Methods and Results—We investigated the role of COX-2 expressed in the myeloid lineage in the development of atherosclerosis using a myeloid-specific COX-2^-/- (COX-2^-M/-M) mouse on a hyperlipidemic apolipoprotein (apo) E^-/- background (COX-2^-M/-M/apoE^-/-). Myeloid COX-2 depletion resulted in significant attenuation of acute inflammation corresponding with decreased PGE₂ levels in an air pouch model. COX-2 depletion in myeloid cells did not influence development of atherosclerosis in COX-2^-M/-M/apoE^-/- when compared to apoE^-/- littermates fed either chow or western diets. The unanticipated lack of contribution of myeloid COX-2 to the development atherosclerosis is not attributable to altered maintenance, differentiation, or mobilization of myeloid and lymphoid populations. Moreover, myeloid COX-2 depletion resulted in unaltered serum prostanoid levels and cellular composition of atherosclerotic lesions of COX-2^-M/-M/apoE^-/- mice.

Conclusions—Our results suggest that COX-2 expression in myeloid cells, including macrophages, does not influence the development of atherosclerosis in mice.

Methods and Results—Adult male swine were fed a hypercholesterolemic or an isocaloric normal diet for 2-week and 6-month periods. Hypercholesterolemia induced localized lipid insudation confined to the aortic side of the leaflet. Transcript profiling of valve endothelial populations showed that the susceptible aortic side was more sensitive to 2-week hypercholesterolemia than the ventricular side (1,325 vs 87 genes were differentially expressed). However, greater sensitivity was not evidence of a dysfunctional phenotype. Instead, pathway analyses identified differential expression of caspase 3–, peroxisome proliferator–activated receptor –, TNF-–, and nuclear factor-B–related pathways that were consistent with a protective endothelial phenotype. This was confirmed at the protein level at 2 weeks and persisted at 6 months.

Conclusions—In a large animal model at high spatial resolution, endothelium on the pathosusceptible side of the aortic valve leaflet is responsive to hypercholesterolemia. Transcript profiles indicative of a protective phenotype were induced and persisted on the side prone to aortic valve sclerosis.

Objective—To determine whether NKT cells are involved in glucose intolerance and adipose tissue inflammation in diet-induced obese mice.

Methods and Results—To determine whether NKT cells are involved in the development of glucose intolerance, male β₂-microglobulin knockout (KO) mice lacking NKT cells and C57BL/6J (wild-type) mice were fed with a high-fat diet (HFD) for 13 weeks. Body weight and visceral obesity were comparable between wild-type and KO mice. However, macrophage infiltration was reduced in adipose tissue and glucose intolerance was significantly ameliorated in KO mice. To further confirm that NKT cells are involved in these abnormalities, -galactosylceramide, 0.1 µg/g body weight, which specifically activates NKT cells, was administered after 13 weeks of HFD feeding. -Galactosylceramide significantly exacerbated glucose intolerance and macrophage infiltration as well as cytokine gene expression in adipose tissue.

Conclusion—NKT cells play a crucial role in the development of adipose tissue inflammation and glucose intolerance in diet-induced obesity.

Objective—To investigate whether TZDs modulate retinal vessel formation in a mouse model of oxygen-induced retinopathy.

Methods and Results—Neonatal mice were subjected to ischemia-induced retinopathy to produce pathological neovascular tuft formation. Pioglitazone, 10 mg/kg per day, rosiglitazone, 10 mg/kg per day, or vehicle was given by gavage once a day from postnatal day 7 to postnatal day 17. Systemic treatment of wild-type (WT) mice with TZDs led to a significant decrease in pathological retinal neovascularization during ischemia compared with vehicle treatment, which was accompanied by increased plasma levels of the fat-derived hormone adiponectin (APN). In contrast to WT mice, TZDs had no effects on ischemia-induced pathological retinal vessel formation in APN-knockout (KO) mice. Pioglitazone reduced tumor necrosis factor (TNF) expression in ischemic retina in WT mice but not in APN-KO mice. Furthermore, pioglitazone increased plasma APN levels in TNF-–KO mice but did not affect ischemia-induced pathological retinal neovascularization in this strain.

Conclusion—These data show that TZDs attenuate pathological retinal microvessel formation through APN-mediated modulation of TNF- production.

