Circulation Research current issue

[Editorials] New Insights Into the Open Artery Hypothesis

Kloner, R. A., Hwang, H. — 2008-07-02

[UltraRapid Communications] Nrf2 Regulates Antioxidant Gene Expression Evoked by Oxidized Phospholipids in Endothelial Cells and Murine Arteries In Vivo

2008-07-02

Besides their well-characterized proinflammatory and proatherogenic effects, oxidized phospholipids, such as oxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-phosphocholine) have been shown to have beneficial responses in vascular cells via induction of antioxidant enzymes such as heme oxygenase-1. We therefore hypothesized that oxPAPC could evoke a general cytoprotective response via activation of antioxidative transcription factor Nrf2. Here, we show that oxPAPC increases nuclear accumulation of Nrf2. Using the small interfering RNA approach, we demonstrate that Nrf2 is critical in mediating the induction of glutamate-cysteine ligase modifier subunit (GCLM) and NAD(P)H quinone oxidoreductase-1 (NQO1) by oxPAPC in human endothelial cells, whereas the contribution to the induction of heme oxygenase-1 was less significant. The induction of GCLM and NQO1 was attenuated by reduction of electrophilic groups with sodium borohydrate, as well as treatment with thiol antioxidant N-acetylcysteine, suggesting that the thiol reactivity of oxPAPC is largely mediating its effect on Nrf2-responsive genes. Moreover, we show that oxidized phospholipid having a highly electrophilic isoprostane ring in its sn-2 position is a potent inducer of Nrf2 target genes. Finally, we demonstrate that the oxPAPC-inducible expression of heme oxygenase-1, GCLM, and NQO1 is lower in Nrf2-null than wild-type mouse carotid arteries in vivo. We suggest that the activation of Nrf2 by oxidized phospholipids provides a mechanism by which their deleterious effects are limited in the vasculature.

[Editorials] A Nucleolar Weapon in Our Fight for Regenerating Adult Hearts: Nucleostemin and Cardiac Stem Cells

Tjwa, M., Dimmeler, S. — 2008-07-02

[Editorials] Endothelial Progenitor Cells and Angiogenesis Join the PPARty

Biscetti, F., Pola, R. — 2008-07-02

[Editorials] eNOS-ER{alpha} Complex Goes to Telomerase

Zeng, L., Xu, Q. — 2008-07-02

[Letters to the Editor] Ca2+-Calmodulin-Dependent Protein Kinase Phosphorylation of Ryanodine Receptor May Contribute to the {beta}-Adrenergic Regulation of Myocardial Contractility Independently of Increases in Heart Rate

Mattiazzi, A., Vittone, L., Mundina-Weilenmann, C. — 2008-07-02

[Letters to the Editor] Response to Mattiazzi et al:

Valdivia, H. H., Houser, S. R. — 2008-07-02

[Editorials] Deck of CArGs

Miano, J. M. — 2008-07-02

[Corrections] Correction

2008-07-02

[Reviews] Emerin and the Nuclear Lamina in Muscle and Cardiac Disease

Holaska, J. M. — 2008-07-02

The human genome is contained within the nucleus and is separated from the cytoplasm by the nuclear envelope. Mutations in the nuclear envelope proteins emerin and lamin A cause a number of diseases including premature aging syndromes, muscular dystrophy, and cardiomyopathy. Emerin and lamin A are implicated in regulating muscle- and heart-specific gene expression and nuclear architecture. For example, lamin A regulates the expression and localization of gap junction and intercalated disc components. Additionally, emerin and lamin A are also required to maintain nuclear envelope integrity. Demonstrating the importance of maintaining nuclear integrity in heart disease, atrioventricular node cells lacking lamin A exhibit increased nuclear deformation and apoptosis. This review highlights the present understanding of lamin A and emerin function in regulating nuclear architecture, gene expression, and cell signaling and discusses putative mechanisms for how specific mutations in lamin A and emerin cause cardiac- or muscle-specific disease.

