Utility of blood pressure genetic risk score in admixed Hispanic samples.

Hypertension is strongly influenced by genetic factors. Although hypertension prevalence in some Hispanic sub-populations is greater than in non-Hispanic whites, genetic studies on hypertension have focused primarily on samples of European descent. A recent meta-analysis of 200 000 individuals of European descent identified 29 common genetic variants that influence blood pressure, and a genetic risk score derived from the 29 variants has been proposed. We sought to evaluate the utility of this genetic risk score in Hispanics. The sample set consists of 1994 Hispanics from 2 cohorts: the Northern Manhattan Study (primarily Dominican/Puerto Rican) and the Miami Cardiovascular Registry (primarily Cuban/South American). Risk scores for systolic and diastolic blood pressure were computed as a weighted sum of the risk alleles, with the regression coefficients reported in the European meta-analysis used as weights. Association of risk score with blood pressure was tested within each cohort, adjusting for age, age2, sex and body mass index. Results were combined using an inverse-variance meta-analysis. The risk score was significantly associated with blood pressure in our combined sample (P=5.65 × 10-4 for systolic and P=1.65 × 10-3 for diastolic) but the magnitude of the effect sizes varied by degree of European, African and Native American admixture. Further studies among other Hispanic sub-populations are needed to elucidate the role of these 29 variants and identify additional genetic and environmental factors contributing to blood pressure variability in Hispanics.

Polymorphisms of the tumor suppressor gene LSAMP are associated with left main coronary artery disease.

Previous association mapping on chromosome 3q13-21 detected evidence for association at the limbic system-associated membrane protein (LSAMP) gene in individuals with late-onset coronary artery disease (CAD). LSAMP has never been implicated in the pathogenesis of CAD. We sought to thoroughly characterize the association and the gene. Non-redundant single nucleotide polymorphisms (SNPs) across the gene were examined in an initial dataset (168 cases with late-onset CAD, 149 controls). Stratification analysis on left main CAD (N = 102) revealed stronger association, which was further validated in a validation dataset (141 cases with left main CAD, 215 controls), a third control dataset (N = 255), and a family-based dataset (N = 2954). A haplotype residing in a novel alternative transcript of the LSAMP gene was significant in all independent case-control datasets (p = 0.0001 to 0.0205) and highly significant in the joint analysis (p = 0.00004). Lower expression of the novel alternative transcript was associated with the risk haplotype (p = 0.0002) and atherosclerosis burden in human aortas (p = 0.0001). Furthermore, silencing LSAMP expression in human aortic smooth muscle cells (SMCs) substantially augmented SMC proliferation (p<0.01). Therefore, the risk conferred by the LSAMP haplotype appears to be mediated by LSAMP down-regulation, which may promote SMC proliferation in the arterial wall and progression of atherosclerosis.

Redox regulation of human Rac1 stability by the proteasome in human aortic endothelial cells.

Rac1 has been shown to activate a NADPH oxidase complex producing superoxide anions in a variety of mammalian cell types. We evaluated the impact of Rac1-induced reactive oxygen species production on the turnover of Rac1 itself in human aortic endothelial cells. The concentration of a constitutively active mutant of Rac1 (Rac1(V12)) was increased by treatment of the cells with diphenylene iodinium (DPI), an inhibitor of the NADPH oxidase. Such an effect was not observed for the dominant negative form of Rac1 (Rac1(N17)). We showed a decrease in proteolytic degradation of Rac1(V12) in the presence of DPI, and showed that short term treatment with H(2)O(2) reverses the effect of DPI. We found that proteasome inhibitors (lactacystin and MG132) increased Rac1(V12) protein level. In support of this finding, we have identified in the primary sequence of Rac1 a potential destruction box domain, which is known to be a signal for protein degradation mediated by the ubiquitin/proteasome system. We show that Rac1(V12) is ubiquitinated before degradation. By contrast Rac1(N17) induces an accumulation of the ubiquitinated form of Rac1. These results suggest that Rac1 activation of NADPH oxidase is necessary for the proteolytic degradation of Rac1 itself.

Redox regulation of vascular smooth muscle cell differentiation.