Methods and Results—Intraluminal thrombus were analyzed by differential proteomics, using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. A 1309-Da peptide was detected in larger amounts in the newly formed luminal thrombus layer relative to older layers. It was identified as being LVVYPWTQRF (known as LVV-Hemorphin 7), a peptide generated from hemoglobin by cathepsin D. By immunohistochemical analysis, we showed that Hemorphin 7 (H7) colocalizes with cathepsin D and cathepsin G in the luminal layer of the intraluminal thrombus. In vitro, cathepsin G was able to generate H7 peptides at pH 7.4, whereas cathepsin D was only active in acidic conditions. Finally, H7 peptides were shown to be increased 3- to 4-fold in sera of abdominal aortic aneurysm patients relative to controls, and their levels were positively correlated with the volume of the thrombus.

Conclusion—Our results suggest that circulating H7 peptides may reflect proteolysis of hemoglobin in the aneurysmal intraluminal thrombus and may be used as a biological marker of pathological vascular remodeling.

Methods and Results— Triglyceride-rich lipoprotein catabolism was decreased in hapoA-II-Tg mice compared to control mice. This suggests that hapoA-II, which was mainly associated with HDL during fasting and postprandially, impairs triglyceride-rich lipoprotein lipolysis. HDL isolated from hapoA-II-Tg mice impaired bovine LPL activity. Two-dimensional gel electrophoresis, mass spectrometry, and immunonephelometry identified a marked deficiency in the HDL content of apoA-I, apoC-III, and apoE in these mice. In normolipidemic women, apoA-II concentration was directly correlated with plasma triglyceride and inversely correlated with HDL-to-apoC-II and apoE/apoC-III ratios. HDL-mediated induction of LPL activity was inversely correlated with apoA-II and directly correlated with HDL-to-apoC-II and apoE/apoC-III ratios. Purified hapoA-II displaced apoC-II, apoC-III, and apoE from human HDL2. Human HDL3 was, compared to HDL2, enriched in apoA-II but poorer in apoC-II, apoC-III, and apoE.

Conclusion—ApoA-II plays a crucial role in triglyceride catabolism by regulating LPL activity, at least in part, through HDL proteome modulation.

Methods and Results—High-fat diet–induced obese C57BL/6 mice, CD11c-deficient mice, and obese humans were studied. CD11c, leukocytes, and chemokines/cytokines were examined in AT and/or blood by flow cytometry, RNase protection assay, quantitative polymerase chain reaction, or enzyme-linked immunosorbent assay. Obese C57BL/6 mice had increased CD11c in AT and blood compared with lean controls. CD11c messenger RNA positively correlated with monocyte chemoattractant protein 1 in human visceral AT. Obese humans with metabolic syndrome had a higher CD11c level in blood monocytes compared with lean humans. Low-fat diet–induced weight loss reduced blood monocyte CD11c in obese mice and humans. Mouse and human monocyte CD11c levels and mouse AT CD11c messenger RNA correlated with insulin resistance. CD11c deficiency in mice did not alter weight gain but decreased inflammation, evidenced by a lower T-cell number and reduced levels of major histocompatibility complex class II, CCL5, CCL4, and interferon in AT, and ameliorated insulin resistance and glucose intolerance associated with diet-induced obesity.

Conclusions—Diet-induced obesity increased CD11c in both AT and blood in mice and humans. CD11c plays an important role in T-cell accumulation and activation in AT, and contributes to insulin resistance associated with obesity.

Methods and Results—We studied the kinetics of 4 very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) types containing: (1) otherApos-CIII-: none of apoCIII, apoAII, apoCI, apoCII, or apoE; (2) otherApos+CIII-: no apoCIII but at least one of the others; (3) otherApos-CIII+: apoCIII, but not any others; and (4) otherApos+CIII+: apoCIII and at least one other. VLDL and IDL otherApos-CIII+ and otherApos-CIII- had similar rates of lipolytic conversion to smaller particles. However, light LDL otherApos-CIII+ compared with otherApos-CIII- had much faster conversion to dense LDL as did light LDL otherApos+CIII+ compared with otherApos+CIII-. VLDL and IDL otherApos-CIII+ had minimal direct removal from circulation, whereas VLDL and IDL otherApos+CIII-, rich in apoE, showed fast clearance. Lipoproteins in fraction otherApos+CIII+ also rich in apoE had very low clearance.