[Molecular Medicine] Endothelial Nitric Oxide Synthase Gene Expression During Murine Embryogenesis: Commencement of Expression in the Embryo Occurs With the Establishment of a Unidirectional Circulatory System

2008-07-02

To elucidate the role of endothelial NO synthase (eNOS)-derived NO during mammalian embryogenesis, we assessed the expression of the eNOS gene during development. Using transgenic eNOS promoter/reporter mice (with β-galactosidase and green fluorescent protein reporters), in situ cRNA hybridization, and immunohistochemistry to assess transcription, steady-state mRNA levels, and protein expression, respectively, we noted that eNOS expression in the developing cardiovascular system was highly restricted to endothelial cells of medium- and large-sized arteries and the endocardium. The onset of transcription of the native eNOS gene and reporters coincided with the establishment of robust, unidirectional blood flow at embryonic day 9.5, as assessed by Doppler ultrasound biomicroscopy. Interestingly, reporter transgene expression and native eNOS mRNA were also observed in discrete regions of the developing skeletal musculature and the apical ectodermal ridge of developing limbs, suggesting a role for eNOS-derived NO in limb development. In vitro studies of promoter/reporter constructs indicated that similar eNOS promoter regions operate in both embryonic skeletal muscle and vascular endothelial cells. In summary, transcriptional activity of the eNOS gene in the murine circulatory system occurred following the establishment of embryonic blood flow. Thus, the eNOS gene is a late-onset gene in endothelial ontogeny.

[Molecular Medicine] Estrogen Receptor-{alpha} and Endothelial Nitric Oxide Synthase Nuclear Complex Regulates Transcription of Human Telomerase

2008-07-02

We report that in endothelial cells, the angiogenic effect of 17β-estradiol (E2) is inhibited by the estrogen receptor (ER) antagonist ICI or the NO synthase (NOS) inhibitor 7-nitroindazole via downregulation of hTERT, the telomerase catalytic subunit, suggesting that E2 and NO are involved in controlling hTERT transcription. Quantitative Real-Time PCR and chromatin immunoprecipitations in E2-treated human umbilical vein endothelial cells, showed recruitment of ERs on the hTERT promoter and concomitant enrichment in histone 3 methylation at Lysine 79, a modification associated with transcription-competent chromatin. Confocal microscopy and re-chromatin immunoprecipitations revealed that on E2 induction, endothelial (e)NOS rapidly localized into the nucleus and associated with ER on the hTERT promoter. Transfections of a constitutively active eNOS mutant (S1177D) strongly induced the hTERT promoter, indicating a direct role of the protein in hTERT transcriptional regulation. Mutation of the estrogen response element in the promoter abolished response to both ERs and active eNOS, demonstrating that the estrogen response element integrity is required for hTERT regulation by these factors. To investigate this novel regulation in a reduced NO environment, pulmonary endothelial cells were isolated from eNOS^–/– mice and grown with/without E2. In wild-type cells, E2 significantly increased telomerase activity. In eNOS^–/– cells, basal telomerase activity was rescued by exogenous eNOS or an NO donor, whereas responsiveness to E2 demanded the active protein. In conclusion, we document the novel findings of a combinatorial eNOS/ER complex at the hTERT estrogen response element site and that active eNOS and ligand-activated ERs cooperate in regulating hTERT expression in the endothelium.

[Molecular Medicine] Heparan Sulfate in Perlecan Promotes Mouse Atherosclerosis: Roles in Lipid Permeability, Lipid Retention, and Smooth Muscle Cell Proliferation

