Circulation Research recent issues

[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.

[UltraRapid Communications] Intercellular Adhesion Molecule-1-Dependent Neutrophil Adhesion to Endothelial Cells Induces Caveolae-Mediated Pulmonary Vascular Hyperpermeability

Hu, G., Vogel, S. M., Schwartz, D. E., Malik, A. B., Minshall, R. D. — 2008-06-19

We investigated the role of caveolae in the mechanism of increased pulmonary vascular permeability and edema formation induced by the activation of polymorphonuclear neutrophils (PMNs). We observed that the increase in lung vascular permeability induced by the activation of PMNs required caveolin-1, the caveolae scaffold protein. The permeability increase induced by PMN activation was blocked in caveolin-1 knockout mice and by suppressing caveolin-1 expression in rats. The response was also dependent on Src phosphorylation of caveolin-1 known to activate caveolae-mediated endocytosis in endothelial cells. To address the role of PMN interaction with endothelial cells, we used an intercellular adhesion molecule (ICAM)-1 blocking monoclonal antibody. Preventing the ICAM-1–mediated PMN binding to endothelial cells abrogated Src phosphorylation of caveolin-1, as well as the increase in endothelial permeability. Direct ICAM-1 activation by crosslinking recapitulated these responses, suggesting that ICAM-1 activates caveolin-1 signaling responsible for caveolae-mediated endothelial hyperpermeability. Our results provide support for the novel concept that a large component of pulmonary vascular hyperpermeability induced by activation of PMNs adherent to the vessel wall is dependent on signaling via caveolin-1 and increased caveolae-mediated transcytosis. Thus, it is important to consider the role of the transendothelial vesicular permeability pathway that contributes to edema formation in developing therapeutic interventions against PMN-mediated inflammatory diseases such as acute lung injury.

[Editorials] As Macrophages Indulge, Atherosclerotic Lesions Bulge

Daugherty, A., Rateri, D. L., Lu, H. — 2008-06-19

[Editorials] A Novel Mechanism of Vascular Smooth Muscle Cell Regulation by Notch: Platelet-Derived Growth Factor Receptor-{beta} Expression?

Weber, D. S. — 2008-06-19

[Editorials] Nitric Oxide-Mediated Zinc Release: A New (Modulatory) Pathway in Hypoxic Pulmonary Vasoconstriction

Weissmann, N. — 2008-06-19

[Editorials] Kruppel-Like Factor 4: Transcriptional Regulator of Proliferation, or Inflammation, or Differentiation, or All Three?

Autieri, M. V. — 2008-06-19

[Reviews] Gene Therapy in Heart Failure

Vinge, L. E., Raake, P. W., Koch, W. J. — 2008-06-19

With increasing knowledge of basic molecular mechanisms governing the development of heart failure (HF), the possibility of specifically targeting key pathological players is evolving. Technology allowing for efficient in vivo transduction of myocardial tissue with long-term expression of a transgene enables translation of basic mechanistic knowledge into potential gene therapy approaches. Gene therapy in HF is in its infancy clinically with the predominant amount of experience being from animal models. Nevertheless, this challenging and promising field is gaining momentum as recent preclinical studies in larger animals have been carried out and, importantly, there are 2 newly initiated phase I clinical trials for HF gene therapy. To put it simply, 2 parameters are needed for achieving success with HF gene therapy: (1) clearly identified detrimental/beneficial molecular targets; and (2) the means to manipulate these targets at a molecular level in a sufficient number of cardiac cells. However, several obstacles do exist on our way to efficient and safe gene transfer to human myocardium. Some of these obstacles are discussed in this review; however, it primarily focuses on the molecular target systems that have been subjected to intense investigation over the last decade in an attempt to make gene therapy for human HF a reality.

[Reviews] Genetic Enhancement of Stem Cell Engraftment, Survival, and Efficacy

Penn, M. S., Mangi, A. A. — 2008-06-19

Cell-based therapies for the prevention and treatment of cardiac dysfunction offer the potential to significantly modulate cardiac function and improve outcomes in patients with cardiovascular disease. To date several clinical studies have suggested the potential efficacy of several different stem cell types; however, the benefits seen in clinical trials have been inconsistent and modest. In parallel, preclinical studies have identified key events in the process of cell-based myocardial repair, including stem cell homing, engraftment, survival, paracrine factor release, and differentiation that need to be optimized to maximize cardiac repair and function. The inconsistent and modest benefits seen in clinical trials combined with the preclinical identification of mediators responsible for key events in cell-based cardiac repair offers the potential for cell-based therapy to advance to cell-based gene therapy in an attempt to optimize these key events in the hope of maximizing clinical benefit. Below we discuss potential key events in cardiac repair and the mediators of these events that could be of potential interest for genetic enhancement of stem cell–based cardiac repair.

