Methods and Results— Femoral arteries of 48 atherosclerotic rabbits underwent balloon stretch injury and were locally treated with either (1) _vβ₃-targeted rapamycin nanoparticles, (2) _vβ₃-targeted nanoparticles without rapamycin, (3) nontargeted rapamycin nanoparticles, or (4) saline. Intramural binding of integrin-targeted paramagnetic nanoparticles was confirmed with MR molecular imaging (1.5 T). MR angiograms were indistinguishable between targeted and control arteries at baseline, but 2 weeks later they showed qualitatively less luminal plaque in the targeted rapamycin treated segments compared with contralateral control vessels. In a first cohort of 19 animals (38 vessel segments), microscopic morphometric analysis of the rapamycin-treated segments revealed a 52% decrease in the neointima/media ratio (P<0.05) compared to control. No differences (P>0.05) were observed among balloon injured vessel segments treated with _vβ₃-targeted nanoparticles without rapamycin, nontargeted nanoparticles with rapamycin, or saline. In a second cohort of 29 animals, endothelial healing followed a parallel pattern over 4 weeks in the vessels treated with _vβ₃-targeted rapamycin nanoparticles and the 3 control groups.

Conclusions— Local intramural delivery of _vβ₃-targeted rapamycin nanoparticles inhibited stenosis without delaying endothelial healing after balloon injury.

Methods and Results— Intramuscular injection of adenovirus harboring AGF into the ischemic limb increased AGF production, which increased blood flow through induction of angiogenesis and arteriogenesis, thereby reducing the necessity for limb amputation. In vitro analysis showed that exposing human umbilical venous endothelial cells to AGF increased nitric oxide (NO) production through activation of an ERK1/2-endothelial NO synthetase (eNOS) signaling pathway. AGF-stimulated eNOS phosphorylation, NO production, and endothelial cell migration were all abolished by specific MEK1/2 inhibitors. Moreover, AGF did not restore blood flow to ischemic hind-limbs of either mice receiving NOS inhibitor L-NAME or eNOS knockout mice.

Conclusion— Activation of an ERK1/2-eNOS-NO pathway is a crucial signaling mechanism by which AGF increases blood flow through induction of angiogenesis and arteriogenesis. Further investigation of the regulation underlying AGF signaling pathway may contribute to develop a new clinical strategy for ischemic vascular diseases.

Methods and Results— Circulating Angptl4 was proteolytically cleaved into NH2-terminal coiled-coil domain (N-Angptl4) and COOH-terminal fibrinogen-like domain (C-Angptl4). Using amino acid sequencing analysis, we identified a major cleavage site between Lys¹⁶⁸ and Leu¹⁶⁹ and a minor cleavage site between Lys¹⁷⁰ and Met¹⁷¹ in mouse Angptl4. C-Angptl4, but not N-Angptl4, potently inhibited both bFGF- and VEGF-induced cell proliferation, migration, and tubule formation in endothelial cells, and prevented neovascularization in mice. Treatment of C-Angptl4 with PNGase F (an N-glycosidase) ablated its N-linked glycosylation, and also significantly attenuated its antiangiogenic activities. C-Angptl4 blocked bFGF-induced activation of ERK1/2 MAP kinase, but had no obvious effect on Akt and P38 MAP kinase. Furthermore, C-Angptl4 abrogated bFGF-induced phosphorylation of Raf-1 and MEK1/2, whereas neither auto-phosphorylation of FGF receptor-1 nor activation of Ras was affected, suggesting that the blockage occurs at the level of Raf-1 activation.

Conclusions— The carboxyl terminus of Angptl4 alone is sufficient to suppress angiogenesis, possibly through inhibiting the Raf/MEK/ERK1/2 MAP kinase pathway in endothelial cells.

