Curt D. Sigmund

Ghrelin Inhibits Proinflammatory Responses and Nuclear Factor-κB Activation in Human Endothelial Cells

Background—Ghrelin is a novel growth hormone–releasing peptide that has been shown to improve cachexia in heart failure and cancer and to ameliorate the hemodynamic and metabolic disturbances in septic shock. Because cytokine-induced inflammation is critical in these pathological states and because the growth hormone secretagogue receptor has been identified in blood vessels, we examined whether ghrelin inhibits proinflammatory responses in human endothelial cells in vitro and after administration of endotoxin to rats in vivo. Methods and Results—Human umbilical vein endothelial cells (HUVECs) were treated with or without tumor necrosis factor-&agr;(TNF-&agr;), and induction of proinflammatory cytokines and mononuclear cell adhesion were determined. Ghrelin (0.1 to 1000 ng/mL) inhibited both basal and TNF-&agr;–induced cytokine release and mononuclear cell binding. Intravenous administration of ghrelin also inhibited endotoxin-induced proinflammatory cytokine production in rats in vivo. Ghrelin inhibited H2O2-induced cytokine release in HUVECs, suggesting that the peptide blocks redox-mediated cellular signaling. Moreover, ghrelin inhibited basal and TNF-&agr;–induced activation of nuclear factor-κB. Des-acyl ghrelin had no effect on TNF-&agr;–induced cytokine production in HUVECs, suggesting that the antiinflammatory effects of ghrelin require interaction with endothelial growth hormone secretagogue receptors. Conclusions—Ghrelin inhibits proinflammatory cytokine production, mononuclear cell binding, and nuclear factor-κB activation in human endothelial cells in vitro and endotoxin-induced cytokine production in vivo. These novel antiinflammatory actions of ghrelin suggest that the peptide could play a modulatory role in atherosclerosis, especially in obese patients, in whom ghrelin levels are reduced.

Viewpoint: Are Studies in Genetically Altered Mice Out of Control?

Because the use of transgenic and gene-targeted models has increased in popularity, the number of reports describing unpredictable phenotypic effects caused by variation in the genetic background used to generate or propagate these models has steadily increased. There are now many examples in which animals containing the same exact genetic manipulation exhibit profoundly different phenotypes when present on diverse genetic backgrounds, demonstrating that genes unrelated, per se, to the ones being targeted can play a significant role in the observed phenotype. Herein, I will discuss (1) the source of genetic variability in mutant mouse models, (2) the appropriateness of using inbred mice as controls, and (3) strategies to help minimize genetic variation between experimental and control mice.

Contrasting blood pressure effects of obesity in leptin-deficient ob/ob mice and agouti yellow obese mice.

OBJECTIVE Recent advances in understanding the neuroendocrine pathways regulating appetite, metabolism and body weight afford an opportunity to explore further the mechanisms by which obesity influences arterial pressure. ob/ob(Lep(ob)/Lep(ob)) mice have a mutation in the ob gene and are leptin-deficient. Leptin possesses pressor actions and has been shown to increase arterial pressure when infused chronically or over-expressed transgenically. In contrast, agouti yellow obese(Ay) mice have overexpression of an agouti peptide that blocks melanocortin receptors. Stimulation of melanocortin receptors by alpha-melanocyte-stimulating hormone decreases arterial pressure. DESIGN AND METHODS This study measured arterial pressure in leptin-deficient ob/ob mice, agouti yellow obese mice and their lean controls to test the hypothesis that the effects of obesity on arterial pressure are importantly influenced by the genetic and neuroendocrine mechanisms causing the obesity. We measured arterial pressure directly in conscious ob/ob mice (n = 14), agouti yellow obese mice (n = 6) and the same number of lean littermates. RESULTS Body weight was nearly twice as high in ob/ob mice as in their lean controls, but mean arterial pressure was significantly lower in ob/ob mice (92+/-3 mmHg) compared with their lean controls (106+/-2 mmHg; P = 0.00017). In contrast, mean arterial pressure was significantly higher in agouti yellow obese mice (124+/-3 mmHg) than in their lean controls (99+/-1 mmHg; P = 0.000002) despite the fact that the agouti mice had milder obesity. CONCLUSIONS This study prompts three conclusions: (1) leptin-deficient ob/ob mice and agouti yellow obese mice have contrasting blood pressure responses to obesity, (2) obesity does not invariably increase arterial pressure in mice, and (3) the arterial pressure response to obesity may depend critically on the underlying genetic and neuroendocrine mechanisms.

