Citations

Abstract —Insulin-like growth factors I and II (IGF-I and -II) and their regulatory proteins are secreted by cells of the cardiovascular system. They are growth promoters for arterial cells and mediators of cardiovascular disease. IGFs are bound to IGF binding proteins (IGFBPs), which modulate IGF ligand-receptor interaction and consequently to IGF action. IGFBPs are in turn posttranslationally modulated by specific proteases. This dynamic balance (IGFs, IGFBPs, and IGFBP proteases) constitutes the IGF axis and ultimately determines the extent of IGF-dependent cellular effects. Dysregulated actions of this axis influence coronary atherosclerosis through effects on vascular smooth muscle cell growth, migration, and extracellular matrix synthesis in the atherosclerotic plaque. IGF-I promotes macrophage chemotaxis, excess LDL cholesterol uptake, and release of proinflammatory cytokines. Endothelial cells also receive the effects of IGFs stimulating their migration and organization forming capillary networks. Neointimal hyperplasia of restenosis after coronary artery injury is also modulated by the IGF axis. IGFs stimulate vascular smooth muscle cell proliferation and migration to form the neointima and upregulate tropoelastin synthesis after disruption of the elastic layer. Understanding IGF axis regulation establishes a scientific basis for strategies directed to limit or reverse plaque growth and vulnerability in atherosclerosis and in the neointimal hyperplasia of restenosis.

Abstract —We investigated whether ADP-ribosyl cyclase (ADPR-cyclase) in rat vascular smooth muscle cells (VSMCs) has enzymatic properties that differ from the well-characterized CD38-antigen ADPR-cyclase, expressed in HL-60 cells. ADPR-cyclase from VSMCs, but not CD38 ADPR-cyclase from HL-60 cells, was inhibited by gangliosides (10 μmol/L) GT 1B , GD 1 , and GM 3 . Preincubation of membranes from CD38 HL-60 cells, but not from VSMCs, with anti-CD38 antibodies increased ADPR-cyclase activity; CD38 antigen was detected both in VSMCs and in HL-60 cells. ADPR-cyclase in VSMC membranes was more sensitive than CD38 HL-60 ADPR-cyclase to inactivation by N- endoglycosidase F and to thermal inactivation at 45°C. The specific activity of ADPR-cyclase in membranes from VSMCs was >20-fold higher than in membranes from CD38 HL-60 cells. Most importantly, VSMC ADPR-cyclase was inhibited by Zn 2+ and Cu 2+ ions; the inhibition by Zn 2+ was dose dependent, noncompetitive, and reversible by EDTA. In contrast, Zn 2+ stimulated the activity of CD38 HL-60 ADPR-cyclase and other known types of ADPR-cyclases. Retinoids act either via the nuclear receptor retinoic acid receptor or retinoid X receptor, including all- trans retinoic acid (atRA), and panagonist 9- cis -retinoic acid–upregulated VSMC ADPR-cyclase; the stimulatory effect of atRA was blocked by actinomycin D and cycloheximide. 1,25(OH) 2 –Vitamin D 3 (calciferol) stimulated VSMC ADPR-cyclase dose dependently at subnanomolar concentrations (ED 50 ≅56 pmol/ l ). Oral administration of atRA to rats resulted in an increase of ADPR-cyclase activity in aorta (≅+60%) and, to a lesser degree, in myocardium of left ventricle (+18%), but atRA had no effect on ADPR-cyclases in lungs, spleen, intestinal smooth muscle, skeletal muscle, liver, or testis. Administration of 3,5,3′-triiodothyronine (T 3 ) to rats resulted in an increase of ADPR-cyclase activity in aorta (≅+89%), but not in liver or brain. We conclude the following: (1) ADPR-cyclase in VSMCs has enzymatic properties distinct from “classic” CD38 ADPR-cyclase, especially sensitivity to inhibition by Zn 2+ and Cu 2+ ; (2) ADPR-cyclase in VSMCs is upregulated by various retinoids, calcitriol, and T 3 in vitro; and (3) administration of atRA and T 3 increases ADPR-cyclase in aorta in vivo. We suggest that the cADPR signaling system plays an important role in the regulation of VSMC functions in response to steroid superfamily hormones.