Methods and Results— Transgenic mice overexpressing PKR1 in cardiomyocytes displayed no spontaneous abnormalities in cardiomyocytes but showed an increased number of epicardial-derived progenitor cells (EPDCs), capillary density, and coronary arterioles. Coculturing EPDCs with H9c2 cardiomyoblasts overexpressing PKR1 promotes EPDC differentiation into endothelial and smooth muscle cells, mimicking our transgenic model. Overexpressing PKR1 in H9c2 cardiomyoblasts or in transgenic hearts upregulated prokineticin-2 levels. Exogenous prokineticin-2 induces significant outgrowth from neonatal and adult epicardial explants, promoting EPDC differentiation. These prokineticin-2 effects were abolished in cardiac explants from mice with PKR1-null mutation. Reduced capillary density and prokineticin-2 levels in PKR1-null mutant hearts supports the hypothesis of an autocrine/paracrine loop between PKR1 and prokineticin-2.

Conclusion— Cardiomyocyte-PKR1 signaling upregulates its own ligand prokineticin-2 that acts as a paracrine factor, triggering EPDCs proliferation/differentiation. This study provides a novel insight for possible therapeutic strategies aiming at restoring pluripotency of adult EPDCs to promote neovasculogenesis by induction of cardiomyocyte PKR1 signaling.

Methods and Results— Expression of 59 genes obtained from coronary, carotid, and thoracic aortic arteries were characterized from diabetic (DM)/hypercholesterolemic (HC) swine (n=52) 1, 3, and 6 months after induction. Lesion development in the 3 arterial beds was quantified and characterized at 1, 3, 6, and 9 months. Progressive lesion development was observed in the coronary>thoracic aorta>>carotid arteries. Genes involved in cholesterol metabolism and insulin pathways were upregulated in coronaries>thoracic aortae>carotids. Inflammatory genes were more markedly upregulated in coronary arteries than the other 2 arteries. Genes implicated in plaque instability (eg, matrix metalloproteinase-9, CCL2 and Lp-PLA₂ mRNAs) were only upregulated at 6 months in coronary arteries.

Conclusions— Variable gene expression, both in regard to the arterial bed and duration of disease, was associated with variable plaque development and progression. These findings may provide further insight into the atherosclerotic process and development of potential therapeutic targets.

Methods and Results— Elastocalcinosis was induced in vivo and ex vivo using warfarin. Hemodynamic parameters, calcium deposition, elastin degradation, transforming growth factor (TGF)-β signaling, and elastase activity were evaluated at different time points in the in vivo model. Metalloproteinases, serine proteases, and cysteine proteases were blocked to measure their relative implication in elastin degradation. Gradual elastocalcinosis was obtained, and paralleled the elastin degradation pattern. Matrix metalloproteinase (MMP)-9 activity was increased at 5 days of warfarin treatment, whereas TGF-β signaling was increased at 7 days. Calcification was significantly elevated after 21 days. Blocking metalloproteinases activation with doxycycline and TGF-β signaling with SB-431542 were able to prevent calcification.

Conclusions— Early MMP-9 activation precedes the increase of TGF-β signaling, and overt vascular elastocalcinosis and stiffness. Modulation of matrix degradation could represent a novel therapeutic avenue to prevent the gradual age-related stiffening of large arteries, leading to isolated systolic hypertension.

Methods and Results— Wild-type (WT), adiponectin knockout (Adipo-KO), and PPAR- knockout (PPAR--KO) mice were infused with Ang II at 1.2 mg/kg/d. Severe cardiac fibrosis and left ventricular dysfunction were observed in Ang II–infused Adipo-KO mice compared to WT mice. Adenovirus-mediated adiponectin treatment improved the above phenotypes and the dysregulation of reactive oxygen species (ROS)-related mRNAs in Adipo-KO mice, whereas such amelioration was not observed in PPAR--KO mice despite adiponectin accumulation in heart tissue. In cultured cardiac fibroblasts, adiponectin improved the reduction of AMP-activated protein kinase (AMPK) activity and elevation of extracellular signal–regulated kinase 1/2 (ERK1/2) activity induced by Ang II. Adiponectin significantly enhanced PPAR- activity, whereas the adiponectin-dependent PPAR- activation was diminished by Compound C, an inhibitor of AMPK.

