BACKGROUND It is unclear whether high-density lipoprotein (HDL) cholesterol concentration plays a causal role in atherosclerosis. A more important factor may be HDL cholesterol efflux capacity, the ability of HDL to accept cholesterol from macrophages, which is a key step in reverse cholesterol transport. We investigated the epidemiology of cholesterol efflux capacity and its association with incident atherosclerotic cardiovascular disease outcomes in a large, multiethnic population cohort. METHODS We measured HDL cholesterol level, HDL particle concentration, and cholesterol efflux capacity at baseline in 2924 adults free from cardiovascular disease who were participants in the Dallas Heart Study, a probability-based population sample. The primary end point was atherosclerotic cardiovascular disease, defined as a first nonfatal myocardial infarction, nonfatal stroke, or coronary revascularization or death from cardiovascular causes. The median follow-up period was 9.4 years. RESULTS In contrast to HDL cholesterol level, which was associated with multiple traditional risk factors and metabolic variables, cholesterol efflux capacity had minimal association with these factors. Baseline HDL cholesterol level was not associated with cardiovascular events in an adjusted analysis (hazard ratio, 1.08; 95% confidence interval [CI], 0.59 to 1.99). In a fully adjusted model that included traditional risk factors, HDL cholesterol level, and HDL particle concentration, there was a 67% reduction in cardiovascular risk in the highest quartile of cholesterol efflux capacity versus the lowest quartile (hazard ratio, 0.33; 95% CI, 0.19 to 0.55). Adding cholesterol efflux capacity to traditional risk factors was associated with improvement in discrimination and reclassification indexes. CONCLUSIONS Cholesterol efflux capacity, a new biomarker that characterizes a key step in reverse cholesterol transport, was inversely associated with the incidence of cardiovascular events in a population-based cohort.

Background-C-reactive protein (CRP), the prototypic marker of inflammation, has been shown to be an independent predictor of cardiovascular events. Endothelial nitric oxide synthase (eNOS) deficiency is a pivotal event in atherogenesis. Methods and Results-We tested the effect of CRP on eNOS expression and bioactivity in cultured human aortic endothelial cells (HAECs). CRP decreased eNOS mRNA, protein abundance, and enzyme activity in HAECs. Furthermore, eNOS bioactivity assayed by cyclic GMP levels was significantly reduced by CRP. Preincubation of cells with CRP also significantly increased the adhesion of monocytes to HAECs. Key Words: inflammation Ⅲ C-reactive protein Ⅲ nitric oxide synthase Ⅲ endothelium I nflammation seems to play a critical role in all stages of atherosclerosis, from the nascent lesion to acute coronary syndromes. 1 C-reactive protein (CRP) is a prototypic marker of inflammation and has been shown in numerous prospective studies to predict both cardiovascular events (CVE) in apparently healthy persons and a poor prognosis after acute coronary syndromes. 2-6 CRP is clearly a risk marker, and much data are evolving to suggest that CRP also promotes atherogenesis. [7][8][9][10][11] In this regard, it has been shown that CRP promotes tissue factor expression in monocytes and also induces adhesion molecule and chemokine expression in human endothelial cells (ECs). 7-10 Recently, CRP was also shown to increase endothelin-1 (ET-1) release from saphenous vein ECs. 11 A critical enzyme present in ECs is endothelial nitric oxide synthase (eNOS). Nitric oxide derived from eNOS promotes arterial vasodilatation and inhibits smooth muscle cell proliferation, LDL oxidation, platelet adhesion and aggregation, and monocyte adhesion to endothelium. [12][13][14] It is believed that endothelial dysfunction (decreased eNOS bioactivity) occurs very early in atherogenesis. Thus, we tested the effect of CRP on eNOS expression, enzymatic activity, and bioactivity in human aortic endothelial cells (HAECs) to ascertain if CRP impaired eNOS activity. Conclusion-CRP causes a direct reduction in eNOS expression MethodsFor all the experiments, HAECs (Clonetics, San Diego, Calif) were used within 5 passages. THP-1 cells, a monocytic cell line (ATCC), were used for adhesion experiments. Purity of recombinant human CRP (Calbiochem) was checked by SDS-PAGE, yielding a single band. Endotoxin was removed from CRP with Detoxigel column (Pierce Biochemicals) and found to be Ͻ0.125 endotoxin units (EU)/mL by Limulus assay (Biowhittaker).HAECs (1x10 6 cells/mL) were used for all assays and incubated with concentrations of CRP ranging from 0 to 50 g/mL. Cell viability as assessed by the 3-(4,5-dimethylthiazol-z-yl)-2,5, diphenyl tetrazolium bromide assay was Ͼ95% with this dose range of CRP. Apoptosis was measured by staining cells with fluorescin isothiocyanate-labeled Annexin V (R&D Systems), followed by flow cytometry.Cells were lysed and 30 g of protein per well were loaded and transferred to membranes. Membranes were blocked...