[Molecular Medicine] Notch Signaling Regulates Platelet-Derived Growth Factor Receptor-{beta} Expression in Vascular Smooth Muscle Cells

2008-06-19

Notch signaling is critically important for proper architecture of the vascular system, and mutations in NOTCH3 are associated with CADASIL, a stroke and dementia syndrome with vascular smooth muscle cell (VSMC) dysfunction. In this report, we link Notch signaling to platelet-derived growth factor (PDGF) signaling, a key determinant of VSMC biology, and show that PDGF receptor (PDGFR)-β is a novel immediate Notch target gene. PDGFR-β expression was upregulated by Notch ligand induction or by activated forms of the Notch receptor. Moreover, upregulation of PDGFR-β expression in response to Notch activation critically required the Notch signal integrator CSL. In primary VSMCs, PDGFR-β expression was robustly upregulated by Notch signaling, leading to an augmented intracellular response to PDGF stimulation. In newborn Notch3-deficient mice, PDGFR-β expression was strongly reduced in the VSMCs that later develop an aberrant morphology. In keeping with this, PDGFR-β upregulation in response to Notch activation was reduced also in Notch3-deficient embryonic stem cells. Finally, in VSMCs from a CADASIL patient carrying a NOTCH3 missense mutation, upregulation of PDGFR-β mRNA and protein in response to ligand-induced Notch activation was significantly reduced. In sum, these data reveal a hierarchy for 2 important signaling systems, Notch and PDGF, in the vasculature and provide insights into how dysregulated Notch signaling perturbs VSMC differentiation and function.

[Molecular Medicine] Deficiency of Adipose Differentiation-Related Protein Impairs Foam Cell Formation and Protects Against Atherosclerosis

Paul, A., Chang, B. H.-J., Li, L., Yechoor, V. K., Chan, L. — 2008-06-19

Foam cells are a hallmark of atherosclerosis. However, it is unclear whether foam cell formation per se protects against atherosclerosis or fuels it. In this study, we investigated the role of adipose differentiation-related protein (ADFP), a major lipid droplet protein (LDP), in the regulation of foam cell formation and atherosclerosis. We show that ADFP expression facilitates foam cell formation induced by modified lipoproteins in mouse macrophages in vitro. We show further that Adfp gene inactivation in apolipoprotein E–deficient (ApoE^–/–) mice reduces the number of lipid droplets in foam cells in atherosclerotic lesions and protects the mice against atherosclerosis. Moreover, transplantation of ADFP-null bone marrow-derived cells effectively attenuated atherosclerosis in ApoE^–/– mice. Deficiency of ADFP did not cause a detectable compensatory increase in the other PAT domain proteins in macrophages in vitro or in vivo. Mechanistically, ADFP enables the macrophage to maintain its lipid content by hindering lipid efflux. We detected no significant difference in lesion composition or in multiple parameters of inflammation in macrophages or in their phagocytic activity between mice with and without ADFP. In conclusion, Adfp inactivation in ApoE^–/– background protects against atherosclerosis and appears to be a relatively pure model of impaired foam cell formation.

[Molecular Medicine] The LIM Protein Leupaxin Is Enriched in Smooth Muscle and Functions As an Serum Response Factor Cofactor to Induce Smooth Muscle Cell Gene Transcription

2008-06-19

Leupaxin is a LIM domain–containing adapter protein belonging to the paxillin family that has been previously reported to be preferentially expressed in hematopoietic cells. Herein, we identified leupaxin in a screen for focal adhesion kinase binding partners in aortic smooth muscle, and we show that leupaxin is enriched in human and mouse vascular smooth muscle and that leupaxin expression is dynamically regulated during development. In addition, our studies reveal that leupaxin can undergo cytoplasmic/nuclear shuttling and functions as an serum response factor cofactor in the nucleus. We found that leupaxin forms a complex with serum response factor and associates with CArG-containing regions of smooth muscle promoters and that ectopic expression of leupaxin induces smooth muscle marker gene expression in both 10T1/2 cells and rat aortic smooth muscle cells. Subsequent studies indicated that enhanced focal adhesion kinase activity (induced by fibronectin or expression of constitutively active focal adhesion kinase) attenuates the nuclear accumulation of leupaxin and limits the ability of leupaxin to enhance serum response factor–dependent gene transcription. Thus, these studies indicate that modulation of the subcellular localization of serum response factor cofactors is 1 mechanism by which extracellular matrix–dependent signals may regulate phenotypic switching of smooth muscle cells.