Conclusion— The present study suggests that adiponectin protects against Ang II–induced cardiac fibrosis possibly through AMPK-dependent PPAR- activation.

Methods and Results— Pravastatin or pitavastatin treatment of obese mice attenuated an increase in mRNA expressions of proinflammatory genes, including MCP1 and IL6, in adipose tissue. The supernatant of TLR4-stimulated RAW264 macrophages strongly induced the expression of these genes in 3T3-L1 adipocytes, which was inhibited by pretreatment of macrophages with either statin. Statins inhibited TLR4-mediated activation of interferon (IFN) regulatory factor (IRF)3 by either lipopolysaccharide (LPS) or palmitic acid, resulting in suppression of IFN-β expression, but not that of NF-B or JNK. Moreover, statins strongly downregulated TLR3-mediated gene expressions by poly(I:C), but not TLR2-stimulation by zymosan A. Neutralization of IFN-β attenuated proinflammatory activities of the macrophage supernatant.

Conclusions— Statins partially attenuated the development of adipose tissue inflammation in obese mice, which might be associated with an inhibitory effect of statins on TLR4-triggered expression of IFN-β via MyD88-independent signaling pathway in macrophages.

Methods and Results— Cultured SMCs were exposed to either angiotensin II (Ang II) or tumor necrosis factor (TNF)-. The lucigenin-enhanced chemiluminescence assay and real-time polymerase chain reaction analysis revealed that AP-1 and mitogen-activated protein kinase inhibitors reduced both Ang II or TNF--dependent upregulation of NADPHox activity and mRNA expression (NOX1, NOX4, p67^phox, p47^phox, p22^phox). Inhibitors of AP-1 significantly diminished the Ang II or TNF--stimulated p22^phox promoter activity and protein level. Transient overexpression of c-Jun/c-Fos upregulated p22^phox promoter activity. Transcription factor pull-down assay and chromatin immunoprecipitation demonstrated the physical interaction of c-Jun protein with predicted AP-1–binding sites in the p22^phox gene promoter.

Conclusions— In SMCs exposed to Ang II or TNF-, inhibition of AP-1–related pathways reduces NADPHox expression and the O₂^– production. The physical interaction of AP-1 with p22^phox gene promoter facilitates NADPHox regulation.

Methods and Results— We investigated the effect of varying glucose concentration on Axl signaling in VSMCs. Glucose exerted powerful effects on Gas6-Axl signaling with greater activation of Akt and mTOR in low glucose, and greater activation of ERK1/2 in high glucose. Plasma membrane distribution and tyrosine phosphorylation of Axl were not affected by glucose. However, coimmunoprecipitation studies demonstrated that glucose changed the interaction of Axl with its binding partners. Specifically, binding of Axl to the p85 subunit of PI3-kinase was increased in low glucose, whereas binding to SHP-2 was increased in high glucose. Furthermore, Gas6-Axl induced migration was increased in high glucose, whereas Gas6-Axl mediated inhibition of apoptosis was greater in low glucose.

Conclusion— This study demonstrates a role for glucose in altering Axl signaling through coupling to binding partners and suggests a mechanism by which Axl contributes to VSMC dysfunction in diabetes.

Methods and Results— Ang II rapidly stimulated phosphorylation of HDAC5 at Ser498 in VSMCs. Knockdown of GIT1 significantly decreased HDAC5 phosphorylation induced by Ang II. The involvement of Src, phospholipase (PLC), and CamK II in GIT1-mediated HDAC5 phosphorylation was demonstrated. The association of GIT1 and CamK II was constitutive but increased after stimulation with Ang II. Moreover, the interaction of GIT1 and CamK II through the ARF GTPase-activating protein (ARF-GAP) and coiled-coil domains of GIT1 was essential for the phosphorylation of HDAC5. Finally, knockdown of GIT1 decreased myocyte enhancer factor 2 transcriptional activity induced by Ang II.

Conclusions— This study identifies a novel function for GIT1 as a mediator of Ang II-induced VSMC gene transcription via a Src-PLC-CamK II-HDAC5 signaling pathway.