Increased Superoxide and Vascular Dysfunction in CuZnSOD-Deficient Mice

Abstract— Increased superoxide is thought to play a major role in vascular dysfunction in a variety of disease states. Superoxide dismutase (SOD) limits increases in superoxide; however, the functional significance of selected isoforms of SOD within the vessel wall are unknown. We tested the hypothesis that selective loss of CuZnSOD results in increased superoxide and altered vascular responsiveness in CuZnSOD-deficient (CuZnSOD−/−) mice compared with wild-type (CuZnSOD+/+) littermates. Total SOD activity was reduced (P <0.05) by approximately 60% and CuZnSOD protein was absent in aorta from CuZnSOD−/− as compared with wild-type mice. Vascular superoxide levels, measured using lucigenin (5 &mgr;mol/L)-enhanced chemiluminescence and hydroethidine (2 &mgr;mol/L)-based confocal microscopy, were increased (approximately 2-fold;P <0.05) in CuZnSOD−/− mice as compared with wild-type mice. Relaxation of the carotid artery in response to acetylcholine and authentic nitric oxide was impaired (P <0.05) in CuZnSOD−/− mice. For example, maximal relaxation to acetylcholine (100 &mgr;mol/L) was 50±6% and 69±5% in CuZnSOD−/− and wild-type mice, respectively. Contractile responses of the carotid artery were enhanced (P <0.05) in CuZnSOD−/− mice in response to phenylephrine and serotonin, but not to potassium chloride or U46619. In vivo, dilatation of cerebral arterioles (baseline diameter=31±1 &mgr;m) to acetylcholine was reduced by approximately 50% in CuZnSOD−/− mice as compared with wild-type mice (P <0.05). These findings provide the first direct insight into the functional importance of CuZnSOD in blood vessels and indicate that this specific isoform of SOD limits increases in superoxide under basal conditions. CuZnSOD-deficiency results in altered responsiveness in both large arteries and microvessels.

PPARγ Agonist Rosiglitazone Improves Vascular Function and Lowers Blood Pressure in Hypertensive Transgenic Mice

Abstract—The peroxisome proliferator activated receptor (PPAR&ggr;) agonist rosiglitazone has been reported to yield cardiovascular benefits in patients by a mechanism that is not completely understood. We tested whether oral rosiglitazone (25 mg/kg per day, 21 days) treatment improves blood pressure and vascular function in a transgenic mouse expressing both human renin and human angiotensinogen transgenes (R+A+). Rosiglitazone decreased systolic (138±5 versus 128±5 mm Hg) and mean blood pressure (145±5 versus 126±7 mm Hg) of R+A+ mice as measured by tail-cuff and indwelling carotid catheters, respectively. Relaxation of carotid arteries to acetylcholine and authentic nitric oxide, but not papaverine, was impaired in R+A+ mice when compared with littermate controls (RA−). There were no effects of rosiglitazone on RA− mice; however, relaxation to acetylcholine (49±10 versus 82±9% at 100 &mgr;mol/L) and nitric oxide (51±11 versus 72±6% at 10 &mgr;mol/L) was significantly improved in treated R+A+ mice. Rosiglitazone treatment of R+A+ mice did not alter the expression of genes, including endothelial nitric oxide synthase (eNOS), angiotensin 1 receptors, and preproendothelin-1, nor did it alter the levels of eNOS or soluble guanylyl cyclase protein. In separate studies, carotid arteries from R+A+ and RA− mice relaxed in a concentration-dependent manner to rosiglitazone, suggesting possible PPAR&ggr;-independent effects in the vasculature. This response was not inhibited with the nitric oxide synthase inhibitor N&ohgr;-nitro-l-arginine methyl ester (200 &mgr;mol/L) or the PPAR&ggr; antagonist bisphenol A diglycidyl ether; 4,4′-isopropylidenediphenol diglycidyl ether (100 &mgr;mol/L). These data suggest that in addition to potential genomic regulation caused by PPAR&ggr; activation, the direct effect of rosiglitazone in blood vessels may contribute to the improved blood pressure and vessel function.

Chronic hypertension and altered baroreflex responses in transgenic mice containing the human renin and human angiotensinogen genes.

We have generated a transgenic model consisting of both the human renin and human angiotensinogen genes to study further the role played by the renin-angiotensin system in regulating arterial pressure. Transgenic mice containing either gene alone were normotensive, whereas mice containing both genes were chronically hypertensive. Plasma renin activity and plasma angiotensin II levels were both markedly elevated in the double transgenic mice compared with either single transgenic or nontransgenic controls. The elevation in blood pressure caused by the human transgenes was independent of the genotype at the endogenous renin locus and was equal in mice homozygous for the Ren-1c allele or in mice containing one copy each of Ren-1c, Ren-1d, or Ren-2. Chronic overproduction of angiotensin II in the double transgenic mice resulted in a resetting of the baroreflex control of heart rate to a higher pressure without significantly changing the gain or sensitivity of the reflex. Moreover, this change was not due to the effects of elevated pressure itself since angiotensin-converting enzyme inhibition had minimal effects on the baroreflex in spontaneously hypertensive BPH-2 control mice, which exhibit non-renin-dependent hypertension. This double transgenic model should provide an excellent tool for further studies on the mechanisms of hypertension initiated by the renin-angiotensin system.

Divergent functions of angiotensin II receptor isoforms in the brain.