Conclusions—The results suggest that apoCIII strongly inhibits hepatic uptake of VLDL and IDL overriding the opposite influence of apoE when both are present. The presence of apoCIII on dense VLDL is not associated with slow conversion to IDL, a lipoprotein lipase-dependent process; but when on light LDL, apoCIII is associated with enhanced conversion to dense LDL, a process involving hepatic lipase.

Methods and Results—We use murine endothelial cell lines of brain and heart origin, which express both subtypes of estrogen receptor, ER and ERβ. Treatment of these cells with 17β-estradiol (E2) led to an increase in transendothelial electric resistance and a most prominent upregulation of the tight junction protein claudin-5 expression. A significant increase of claudin-5 promoter activity, mRNA, and protein levels was detected in cells from both vascular beds. In protein lysates and in immunoreactions on brain sections from ovariectomized E2-treated mice, we noticed an increase in claudin-5 protein and mRNA content. Treatment of cells with a specific ERβ agonist, diarylpropionitrile, revealed the same effect as E2 stimulation. Moreover, we detected significantly lower claudin-5 mRNA and protein content in ERβ knockout mice.

Conclusions—We describe claudin-5 as a novel estrogen target in vascular endothelium and show in vivo (brain endothelium) and in vitro (brain and heart endothelium) effects of estrogen on claudin-5 levels. The estrogen-induced increase in junctional protein levels may lead to an improvement in vascular structural integrity and barrier function of vascular endothelium.

Methods and Results—We could extend the survival of the Tie1-deficient embryos in the ICR background, which allowed us to study their lymphatic vessel development. At embryonic day 14.5, the Tie1^-/- embryos had edema and hemorrhages and began to die. Immunohistochemical analysis revealed that they have abnormal lymph sacs. Tie1^-/- mutants were swollen already at embryonic day 12.5 without signs of hemorrhage. Their lymph sacs were abnormally patterned, suggesting that lymphatic malformations precede the blood vascular defects. We generated mice with a conditional Cre/loxP Tie1^neo locus and found that the homozygous Tie1^neo/neo hypomorphic embryos survived until embryonic day 15.5 with lymphatic malformations resembling those seen in the Tie1^-/- mutants.

Conclusion—Our data show that loss of Tie1 results in lymphatic vascular abnormalities that precede the blood vessel phenotype. These findings indicate that Tie1 is involved in lymphangiogenesis and suggest differential requirements for Tie1 signaling in the 2 vascular compartments.

Methods and Results—We genotyped polymorphisms at nine established obesity loci in 1,395 women with diabetes from the Nurses' Health Study: 449 women developed CVD, and 946 women did not develop CVD. A genetic risk score (GRS) was derived by summing risk alleles for each individual. Four polymorphisms (rs9939609 [FTO], rs11084753 [KCTD15], rs10838738 [MTCH2], and rs10938397 [GNPDA2]) showed nominally significant associations with CVD. The GRS combining all obesity loci was linearly related to CVD risk (P = 0.013 for trend). The odds ratio was 1.08 per risk allele (95% confidence interval, 1.02–1.15; P = 0.01) after adjustment for body mass index and other conventional risk factors. Women with the highest quartile of GRS had 53% (95% confidence interval, 6%–122%) increased CVD risk, compared with those in the lowest quartile of GRS (P = 0.024). In addition, a higher GRS was associated with lower adiponectin levels (P = 0.02). Further adjustment for body mass index and other covariates did not change the association (P = 0.006). A higher GRS was also correlated with lower levels of high-density lipoprotein (P = 0.01).

Conclusion—Obesity-predisposing variants may jointly affect CVD risk among women with diabetes.

Methods and Results—Loss of SCD1 in both genetic (Agouti) and diet-induced obesity (high-fat diet) mouse models prevented inflammation in white adipose tissue and improved its basal insulin signaling. In SCD1-deficient mice, white adipose tissue exhibited lower inflammation, with a reduced response to lipopolysaccharide in isolated adipocytes, but not in peritoneal macrophages. Mimicking the in vivo paracrine regulation of white adipose tissue inflammation, SCD1-deficient adipocyte-conditioned medium attenuated the induction of tumor necrosis factor (TNF) /interleukin 1β gene expression in RAW264.7 macrophages and reduced the adhesion response in endothelial cells. We further demonstrated that the adipocyte-derived oleate (18:1n9), but not palmitoleate (16:1n7), mediated the inflammation in macrophages and adhesion responses in endothelial cells.