[Molecular Medicine] A Specific CD36-Dependent Signaling Pathway Is Required for Platelet Activation by Oxidized Low-Density Lipoprotein

Chen, K., Febbraio, M., Li, W., Silverstein, R. L. — 2008-06-19

Platelet hyperactivity associated with hyperlipidemia may contribute to development of a prothrombotic state. We previously showed that oxidized low-density lipoprotein (oxLDL) formed in the setting of hyperlipidemia and atherosclerosis activated platelets in a CD36-dependent manner. We now show that mitogen-activated protein kinase c-Jun N-terminal kinase (JNK)2 and its upstream activator MKK4 were phosphorylated in platelets exposed to oxLDL. Using apoE^–/– mice as a model of hyperlipidemia, we showed that JNK was constitutively phosphorylated in platelets in a CD36-dependent manner. Inhibition of src kinase activity reduced JNK phosphorylation by oxLDL. Immunoprecipitations revealed that active phosphorylated forms of src kinases Fyn and Lyn were recruited to CD36 in platelets exposed to oxLDL. Pharmacological inhibition of the mitogen-activated protein kinase JNK or src family kinases abolished platelet activation by oxLDL in vitro. Using a murine carotid artery thrombosis model we demonstrated CD36-dependent phosphorylation of platelet JNK within thrombi. Furthermore, pharmacological inhibition of JNK prolonged thrombosis times in wild-type but not cd36-null mice in vivo. These findings suggest that a specific CD36-dependent signaling pathway is required for platelet activation by oxLDL and may provide insights related to development of novel antiplatelet therapies more relevant to atherothrombosis than to normal hemostasis.

[Molecular Medicine] Inhibition of Endothelial Nitric Oxide Synthase Activity by Proline-Rich Tyrosine Kinase 2 in Response to Fluid Shear Stress and Insulin

Fisslthaler, B., Loot, A. E., Mohamed, A., Busse, R., Fleming, I. — 2008-06-19

In native and primary cultures of endothelial cells, fluid shear stress elicits the tyrosine phosphorylation of the endothelial NO synthase (eNOS), however, the consequences of this modification on enzyme activity are unclear. We found that fluid shear stress induces the association of eNOS with the proline-rich tyrosine kinase 2 (PYK2) in endothelial cells and that the eNOS immunoprecipitated from eNOS- and PYK2-overexpressing HEK293 cells was tyrosine-phosphorylated on Tyr657. In mouse carotid arteries, the overexpression of wild-type PYK2, but not a dominant-negative PYK2, decreased eNOS activity (50%), whereas in murine lung endothelial cells, the downregulation of PYK2 (small interfering RNA) increased ionomycin-induced NO production. Mutation of Tyr657 to the phosphomimetic residues aspartate (D) or glutamate (E) abolished enzyme activity, whereas a nonphosphorylatable mutant (phenylalanine [F]) showed activity comparable to the wild-type enzyme. Moreover, normal flow-induced vasodilatation was apparent in carotid arteries from eNOS^–/– mice overexpressing either the wild-type eNOS or the Y657F mutant, whereas no flow-induced vasodilatation was apparent in arteries expressing the Y657E eNOS mutant. Insulin also activated PYK2 and stimulated eNOS in endothelial cells expressing the Y657F mutant but not wild-type eNOS. These data indicate that PYK2 mediates the tyrosine phosphorylation of eNOS on Tyr657 in response to fluid shear stress and insulin stimulation and that this modification attenuates the activity of the enzyme. The PYK2-dependent inhibition of NO production may serve to keep eNOS activity low and limit the detrimental consequences of maintained high NO output, ie, the generation of peroxynitrite.

[Molecular Medicine] Chronic Apoptosis of Vascular Smooth Muscle Cells Accelerates Atherosclerosis and Promotes Calcification and Medial Degeneration

2008-06-19

Vascular smooth muscle cell (VSMC) accumulation is implicated in plaque development. In contrast, VSMC apoptosis is implicated in plaque rupture, coagulation, vessel remodeling, medial atrophy, aneurysm formation, and calcification. Although VSMC apoptosis accompanies multiple pathologies, there is little proof of direct causality, particularly with the low levels of VSMC apoptosis seen in vivo. Using a mouse model of inducible VSMC–specific apoptosis, we demonstrate that low-level VSMC apoptosis during either atherogenesis or within established plaques of apolipoprotein (Apo)E^–/– mice accelerates plaque growth by two-fold, associated with features of plaque vulnerability including a thin fibrous cap and expanded necrotic core. Chronic VSMC apoptosis induced development of calcified plaques in younger animals and promoted calcification within established plaques. In addition, VSMC apoptosis induced medial expansion, associated with increased elastic lamina breaks, and abnormal matrix deposition reminiscent of cystic medial necrosis in humans. VSMC apoptosis prevented outward remodeling associated with atherosclerosis resulting in marked vessel stenosis. We conclude that VSMC apoptosis is sufficient to accelerate atherosclerosis, promote plaque calcification and medial degeneration, prevent expansive remodeling, and promote stenosis in atherosclerosis.