The renin-angiotensin system (RAS) plays a critical role in cardiovascular and fluid homeostasis. The major biologically active peptide of the RAS is angiotensin II, which acts through G protein-coupled receptors of two pharmacological classes, AT(1) and AT(2). AT(1) receptors, expressed in brain and peripheral tissues, mediate most classically recognized actions of the RAS, including blood pressure homeostasis and regulation of drinking and water balance. In rodents, two highly homologous AT(1) receptor isoforms, termed AT(1A) and AT(1B) receptors, are expressed at different levels in major forebrain cardiovascular and fluid regulatory centers, with AT(1A) expression generally exceeding AT(1B) expression, but the relative contributions of these receptor subtypes to central angiotensin II responses are not known. We used gene targeting in combination with a unique system for maintaining catheters in the cerebral ventricles of conscious mice to test whether there are differential roles for AT(1A) and AT(1B) receptors in responses elicited by angiotensin II in the brain. Here we show that the blood pressure increase elicited by centrally administered angiotensin II can be selectively ascribed to the AT(1A) receptor. However, the drinking response requires the presence of AT(1B) receptors. To our knowledge, this is the first demonstration of a primary and nonredundant physiological function for AT(1B) receptors.

Hypothalamic PI3K and MAPK differentially mediate regional sympathetic activation to insulin

The action of insulin in the central nervous system produces sympathetic nervous system activation (also called sympathoactivation), although the neuronal intracellular mechanisms that mediate this are unclear. We hypothesized that PI3K and MAPK, the major pathways involved in insulin receptor signaling, mediate sympathetic nerve responses to insulin. Intracerebroventricular administration of insulin in rat increased multifiber sympathetic nerve activity to the hindlimb, brown adipose tissue (BAT), adrenal gland, and kidney. Ex vivo biochemical studies of mediobasal hypothalamic tissue revealed that insulin stimulated the association of insulin receptor substrate-1 with the p85alpha subunit of PI3K and also tyrosine phosphorylation of p42 and p44 subunits of MAPK in the hypothalamus. In order to determine whether PI3K and/or MAPK were involved in insulin-mediated sympathoactivation, we tested the effect of specific inhibitors of PI3K (LY294002 and wortmannin) and MAPK (PD98059 and U0126) on regional sympathetic responses to insulin. Interestingly, regional sympathoactivation to insulin was differentially affected by blockade of PI3K and MAPK. Inhibition of PI3K specifically blocked insulin-induced sympathoactivation to the hindlimb, while inhibition of MAPK specifically blocked insulin-induced sympathoactivation to BAT. Sympathoactivation to corticotrophin-releasing factor, however, was not affected by inhibition of PI3K and MAPK. These data demonstrate that PI3K and MAPK are specific and regionally selective mediators of the action of insulin on the sympathetic nervous system.

Oxidation of CaMKII determines the cardiotoxic effects of aldosterone

Excessive activation of the β-adrenergic, angiotensin II (Ang II) and aldosterone signaling pathways promotes mortality after myocardial infarction, and antagonists targeting these pathways are core therapies for treating this condition. Catecholamines and Ang II activate the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII), the inhibition of which prevents isoproterenol-mediated and Ang II–mediated cardiomyopathy. Here we show that aldosterone exerts direct toxic actions on myocardium by oxidative activation of CaMKII, causing cardiac rupture and increased mortality in mice after myocardial infarction. Aldosterone induces CaMKII oxidation by recruiting NADPH oxidase, and this oxidized and activated CaMKII promotes matrix metalloproteinase 9 (MMP9) expression in cardiomyocytes. Myocardial CaMKII inhibition, overexpression of methionine sulfoxide reductase A (an enzyme that reduces oxidized CaMKII) or NADPH oxidase deficiency prevented aldosterone-enhanced cardiac rupture after myocardial infarction. These findings show that oxidized myocardial CaMKII mediates the cardiotoxic effects of aldosterone on the cardiac matrix and establish CaMKII as a nodal signal for the neurohumoral pathways associated with poor outcomes after myocardial infarction.

[46] Antibiotic resistance mutations in ribosomal RNA genes of Escherichia coli

Publisher Summary This chapter outlines the strategies for selecting mutants. The chapter also describes procedures to map the mutations to small regions of rRNA operons, facilitating identification of the mutations by DNA sequencing. The chapter also describes a primer extension method using rRNA templates and DNA oligonucleotide primers that permits convenient and accurate quantitation of the percent mutant rRNA in an rRNA preparation. This primer extension method is useful for determining the structural and functional capabilities of ribosomes synthesized from a single mutant rRNA operon (e.g., by analysis of total cellular ribosomes or ribosomes on polysomes) and provides a convenient way to determine if newly isolated mutations have base changes at the same position as previously isolated mutations. It also describes how a similar primer extension method can be used to accurately determine the copy number of mutant rRNA genes (e.g., plasmid copy number), permitting reliable quantitative analysis of rRNA gene expression.