1, but lowering of serum free fatty acid levels is not. Furthermore, G protein–biased ligands that activate GPR109A in a β-arrestin–independent fashion may represent an improved therapeutic option for the treatment of dyslipidemia.
Authors
Robert W. Walters, Arun K. Shukla, Jeffrey J. Kovacs, Jonathan D. Violin, Scott M. DeWire, Christopher M. Lam, J. Ruthie Chen, Michael J. Muehlbauer, Erin J. Whalen, Robert J. Lefkowitz
Abstract
How Ca2+-dependent signaling effectors are regulated in cardiomyocytes, given the extreme cytoplasmic Ca2+ concentration changes that underlie contraction, remains unknown. Cardiomyocyte plasma membrane Ca2+-ATPase (PMCA) extrudes Ca2+ but has little effect on excitation-contraction coupling, suggesting its potential role in controlling Ca2+-dependent signaling effectors such as calcineurin. We generated cardiac-specific inducible PMCA4b transgenic mice that displayed normal global Ca2+ transient and cellular contraction levels and reduced cardiac hypertrophy following transverse aortic constriction (TAC) or phenylephrine/Ang II infusion, but showed no reduction in exercise-induced hypertrophy. Transgenic mice were protected from decompensation and fibrosis following long-term TAC. The PMCA4b transgene reduced the hypertrophic augmentation associated with transient receptor potential canonical 3 channel overexpression, but not that associated with activated calcineurin. Furthermore, Pmca4 gene–targeted mice showed increased cardiac hypertrophy and heart failure events after TAC. Physical associations between PMCA4b and calcineurin were enhanced by TAC and by agonist stimulation of cultured neonatal cardiomyocytes. PMCA4b reduced calcineurin nuclear factor of activated T cell–luciferase activity after TAC and in cultured neonatal cardiomyocytes after agonist stimulation. PMCA4b overexpression inhibited cultured cardiomyocyte hypertrophy following agonist stimulation, but much less so in a Ca2+ pumping–deficient PMCA4b mutant. Thus, Pmca4b likely reduces the local Ca2+ signals involved in reactive cardiomyocyte hypertrophy via calcineurin regulation.
Authors
Xu Wu, Baojun Chang, N. Scott Blair, Michelle Sargent, Allen J. York, Jeffrey Robbins, Gary E. Shull, Jeffery D. Molkentin
Abstract
ER stress occurs in macrophage-rich areas of advanced atherosclerotic lesions and contributes to macrophage apoptosis and subsequent plaque necrosis. Therefore, signaling pathways that alter ER stress–induced apoptosis may affect advanced atherosclerosis. Here we placed Apoe–/– mice deficient in macrophage p38α MAPK on a Western diet and found that they had a marked increase in macrophage apoptosis and plaque necrosis. The macrophage p38α–deficient lesions also exhibited a significant reduction in collagen content and a marked thinning of the fibrous cap, which suggests that plaque progression was advanced in these mice. Consistent with our in vivo data, we found that ER stress–induced apoptosis in cultured primary mouse macrophages was markedly accelerated under conditions of p38 inhibition. Pharmacological inhibition or genetic ablation of p38 suppressed activation of Akt in cultured macrophages and in atherosclerotic lesions. In addition, inhibition of Akt enhanced ER stress–induced macrophage apoptosis, and expression of a constitutively active myristoylated Akt blocked the enhancement of ER stress–induced apoptosis that occurred with p38 inhibition in cultured cells. Our results demonstrate that p38α MAPK may play a critical role in suppressing ER stress–induced macrophage apoptosis in vitro and advanced lesional macrophage apoptosis in vivo.
Authors
Tracie A. Seimon, Yibin Wang, Seongah Han, Takafumi Senokuchi, Dorien M. Schrijvers, George Kuriakose, Alan R. Tall, Ira A. Tabas
Citation Information: J Clin Invest. 2009. https://doi.org/10.1172/JCI35814.
Abstract
Myocardial Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibition improves cardiac function following myocardial infarction (MI), but the CaMKII-dependent pathways that participate in myocardial stress responses are incompletely understood. To address this issue, we sought to determine the transcriptional consequences of myocardial CaMKII inhibition after MI. We performed gene expression profiling in mouse hearts with cardiomyocyte-delimited transgenic expression of either a CaMKII inhibitory peptide (AC3-I) or a scrambled control peptide (AC3-C) following MI. Of the 8,600 mRNAs examined, 156 were substantially modulated by MI, and nearly half of these showed markedly altered responses to MI with CaMKII inhibition. CaMKII inhibition substantially reduced the MI-triggered upregulation of a constellation of proinflammatory genes. We studied 1 of these proinflammatory genes, complement factor B (Cfb), in detail, because complement proteins secreted by cells other than cardiomyocytes can induce sarcolemmal injury during MI. CFB protein expression in cardiomyocytes was triggered by CaMKII activation of the NF-κB pathway during both MI and exposure to bacterial endotoxin. CaMKII inhibition suppressed NF-κB activity in vitro and in vivo and reduced Cfb expression and sarcolemmal injury. The Cfb–/– mice were partially protected from the adverse consequences of MI. Our findings demonstrate what we believe is a novel target for CaMKII in myocardial injury and suggest that CaMKII is broadly important for the genetic effects of MI in cardiomyocytes.