Experiments were performed to determine the role of reactive oxygen species (ROS) in regulating vascular smooth muscle cell (VSMC) phenotype. After quiescence, cultured human VSMCs increased their expression of differentiation proteins (alpha-actin, calponin, and SM1 and SM2 myosin), but not beta-actin. ROS activity, determined using the H(2)O(2)-sensitive probe dichlorodihydrofluorescein (DCF), remained high in quiescent cells and was inhibited by catalase (3000 U/mL) or by N-acetylcysteine (NAC, 2 to 20 mmol/L). A superoxide dismutase mimic (SOD; MnTMPyP, 25 micromol/L) or SOD plus low concentrations of NAC (SODNAC2, 2 mmol/L) increased DCF fluorescence, which was inhibited by catalase or by NAC (10 to 20 mmol/L). Inhibition of ROS activity (by catalase or NAC) decreased the baseline expression of differentiation proteins, whereas elevation of ROS (by SOD or SODNAC2) increased expression of the differentiation markers. The latter effect was blocked by catalase or by NAC (10 to 20 mmol/L). None of the treatments altered beta-actin expression. SODNAC2-treated cells demonstrated contractions to endothelin that were absent in proliferating cells. p38 Mitogen-activated protein kinase (MAPK) activity was decreased when ROS activity was reduced (NAC, 10 mmol/L) and was augmented when ROS activity was increased (SODNAC2). Inhibition of p38 MAPK with pyridyl imidazole compound (SB202190, 2 to 10 micromol/L) reduced expression of differentiation proteins occurring under basal conditions and in response to SODNAC2. Transduction of VSMCs with an adenovirus encoding constitutively active MKK6, an activator of p38 MAPK, increased expression of differentiation proteins, whereas transduction with an adenovirus encoding dominant-negative p38 MAPK decreased expression of the differentiation proteins. These findings demonstrate that ROS can increase VSMC differentiation through a p38 MAPK-dependent pathway.

Redox signaling of the arteriolar myogenic response.

Arteriolar vascular smooth muscle cells (VSMCs) are mechanosensitive, constricting to elevations in transmural pressure (P(TM)). The goal of the present study was to determine using mouse isolated tail arterioles and arteries whether oxidant signaling regulates this myogenic response. In response to P(TM) elevation, VSMCs of arterioles but not arteries generated constriction and increased reactive oxygen species (ROS) activity (using the H(2)O(2)-sensitive probe dichlorodihydrofluorescein). Arterioles had increased expression of NADPH oxidase components compared with arteries. Inhibition of NADPH oxidase, using mice with targeted impairment of enzyme components (p47(phox) or rac1) or diphenyleneiodonium, prevented the pressure-induced generation of ROS. When ROS activity was inhibited, either by inhibiting NADPH oxidase or with N-acetylcysteine, the myogenic constriction was abolished. The myogenic constriction was also inhibited by catalase, which inactivates H(2)O(2), but was unaffected by a cell-permeant mimic of superoxide dismutase (MnTMPyP). alpha(1)-Adrenergic constriction was not associated with altered ROS activity and was not affected by inhibition of NADPH oxidase or ROS. Exogenous H(2)O(2) constricted VSMCs of arterioles but not arteries. Thus, NADPH oxidase and ROS, in particular H(2)O(2), contribute to the myogenic response of arteriolar VSMCs.

The GPIIIa (beta3 integrin) PlA polymorphism in the early development of coronary atherosclerosis.

The GPIIIa (beta3 integrin) is an integral part of two glycoprotein receptors - the GP(IIb/IIIa) fibrinogen receptors in platelets and the GP(V/IIIa) vitronectin receptors in endothelium and vascular smooth muscle cells (vSMC). The PlA polymorphism of the gene for GPIIIa (beta3 integrin) has been suggested to play an important role in the progression of coronary artery disease (CAD) and in coronary thrombosis. Whether the action of the PlA polymorphism is due to differences in platelet aggregability or function of the vSMC and endothelial GPIIIa is not known. The association of the PlA polymorphism with the early, non-complicated atherosclerosis and CAD was studied in the Helsinki Sudden Death Study (HSDS) comprising two independent, autopsy series of altogether 700 middle-aged Caucasian Finnish men (33-70 year) suffering sudden out-of-hospital death. The burden of complicated lesions was greater in men with the A2 allele (heterozygotes or homozygotes for A2) (P=0.01) compared with PlA1/A1 homozygotes in the entire series. To further estimate the role of platelet-independent GPIIIa receptors, we excluded all cases with coronary thrombosis and thrombus-overlaid complicated lesions. In this subset of men, fibrous coronary lesions were more frequent (OR 2.9; P<0.01) in the coronary arteries of PlA1/A1 homozygotes compared with men with the PlA2 allele. Moreover, men with the PlA1/A1 genotype also had more stenotic coronary arteries (P<0.05) compared with men with the A2 allele at this early, non-complicated stage of atherosclerosis. The findings of this study suggest that Pl(A1/A1) homozygotes may be prone to early atherosclerosis and more rapid progression of stable CAD whereas carriers of the PlA2 allele are more prone to thrombotic complications. We hypothesize that the PlA polymorphism may account for the early atherosclerosis by affecting the function of endothelial and vSMC GP(V/IIIa) receptors, whereas the PlA polymorphism on platelet GP(IIb/IIIa) receptors may play a major role in coronary thrombosis.