[Molecular Medicine] The Trafficking Protein GABARAP Binds to and Enhances Plasma Membrane Expression and Function of the Angiotensin II Type 1 Receptor

Cook, J. L., Re, R. N., deHaro, D. L., Abadie, J. M., Peters, M., Alam, J. — 2008-06-19

Proteins that bind to the intracellular expanses, particularly cytoplasmic tail regions, of heptahelical integral membrane receptors are of particular interest in that they can mediate or modulate trafficking or intracellular signaling. In an effort to distinguish new proteins that might promote angiotensin II type 1 (AT₁) receptor intracellular events, we screened a yeast 2-hybrid mouse brain library with the rat AT_1A receptor (AT₁R) carboxyl terminus and identified GABARAP, a protein involved in intracellular trafficking of the GABA_A receptor, as a binding partner for the AT₁R. Interaction of GABARAP with the AT₁R carboxyl terminus was further substantiated using GST pull-down assays, and binding of the full-length tagged AT₁R to GABARAP was verified using coimmunoprecipitation. Bioluminescence resonance energy transfer assays further confirmed specific interaction of GABARAP with AT₁R. Moreover, GABARAP clearly increased the steady-state level of plasma membrane-associated AT₁R in PC-12 cells. Cotransfection of GABARAP with an AT₁R fluorescent fusion protein increased PC-12 cell surface expression of the AT₁R more than 6-fold when standardized to the level of intracellular expression. Furthermore, GABARAP overexpression in CHO-K1 cells engineered to express AT₁R increased angiotensin II binding sites 3.7-fold and angiotensin II–induced phospho–extracellular signal-regulated kinase 1/2 and cellular proliferation significantly over levels obtained with AT₁R overexpression alone. In addition, small interfering RNA–mediated knockdown of GABARAP reduced the steady-state levels of the AT₁R fluorescent fusion protein by 43% and its cell surface expression by 84%. Immunoblot analyses confirmed the quantitative image data. We conclude that GABARAP binds to and promotes trafficking of the AT₁R to the plasma membrane.

[Molecular Medicine] Conditional Deletion of Kruppel-Like Factor 4 Delays Downregulation of Smooth Muscle Cell Differentiation Markers but Accelerates Neointimal Formation Following Vascular Injury

Yoshida, T., Kaestner, K. H., Owens, G. K. — 2008-06-19

Phenotypic switching of smooth muscle cells (SMCs) plays a key role in vascular proliferative diseases. We previously showed that Krüppel-like factor 4 (Klf4) suppressed SMC differentiation markers in cultured SMCs. Here, we derive mice deficient for Klf4 by conditional gene ablation and analyze their vascular phenotype following carotid injury. Klf4 expression was rapidly induced in SMCs of control mice after vascular injury but not in Klf4-deficient mice. Injury-induced repression of SMC differentiation markers was transiently delayed in Klf4-deficient mice. Klf4 mutant mice exhibited enhanced neointimal formation in response to vascular injury caused by increased cellular proliferation in the media but not an altered apoptotic rate. Consistent with these findings, cultured SMCs overexpressing Klf4 showed reduced cellular proliferation, in part, through the induction of the cell cycle inhibitor, p21^WAF1/Cip1 via increased binding of Klf4 and p53 to the p21^WAF1/Cip1 promoter/enhancer. In vivo chromatin immunoprecipitation assays also showed increased Klf4 binding to the promoter/enhancer regions of the p21^WAF1/Cip1 gene and SMC differentiation marker genes following vascular injury. Taken together, we have demonstrated that Klf4 plays a critical role in regulating expression of SMC differentiation markers and proliferation of SMCs in vivo in response to vascular injury.

[Molecular Medicine] Aryl Hydrocarbon Receptor Is Activated by Glucose and Regulates the Thrombospondin-1 Gene Promoter in Endothelial Cells

Dabir, P., Marinic, T. E., Krukovets, I., Stenina, O. I. — 2008-06-19

Hyperglycemia is an independent risk factor for development of diabetic vascular complications. The molecular mechanisms that are activated by glucose in vascular cells and could explain the development of vascular complications are still poorly understood. A putative binding site for the transcription factor aryl hydrocarbon receptor (AhR) was identified in the glucose-responsive fragment of the promoter of thrombospondin-1, a potent antiangiogenic and proatherogenic protein involved in development of diabetic vascular complications. AhR was expressed in aortic endothelial cells (ECs), activated, and bound to the promoter in response to high glucose stimulation of ECs. The constitutively active form of AhR induced activation of the thrombospondin-1 gene promoter. In response to high glucose stimulation, AhR was found in complex with Egr-1 and activator protein-2, which are 2 other nuclear transcription factors activated by glucose in ECs that have not been previously detected in complex with AhR. The activity of the DNA-binding complex was regulated by glucose through the activation of hexosamine pathway and intracellular glycosylation. This is the first report of activation of AhR (a receptor for xenobiotic compounds) by a physiological stimulus. This report links the activation of AhR to the pathological effects of hyperglycemia in the vasculature.