Conclusions—Loss of SCD1 attenuates adipocyte inflammation and its paracrine regulation of inflammation in macrophages and endothelial cells. The reduced oleate level is linked to the inflammation-modulating effects of SCD1 deficiency.

Methods and Results—We studied 47 definite IST patients, including 15 with acute, 26 subacute and 6 late IST, and 48 controls matched for demographics, cardiovascular risk factors, concomitant treatment and angiographic/stent parameters. Plasma clot permeability (K_s), which indicates a pore size, turbidity (lag phase, indicating the rate of fibrin clot formation, Abs_max, maximum absorbance of a fibrin gel, reflecting the fiber thickness), lysis time (t_50%) and maximum rate of D-dimer release from clots (D-D_rate) were determined 2 to 73 (median 14.7) months after IST. Patients with IST had 21% lower K_s, 14% higher Abs_max, 11% lower D-D_rate, 30% longer t_50% (all P<0.0001) and 5% shorter lag phase compared to controls (P=0.042). There were no correlations between clot variables and the time of IST or that from IST to blood sampling. Multiple regression analysis showed that K_s (odds ratio=0.36 per 0.1 µm², P<0.001), D-D_rate (odds ratio=0.16 per 0.01 mg/L/min, P<0.001) and stent length (odds ratio=1.1 per 1 mm, P=0.043) were independent predictors of IST (R²=0.58, P<0.001).

Conclusions—IST patients tend to form dense fibrin clots resistant to lysis, and altered plasma fibrin clot features might contribute to the occurrence of IST.

Methods and Results—In vitro, leptin specifically promoted CAC adhesion to tubular endothelial structures and migration along outgrowing sprouts of endothelial cells. In vivo, stimulation of CACs with leptin increased their capacity to promote new vessel formation in the chorioallantoic membrane of chicken embryos and to improve neovascularization of ischemic murine hind limbs. These effects required the phosphorylation of vβ5 integrins, which depended on the interaction of leptin with its receptor ObR, and on Janus kinase (JAK) 2– and PLC-mediated activation of Src kinase. Protein tyrosine phosphatase 1B, a negative regulator of leptin signaling, was overexpressed in CACs from obese, hyperleptinemic individuals, and this was associated with insensitivity of CACs to the angiogenic effects of leptin. Weight loss (by 30±15 kg) normalized protein tyrosine phosphatase 1B expression in CACs and restored their responsiveness to leptin. A similar dose-dependent response was found after incubation of CACs from obese subjects with a protein tyrosine phosphatase 1B inhibitor ex vivo.

Conclusion—Our results point to the ObR–Src kinase–vβ5 cross talk as a distinct novel component of the network of specific interactions between integrins and cytokine receptors in angiogenesis.

Methods and Results—We analyzed allelic variants of USF1 and APOA5 in families (n=516) ascertained for atherogenic dyslipidemia and in an autopsy series of middle-aged men (n=300) with detailed quantitative measurements of atherosclerotic lesions. The impact of previously associated APOA5 variants on TGs was observed in the dyslipidemic families, and variant rs3135506 was associated with size of fibrotic aortic lesions in the autopsy series. The USF1 variant rs2516839, associated previously with atherosclerotic lesions, showed an effect on TGs in members of the dyslipidemic families with documented coronary artery disease. We provide preliminary evidence of gene-gene interaction between these variants in an autopsy series with a fibrotic lesion area in the abdominal aorta (P=0.0028), with TGs in dyslipidemic coronary artery disease subjects (P=0.03), and with high-density lipoprotein cholesterol (P=0.008) in a large population cohort of coronary artery disease patients (n=1065) in which the interaction for TGs was not replicated.

Conclusion—Our findings in these unique samples reinforce the roles of APOA5 and USF1 variants on cardiovascular phenotypes and suggest that both genes contribute to lipid levels and aortic atherosclerosis individually and possibly through epistatic effects.

Methods and Results—CCL5 levels measured by enzyme immunoassay in serum and by real-time polymerase chain reaction in WAT were higher in obese compared to lean subjects. CCL5, but not CCL2, secretion was higher in visceral compared to subcutaneous WAT. CCL5 mRNA expression was positively correlated with the inflammatory macrophage markers as CD11b, tumor necrosis factor-, and IL-6 in visceral WAT (n=24 obese subjects), and was higher in macrophages than other WAT cells. We found that CCL5 triggered adhesion and transmigration of blood monocytes to/through endothelial cells of human WAT. Whereas in obese WAT apoptotic macrophages were located around necrotic adipocytes, we demonstrated that CCL5, but not CCL2, protected macrophages from free cholesterol-induced apoptosis via activation of the Akt/Erk pathways.