The PlA polymorphism of glycoprotein IIIa functions as a modifier for the effect of estrogen on platelet aggregation.

BACKGROUND: Although estrogen has been shown to contribute to retardation of the development of coronary heart disease in premenopausal women, the efficacy of hormone replacement therapy for coronary heart disease prevention in women with established coronary heart disease remains controversial. Hence, additional research is needed to clarify the effects of hormone replacement therapy on the cardiovascular and clotting systems. We investigated the effect of estrogen on platelet aggregation induced by standard agonists (epinephrine and adenosine diphosphate), with and without the platelet antagonist aspirin. Furthermore, we analyzed our data according to the presence or absence of a prevalent polymorphism of the glycoprotein (GP) IIIa subunit of the platelet fibrinogen receptor GPIIb-IIIa, PlA2. METHODS AND RESULTS: The effect of estrogen on aggregation of platelets was studied in healthy men (n = 20, 10 PlA1/A1 and 10 PlA1/A2) and premenopausal healthy women (n = 10, 5 PlA1/A1 and 5 PlA1/A2). The PlA1/A1 and PlA1/A2 individuals were matched for age and race. Platelet response to agonists was investigated in the presence of (1) estrogen (10(-11) to 10(-8) mol/L), (2) aspirin (0.056 to 56 micromol/L), (3) estrogen plus aspirin, and (4) estrogen plus ICI 182 780 (ICI, 10(-9) mol/L, an inhibitor of the estrogen receptor). We found that physiologic concentrations of estrogen strongly and significantly inhibited the aggregation of PlA1/A2 platelets (P<.005 for epinephrine and P<.05 for adenosine diphosphate, induced aggregation, respectively) in both men and women. Concentrations of estrogen that were 1000-fold greater were required to observe the same level of inhibition with PlA1/A1 platelets. In the presence of aspirin, estrogen failed to provide additional inhibitory effect on aggregation of both PlA1/A1 and PlA1/A2 platelets. The estrogen-specific inhibitor ICI blocked the effect of estrogen on aggregation, suggesting that this effect is mediated by the estrogen receptor. CONCLUSIONS: Estrogen inhibits the aggregation of platelets, but such inhibition is highly dependent on the presence or absence of the PlA2 polymorphism of GPIIIa. However, in the presence of aspirin, the inhibitory effect of estrogen on aggregation was no longer detectable.

Contribution of monocytes/macrophages to compensatory neovascularization: the drilling of metalloelastase-positive tunnels in ischemic myocardium.

In a transgenic model of ischemic cardiomyopathy in which monocytes are attracted to the myocardium by the targeted overexpression of monocyte chemoattractant protein-1 (MCP-1), we have observed the presence of endothelial NO synthase and platelet endothelial cell adhesion molecule-1-negative tunnels, occasionally containing blood-derived cells, that probe the cardiac tissue. Immunohistochemical data show that monocytes/macrophages (MCs/Mphs) drill tunnels using the broad-spectrum mouse macrophage metalloelastase. 5-Bromo-2'-deoxyuridine incorporation and neo-endothelial markers present in the microvasculature of MCP-1 mouse hearts suggest an active angiogenic process. Further studies will be required to establish that the MC-/Mph-drilled tunnels evolve to become capillaries, connected to the existing vessels and colonized by circulating endothelial cell progenitors. This possibility is supported by the availability of these cells, which is demonstrated by cell tagging with beta-galactosidase placed under an active endothelial Tie-2 promoter. This phenomenon might represent another mechanism, in addition to the secretion of the angiogenic factors, by which MCs/MPhs may participate in the elaboration of new blood vessels in adult tissues.