Methods and Results— The effect of adiponectin on transendothelial electric resistance (TEER) and diffusion of albumin through human umbilical vein and bovine aortic endothelial cell monolayers induced by Ang II (100 nmol/L) or TNF- (5 ng/mL) was measured. Treatment with the globular domain of adiponectin (3 µg/mL) for 16 hours abrogated the adverse TEER effect of TNF- (–35 versus –12 /cm² at 45 minutes, P<0.05) and Ang II (–25 versus –5 /cm² at 45 minutes, P<0.01) and partially suppressed the increased diffusion of albumin with Ang II (40% versus 10% change, P<0.05) or TNF- (40% versus 20% change, P<0.05). Full-length adiponectin also suppressed Ang II–induced monolayer hyperpermeability. Adiponectin treatment also suppressed Ang II–induced increased actin stress fiber development, intercellular gap formation, and β-tubulin disassembly. Adiponectin increased cAMP levels, and its effects were abrogated by inhibition of adenylyl cyclase or cAMP-dependent protein kinase signaling.

Conclusions— Adiponectin protects the endothelial monolayer from Ang II or TNF--induced hyperpermeability by modulating microtubule and cytoskeleton stability via a cAMP/ PKA signaling cascade.

Methods and Results— Cells were pretreated with STC1 for 30 minutes followed by treatment with TNF- (2 ng/mL) for 24 hours. Monolayer permeability was studied using a transwell system. STC1 pretreatment significantly blocked TNF-–induced monolayer permeability in a concentration- and time-dependent manner. STC1 effectively blocked TNF-–induced downregulation of endothelial tight junction proteins zonula occluden-1 and claudin-1 at both mRNA and protein levels. STC1 also significantly decreased TNF-–induced superoxide anion production. The inhibitory effect of STC1 was specific to TNF-, as it failed to inhibit VEGF-induced endothelial permeability. Furthermore, STC1 partially blocked NF-B and JNK activation in TNF-–treated endothelial cells. JNK inhibitor and antioxidant also effectively blocked TNF-–induced NF-B activation and monolayer permeability in HCAECs.

Conclusions— STC1 maintains endothelial permeability in TNF-–treated HCAECs through preservation of tight junction protein expression, suppression of superoxide anion production, and inhibition of the activation of NFB and JNK, suggesting an important role for STC1 in regulating endothelial functions during cardiovascular inflammation.

Methods and Results— Adenosine-induced Ca²⁺ influx was markedly reduced by L-cis-diltiazem and LY-83583, two selective inhibitors for cyclic nucleotide-gated (CNG) channels, in H5V endothelial cells and primary cultured bovine aortic endothelial cells (BAECs). The Ca²⁺ influx was also inhibited by 2 adenylyl cyclase inhibitors MDL-12330A and SQ-22536, and by 2 A_2B receptor inhibitors MRS-1754 and 8-SPT, but not by an A_2A receptor inhibitor SCH-58261 or a guanylyl cyclase inhibitor ODQ. Patch clamp experiments recorded an adenosine-induced current that could be inhibited by L-cis-diltiazem and LY-83583. A CNGA2-specific siRNA markedly decreased the Ca²⁺ influx and the cation current in H5V cells. Furthermore, L-cis-diltiazem inhibited the endothelial Ca²⁺ influx in mouse aortic strips, and it also reduced 5-N-ethylcarboxamidoadenosine (NECA, an A₂ adenosine receptor agonist)-induced vasorelaxation.

Conclusion— CNGA2 channels play a key role in adenosine-induced endothelial Ca²⁺ influx and vasorelaxation. It is likely that adenosine acts through A_2B receptors and adenylyl cyclases to stimulate CNGA2.