[Molecular Medicine] A Novel Human-Specific Soluble Vascular Endothelial Growth Factor Receptor 1: Cell Type-Specific Splicing and Implications to Vascular Endothelial Growth Factor Homeostasis and Preeclampsia

2008-06-19

A human-specific splicing variant of vascular endothelial growth factor (VEGF) receptor 1 (Flt1) was discovered, producing a soluble receptor (designated sFlt1-14) that is qualitatively different from the previously described soluble receptor (sFlt1) and functioning as a potent VEGF inhibitor. sFlt1-14 is generated in a cell type-specific fashion, primarily in nonendothelial cells. Notably, in vascular smooth muscle cells, all Flt1 messenger RNA is converted to sFlt1-14, whereas endothelial cells of the same human vessel express sFlt1. sFlt1-14 expression by vascular smooth muscle cells is dynamically regulated as evidenced by its upregulation on coculture with endothelial cells or by direct exposure to VEGF. Increased production of soluble VEGF receptors during pregnancy is entirely attributable to induced expression of placental sFlt1-14 starting by the end of the first trimester. Expression is dramatically elevated in the placenta of women with preeclampsia, specifically induced in abnormal clusters of degenerative syncytiotrophoblasts known as syncytial knots, where it may undergo further messenger RNA editing. sFlt1-14 is the predominant VEGF-inhibiting protein produced by the preeclamptic placenta, accumulates in the circulation, and hence is capable of neutralizing VEGF in distant organs affected in preeclampsia. Together, these findings revealed a new natural VEGF inhibitor that has evolved in humans, possibly to protect nonendothelial cells from adverse VEGF signaling. Furthermore, the study uncovered the identity of a VEGF-blocking protein implicated in preeclampsia.

[Integrative Physiology] Nitric Oxide-Mediated Zinc Release Contributes to Hypoxic Regulation of Pulmonary Vascular Tone

2008-06-19

The metal binding protein metallothionein (MT) is a target for nitric oxide (NO), causing release of bound zinc that affects myogenic reflex in systemic resistance vessels. Here, we investigate a role for NO-induced zinc release in pulmonary vasoregulation. We show that acute hypoxia causes reversible constriction of intraacinar arteries (<50 µm/L) in isolated perfused mouse lung (IPL). We further demonstrate that isolated pulmonary (but not aortic) endothelial cells constrict in hypoxia. Hypoxia also causes NO-dependent increases in labile zinc in mouse lung endothelial cells and endothelium of IPL. The latter observation is dependent on MT because it is not apparent in IPL of MT^–/– mice. Data from NO-sensitive fluorescence resonance energy transfer–based reporters support hypoxia-induced NO production in pulmonary endothelium. Furthermore, hypoxic constriction is blunted in IPL of MT^–/– mice and in wild-type mice, or rats, treated with the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), suggesting a role for chelatable zinc in modulating HPV. Finally, the NO donor DETAnonoate causes further vasoconstriction in hypoxic IPL in which NO vasodilatory pathways are inhibited. Collectively, these data suggest that zinc thiolate signaling is a component of the effects of acute hypoxia-mediated NO biosynthesis and that this pathway may contribute to constriction in the pulmonary vasculature.

[Author Index] Volume 102, June 20, 2008

2008-06-19

[UltraRapid Communications] Cell-Free Embryonic Stem Cell Extract-Mediated Derivation of Multipotent Stem Cells From NIH3T3 Fibroblasts for Functional and Anatomical Ischemic Tissue Repair

2008-06-05

The oocyte-independent source for the generation of pluripotent stem cells is among the ultimate goals in regenerative medicine. We report that on exposure to mouse embryonic stem cell (mESC) extracts, reversibly permeabilized NIH3T3 cells undergo dedifferentiation followed by stimulus-induced redifferentiation into multiple lineage cell types. Genome-wide expression profiling revealed significant differences between NIH3T3 control and ESC extract–treated NIH3T3 cells including the reactivation of ESC-specific transcripts. Epigenetically, ESC extracts induced CpG demethylation of Oct4 promoter, hyperacetylation of histones 3 and 4, and decreased lysine 9 (K-9) dimethylation of histone 3. In mouse models of surgically induced hindlimb ischemia or acute myocardial infarction transplantation of reprogrammed NIH3T3 cells significantly improved postinjury physiological functions and showed anatomic evidence of engraftment and transdifferentiation into skeletal muscle, endothelial cell, and cardiomyocytes. These data provide evidence for the generation of functional multipotent stem-like cells from terminally differentiated somatic cells without the introduction of retroviral mediated transgenes or ESC fusion.

[Letters to the Editor] Is LPL Deficiency Atherogenic?

Ebara, T., Murase, T., Okubo, M. — 2008-06-05

[Letters to the Editor] Response to the Letter by Ebara et al

Liu, G., Hayden, M. R. — 2008-06-05

[Editorials] Teed Off: Cardiac Conduction System Development Requires T-box Transcription Factors

Yutzey, K. E. — 2008-06-05

[Editorials] Effects of Heart Disease on Cardiac Ion Current Density Versus Current Amplitude: Important Conceptual Subtleties in the Language of Arrhythmogenic Ion Channel Remodeling

