Neuropeptide Y gene polymorphisms confer risk of early-onset atherosclerosis.

Neuropeptide Y (NPY) is a strong candidate gene for coronary artery disease (CAD). We have previously identified genetic linkage to familial CAD in the genomic region of NPY. We performed follow-up genetic, biostatistical, and functional analysis of NPY in early-onset CAD. In familial CAD (GENECARD, N = 420 families), we found increased microsatellite linkage to chromosome 7p14 (OSA LOD = 4.2, p = 0.004) in 97 earliest age-of-onset families. Tagged NPY SNPs demonstrated linkage to CAD of a 6-SNP block (LOD = 1.58-2.72), family-based association of this block with CAD (p = 0.02), and stronger linkage to CAD in the earliest age-of-onset families. Association of this 6-SNP block with CAD was validated in: (a) 556 non-familial early-onset CAD cases and 256 controls (OR 1.46-1.65, p = 0.01-0.05), showing stronger association in youngest cases (OR 1.84-2.20, p = 0.0004-0.09); and (b) GENECARD probands versus non-familial controls (OR 1.79-2.06, p = 0.003-0.02). A promoter SNP (rs16147) within this 6-SNP block was associated with higher plasma NPY levels (p = 0.04). To assess a causal role of NPY in atherosclerosis, we applied the NPY1-receptor-antagonist BIBP-3226 adventitially to endothelium-denuded carotid arteries of apolipoprotein E-deficient mice; treatment reduced atherosclerotic neointimal area by 50% (p = 0.03). Thus, NPY variants associate with atherosclerosis in two independent datasets (with strong age-of-onset effects) and show allele-specific expression with NPY levels, while NPY receptor antagonism reduces atherosclerosis in mice. We conclude that NPY contributes to atherosclerosis pathogenesis.

GATA2 is associated with familial early-onset coronary artery disease.

The transcription factor GATA2 plays an essential role in the establishment and maintenance of adult hematopoiesis. It is expressed in hematopoietic stem cells, as well as the cells that make up the aortic vasculature, namely aortic endothelial cells and smooth muscle cells. We have shown that GATA2 expression is predictive of location within the thoracic aorta; location is suggested to be a surrogate for disease susceptibility. The GATA2 gene maps beneath the Chromosome 3q linkage peak from our family-based sample set (GENECARD) study of early-onset coronary artery disease. Given these observations, we investigated the relationship of several known and novel polymorphisms within GATA2 to coronary artery disease. We identified five single nucleotide polymorphisms that were significantly associated with early-onset coronary artery disease in GENECARD. These results were validated by identifying significant association of two of these single nucleotide polymorphisms in an independent case-control sample set that was phenotypically similar to the GENECARD families. These observations identify GATA2 as a novel susceptibility gene for coronary artery disease and suggest that the study of this transcription factor and its downstream targets may uncover a regulatory network important for coronary artery disease inheritance.

Genetic and functional association of FAM5C with myocardial infarction.

BACKGROUND: We previously identified a 40 Mb region of linkage on chromosome 1q in our early onset coronary artery disease (CAD) genome-wide linkage scan (GENECARD) with modest evidence for linkage (n = 420, LOD 0.95). When the data are stratified by acute coronary syndrome (ACS), this modest maximum in the overall group became a well-defined LOD peak (maximum LOD of 2.17, D1S1589/D1S518). This peak overlaps a recently identified inflammatory biomarker (MCP-1) linkage region from the Framingham Heart Study (maximum LOD of 4.27, D1S1589) and a region of linkage to metabolic syndrome from the IRAS study (maximum LOD of 2.59, D1S1589/D1S518). The overlap of genetic screens in independent data sets provides evidence for the existence of a gene or genes for CAD in this region. METHODS: A peak-wide association screen (457 SNPs) was conducted of a region 1 LOD score down from the peak marker (168-198 Mb) in a linkage peak for acute coronary syndrome (ACS) on chromosome 1, within a family-based early onset coronary artery disease (CAD) sample (GENECARD). RESULTS: Polymorphisms were identified within the 'family with sequence similarity 5, member C' gene (FAM5C) that show genetic linkage to and are associated with myocardial infarction (MI) in GENECARD. The association was confirmed in an independent CAD case-control sample (CATHGEN) and strong association with MI was identified with single nucleotide polymorphisms (SNPs) in the 3' end of FAM5C. FAM5C genotypes were also correlated with expression of the gene in human aorta. Expression levels of FAM5C decreased with increasing passage of proliferating aortic smooth muscle cells (SMC) suggesting a role for this molecule in smooth muscle cell proliferation and senescence. CONCLUSION: These data implicate FAM5C alleles in the risk of myocardial infarction and suggest further functional studies of FAM5C are required to identify the gene's contribution to atherosclerosis.