Methods and Results— Transfection of bovine aortic endothelial cells with small interfering RNA (siRNA) targeting either PKC-, , or caused a reduction in the cognate PKC protein by 76% to 89% without changing expression of nontargeted isoforms. Downregulation of PKC- abrogated VEGF-stimulated phosphorylation of Akt at Ser473 and eNOS at Ser1179 and decreased VEGF-stimulated NO synthase activity in intact cells. In contrast, PKC- knockdown increased Akt and eNOS phosphorylation, whereas PKC knockdown had no significant effect. PKC- knockdown also decreased VEGF-stimulated Erk1/2 phosphorylation and abolished VEGF-stimulated DNA synthesis. Consistent with an effect on several pathways of VEGF signaling, VEGF receptor-2 (VEGFR2) tyrosine phosphorylation and expression of VEGFR2 protein and mRNA was decreased by 81, 90, and 84%, respectively, during knockdown of PKC-, but increased during PKC- knockdown.

Conclusions— By regulating VEGFR2 expression and activation, PKC- expression is critical for activation of Akt and eNOS by VEGF and contributes to VEGF-stimulated Erk activation, whereas PKC- has opposite effects.

Methods and Results— To identify possible mechanisms involved in these effects we investigated the modulation of the expression of acute phase proteins of the pentraxin superfamily, such as C-reactive protein (CRP), serum amyloid P component protein (SAP), and the long pentraxin 3 (PTX3) by HDL in human endothelial cells. HDL induced PTX3 mRNA expression and protein release, whereas no effect was observed on CRP and SAP expression. This effect was mainly dependent on the activation of the lysosphingolipids receptors-PI3K/Akt axis and was mimicked by sphingosine 1 phosphate and other S1P mimetics. This observation was confirmed in vivo; indeed an increased expression of PTX3 mRNA was detected in the aorta of transgenic mice overexpressing human apoA-I, compared to apoA-I knock-out mice. Furthermore, plasma levels of PTX3 significantly increased in C57BL/6 mice injected with HDL.

Conclusions— These data suggest that part of the atheroprotective effects of HDL could result from the modulation of molecules that act as sensors of the immunoinflammatory balance in the vascular wall.

Methods and Results— Synthetic PPAR- ligands increase CXCR2 but not CXCR1 gene expression in a PPAR--dependent manner in primary human macrophages in vitro and in atherosclerotic plaques in vivo. The increase of CXCR2 mRNA was paralleled by an increase in membrane protein expression. EMSA, ChIP, and transient transfection assays indicate that PPAR- activates the CXCR2 promoter by binding to a PPAR response element (PPRE). Finally, human macrophages acquire responsiveness to the CXCR2 ligands (IL-8 and Groβ), as measured by superoxide anion production, after induction of CXCR2 expression by PPAR- ligands.

Conclusions— Our results provide a novel mechanism via which PPAR- can enhance the immune response in human macrophages.

Methods and Results— We studied the relation between coagulation activation and aortic diameter in Marfan patients (MFS) with various aortic diameters (n=52). We then studied patients presenting large aneurysms associated with bicuspid aortic valve (BAV) and degenerative form. Lastly, we used immunochemistry and biochemistry to investigate prothrombin/thrombin retention within the aortic wall. Microparticles, sGPV, tissue factor, and TAT complexes were increased in plasma from MFS with large aneurysms (≥45 mm) compared to MFS with limited aortic dilatation (<45 mm). Similar elevations were observed in all patients with large aortic aneurysms, regardless of the etiology, the site of maximal aortic dilation, associated valvulopathy, risk factors, or treatments. P-selectin and platelet-bound fibrinogen were also increased, demonstrating platelet activation in large aneurysms. Significant increase in sCD146 plasma concentration suggested alteration of endothelium.

Conclusions— Platelet activation occurs in patients with large aneurysms of the ascending aorta, is dependent on aortic dilation, and is associated with thrombin generation, part of which appears to be retained in mucoid degeneration areas.

Methods and Results— We enrolled 26 consecutive patients with ACS, 29 patients with SA, and 25 Controls. A significantly greater number of total and TF positive platelet-monocyte aggregates was found by flow cytometry in blood of ACS patients than in either SA patients (3-fold) or Controls (5-fold). ACS patients also had a significantly higher amount of TF-positive platelets than SA or Controls (>3-fold) and significantly higher thrombin generation capacity. TF mRNA expression in platelets was significantly higher in ACS patients than in SA or Controls.