Abstract—Synthesis of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) is critical for normal vascular homeostasis. eNOS function is rapidly regulated by agonists and blood flow and chronically by factors that regulate mRNA stability and gene transcription. Recently, localization of eNOS to specialized plasma membrane invaginations termed caveolae has been proposed to be required for maximal eNOS activity. Because caveolae are highly enriched in cholesterol, and hypercholesterolemia is associated with increased NO production, we first studied the effects of cholesterol loading on eNOS localization and NO production in cultured bovine aortic endothelial cells (BAECs). Caveolae-enriched fractions were prepared by OptiPrep gradient density centrifugation. Treatment of BAECs with 30 μg/mL cholesterol for 24 hours stimulated significant increases in total eNOS protein expression (1.50-fold), eNOS associated with caveolae-enriched membranes (2.23-fold), and calcium ionophore-stimulated NO production (1.56-fold). Because reactive oxygen species (ROS) contribute to endothelial dysfunction in hypercholesterolemia, we next studied the effects of ROS on eNOS localization and caveolae number. Treatment of BAECs for 24 hours with 1 μmol/L LY83583, a superoxide-generating napthoquinolinedione, decreased caveolae number measured by electron microscopy and prevented the cholesterol-mediated increases in eNOS expression. In vitro exposure of caveolae-enriched membranes to ROS (xanthine plus xanthine oxidase) dissociated caveolin more readily than eNOS from the membranes. These results show that cholesterol treatment increases eNOS expression, whereas ROS treatment decreases eNOS expression and the association of eNOS with caveolin in caveolae-enriched membranes. Our data suggest that oxidative stress modulates endothelial function by regulating caveolae formation, eNOS expression, and eNOS-caveolin interactions.

Abstract —Giant cell arteritis (GCA) is an inflammatory vasculopathy in which T cells and macrophages infiltrate the wall of medium and large arteries. Clinical consequences such as blindness and stroke are related to arterial occlusion. Formation of aortic aneurysms may result from necrosis of smooth muscle cells and fragmentation of elastic membranes. The molecular mechanisms of arterial wall injury in GCA are not understood. To identify mechanisms of arterial damage, gene expression in inflamed and unaffected temporal artery specimens was compared by differential display polymerase chain reaction. Genes differentially expressed in arterial lesions included 3 products encoded by the mitochondrial genome. Immunohistochemistry with antibodies specific for a 65-kDa mitochondrial antigen revealed that increased expression of mitochondrial products was characteristic of multinucleated giant cells and of CD68+ macrophages that cluster in the media and at the media-intima junction. 4-Hydroxy-2-nonenal adducts, products of lipid peroxidation, were detected on smooth muscle cells and on tissue infiltrating cells, in close proximity to multinucleated giant cells and CD68+ macrophages. Also, giant cells and macrophages with overexpression of mitochondrial products were able to synthesize metalloproteinase-2. Our data suggest that in the vascular lesions characteristic for GCA, a subset of macrophages has the potential to support several pathways of arterial injury, including the release of reactive oxygen species and the production of metalloproteinase-2. This macrophage subset is topographically defined and is also identified by overexpression of mitochondrial genes. Because these macrophages have a high potential to promote several mechanisms of arterial wall damage, they should be therapeutically targeted to prevent blood vessel destruction.