Endothelial nitric-oxide synthase (eNOS) generates the key signaling molecule nitric oxide in response to intralumenal hormonal and mechanical stimuli. We designed studies to determine whether eNOS is localized to plasmalemmal microdomains implicated in signal transduction called caveolae. Using immunoblot analysis, eNOS protein was detected in caveolar membrane fractions isolated from endothelial cell plasma membranes by a newly developed detergent-free method; eNOS protein was not found in noncaveolar plasma membrane. Similarly, NOS enzymatic activity was 9.4-fold enriched in caveolar membrane versus whole plasma membrane, whereas it was undetectable in noncaveolar plasma membrane. 51-86% of total NOS activity in postnuclear supernatant was recovered in plasma membrane, and 57-100% of activity in plasma membrane was recovered in caveolae. Immunoelectron microscopy showed that eNOS heavily decorated endothelial caveolae, whereas coated pits and smooth plasma membrane were devoid of gold particles. Furthermore, eNOS was targeted to caveolae in COS-7 cells transfected with wild-type eNOS cDNA. Studies with eNOS mutants revealed that both myristoylation and palmitoylation are required to target the enzyme to caveolae and that each acylation process enhances targeting by 10-fold. Thus, acylation targets eNOS to plasmalemmal caveolae. Localization to this microdomain is likely to optimize eNOS activation and the extracellular release of nitric oxide.The endothelial isoform of nitric-oxide synthase (eNOS) 1 is one of three isoenzymes that converts L-arginine to L-citrulline plus the key signaling molecule nitric oxide (NO). eNOS is acutely activated by increases in endothelial intracellular calcium induced by the stimulation of diverse G-protein-coupled cell surface receptors and by physical stimuli such as hemodynamic shear stress and varying oxygenation. Endotheliumderived NO regulates blood pressure, platelet aggregation, and vascular smooth muscle mitogenesis. Diminished NO production has been implicated in the pathogenesis of a variety of vascular disorders including atherosclerosis and pulmonary hypertension (1-4).eNOS is a unique isoform of the enzyme in that it is primarily localized to the particulate subcellular fraction (1-4). Studies evaluating NOS enzymatic activity have demonstrated that functional eNOS is primarily associated with the plasma membrane (5). Since eNOS activity is acutely regulated by intralumenal factors and the NO produced is a labile, cytotoxic messenger molecule (1-4), the intracellular site of NO synthesis is likely to have a major influence on its biological activity in the vascular wall. We therefore sought to determine the specific subcellular location to which eNOS is targeted and designed studies to delineate whether the enzyme is localized to plasmalemmal caveolae. Caveolae are plasmalemmal microdomains that have been implicated in the transcytosis of macromolecules, the uptake of small molecules by potocytosis, and the compartmentalization of signaling molecules (6 -9). The ...

Atherosclerosis is the primary cause of cardiovascular disease, and the risk for atherosclerosis is inversely proportional to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL is atheroprotective are complex and not well understood. Here we show that HDL stimulates endothelial nitric oxide synthase (eNOS) in cultured endothelial cells. In contrast, eNOS is not activated by purified forms of the major HDL apolipoproteins ApoA-I and ApoA-II or by low-density lipoprotein. Heterologous expression experiments in Chinese hamster ovary cells reveal that scavenger receptor-BI (SR-BI) mediates the effects of HDL on the enzyme. HDL activation of eNOS is demonstrable in isolated endothelial-cell caveolae where SR-BI and eNOS are colocalized, and the response in isolated plasma membranes is blocked by antibodies to ApoA-I and SR-BI, but not by antibody to ApoA-II. HDL also enhances endothelium- and nitric-oxide-dependent relaxation in aortae from wild-type mice, but not in aortae from homozygous null SR-BI knockout mice. Thus, HDL activates eNOS via SR-BI through a process that requires ApoA-I binding. The resulting increase in nitric-oxide production might be critical to the atheroprotective properties of HDL and ApoA-I.

During the production process, the word caspase was misspelled in the title; the correct title appears above. Also, in the legend for Table 1 the mu symbol (µ) was formatted incorrectly; the correct legend appears below. We regret the error. Table 1 Jurkat cells (J16) were preincubated for 2 h with zVAD-fmk (50 µM), DEVD-CHO (100 µM) or left untreated and then exposed to etoposide (10 µg/ml) or IR (30 Gy). After 16 h incubation, Cer content, nuclear fragmentation, mitochondrial transmembrane potential and cell viability were determined in parallel samples as described in the Methods section. The results are representative of two independent experiments.