Conclusions— In ACS patients the greater expression of TF in platelets and PLA strengthens the link between platelet activation, blood coagulation, and thrombus formation and may further contribute to the hypercoagulability associated with the disease.

Methods and Results— Blood samples from 249 consecutive patients hospitalized for acute MI <24 hours were taken on admission. Serum levels of ADMA and its stereoisomer, symmetrical dimethylarginine (SDMA), were determined using high-performance liquid chromatography. The independent predictors of ADMA were glomerular filtration rate, female sex, and SDMA (R²=0. 25). Baseline ADMA levels were higher in patients who had died than in patients who were alive at 1 year follow-up (1.23 [0.98 to 1.56] versus 0.95 [0.77 to 1.20] µmol/L, P<0.001). By Cox multivariate analysis, the higher tertile of ADMA (median [interquartile range]: 1.45 [1.24 to 1.70] µmol/L) was a predictor for mortality (Hazard Ratio [95% CI], 4.83 [1.59 to 14.71]), when compared to lower tertiles, even when adjusted for potential confounders, such as acute therapy, biological, and clinical factors.

Conclusion— Our study suggests that the baseline ADMA level has a strong prognostic value for mortality after MI, beyond traditional risk factors and biomarkers.

Methods and Results— We measured baseline ADMA of 880 women in the Population Study of Women in Gothenburg using high-performance liquid chromatography. After adjustment for traditional risk factors, creatinine clearance, and homocysteine using Cox models, the HR (95% CI in parentheses) of CVD end points at 24 years for a 0.15 µmol/L (1 SD) increase in ADMA were: all-cause mortality 1.12 (0.96, 1.32), fatal CVD 1.30 (1.04, 1.62), total CVD events 1.29 (1.09, 1.53). The top quintile (ADMA ≥0.71 µmol/L) compared with the bottom four-fifths, conferred a cumulative risk 22 versus 14%, relative risk 1.75 (95% CI 1.18, 2.59) and population attributable risk 12.7% of total CVD events, and further identified individuals who are at higher than expected risk based on the SCORE and Framingham systems.

Conclusions— A 0.15 µmol/L increase in baseline ADMA levels is associated with approximately 30% increase in incident cardiovascular risk at 24 years in women after adjustment. ADMA levels ≥0.71 µmol/L enhances CVD risk assessment in women.

Methods and Results— With this study, we intended to identify the influence of telomere length on bone marrow functionality in 50 patients with coronary artery disease (CAD) and previous myocardial infarction. Mean telomere length (mTL) was measured simultaneously in peripheral blood leukocytes and mononuclear bone marrow cells (BMC), using the flow-FISH method. Telomere erosion already occurred at the bone marrow level, whereby age (39 bp/yr, P=0.025) and the number of affected vessels (434 bp/vessel, P=0.029) were the only independent predictors. Lymphocytes demonstrated significant TL shortening between BMCs and peripheral blood in CAD patients (–1011±897 bp) as opposed to an increase in a young control group (+235±459 bp, P<0.001). SDF- and VEGF-specific migration of BMCs correlated with mTL of lymphocytes (r=0.42, P<0.001) and was significantly reduced in CAD patients. Finally, the telomere length difference between granulocytes and lymphocytes was the most determinant for telomere-associated bone marrow impairment (P<0.001).

Conclusion— In patients with CAD, telomere shortening of BMCs is dependent on both age and the extent of CAD and correlates with bone marrow cell functionality.

Methods and Results— We examined IRF5 mRNA expression in carotid atherosclerotic tissue (n=99) and the case-control association between SNPs in the IRF5 gene with myocardial infarction (MI) (n=376+387) and unstable coronary artery disease (CAD) (n=3101+445). Among unstable CAD patients, association of IRF5 SNPs with recurrent coronary events (n=401) was also investigated. The IRF5 mRNA expression was increased in atherosclerotic tissue compared with control tissue (P<0.001). Significant associations with IRF5 expression was observed for 6 of 10 SNPs in the study. However, the IRF5 SNPs examined were neither associated with the risk of precocious MI, nor with unstable CAD or risk of recurrent cardiovascular events in unstable CAD patients.