Abstract —A rapidly emerging body of literature implicates a pivotal role for the Ca 2+ -calmodulin–dependent phosphatase calcineurin as a cellular target for a variety of Ca 2+ -dependent signaling pathways culminating in left ventricular hypertrophy (LVH). Most of the recent experimental support for this hypothesis is derived from in vitro studies or in vivo studies in transgenic mice expressing activated calcineurin or mutant sarcomeric proteins. The aim of the present study was to test whether calcineurin inhibitors, cyclosporin A (CsA) and FK 506, prevent pressure-overload LVH using 2 standard rat models: (1) the spontaneously hypertensive rat (SHR) and (2) aortic banding. The major new findings are 2-fold. First, in SHR, LVH (left ventricular weight to body weight ratio) was unaffected by a dose of CsA (5 mg · kg −1 · d −1 ) that was sufficient to raise blood pressure and to inhibit calcineurin-mediated transcriptional activation in skeletal muscle. Second, in rats with aortic banding, LVH was unaffected by FK 506 (0.3 mg · kg −1 · d −1 ) or even higher doses of CsA (10 and 20 mg · kg −1 · d −1 ) that were sufficient to inhibit 90% of total calcineurin phosphatase activity in the hypertrophied myocardium. In the latter experiments, CsA blocked neither the elevated left ventricular end-diastolic pressures, a measure of diastolic function, nor the induction in atrial natriuretic peptide mRNA in the hypertrophic ventricles. Thus, in numerous experiments, systemic administration of potent calcineurin inhibitors did not prevent the development of LVH in 2 classic models of pressure-overload hypertrophy. These results demonstrate that pressure-overload hypertrophy can arise through calcineurin-independent pathways.

Abstract —Communication between the cytoplasm and nucleoplasm of cardiac cells occurs by molecular transport through nuclear pores. In lower eukaryotes, nuclear transport requires the maintenance of cellular energetics and ion homeostasis. Although heart muscle is particularly sensitive to metabolic stress, the regulation of nuclear transport through nuclear pores in cardiomyocytes has not yet been characterized. With the use of laser confocal and atomic force microscopy, we observed nuclear transport in cardiomyocytes and the structure of individual nuclear pores under different cellular conditions. In response to the depletion of Ca 2+ stores or ATP/GTP pools, the cardiac nuclear pore complex adopted 2 distinct conformations that led to different patterns of nuclear import regulation. Depletion of Ca 2+ indiscriminately prevented the nuclear import of macromolecules through closure of the nuclear pore opening. Depletion of ATP/GTP only blocked facilitated transport through a simultaneous closure of the pore and relaxation of the entire complex, which allowed other molecules to pass into the nucleus through peripheral routes. The current study of the structural plasticity of the cardiac nuclear pore complex, which was observed in response to changes in cellular conditions, identifies a gating mechanism for molecular translocation across the nuclear envelope of cardiac cells. The cardiac nuclear pore complex serves as a conduit that differentially regulates nuclear transport of macromolecules and provides a mechanism for the control of nucleocytoplasmic communication in cardiac cells, in particular under stress conditions associated with disturbances in cellular bioenergetics and Ca 2+ homeostasis.

Abstract —Although the downregulation of creatine kinase activity has been associated with heart failure, creatine kinase–deficient transgenic hearts have a preserved contractile function. This suggests the existence of alternative phosphotransfer pathways in the myocardium, the identity of which is still unknown. In this study, we examined the contribution of adenylate kinase–catalyzed phosphotransfer to myocardial energetics. In the isolated mitochondria/actomyosin system, which possesses endogenous adenylate kinase activity in both compartments, substrates for adenylate kinase promoted the rate and amplitude of actomyosin contraction that was further enhanced by purified adenylate kinase. Inhibition of adenylate kinase activity diminished both actomyosin contraction and mitochondrial respiration, which indicated reduced energy flow between mitochondria and myofibrils. In intact myocardium, the net adenylate kinase–catalyzed phosphotransfer rate was 10% of the total ATP turnover rate as measured by 18 O-phosphoryl labeling in conjunction with gas chromatography and mass spectrometry. In pacing-induced failing heart, adenylate kinase–catalyzed phosphotransfer increased by 134% and contributed 21% to the total ATP turnover. Concomitantly, the contribution by creatine kinase dropped from 89% in normal hearts to 40% in failing hearts. These phosphotransfer changes were associated with reduced levels of metabolically active ATP but maintained overall ATP turnover rate. Thus, this study provides evidence that adenylate kinase facilitates the transfer of high-energy phosphoryls and signal communication between mitochondria and actomyosin in cardiac muscle, with an increased contribution to cellular phosphotransfer in heart failure. This phosphotransfer function renders adenylate kinase an important component for optimal myocardial bioenergetics and a compensatory mechanism in response to impaired intracellular energy flux in the failing heart.