Nattel, S. — 2008-06-05

[Editorials] The Ins and Outs of Calcium in Heart Failure

O'Rourke, B. — 2008-06-05

[Editorials] Holt-Oram Syndrome and Atrial Fibrillation: Opening the (T)-Box

Cerbai, E., Sartiani, L. — 2008-06-05

[Reviews] Nuclear Shape, Mechanics, and Mechanotransduction

Dahl, K. N., Ribeiro, A. J.S., Lammerding, J. — 2008-06-05

In eukaryotic cells, the nucleus contains the genome and is the site of transcriptional regulation. The nucleus is the largest and stiffest organelle and is exposed to mechanical forces transmitted through the cytoskeleton from outside the cell and from force generation within the cell. Here, we discuss the effect of intra- and extracellular forces on nuclear shape and structure and how these force-induced changes could be implicated in nuclear mechanotransduction, ie, force-induced changes in cell signaling and gene transcription. We review mechanical studies of the nucleus and nuclear structural proteins, such as lamins. Dramatic changes in nuclear shape, organization, and stiffness are seen in cells where lamin proteins are mutated or absent, as in genetically engineered mice, RNA interference studies, or human disease. We examine the different mechanical pathways from the force-responsive cytoskeleton to the nucleus. We also highlight studies that link changes in nuclear shape with cell function during developmental, physiological, and pathological modifications. Together, these studies suggest that the nucleus itself may play an important role in the response of the cell to force.

[Reviews] Aging and Disease as Modifiers of Efficacy of Cell Therapy

Dimmeler, S., Leri, A. — 2008-06-05

Cell therapy is a promising option for treating ischemic diseases and heart failure. Adult stem and progenitor cells from various sources have experimentally been shown to augment the functional recovery after ischemia, and clinical trials have confirmed that autologous cell therapy using bone marrow—derived or circulating blood–derived progenitor cells is safe and provides beneficial effects. However, aging and risk factors for coronary artery disease affect the functional activity of the endogenous stem/progenitor cell pools, thereby at least partially limiting the therapeutic potential of the applied cells. In addition, age and disease affect the tissue environment, in which the cells are infused or injected. The present review article will summarize current evidence for cell impairment during aging and disease but also discuss novel approaches how to reverse the dysfunction of cells or to refresh the target tissue. Pretreatment of cells or the target tissue by small molecules, polymers, growth factors, or a combination thereof may provide useful approaches for enhancement of cell therapy for cardiovascular diseases.

[Molecular Medicine] Hypoxia-Inducible Transcription Factor-1{alpha} Triggers an Autocrine Survival Pathway During Embryonic Cardiac Outflow Tract Remodeling

Liu, H., Fisher, S. A. — 2008-06-05

The cardiac outflow tract (OFT) of birds and mammals undergoes complex remodeling in the transition to a dual circulation. We have previously suggested a role of myocardial hypoxia and hypoxia inducible factor (HIF)-1 in the apoptosis-dependent remodeling of the OFT. In the present study, we transduced recombinant adenovirus-mediated HIF-1 in embryonic chick OFT myocardium to test its role in OFT remodeling. HIF-1 reduced the prevalence of apoptosis in OFT cardiomyocytes at stages 25 and 30, as determined by lysosome accumulation and caspase-3 activity. Associated conotruncal defects included malrotation of the aorta and excessive infundibular myocardium. HIF-1 targets induced in these gain-of-function experiments included vascular endothelial growth factor (VEGF), inducible nitric oxide synthase, and stromal cell–derived factor-1. To test the role of VEGF in this context, an adenovirus expressing secreted Flk1 (VEGF receptor 2) that binds and blocks VEGF signaling was targeted to the OFT myocardium. This caused increased cell death in the OFT myocardium at stages 25 and 30. Associated conotruncal heart defects included malrotation of the aorta, defects in the subpulmonic infundibulum associated with a small right ventricle, and increased OFT mesenchyme with failure of semilunar valve formation. We conclude that hypoxia signaling through HIF-1 and VEGF provides an autocrine survival signal in the developing cardiac OFT and that perturbation in this pathway causes OFT defects that model congenital human conotruncal heart defects.

[Molecular Medicine] Transcription Factor Tbx3 Is Required for the Specification of the Atrioventricular Conduction System

2008-06-05

The cardiac conduction system consists of distinctive heart muscle cells that initiate and propagate the electric impulse required for coordinated contraction. The conduction system expresses the transcriptional repressor Tbx3, which is required for vertebrate development and controls the formation of the sinus node. In humans, mutations in Tbx3 cause ulnar–mammary syndrome. Here, we investigated the role of Tbx3 in the molecular specification of the atrioventricular conduction system. Expression analysis revealed early delineation of the atrioventricular bundle and proximal bundle branches by Tbx3 expression in human, mouse, and chicken. Tbx3-deficient mice, which die between embryonic day 12.5 and 15.5, ectopically expressed genes for connexin (Cx)43, atrial natriuretic factor (Nppa), Tbx18, and Tbx20 in the atrioventricular bundle and proximal bundle branches. Cx40 was precociously upregulated in the atrioventricular bundle of Tbx3 mutants. Moreover, the atrioventricular bundle and branches failed to exit the cell cycle in Tbx3 mutant embryos. Finally, Tbx3-deficient embryos developed outflow tract malformations and ventricular septal defects. These data reveal that Tbx3 is required for the molecular specification of the atrioventricular bundle and bundle branches and for the development of the ventricular septum and outflow tract. Our data suggest a mechanism in which Tbx3 represses differentiation into ventricular working myocardium, thereby imposing the conduction system phenotype on cells within its expression domain.