Inhibition of cortisol production with metyrapone prevents mental stress-induced endothelial dysfunction and baroreflex impairment.

OBJECTIVES: This study was designed to investigate the role of cortisol in stress-induced endothelial dysfunction and impaired baroreflex sensitivity (BRS) by blocking cortisol production with metyrapone before subjecting healthy volunteers to mental stress. BACKGROUND: Mental stress raises cortisol levels and is associated with increased coronary heart disease (CHD) morbidity and mortality, especially from sudden cardiac death. It also causes endothelial dysfunction and impaired BRS. METHODS: We measured brachial artery flow-mediated dilation (FMD), a measure of endothelial function, and BRS in 36 subjects without CHD risk factors who were then randomized in a double-blind fashion to oral metyrapone 750 mg x 2 or placebo. Five hours later we subjected subjects to mental stress and then remeasured endothelial function and BRS. RESULTS: Prestress cortisol levels were significantly higher in the placebo group at 270.5 (30.9) nmol/l versus 89.1 (11.8) nmol/l (p = 0.01), and the increase with stress was higher at 57.9 (17.9) nmol/l versus 11.2 (2.2) nmol/l (p < 0.001). In the placebo group, compared to baseline, FMD and BRS fell significantly from 4.5% (0.7%) to 1.4% (1.1%) (p = 0.02) and 21.4 (2.3) ms/mmHg to 16.3 (1.5) ms/mmHg (p = 0.04), respectively. In the metyrapone group, FMD and BRS were unchanged from baseline: 4.3% (0.9%) versus 5.1% (0.8%) (p = 0.48) and 26.4 (2.9) ms/mmHg versus 24.9 (2.6) ms/mmHg (p = 0.62), respectively. Analysis of covariation showed a significant effect of metyrapone on change in both FMD (p = 0.009) and BRS (p = 0.024). CONCLUSIONS: Stress-related endothelial dysfunction and BRS impairment can be prevented by blocking cortisol production with metyrapone, demonstrating a direct or facilitative role for cortisol in these phenomena and suggesting mechanisms by which stress contributes to CHD and sudden cardiac death.

Red blood cell nitric oxide as an endocrine vasoregulator: a potential role in congestive heart failure.

BACKGROUND: A respiratory cycle for nitric oxide (NO) would involve the formation of vasoactive metabolites between NO and hemoglobin during pulmonary oxygenation. We investigated the role of these metabolites in hypoxic tissue in vitro and in vivo in healthy subjects and patients with congestive heart failure (CHF). METHODS AND RESULTS: We investigated the capacity for red blood cells (RBCs) to dilate preconstricted aortic rings under various O2 tensions. RBCs induced cyclic guanylyl monophosphate-dependent vasorelaxation during hypoxia (35+/-4% at 1% O2, 4.7+/-1.6% at 95% O2; P<0.05). RBC-induced relaxations during hypoxia correlated with S-nitrosohemoglobin (SNO-Hb) (R2=0.88) but not iron nitrosylhemoglobin (HbFeNO) content. Relaxation responses for RBCs were compared with S-nitrosoglutathione across a range of O2 tensions. The fold increases in relaxation evoked by RBCs were significantly greater at 1% and 2% O2 compared with relaxations induced at 95% (P<0.05), consistent with an allosteric mechanism of hypoxic vasodilation. We also measured transpulmonary gradients of NO metabolites in healthy control subjects and in patients with CHF. In CHF patients but not control subjects, levels of SNO-Hb increase from 0.00293+/-0.00089 to 0.00585+/-0.00137 mol NO/mol hemoglobin tetramer (P=0.005), whereas HbFeNO decreases from 0.00361+/-0.00109 to 0.00081+/-0.00040 mol NO/mol hemoglobin tetramer (P=0.03) as hemoglobin is oxygenated in the pulmonary circulation. These metabolite gradients correlated with the hemoglobin O2 saturation gradient (P<0.05) and inversely with cardiac index (P<0.05) for both CHF patients and control subjects. CONCLUSIONS: We confirm that RBC-bound NO mediates hypoxic vasodilation in vitro. Transpulmonary gradients of hemoglobin-bound NO are evident in CHF patients and are inversely dependent on cardiac index. Hemoglobin may transport and release NO bioactivity to areas of tissue hypoxia or during increased peripheral oxygen extraction via an allosteric mechanism.