2008-07-02

Heparan sulfate (HS) has been proposed to be antiatherogenic through inhibition of lipoprotein retention, inflammation, and smooth muscle cell proliferation. Perlecan is the predominant HS proteoglycan in the artery wall. Here, we investigated the role of perlecan HS chains using apoE null (ApoE0) mice that were cross-bred with mice expressing HS-deficient perlecan (Hspg2^3/3). Morphometry of cross-sections from aortic roots and en face preparations of whole aortas revealed a significant decrease in lesion formation in ApoE0/Hspg2^3/3 mice at both 15 and 33 weeks. In vitro, binding of labeled mouse triglyceride-rich lipoproteins and human LDL to total extracellular matrix, as well as to purified proteoglycans, prepared from ApoE0/Hspg2^3/3 smooth muscle cells was reduced. In vivo, at 20 minutes influx of human ¹²⁵I-LDL or mouse triglyceride-rich lipoproteins into the aortic wall was increased in ApoE0/Hspg2^3/3 mice compared to ApoE0 mice. However, at 72 hours accumulation of ¹²⁵I-LDL was similar in ApoE0/Hspg2^3/3 and ApoE0 mice. Immunohistochemistry of lesions from ApoE0/Hspg2^3/3 mice showed decreased staining for apoB and increased smooth muscle -actin content, whereas accumulation of CD68-positive inflammatory cells was unchanged. We conclude that the perlecan HS chains are proatherogenic in mice, possibly through increased lipoprotein retention, altered vascular permeability, or other mechanisms. The ability of HS to inhibit smooth muscle cell growth may also influence development as well as instability of lesions.

[Molecular Medicine] Proteolytic Processing of cGMP-Dependent Protein Kinase I Mediates Nuclear cGMP Signaling in Vascular Smooth Muscle Cells

Sugiura, T., Nakanishi, H., Roberts, J. D. — 2008-07-02

Cyclic GMP modulates gene expression in vascular smooth muscle cells (SMCs) in part by stimulating cGMP-dependent protein kinase I (PKGI) and the phosphorylation of transcription factors. In some cells, cGMP increases nuclear translocation of PKGI and PKGI-dependent phosphorylation of transcription regulators; however, these observations have been variable, and the mechanisms mediating nuclear PKGI translocation are incompletely understood. We tested the hypothesis that proteolytic cleavage of PKGI is required for cGMP-stimulated nuclear compartmentation of PKGI and phosphorylation of transcription factors. We detected an NH₂-terminal PKGI fragment with leucine zipper domain immunoreactivity in the cytosol and endoplasmic reticulum of SMCs, but only a COOH-terminal PKGI fragment containing the catalytic region (now termed PKGI) was observed in the Golgi apparatus (GA) and nucleoplasm. Posttranslational PKGI processing in the GA was critical for nuclear compartmentation of PKGI because GA disruption with nocodazol or brefeldin A inhibited PKGI nuclear localization. PKGI immunoreactivity was particularly abundant in the nucleolus of interphase SMCs where its colocalization with the nucleolar dense fibrillar component protein fibrillarin closely matched the level of nucleolar assembly. Purified nucleolar PKGI enzyme activity was insensitive to cGMP stimulation, which is consistent with its lack of the NH₂-terminal autoinhibitory domain. Mutation of a putative proteolytic cleavage region in PKGI inhibited cGMP-mediated phosphorylation of cAMP response element-binding protein, cAMP response element-dependent transcription, and nuclear localization of PKGI. These observations suggest that posttranslational modification of PKGI critically influences the nuclear translocation of PKGI and activities of cGMP in SMCs.

[Molecular Medicine] Smooth Muscle Expression of Lipoma Preferred Partner Is Mediated by an Alternative Intronic Promoter That Is Regulated by Serum Response Factor/Myocardin