Conclusions—CCL5 could participate in the inflammation of obese WAT by recruiting blood monocytes and exerting antiapoptotic properties on WAT macrophages. This specific role of CCL5 on macrophage survival with maintenance of their lipid scavenging function should be taken into account for future therapeutic strategies in obesity-related diseases.

Methods and Results—Fibulin-2 is distinctly located in the subendothelial matrix, whereas fibulin-5 is observed throughout the vessel wall. All of the elastic laminae, including the internal elastic lamina (IEL), were severely disorganized in DKO mice, which was not observed in single knockout mice for Fbln2 or Fbln5. Furthermore, DKO vessels displayed upregulation of vascular adhesion molecules, tissue factor expression, and thrombus formation with marked dilation and thinning of the vessel wall after carotid artery ligation-injury.

Conclusions—Fibulin-2 and fibulin-5 cooperatively function to form the IEL during postnatal development by directing the assembly of elastic fibers, and are responsible for maintenance of the adult vessel wall after injury. The DKO mouse will serve as a unique animal model to test the effect of vessel integrity during various pathological insults.

Results and Methods—The effects of increased HO-1 expression, as well as biliverdin, bilirubin, and carbon monoxide (CO), were studied in in vitro models of VSMC migration. Induction of HO-1 or CO, but not biliverdin or bilirubin, inhibited VSMC migration. This effect was mediated by the inhibition of Nox1 as determined by a range of approaches, including detection of intracellular superoxide, nicotinamide adenine dinucleotide phosphate oxidase activity measurements, and siRNA experiments. Furthermore, CO decreased platelet-derived growth factor-stimulated, redox-sensitive signaling pathways.

Conclusion—Herein, we demonstrate that increased HO-1 expression and CO decreases platelet-derived growth factor-stimulated VSMC migration via inhibition of Nox1 enzymatic activity. These studies reveal a novel mechanism by which HO-1 and CO may mediate their beneficial effects in arterial inflammation and injury.

Methods and Results—To enhance endothelial cell differentiation above a baseline of 2% in embryoid body (EB) spontaneous differentiation, 3 alternate culture conditions were compared. Vascular endothelial growth factor (VEGF) treatment of EBs showed the best induction, with markedly increased expression of endothelial cell proteins CD31, VE-Cadherin, and von Willebrand Factor, but not the hematopoietic cell marker CD45. CD31 expression peaked around days 10 to 14. Continuous VEGF treatment resulted in a 4- to 5-fold enrichment of CD31⁺ cells but did not increase endothelial proliferation rates, suggesting a primary effect on differentiation. CD31⁺ cells purified from differentiating EBs upregulated ICAM-1 and VCAM-1 in response to TNF, confirming their ability to function as endothelial cells. These cells also expressed multiple endothelial genes and formed lumenized vessels when seeded onto porous poly(2-hydroxyethyl methacrylate) scaffolds and implanted in vivo subcutaneously in athymic rats. Collagen gel constructs containing hESC-derived endothelial cells and implanted into infarcted nude rat hearts formed robust networks of patent vessels filled with host blood cells.

Conclusions—VEGF induces functional endothelial cells from hESCs independent of endothelial cell proliferation. These enrichment methods increase endothelial cell yield, enabling applications for revascularization as well as basic studies of human endothelial biology. We demonstrate the ability of hESC-derived endothelial cells to facilitate vascularization of tissue-engineered implants.

Methods and Results—The relative contributions of genetic and environmental influences to the prostanoid biosynthetic pathway-related phenotypes, cyclooxygenase isoenzymes, microsomal-PGE-synthase-1 and TxA-synthase expression, and thromboxane-A₂ and prostaglandin-E₂ production by stimulated whole blood, were assessed in a sample of 308 individuals in 15 extended families. The effects of measured covariates (such as sex, age, and smoking), genes, and environmental variables shared by members of a household were quantified. Heritabilities ranging from 0.406 to 0.634 for enzyme expression and from 0.283 to 0. 751 for prostanoid production were found.