Peakwide mapping on chromosome 3q13 identifies the kalirin gene as a novel candidate gene for coronary artery disease.

A susceptibility locus for coronary artery disease (CAD) has been mapped to chromosome 3q13-21 in a linkage study of early-onset CAD. We completed an association-mapping study across the 1-LOD-unit-down supporting interval, using two independent white case-control data sets (CATHGEN, initial and validation) to evaluate association under the peak. Single-nucleotide polymorphisms (SNPs) evenly spaced at 100-kb intervals were screened in the initial data set (N=468). Promising SNPs (P<.1) were then examined in the validation data set (N=514). Significant findings (P<.05) in the combined initial and validation data sets were further evaluated in multiple independent data sets, including a family-based data set (N=2,954), an African American case-control data set (N=190), and an additional white control data set (N=255). The association between genotype and aortic atherosclerosis was examined in 145 human aortas. The peakwide survey found evidence of association in SNPs from multiple genes. The strongest associations were found in three SNPs from the kalirin (KALRN) gene, especially in patients with early-onset CAD (P=.00001-00028 in the combined CATHGEN data sets). In-depth investigation of the gene found that an intronic SNP, rs9289231, was associated with early-onset CAD in all white data sets examined (P<.05). In the joint analysis of all white early-onset CAD cases (N=332) and controls (N=546), rs9289231 was highly significant (P=.00008), with an odds-ratio estimate of 2.1. Furthermore, the risk allele of this SNP was associated with atherosclerosis burden (P=.03) in 145 human aortas. KALRN is a protein with many functions, including the inhibition of inducible nitric oxide synthase and guanine-exchange-factor activity. KALRN and two other associated genes identified in this study (CDGAP and MYLK) belong to the Rho GTPase-signaling pathway. Our data suggest the importance of the KALRN gene and the Rho GTPase-signaling pathway in the pathogenesis of CAD.

Metyrapone improves endothelial dysfunction in patients with treated depression.

OBJECTIVES: This study sought to examine the effect of metyrapone on endothelial dysfunction in patients with treated recurrent major depression. BACKGROUND: Depression is an independent risk factor for the development of coronary heart disease, and patients with depression have endothelial dysfunction, an atherogenic abnormality. This abnormality may be attributable to abnormal hypothalamic-pituitary-adrenal (HPA) axis function, a feature of depression, resulting in increased exposure to cortisol. Cortisol administration produces endothelial dysfunction in healthy subjects. METHODS: We measured endothelial function using flow-mediated dilation (FMD) of the brachial artery in 30 patients with depression and in 36 matched control subjects. Patients were randomized (double blind) to metyrapone (an inhibitor of cortisol synthesis) or placebo, and FMD was remeasured 6 h later. RESULTS: At baseline, FMD was impaired in patients versus control subjects (mean [standard error]), -1.27% [0.91%] vs. 4.37% [0.59%] (p < 0.001). The FMD was similar in the placebo and the metyrapone patient groups at baseline (0.17% [1.04%] vs. -2.72% [1.30%], p = 0.11). Metyrapone significantly reduced plasma cortisol levels. There was a significant improvement in FMD in the metyrapone group from -2.72% [1.30%] to 3.82% [0.99%] (p < 0.001), whereas the change in the placebo group, from 0.17% [1.04%] to 1.15% [1.14%], was not significant. Analysis of covariation showed that the effect of metyrapone was significant (p = 0.034). CONCLUSIONS: Inhibition of cortisol production by metyrapone ameliorates the endothelial dysfunction seen in depression, suggesting that the mechanism of the endothelial dysfunction may involve cortisol.

Compression and expansion wavefront travel in canine ascending aortic flow: wave intensity analysis.