Abstract —Tissue factor pathway inhibitor (TFPI) in vivo is thought to be synthesized mainly by endothelial cells. To date, no significant regulator of TFPI synthesis has been described. Vascular smooth muscle cells (VSMC) express tissue factor in vitro and in vivo, which may contribute to vascular thrombosis. We hypothesized that VSMC might also express TFPI. To determine this, we examined growth-arrested coronary VSMC in culture and found that VSMC secreted an amount of TFPI similar to that seen in endothelial cells. Immunohistochemistry of normal human coronary arteries showed TFPI staining throughout the media and intima of the vessel with localization to VSMC and endothelial cells. To determine regulation of TFPI expression in VSMC, we examined the effects of serum stimulation on TFPI secretion and found that FBS induced a 5-fold increase in TFPI antigen and activity levels in conditioned medium at 48 hours ( P <0.001) when compared with serum-free conditions. A similar stimulatory effect was seen with 10% pooled human serum. Moreover, epidermal growth factor and platelet-derived growth factor-B increased TFPI secretion by 4- to 5-fold and 2- to 3-fold, respectively ( P <0.05), and these growth factors accounted for ≈50% of the TFPI secretion effects of human serum. The serum effect was associated with a 3-fold increase in TFPI mRNA 24 hours after release from growth arrest and a 50% decrease in TFPI secretion after treatment with actinomycin D. Taken together, this study suggests that there is significant TFPI expression in VSMC in culture and in VSMC within the intima and media of the normal coronary artery wall. We present the first evidence for TFPI regulation by serum in VSMC and more specifically by its constituent growth factors, epidermal growth factor and platelet-derived growth factor-B.

Abstract—A defining property of ATP-sensitive K+(KATP) channels is inhibition by sulfonylurea drugs, yet the response of cardiac KATPchannels toward sulfonylureas during myocardial ischemia is not consistent. Altered channel sensitivity toward sulfonylureas has, in part, been ascribed to antagonism by cytosolic nucleotide diphosphates, although the mechanism of interaction remains unclear. Herein, in inside-out patches excised from cardiomyocytes, we observed a dual response of KATPchannels toward the sulfonylurea drug, glyburide, in the presence of cytosolic UDP. Specifically, glyburide failed to inhibit spontaneous KATPchannel activity in the presence of UDP but inhibited UDP-induced channel activity after rundown of spontaneous channel openings. Such behavior of KATPchannels cannot be explained by differences in the level of channel activity or by UDP-induced displacement of glyburide. Rather, the dual response toward the sulfonylurea could be attributed to a property of KATPchannels to switch between operative conditions (spontaneous versus UDP-induced) each associated with a distinct responsiveness toward ligands. Conversion of post-rundown KATPchannels to the spontaneously operative channel condition, by Mg-ATP, restored the ability of UDP to antagonize the inhibitory action of glyburide lost after rundown, suggesting that the response of the channel to glyburide is phosphorylation dependent. The existence of distinct operative conditions of cardiac KATPchannels could be the basis for the inconsistent response of the channel toward sulfonylurea drugs and should be considered when sulfonylureas are used to implicate the opening of KATPchannels in the myocardium.