[Molecular Medicine] H-Ras Regulates Angiogenesis and Vascular Permeability by Activation of Distinct Downstream Effectors

Serban, D., Leng, J., Cheresh, D. — 2008-06-05

Angiogenesis and vascular permeability occur following endothelium activation by vascular endothelial growth factor (VEGF). Downstream mechanisms that define these vascular responses remain unknown. H-Ras activation has been associated with the angiogenic response. However, active H-Ras initiates a wide spectrum of other biological responses through multiple downstream effectors. To identify vascular signaling by H-Ras and the immediate effectors we activated the extracellular signal regulated kinase/mitogen-activated protein kinase or phosphatidylinositol 3-kinase (PI3K) pathways in chicken and mouse endothelial tissues by ectopic expression of the Ras effector mutants H-RasV12S35 or H-RasV12C40, respectively. Constitutive activation of the extracellular signal-regulate kinase/mitogen-activated protein kinase pathway by H-RasV12S35 was sufficient to induce angiogenesis and not vascular permeability, whereas activation of the PI3K pathway by H-RasV12C40 was required for both angiogenesis and vascular permeability. Pharmacological inhibition of PI3K (/β) suppressed both Ras- or VEGF-mediated vascular response in vivo and survival of primary human endothelial cells in vitro. However, inhibition of PI3K (/) suppressed Ras- or VEGF-mediated vascular permeability in vivo, with no effect on survival of primary endothelial cells. This was supported by genetic studies because PI3K p110 knockout mice showed impaired vascular permeability response to VEGF or H-RasV12C40 treatment yet produced a wild-type angiogenic response to H-RasV12S35. We conclude that downstream of VEGF, H-Ras serves as a cellular switch that controls neovascularization and vascular permeability by activation of distinct effectors.

[Molecular Medicine] Functional Mineralocorticoid Receptors in Human Vascular Endothelial Cells Regulate Intercellular Adhesion Molecule-1 Expression and Promote Leukocyte Adhesion

2008-06-05

In clinical trials, aldosterone antagonists decrease cardiovascular mortality and ischemia by unknown mechanisms. The steroid hormone aldosterone acts by binding to the mineralocorticoid receptor (MR), a ligand-activated transcription factor. In humans, aldosterone causes MR-dependent endothelial cell (EC) dysfunction and in animal models, aldosterone increases vascular macrophage infiltration and atherosclerosis. MR antagonists inhibit these effects without changing blood pressure, suggesting a direct role for vascular MR in EC function and atherosclerosis. Whether human vascular ECs express functional MR is not known. Here, we show that human coronary artery and aortic ECs express MR mRNA and protein and that EC MR mediates aldosterone-dependent gene transcription. Human ECs also express the enzyme 11-β-hydroxysteroid dehydrogenase-2 (11βHSD2), and inhibition of 11βHSD2 in aortic ECs enhances gene transactivation by cortisol, supporting that EC 11βHSD2 is functional. Furthermore, aldosterone stimulates transcription of the proatherogenic leukocyte–EC adhesion molecule intercellular adhesion molecule (ICAM)1 gene and protein expression on human coronary artery ECs, an effect inhibited by the MR antagonist spironolactone and by MR knock down with small interfering RNA. Cell adhesion assays demonstrate that aldosterone promotes leukocyte–EC adhesion, an effect that is inhibited by spironolactone and ICAM1 blocking antibody, supporting that aldosterone induction of EC ICAM1 surface expression via MR mediates leukocyte–EC adhesion. These data show that aldosterone activates endogenous EC MR and proatherogenic gene expression in clinically important human ECs. These studies describe a novel mechanism by which aldosterone may influence ischemic cardiovascular events and support a new explanation for the decrease in ischemic events in patients treated with aldosterone antagonists.

[Molecular Medicine] Adventitial Mast Cells Contribute to Pathogenesis in the Progression of Abdominal Aortic Aneurysm

2008-06-05

Abdominal aortic aneurysm (AAA) is histologically characterized by medial degeneration and various degrees of chronic adventitial inflammation, although the mechanisms for progression of aneurysm are poorly understood. In the present study, we carried out histological study of AAA tissues of patients, and interventional animal and cell culture experiments to investigate a role of mast cells in the pathogenesis of AAA. The number of mast cells was found to increase in the outer media or adventitia of human AAA, showing a positive correlation between the cell number and the AAA diameter. Aneurysmal dilatation of the aorta was seen in the control (+/+) rats following periaortic application of calcium chloride (CaCl₂) treatment but not in the mast cell–deficient mutant Ws/Ws rats. The AAA formation was accompanied by accumulation of mast cells, T lymphocytes and by activated matrix metalloproteinase 9, reduced elastin levels and augmented angiogenesis in the aortic tissue, but these changes were much less in the Ws/Ws rats than in the controls. Similarly, mast cells were accumulated and activated at the adventitia of aneurysmal aorta in the apolipoprotein E–deficient mice. The pharmacological intervention with the tranilast, an inhibitor of mast cell degranulation, attenuated AAA development in these rodent models. In the cell culture experiment, a mast cell directly augmented matrix metalloproteinase 9 activity produced by the monocyte/macrophage. Collectively, these data suggest that adventitial mast cells play a critical role in the progression of AAA.