Conclusions—These results demonstrate clearly the importance of genetic factors in determining variation in phenotypes that are components of the prostanoid biosynthetic pathways. The presence of such strong genetic effects suggest that it will be possible to localize previously unknown genes that influence quantitative variation in these phenotypes, some of which affect multiple aspects of cell biology, with important clinical implications.

Methods and Results—LDLr^-/-, Apob^100/100 mice were fed diets enriched in saturated fat or fish oil in conjunction with antisense oligonucleotide (ASO) treatment to inhibit SCD1. As previously reported, in SFA-fed mice, SCD1 inhibition dramatically protected against development of the metabolic syndrome, yet promoted atherosclerosis. In contrast, in mice fed fish oil, SCD1 inhibition did not result in augmented macrophage inflammatory response or severe atherosclerosis. In fact, the combined therapy of dietary fish oil and SCD1 ASO treatment effectively prevented both the metabolic syndrome and atherosclerosis.

Conclusions—SCD1 ASO treatment in conjunction with dietary fish oil supplementation is an effective combination therapy to comprehensively combat the metabolic syndrome and atherosclerosis in mice.

Methods and Results—Plasma levels of squalene, desmosterol, and lathosterol (cholesterol synthesis markers) and campesterol, sitosterol, and cholestanol (cholesterol absorption markers) were measured by gas-liquid chromatography in 103 FCH patients and 240 normolipidemic relatives (NLR). Squalene, desmosterol, and lathosterol levels were 6% (0.078), 31%, (P<0.001) and 51% (P<0.001) higher in FCH as compared to NLR, and these differences were especially pronounced in women. An interaction with obesity was also noted for a subset of these markers. We did not observe any apparent differences for the cholesterol absorption markers among FCH patients and NLR.

Conclusions—Our data indicate that both men and women with FCH have alterations in the cholesterol synthesis pathway, resulting in 51% higher levels of lathosterol (and additionally desmosterol in women). Plasma levels of the cholesterol precursor sterol squalene were only slightly increased (6%), suggesting enhanced conversion of squalene to lathosterol in this disorder.

Methods and Results—Mice deficient for the LDL-receptor and the Apobec-1 editing peptide (DKO mice) were studied as an age-dependent model of atherosclerosis. At 10, 20, and 40 weeks of age, ultrasound molecular imaging of the proximal thoracic aorta was performed with contrast agents targeted to P-selectin and VCAM-1. Atherosclerotic lesion severity and content were assessed by ultrahigh frequency ultrasound, histology, and immunohistochemistry. In wild-type mice at all ages, there was neither aortic thickening nor targeted tracer signal enhancement. In DKO mice, lesions progressed from sparse mild intimal thickening at 10 weeks to widespread severe lesions with luminal encroachment at 40 weeks. Molecular imaging for P-selectin and VCAM-1 demonstrated selective signal enhancement (P<0.01 versus nontargeted agent) at all ages for DKO mice. P-selectin and VCAM-1 signal in DKO mice were greater by 3-fold at 10 weeks, 4- to 6-fold at 20 weeks, and 9- to 10-fold at 40 weeks compared to wild-type mice. En face microscopy demonstrated preferential attachment of targeted microbubbles to regions of lesion formation.

Conclusions—Noninvasive ultrasound molecular imaging of endothelial activation can detect lesion-prone vascular phenotype before the appearance of obstructive atherosclerotic lesions.

Methods and Results—We used data from the E3N French prospective cohort of women born between 1925 and 1950 and biennially followed by questionnaires from 1990. Study population consisted of 80 308 postmenopausal women (average follow-up: 10.1 years) including 549 documented idiopathic first venous thromboembolism. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox proportional models. Compared to never-users, past-users of hormone therapy had no increased thrombotic risk (HR=1.1; 95% CI: 0.8 to 1.5). Oral not transdermal estrogens were associated with increased thrombotic risk (HR=1.7; 95% CI: 1.1 to 2.8 and HR=1.1; 95% CI: 0.8 to 1.8; homogeneity: P=0.01). The thrombotic risk significantly differed by concomitant progestogens type (homogeneity: P<0.01): there was no significant association with progesterone, pregnanes, and nortestosterones (HR=0.9; 95% CI: 0.6 to 1.5, HR=1.3; 95% CI: 0.9 to 2.0 and HR=1.4; 95% CI: 0.7 to 2.4). However, norpregnanes were associated with increased thrombotic risk (HR=1.8; 95% CI: 1.2 to 2.7).