The forces underlying left ventricular ejection were investigated by applying a wavefront analysis to blood pressure (P) and velocity (U) waveforms measured in the ascending aorta of anesthetized dogs (n = 13). Wavefronts travel forward (to the periphery) and/or backward (to the heart) after peripheral reflection. They are characterized by the rate of pressure change they cause, i.e., the time derivative of pressure (dP/dt): compression wavefronts have dP/dt > 0: expansion wavefronts have dP/dt < 0. Wave intensity is defined as (dP/dt)(dU/dt), where dU/dt is the time derivative of U. Forward wavefronts contribute positively to wave intensity and backward wavefronts contribute negatively. Therefore, wave intensity indicates whether the effects of forward wavefronts are predominant or whether those of backward wavefronts predominate in the formation of pressure and velocity waveforms. Under control conditions, wave intensity was positive in early and late systole, indicating that forward compression and expansion wavefronts dominate aortic acceleration and deceleration, respectively. Compression wave intensity was increased during inotropic stimulation by dobutamine (10-15 microg/kg per min i.v. infusion; +161% +/- 31% mean change in peak value +/- SEM (%), P < 0.05), and was reduced during beta-blockade by propranolol (1 mg/kg i.v. injection; -58% +/- 7%, P < 0.05). Expansion wave intensity was unchanged by dobutamine and propranolol (n = 6). In a separate group of animals (n = 7), expansion wave intensity was reduced during vasodilatation by nitroglycerin (0.5mg i.v. injection and 0.02 microg/kg per min infusion; -32% +/- 12%, P < 0.05), but was unchanged during vasoconstriction by methoxamine (2 mg i.v. injection). However, methoxamine reduced compression wave intensity (-46% +/- 14%, P < 0.05). These results indicate that (1) compression and expansion wavefronts generated by the left ventricle dominate acceleration and deceleration in the ascending aorta, (2) compression wave intensity is related to the inotropic state of the left ventricle, but is reduced during vasoconstriction, and (3) expansion wave intensity is reduced during vasodilatation. This time domain analysis of traveling wavefronts readily provides information concerning the dynamics of the ventriculoarterial interaction.

Wave intensity analysis from the common carotid artery: a new noninvasive index of cerebral vasomotor tone.

Cerebral vasomotor tone is difficult to assess in patients. Wave intensity analysis has been applied to resolve complex upstream and downstream events within the vascular system. We hypothesized that the backward-traveling wave measured in the common carotid artery was caused by reflection from the cerebrovascular "beach", and that the magnitude of this reflected wave would be altered by changes in cerebral vasomotor tone. We measured common carotid arterial diameter and velocity of flow to calculate wave intensity in ten healthy male volunteers (age mean 31 +/- 3 years). Applying a rebreathing technique, we were able to increase the inspired carbon dioxide concentration to a mean of 5.9% +/- 1.7% and to compare baseline wave intensity readings to those recorded during hypercapnia. The magnitude of the reflected wave decreased significantly after CO(2) rebreathing, from -43.0 +/- 27.1 to -25.0 +/- 16.9 mmHg m s(-2), P = 0.02. This reduction in negative wave reflections in mid-systole during hypercapnia remained significant when it was analyzed as the reflection coefficient (the magnitude of the reflected wave normalized for the magnitude of the initiating forward wave, which fell from -2.8 +/- 1.5 to -1.6 +/- 1.4 ms (P = 0.01). Carotid wave reflection was significantly decreased during cerebral vasodilatation induced by increased arterial pCO(2). Wave intensity may provide a simple noninvasive means of assessing changes in cerebral vasomotor tone in vivo.

A genomewide scan for early-onset coronary artery disease in 438 families: the GENECARD Study.

A family history of coronary artery disease (CAD), especially when the disease occurs at a young age, is a potent risk factor for CAD. DNA collection in families in which two or more siblings are affected at an early age allows identification of genetic factors for CAD by linkage analysis. We performed a genomewide scan in 1,168 individuals from 438 families, including 493 affected sibling pairs with documented onset of CAD before 51 years of age in men and before 56 years of age in women. We prospectively defined three phenotypic subsets of families: (1) acute coronary syndrome in two or more siblings; (2) absence of type 2 diabetes in all affected siblings; and (3) atherogenic dyslipidemia in any one sibling. Genotypes were analyzed for 395 microsatellite markers. Regions were defined as providing evidence for linkage if they provided parametric two-point LOD scores >1.5, together with nonparametric multipoint LOD scores >1.0. Regions on chromosomes 3q13 (multipoint LOD = 3.3; empirical P value <.001) and 5q31 (multipoint LOD = 1.4; empirical P value <.081) met these criteria in the entire data set, and regions on chromosomes 1q25, 3q13, 7p14, and 19p13 met these criteria in one or more of the subsets. Two regions, 3q13 and 1q25, met the criteria for genomewide significance. We have identified a region on chromosome 3q13 that is linked to early-onset CAD, as well as additional regions of interest that will require further analysis. These data provide initial areas of the human genome where further investigation may reveal susceptibility genes for early-onset CAD.