Conclusions— IRF5 mRNA is expressed in cells in atherosclerotic tissue and its expression is modified by SNPs in the IRF5 gene. Genetic variation at the IRF5 locus was, however, not associated with CAD or related phenotypes.

Methods and Results— We analyzed the USF1 in 2 autopsy series of altogether 700 middle-aged men (the Helsinki Sudden Death Study) with quantitative morphometric measurements of coronary atherosclerosis. SNP rs2516839, tagging common USF1 haplotypes, associated with the presence of several types of atherosclerotic lesions, particularly with the proportion of advanced atherosclerotic plaques (P=0.02) and area of calcified lesions (P<0.001) of the coronary arteries. Importantly, carriers of risk alleles of rs2516839 also showed a 2-fold risk for sudden cardiac death (genotype TT versus CC; OR 2.10, 95% CI 1.17 to 3.75, P=0.04). The risk effect of rs2516839 was present also in aorta samples of the men.

Conclusions— Our findings in this unique study sample suggest that USF1 contributes to atherosclerosis, the pathological arterial wall phenotype resulting in coronary heart disease and in its most dramatic consequence—sudden cardiac death.

Methods and Results— We genotyped 10 592 individuals from the Danish general population, the Copenhagen City Heart Study. During 24 years of follow-up, 557 individuals developed ischemic cerebrovascular disease. The allele frequency was 0.07 for –1072 A and 0.29 for –444 C. Cumulative incidence for ischemic cerebrovascular disease was higher for –1072 AA versus GG genotype (log-rank: P=0.002), and lower for –444 CC versus AA genotype (log-rank: P=0.008). Combined genotypes showed corresponding cumulative incidence differences (log-rank: P=0.003). Multifactorially adjusted hazard ratios for ischemic cerebrovascular disease were 2.8(1.4 to 5.7) for –1072 AA versus GG genotype, 0.6(0.4 to 0.9) for –444 CC versus AA genotype, 2.5(1.2 to 5.4) for combined AA-AA versus GG-AA genotype, and 0.6(0.4 to 0.9) for combined GG-CC versus GG-AA genotype. Genotype did not associate with risk of deep venous thrombosis or severe carotid atherosclerosis, or with levels of platelets and coagulation factors.

Conclusions— Leukotriene C₄ synthase –1072 AA genotype predict increased risk, whereas –444 CC genotype predict decreased risk of ischemic cerebrovascular disease.

Methods and Results— As measured by flow-cytometry in 210 healthy subjects, CD34⁺KDR⁺ EPCs were higher in fertile women than in men, but were not different between postmenopausal women and age-matched men. These gender gradients mirrored differences in cardiovascular profile, carotid intima-media thickness, and brachial artery flow-mediated dilation. Moreover, EPCs and soluble c-kit ligand varied in phase with menstrual cycle in ovulatory women, suggesting cyclic bone marrow mobilization. Experimentally, hysterectomy in rats was followed by an increase in circulating EPCs. EPCs cultured from female healthy donors were more clonogenic and adherent than male EPCs. Treatment with 17β-estradiol stimulated EPC proliferation and adhesion, via estrogen receptors. Finally, we show that the proangiogenic potential of female EPCs was higher than that of male EPCs in vivo.

Conclusions— EPCs are mobilized cyclically in fertile women, likely to provide a pool of cells for endometrial homeostasis. The resulting higher EPC levels in women than in men reflect the cardiovascular profile and could represent one mechanism of protection in the fertile female population.