Conclusions—In this large study, we found that route of estrogen administration and concomitant progestogens type are 2 important determinants of thrombotic risk among postmenopausal women using hormone therapy. Transdermal estrogens alone or combined with progesterone might be safe with respect to thrombotic risk.

Methods and Results—Ado, under hypoxia, stimulates HIF-1 accumulation by activating all adenosine receptors (ARs). HIF-1 modulation involved extracellular signal-regulated kinase 1/2 (ERK 1/2), p38 mitogen-activated protein kinase (p38 MAPK), and protein kinase B (Akt) phosphorylation in the case of A₁, A_2A, A_2B, and ERK 1/2 phosphorylation in the case of A₃ receptors. Ado, through the activation of A₃ and A_2B receptors, stimulates vascular endothelial growth factor (VEGF) secretion in a HIF-1–dependent way. Furthermore, ado, through the A_2B subtype, induces an increase of Interleukin-8 (IL-8) secretion in a ERK 1/2, p38, and Akt kinase–dependent but not HIF-1–mediated way. Finally, ado stimulates FC formation, and this effect is strongly reduced by A₃ and A_2B blockers and by HIF-1 silencing.

Conclusions—This study provides the first evidence that A_3, A_2B, or mixed A₃/A_2B antagonists may be useful to block important steps in the atherosclerotic plaque development ado-induced.

Methods and Results—Cultured SMCs were exposed to interferon (IFN) for 24 hours. Using lucigenin-enhanced chemiluminescence and dihydroethidium assays, real-time polymerase chain reaction, and Western blot analysis, we found that JAK/STAT inhibitors significantly diminished the IFN-dependent upregulation of Nox activity, Nox1 and Nox4 expression. In silico analysis revealed the presence of highly conserved GAS elements within human Nox1, Nox4, p22phox, p47phox, and p67phox promoters. Transient overexpression of STAT1/STAT3 augmented the promoter activities of each subunit. JAK/STAT blockade reduced the Nox subunits transcription. Chromatin immunoprecipitation demonstrated the physical interaction of STAT1/STAT3 proteins with the predicted GAS elements from Nox1 and Nox4 promoters.

Conclusions—JAK/STAT is a key regulator of Nox1 and Nox4 in human vascular SMCs. Inhibition of JAK/STAT pathway and the consequent Nox-dependent oxidative stress may be an efficient therapeutic strategy to reduce atherogenesis.

Methods and Results—A wire-induced vascular injury was performed in the femoral artery of BM-chimeric mice whose BM was repopulated with AT₁-deficient (BM-Agtr1^-/-) or wild-type (BM-Agtr1^+/+) cells. Neointima formation was profoundly reduced by 38% in BM-Agtr1^-/- mice. Although the number of circulating EPCs (Sca-1⁺Flk-1⁺) and extent of reendothelialization did not differ between the 2 groups, the numbers of both circulating VPCs (c-Kit^-Sca-1⁺Lin^-) and tissue VPCs (Sca-1⁺CD31^-) incorporated into neointima were markedly decreased in BM-Agtr1^-/- mice. The accumulation of aggregated platelets and their content of stromal cell–derived factor-1 (SDF-1) were significantly reduced in BM-Agtr1^-/- mice, accompanied by a decrease in the serum level of SDF-1. Thrombin-induced platelets aggregation was dose-dependently inhibited (45% at 0.1 IU/mL, P<0.05) in Agtr1^-/- platelets compared with Agtr1^+/+ platelets, accompanied by the reduced expression and release of SDF-1.

Conclusions—The BM-AT₁ receptor promotes neointima formation by regulating the mobilization and homing of BM-derived VPCs in a platelet-derived SDF-1–dependent manner without affecting EPC-mediated reendothelialization.

Methods and Results—Measures of body composition such as BMI, waist circumference (WC), hip circumference (HC), and waist-hip ratio (WHR) were registered in 6708 subjects aged 25 to 84 years, who participated in the Tromsø Study (1994–1995). Incident VTE-events were registered during follow-up until September 1, 2007. There were 222 VTE-events during a median of 12.3 years of follow-up. All measures of obesity exhibited significantly increased HR for VTE in multivariable models with highest risk estimates for WC in both genders. The risk of VTE increased across quartiles of BMI, WC, and HC in both genders, but not for WHR. WC identified more subjects at risk using established criteria for obesity. WC had the highest area under the curve in both genders in ROC analysis, and WC above ROC-derived cut-off values (WC ≥85 cm in women and ≥95 cm in men) were associated with HRs of 1.92 (95% CI: 1.05 to 3.48) in women and 2.78 (95% CI: 1.47 to 5.27) in men.