[Cellular Biology] Interaction of {alpha}1-Adrenoceptor Subtypes With Different G Proteins Induces Opposite Effects on Cardiac L-type Ca2+ Channel

2008-06-05

We examined the effect of ₁-adrenoceptor subtype-specific stimulation on L-type Ca²⁺ current (I_Ca) and elucidated the subtype-specific intracellular mechanisms for the regulation of L-type Ca²⁺ channels in isolated rat ventricular myocytes. We confirmed the protein expression of _1A- and _1B-adrenoceptor subtypes at the transverse tubules (T-tubules) and found that simultaneous stimulation of these 2 receptor subtypes by nonsubtype selective agonist, phenylephrine, showed 2 opposite effects on I_Ca (transient decrease followed by sustained increase). However, selective _1A-adrenoceptor stimulation (≥0.1 µmol/L A61603) only potentiated I_Ca, and selective _1B-adrenoceptor stimulation (10 µmol/L phenylephrine with 2 µ mol/L WB4101) only decreased I_Ca. The positive effect by _1A-adrenoceptor stimulation was blocked by the inhibition of phospholipase C (PLC), protein kinase C (PKC), or Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). The negative effect by _1B-adrenoceptor stimulation disappeared after the treatment of pertussis toxin or by the prepulse depolarization, but was not attriburable to the inhibition of cAMP-dependent pathway. The translocation of PKC and to the T-tubules was observed only after _1A-adrenoceptor stimulation, but not after _1B-adrenoceptor stimulation. Immunoprecipitaion analysis revealed that _1A-adrenoceptor was associated with G_q/11, but _1B-adrenoceptor interacted with one of the pertussis toxin-sensitive G proteins, G_o. These findings demonstrated that the interactions of ₁-adrenoceptor subtypes with different G proteins elicit the formation of separate signaling cascades, which produce the opposite effects on I_Ca. The coupling of _1A-adrenoceptor with G_q/11-PLC-PKC-CaMKII pathway potentiates I_Ca. In contrast, _1B-adrenoceptor interacts with G_o, of which the β-complex might directly inhibit the channel activity at T-tubules.

[Cellular Biology] Arrhythmogenic Effects of {beta}2-Adrenergic Stimulation in the Failing Heart Are Attributable to Enhanced Sarcoplasmic Reticulum Ca Load

2008-06-05

Ventricular tachycardia in heart failure (HF) can initiate by nonreentrant mechanisms such as delayed afterdepolarizations. In an arrhythmogenic rabbit model of HF, we have shown that isoproterenol induces ventricular tachycardia in vivo and aftercontractions and transient inward currents in HF myocytes. To determine whether β₂-adrenergic receptor (β₂-AR) stimulation contributes, we performed in vivo drug infusion, in vitro myocyte and biochemical studies. Intravenous zinterol (2.5 µg/kg) led to ventricular arrhythmias, including ventricular tachycardia up to 13 beats long in 4 of 6 HF rabbits (versus 0 of 5 controls, P<0.01), an effect blocked by β₂-AR antagonist ICI-118,551 (0.2 mg/kg). In field-stimulated myocytes (0.5 to 4 Hz, 37°C), β₂-AR stimulation (1 µmol/L zinterol+300 nmol/L β₁-AR antagonist CGP-29712A) induced aftercontractions and Ca aftertransients in 88% of HF versus 0% of control myocytes (P<0.01). β₂-AR stimulation in HF (but not control) myocytes increased Ca transient amplitude (by 29%), sarcoplasmic reticulum (SR) Ca load (by 28%), the rate of [Ca]_i decline (by 28%; n=12, all P<0.05), and phospholamban phosphorylation at Ser16, but Ca current was unchanged. All of these effects in HF myocytes were blocked by ICI-118,551 (100 nmol/L). Although total β-AR expression was reduced by 47% in HF rabbit left ventricle, β₂-AR number was unchanged, indicating more potent β₂-AR–dependent SR Ca uptake and arrhythmogenesis in HF. Human HF myocytes showed similar β₂-AR–induced aftercontractions, aftertransients, and enhanced Ca transient amplitude, SR Ca load and twitch [Ca]_i decline rate. Thus, β₂-AR stimulation is arrhythmogenic in HF, mediated by SR Ca overload-induced spontaneous SR Ca release and aftercontractions.