2008-07-02

Lipoma preferred partner (LPP) was recently recognized as a smooth muscle marker that plays a role in smooth muscle cell migration. In this report, we focus on the transcriptional regulation of the LPP gene. In particular, we investigate whether LPP is directly regulated by serum response factor (SRF). We show that the LPP gene contains 3 evolutionarily conserved CArG boxes and that 1 of these is part of an alternative promoter in intron 2. Quantitative RT-PCR shows that this alternative promoter directs transcription specifically to smooth muscle containing tissues in vivo. By using chromatin immunoprecipitation, we demonstrate that 2 of the CArG boxes, including the promoter-associated CArG box, bind to endogenous SRF in cultured aortic smooth muscle cells. Electrophoretic mobility-shift assays show that the conserved CArG boxes bind SRF in vitro. In reporter experiments, we show that the alternative promoter has transcriptional capacity that is dependent on SRF/myocardin and that the promoter associated CArG box is required for that activity. Finally, we show by quantitative RT-PCR that the alternative promoter is strongly downregulated in SRF-deficient embryonic stem cells and in smooth muscle tissues derived from conditional SRF knockout mice. Collectively, our data demonstrate that expression of LPP in smooth muscle is mediated by an alternative promoter that is regulated by SRF/myocardin.

[Cellular Biology] {beta}-Arrestins Regulate Atherosclerosis and Neointimal Hyperplasia by Controlling Smooth Muscle Cell Proliferation and Migration

2008-07-02

Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins β-arrestin1 and -2 might regulate this pathological process. Deficiency of β-arrestin2 in ldlr^–/– mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that β-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, β-arrestin1^–/–, and β-arrestin2^–/– mice. Neointimal hyperplasia was enhanced in β-arrestin1^–/– mice, and diminished in β-arrestin2^–/– mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in β-arrestin2^–/– mice was not altered by transplantation with either wild-type or β-arrestin2^–/– bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in β-arrestin1^–/– and decreased in β-arrestin2^–/– mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in β-arrestin1^–/– SMCs and less in β-arrestin2^–/– SMCs. Proliferation was less than wild type in β-arrestin2^–/– SMCs but not in β-arrestin2^–/– endothelial cells. We conclude that β-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by β-arrestin2 and β-arrestin1. These findings identify inhibition of β-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.

[Cellular Biology] Angiogenic Function of Prostacyclin Biosynthesis in Human Endothelial Progenitor Cells

He, T., Lu, T., d'Uscio, L. V., Lam, C.-F., Lee, H.-C., Katusic, Z. S. — 2008-07-02

The role of prostaglandin production in the control of regenerative function of endothelial progenitor cells (EPCs) has not been studied. We hypothesized that activation of cyclooxygenase (COX) enzymatic activity and the subsequent production of prostacyclin (PGI₂) is an important mechanism responsible for the regenerative function of EPCs. In the present study, we detected high levels of COX-1 protein expression and PGI₂ biosynthesis in human EPCs outgrown from blood mononuclear cells. Expression of COX-2 protein was almost undetectable under basal conditions but significantly elevated after treatment with tumor necrosis factor-. Condition medium derived from EPCs hyperpolarized human coronary artery smooth muscle cells, similar to the effect of the PGI₂ analog iloprost. The proliferation and in vitro tube formation by EPCs were inhibited by the COX inhibitor indomethacin or by genetic inactivation of COX-1 or PGI₂ synthase with small interfering (si)RNA. Impaired tube formation and cell proliferation induced by inactivation of COX-1 were rescued by the treatment with iloprost or the selective peroxisome proliferator–activated receptor (PPAR) agonist GW501516 but not by the selective PGI₂ receptor agonist cicaprost. Downregulation of PPAR by siRNA also reduced angiogenic capacity of EPCs. Iloprost failed to reverse PPAR siRNA-induced impairment of angiogenesis. Furthermore, transfection of PGI₂ synthase siRNA, COX-1 siRNA, or PPAR siRNA into EPCs decreased the capillary formation in vivo after transplantation of human EPCs into the nude mice. These results suggest that activation of COX-1/PGI₂/PPAR pathway is an important mechanism underlying proangiogenic function of EPCs.