Endothelial dysfunction in a murine model of mild hyperhomocyst(e)inemia.

Homocysteine is a risk factor for the development of atherosclerosis and its thrombotic complications. We have employed an animal model to explore the hypothesis that an increase in reactive oxygen species and a subsequent loss of nitric oxide bioactivity contribute to endothelial dysfunction in mild hyperhomocysteinemia. We examined endothelial function and in vivo oxidant burden in mice heterozygous for a deletion in the cystathionine beta-synthase (CBS) gene, by studying isolated, precontracted aortic rings and mesenteric arterioles in situ. CBS(-/+) mice demonstrated impaired acetylcholine-induced aortic relaxation and a paradoxical vasoconstriction of mesenteric microvessels in response to superfusion of methacholine and bradykinin. Cyclic GMP accumulation following acetylcholine treatment was also impaired in isolated aortic segments from CBS(-/+) mice, but aortic relaxation and mesenteric arteriolar dilation in response to sodium nitroprusside were similar to wild-type. Plasma levels of 8-epi-PGF(2alpha) (8-IP) were somewhat increased in CBS(-/+) mice, but liver levels of 8-IP and phospholipid hydroperoxides, another marker of oxidative stress, were normal. Aortic tissue from CBS(-/+) mice also demonstrated greater superoxide production and greater immunostaining for 3-nitrotyrosine, particularly on the endothelial surface. Importantly, endothelial dysfunction appears early in CBS(-/+) mice in the absence of structural arterial abnormalities. Hence, mild hyperhomocysteinemia due to reduced CBS expression impairs endothelium-dependent vasodilation, likely due to impaired nitric oxide bioactivity, and increased oxidative stress apparently contributes to inactivating nitric oxide in chronic, mild hyperhomocysteinemia.

Plant rac proteins induce superoxide production in mammalian cells.

The small GTP-binding protein family including Rac proteins represents a paradigm for signaling molecules shared by animal and plants. In mammalian cells, Rac induces the activation of NADPH oxidase leading to superoxide production. In plants, evidence suggests that resistance to pathogens depends on superoxide that is generated via NADPH oxidase-like enzymes. We have identified four closely related Rho/Rac genes from Zea mays that exhibit a high degree of homology to the human Rac. We hypothesized that these plant Rac proteins could function as their mammalian counterpart and activate an enzymatic complex that leads to superoxide production. Here, we show that like human Rac1, activated Zea mays Rac genes can induce superoxide production, when expressed in a mammalian system: NIH 3T3 cells. Our results suggest that in plants, Rac proteins can function as activators of oxidative burst and indicate the remarkable functional and structural conservation of Rho/Rac proteins between plant and animal kingdoms during evolution.

Sample size to test for interaction between a specific exposure and a second risk factor in a pair-matched case-control study.

We discuss a sample size calculation for a pair-matched case-control study to test for interaction between a specific exposure and a second risk factor. The second risk factor could be either binary or continuous. An algorithm for the calculation of sample size is suggested which is based on a logistic regression model that relates the logarithm of the disease-exposure odds ratio to the second risk factor. This problem is motivated by a study comparing the prevalence of GP-IIIa Pl(A2) polymorphism (the exposure) in individuals with and without myocardial infarction (case-control). One of the hypotheses in this study is whether or not there is an interaction between the prevalence of GP-IIIa Pl(A2) polymorphism and a second risk factor such as smoking status and homocysteine level. We introduce the algorithm in detail with several numerical examples.

Microtubule binding to Smads may regulate TGF beta activity.

Smad proteins are intracellular signaling effectors of the TGF beta superfamily. We show that endogenous Smad2, 3, and 4 bind microtubules (MTs) in several cell lines. Binding of Smads to MTs does not require TGF beta stimulation. TGF beta triggers dissociation from MTs, phosphorylation, and nuclear translocation of Smad2 and 3, with consequent activation of transcription in CCL64 cells. Destabilization of the MT network by nocodazole, colchicine, or a tubulin mutant disrupts the complex between Smads and MTs and increases TGF beta-induced Smad2 phosphorylation and transcriptional response in CCL64 cells. These data demonstrate that MTs may serve as a cytoplasmic sequestering network for Smads, controlling Smad2 association with and phosphorylation by activated TGF beta receptor I, and suggest a novel mechanism for the MT network to negatively regulate TGF beta function.