Methods and Results— GRO levels were examined by enzyme immunoassay, real-time quantitative RT-PCR, and cDNA microarrays. The in vitro effect of statins on GRO was examined in endothelial cells and THP-1 macrophages. Our main findings were: (1) GRO was among the 10 most differentially expressed transcripts comparing peripheral blood mononuclear cells (PBMCs) from patients with coronary artery disease (CAD) and healthy controls. (2) Both patients with stable (n=41) and particularly those with unstable (n=47) angina had increased plasma levels of GRO comparing controls (n=20). (3) We found increased expression of GRO within symptomatic carotid plaques, located to macrophages and endothelial cells. (4) GRO enhanced the release of matrix metalloproteinases in vascular smooth muscle cells, and increased the binding of acetylated LDL in macrophages. (5) Atorvastatin downregulated GRO levels as shown both in vitro in endothelial cells and macrophages and in vivo in PBMCs from CAD patients. (6) The effect on GRO in endothelial cells involved increased storage and reduced secretion of GRO.

Conclusions— GRO could be involved in atherogenesis and plaque destabilization, potentially contributing to inflammation, matrix degradation, and lipid accumulation within the atherosclerotic lesion.

Methods and Results— Study subjects were participants of the longitudinal Cardiovascular Risk in Young Finns Study started in 1980 (n=2265, age 3 to 18 years). To phenotype type IIa, IIb, and IV dyslipidemias and hypoHDL-cholesterolemia, we calculated age and sex-specific z scores for lipid values for each subject in 1980, 1983, 1986, and 2001. Subjects with mean z score over 90th percentile for LDL-cholesterol or triglycerides were considered having type IIa or IV dyslipidemia. Subjects with mean z score over 90th percentile for LDL-cholesterol and triglycerides had type IIb dyslipidemia, and those with mean z score below 10th percentile for HDL-cholesterol had hypoHDL-cholesterolemia. Compared to controls, subjects with type IIb dyslipidemia had increased carotid IMT (P<0.01). This difference remained significant when adjusted with other risk factors (P<0.05). Carotid IMT also increased significantly more with increasing number of nonlipid risk factors (P<0.001) or presence of the metabolic syndrome (P<0.05) in subjects with type IIb than in controls. Subjects with type IIb or type IV dyslipidemia had decreased carotid elasticity (P<0.05), but these differences became nonsignificant (P>0.3) when adjusted with blood pressure.

Conclusions— Our findings suggest that type IIb dyslipidemia has deleterious effects on vasculature already since childhood. Subjects with type IIb dyslipidemia are more vulnerable to the effects of cardiovascular risk factors and metabolic syndrome.

Methods and Results— The study involved 1570 older men and women without coronary heart disease who participated in the Rotterdam Study. Coffee intake was assessed with a semiquantitative food frequency questionnaire. Coronary calcification was detected with electron beam computed tomography. Severe calcification was defined as an Agatson calcium score >400. Sex-specific odds ratios (ORs) with 95% confidence intervals (95% CI) were obtained by logistic regression with adjustment for age, smoking, body mass index, education, and intake of energy and alcohol. In multivariable analysis, coronary calcification in women was significantly reduced for moderate (>3 to 4 cups) and high (>4 cups) coffee intake, compared with a daily intake of 3 cups or less (OR of 0.41 [95% CI: 0.25 to 0.65] and 0.54 [0.33 to 0.87], respectively). The association persisted after additional adjustment for tea and other dietary confounders, and was not modified by smoking. A nonsignificant inverse relationship was also found in men who smoked, whereas in nonsmoking men a direct association was observed.

Conclusion— The present study suggests a beneficial effect of coffee drinking against coronary calcification, particularly in women. More research is needed to confirm these findings and to clarify possible effect modification by gender and smoking.

Methods and Results— Metal ions were quantified by EPR and inductively coupled plasma mass spectroscopy. Native and oxidized protein side-chains were quantified by high-performance liquid chromatography. Elevated levels of zinc (6-fold) were detected in advanced lesions compared to healthy tissue or early lesions. Zinc did not correlate negatively with iron or copper levels suggesting that zinc does not displace these metal ions. Highly significant positive correlations (P<0.005) were detected between zinc and calcium levels.

Conclusions— Zinc did not correlate with low iron levels and reduced protein oxidation. These data indicate that zinc does not prevent protein oxidation in advanced lesions. The reported protective effect of zinc accumulation is proposed to be associated with lesion calcification.