[Integrative Physiology] Myocardial Induction of Nucleostemin in Response to Postnatal Growth and Pathological Challenge

2008-07-02

Stem cell–specific proteins and regulatory pathways that determine self-renewal and differentiation have become of fundamental importance in understanding regenerative and reparative processes in the myocardium. One such regulatory protein, named nucleostemin, has been studied in the context of stem cells and several cancer cell lines, where expression is associated with proliferation and maintenance of a primitive cellular phenotype. We find nucleostemin is present in young myocardium and is also induced following cardiomyopathic injury. Nucleostemin expression in cardiomyocytes is induced by fibroblast growth factor-2 and accumulates in response to Pim-1 kinase activity. Cardiac stem cells also express nucleostemin that is diminished in response to commitment to a differentiated phenotype. Overexpression of nucleostemin in cultured cardiac stem cells increases proliferation while preserving telomere length, providing a mechanistic basis for potential actions of nucleostemin in promotion of cell survival and proliferation as seen in other cell types.

[Integrative Physiology] Mechanisms by Which Late Coronary Reperfusion Mitigates Postinfarction Cardiac Remodeling

2008-07-02

Although recanalization of the infarct-related artery late after myocardial infarction (MI) is known to reduce both cardiac remodeling and mortality, the mechanisms responsible are not yet fully understood. We compared infarcted rat hearts in which the infarct-related coronary artery was opened 24 hours after infarction (late reperfusion [LR] group) with those having a permanently occluded artery. Left ventricular dilatation and dysfunction were significantly mitigated in the LR group 1, 2, and 4 weeks post-MI. Attributable, in large part, to the greater number of cells present, the infarcted wall was significantly thicker in the LR group, which likely reduced wall stress and mitigated cardiac dysfunction. Granulation tissue cell proliferation was increased to a greater degree in the LR group 4 days post-MI, whereas the incidence of apoptosis was significantly lower throughout the subacute stage (4 days, 1 week, and 2 weeks post-MI), further suggesting preservation of granulation tissue cells contributes to the thick, cell-rich scar. Functionally, myocardial debris was more rapidly removed from the infarcted areas in the LR group during subacute stages, and stouter collagen was more rapidly synthesized in those areas. Direct acceleration of Fas-mediated apoptosis by hypoxia was confirmed in vitro using infarct tissue-derived myofibroblasts. In salvaged cardiomyocytes, degenerative changes, but not apoptosis, were mitigated in the LR group, accompanied by restoration of GATA-4 and sarcomeric protein expression. Along with various mechanisms proposed earlier, the present findings appear to provide an additional pathophysiological basis for the benefits of late reperfusion.

[Integrative Physiology] Local Activation or Implantation of Cardiac Progenitor Cells Rescues Scarred Infarcted Myocardium Improving Cardiac Function

2008-07-02

Ischemic heart disease is characterized chronically by a healed infarct, foci of myocardial scarring, cavitary dilation, and impaired ventricular performance. These alterations can only be reversed by replacement of scarred tissue with functionally competent myocardium. We tested whether cardiac progenitor cells (CPCs) implanted in proximity of healed infarcts or resident CPCs stimulated locally by hepatocyte growth factor and insulin-like growth factor-1 invade the scarred myocardium and generate myocytes and coronary vessels improving the hemodynamics of the infarcted heart. Hepatocyte growth factor is a powerful chemoattractant of CPCs, and insulin-like growth factor-1 promotes their proliferation and survival. Injection of CPCs or growth factors led to the replacement of 42% of the scar with newly formed myocardium, attenuated ventricular dilation and prevented the chronic decline in function of the infarcted heart. Cardiac repair was mediated by the ability of CPCs to synthesize matrix metalloproteinases that degraded collagen proteins, forming tunnels within the fibrotic tissue during their migration across the scarred myocardium. New myocytes had a 2n karyotype and possessed 2 sex chromosomes, excluding cell fusion. Clinically, CPCs represent an ideal candidate cell for cardiac repair in patients with chronic heart failure. CPCs may be isolated from myocardial biopsies and, following their expansion in vitro, administered back to the same patients avoiding the adverse effects associated with the use of nonautologous cells. Alternatively, growth factors may be delivered locally to stimulate resident CPCs and promote myocardial regeneration. These forms of treatments could be repeated over time to reduce progressively tissue scarring and expand the working myocardium.