Authors
Madhu V. Singh, Ann Kapoun, Linda Higgins, William Kutschke, Joshua M. Thurman, Rong Zhang, Minati Singh, Jinying Yang, Xiaoqun Guan, John S. Lowe, Robert M. Weiss, Kathy Zimmermann, Fiona E. Yull, Timothy S. Blackwell, Peter J. Mohler, Mark E. Anderson
Abstract
Type 2 diabetes is associated with accelerated atherogenesis, which may result from a combination of factors, including dyslipidemia characterized by increased VLDL secretion, and insulin resistance. To assess the hypothesis that both hepatic and peripheral insulin resistance contribute to atherogenesis, we crossed mice deficient for the LDL receptor (Ldlr–/– mice) with mice that express low levels of IR in the liver and lack IR in peripheral tissues (the L1B6 mouse strain). Unexpectedly, compared with Ldlr–/– controls, L1B6Ldlr–/– mice fed a Western diet showed reduced VLDL and LDL levels, reduced atherosclerosis, decreased hepatic AKT signaling, decreased expression of genes associated with lipogenesis, and diminished VLDL apoB and lipid secretion. Adenovirus-mediated hepatic expression of either constitutively active AKT or dominant negative glycogen synthase kinase (GSK) markedly increased VLDL and LDL levels such that they were similar in both Ldlr–/– and L1B6Ldlr–/– mice. Knocking down expression of hepatic IR by adenovirus-mediated shRNA decreased VLDL triglyceride and apoB secretion in Ldlr–/– mice. Furthermore, knocking down hepatic IR expression in either WT or ob/ob mice reduced VLDL secretion but also resulted in decreased hepatic Ldlr protein. These findings suggest a dual action of hepatic IR on lipoprotein levels, in which the ability to increase VLDL apoB and lipid secretion via AKT/GSK is offset by upregulation of Ldlr.
Authors
Seongah Han, Chien-Ping Liang, Marit Westerterp, Takafumi Senokuchi, Carrie L. Welch, Qizhi Wang, Michihiro Matsumoto, Domenico Accili, Alan R. Tall
Citation Information: J Clin Invest. 2009. https://doi.org/10.1172/JCI35620.
Abstract
The heart initially compensates for hypertension-mediated pressure overload by enhancing its contractile force and developing hypertrophy without dilation. Gq protein–coupled receptor pathways become activated and can depress function, leading to cardiac failure. Initial adaptation mechanisms to reduce cardiac damage during such stimulation remain largely unknown. Here we have shown that this initial adaptation requires regulator of G protein signaling 2 (RGS2). Mice lacking RGS2 had a normal basal cardiac phenotype, yet responded rapidly to pressure overload, with increased myocardial Gq signaling, marked cardiac hypertrophy and failure, and early mortality. Swimming exercise, which is not accompanied by Gq activation, induced a normal cardiac response, while Rgs2 deletion in Gαq-overexpressing hearts exacerbated hypertrophy and dilation. In vascular smooth muscle, RGS2 is activated by cGMP-dependent protein kinase (PKG), suppressing Gq-stimulated vascular contraction. In normal mice, but not Rgs2–/– mice, PKG activation by the chronic inhibition of cGMP-selective phosphodiesterase 5 (PDE5) suppressed maladaptive cardiac hypertrophy, inhibiting Gq-coupled stimuli. Importantly, PKG was similarly activated by PDE5 inhibition in myocardium from both genotypes, but PKG plasma membrane translocation was more transient in Rgs2–/– myocytes than in controls and was unaffected by PDE5 inhibition. Thus, RGS2 is required for early myocardial compensation to pressure overload and mediates the initial antihypertrophic and cardioprotective effects of PDE5 inhibitors.
Authors
Eiki Takimoto, Norimichi Koitabashi, Steven Hsu, Elizabeth A. Ketner, Manling Zhang, Takahiro Nagayama, Djahida Bedja, Kathleen L. Gabrielson, Robert Blanton, David P. Siderovski, Michael E. Mendelsohn, David A. Kass
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