Conclusions—Our findings indicate that WC is the preferable anthropometric measure of obesity to identify subjects at risk and to predict risk of VTE.

Methods and Results—Gpihbp1^-/- mice on a chow diet have plasma triglyceride and cholesterol levels of 2812±209 and 319±27 mg/dL, respectively. Even though nearly all of the lipids were contained in large lipoproteins (50 to 135 nm), the mice developed progressive aortic atherosclerosis. In other experiments, we found that both Gpihbp1-deficient "apo-B48–only" mice and Gpihbp1-deficient "apo-B100–only" mice manifest severe chylomicronemia. Thus, GPIHBP1 is required for the processing of both apo-B48– and apo-B100–containing lipoproteins.

Conclusions—Chylomicronemia causes atherosclerosis in mice. Also, we found that GPIHBP1 is required for the lipolytic processing of both apo-B48– and apo-B100–containing lipoproteins.

Methods and Results—From young healthy males smokers (n=34) and nonsmokers (n=34) a baseline blood was drawn, and smokers had another blood draw after smoking 2 regular cigarettes. Using thromboelastography (TEG) the degree of platelet-fibrin interaction was measured. In additional experiments, abciximab (20 µg/mL) was added to the smokers samples (n=27) to reduce the effects of platelet function from the TEG parameters. The maximum clot strength (G) obtained with abciximab measured mainly the contribution of fibrinogen to clot strength (GF). By subtracting GF from G, the contribution of platelets to clot strength (GP) was presumed. A significant difference was found for all TEG parameters between nonsmokers versus postsmoking and pre- versus postsmoking samples. Postsmoking both GF and GP were significantly higher as compared to presmoking. On electron microscopy and turbidity analysis, postsmoking fibrin clots were significantly different compared to presmoking and nonsmoking samples.

Conclusions—Acute CSE changes clot dynamics and alters fibrin architecture. Both functional changes in fibrinogen and platelets appear to contribute to heightened thrombogenicity after acute CSE.

For the paper by Seiichiro Takahashi, Markus Moser, Eloi Montanez, Takanari Nakano, Makoto Seo, Steffen Backert, Ikuo Inoue, Takuya Awata, Sigehiro Katayama, Tsugikazu Komoda, and Reinhard Fässler (The fibronectin RGD motif is required for multiple angiogenic events during early embryonic development. Arterioscler Thromb Vasc Biol. 2009 August 27 [Epub ahead of print]; DOI: 10.1161/ATVBAHA.108.181164), after an investigation by the Saitama Medical University Internal Investigation Committee, the Committee concluded that it was unethical for Dr. Takahashi to publish this paper for the following reasons:

1. Dr. Takahashi admitted that he forged the signatures of coauthors Markus Moser, Eloi Montanez, and Reinhard Fässler that appeared on the Authorship Responsibility and Copyright Transfer Agreement.

2. Signatures of the other coauthors were made by themselves, not by Dr. Takahashi.

3. None of the Japanese coauthors knew before publication that the signatures of Dr. Moser, Dr. Montanez, and Dr. Fässler were forged by Dr. Takahashi.

4. None of the coauthors advised Dr. Takahashi that the signatures of Dr. Moser, Dr. Montanez, and Dr. Fässler should be faked by Dr. Takahashi.

5. Neither Dr. Moser, Dr. Montanez, nor Dr. Fässler read the submitted version of original manuscript.

6. Some of the Japanese coauthors did not check the boxes that appear on 1-E of the Authorship Responsibility and Copyright Transfer Agreement. In such cases, Dr. Takahashi did that instead, without permission.

7. Dr. Takahashi sent an e-mail message to Dr. Moser, Dr. Montanez, and Dr. Fässler, using fake e-mail addresses, to pretend that the German coauthors knew about the paper. Some of the Japanese coauthors believed this message and signed the Authorship Responsibility and Copyright Transfer Agreement.

The editors, therefore, hereby retract the paper.