The cardioprotective effects of estrogen are mediated by receptors expressed in vascular cells. Here we show that 27-hydroxycholesterol (27HC), an abundant cholesterol metabolite that is elevated with hypercholesterolemia and found in atherosclerotic lesions, is a competitive antagonist of estrogen receptor action in the vasculature. 27HC inhibited both the transcription-mediated and the non-transcription-mediated estrogen-dependent production of nitric oxide by vascular cells, resulting in reduced estrogen-induced vasorelaxation of rat aorta. Furthermore, increasing 27HC levels in mice by diet-induced hypercholesterolemia, pharmacologic administration or genetic manipulation (by knocking out the gene encoding the catabolic enzyme CYP7B1) decreased estrogen-dependent expression of vascular nitric oxide synthase and repressed carotid artery reendothelialization. As well as antiestrogenic effects, there were proestrogenic actions of 27HC that were cell-type specific, indicating that 27HC functions as an endogenous selective estrogen receptor modulator (SERM). Taken together, these studies point to 27HC as a contributing factor in the loss of estrogen protection from vascular disease.

Summary To date estrogen is the only known endogenous estrogen receptor (ER) ligand that promotes ER+ breast tumor growth. We report that the cholesterol metabolite 27-hydroxycholesterol (27HC) stimulates MCF-7 cell xenograft growth in mice. More importantly, in ER+ breast cancer patients, 27HC content in normal breast tissue is increased compared to that in cancer-free controls, and tumor 27HC content is further elevated. Increased tumor 27HC is correlated with diminished expression of CYP7B1, the 27HC metabolizing enzyme, and reduced expression of CYP7B1 in tumors is associated with poorer patient survival. Moreover, 27HC is produced by MCF-7 cells and it stimulates cell-autonomous, ER-dependent and GDNF-RET-dependent cell proliferation. Thus, 27HC is a locally-modulated, non-aromatized ER ligand that promotes ER+ breast tumor growth.

Abstract-Estrogen causes nitric oxide (NO)-dependent vasodilation due to estrogen receptor (ER) ␣-mediated, nongenomic activation of endothelial NO synthase (eNOS). The subcellular site of interaction between ER␣ and eNOS was determined in studies of isolated endothelial cell plasma membranes. Estradiol (E 2 , 10 -8 mol/L) caused an increase in eNOS activity in plasma membranes in the absence of added calcium, calmodulin, or eNOS cofactors, which was blocked by ICI 182,780 and ER␣ antibody. Immunoidentification studies detected the same 67-kDa protein in endothelial cell nucleus, cytosol, and plasma membrane. Plasma membranes from COS-7 cells expressing eNOS and ER␣ displayed ER-mediated eNOS stimulation, whereas membranes from cells expressing eNOS alone or ER␣ plus a myristoylation-deficient mutant eNOS were insensitive. Fractionation of endothelial cell plasma membranes revealed ER␣ protein in caveolae, and E 2 caused stimulation of eNOS in isolated caveolae that was ER-dependent; noncaveolae membranes were insensitive. Acetylcholine and bradykinin also activated eNOS in isolated caveolae. Furthermore, the effect of E 2 on eNOS in caveolae was prevented by calcium chelation. Thus, a subpopulation of ER␣ is localized to endothelial cell caveolae where they are coupled to eNOS in a functional signaling module that may regulate the local calcium environment. The full text of this article is available at http://www.circresaha.org. (Circ Res. 2000;87:e44-e52.) Key Words: acetylcholine Ⅲ bradykinin Ⅲ caveolin Ⅲ cell membrane Ⅲ endothelium Ⅲ estrogens T he hormone estrogen classically exerts its effects by modifying gene expression through the activation of estrogen receptors (ERs), which serve as transcription factors. 1-3 However, there are also rapid, presumably nongenomic effects of estrogen in a variety of tissues including the vasculature. 4 -6 Estrogen has important atheroprotective properties that are at least partially related to its capacity to enhance the bioavailability of nitric oxide (NO). [5][6][7] NO is a potent regulator of blood pressure, platelet aggregation, leukocyte adhesion, and vascular smooth muscle mitogenesis that is produced in the vascular wall primarily by the endothelial isoform of NO synthase (eNOS) on the conversion of the substrate L-arginine to L-citrulline. 8 The function of the L-arginine/eNOS system is altered in a variety of vascular disorders. 9 We have previously shown that estrogen rapidly stimulates eNOS activity in endothelial cells, that the response is attenuated by ER antagonism but not by inhibiting gene transcription, and that ER␣ is expressed in endothelium. 10,11 We have also shown that the overexpression of ER␣ in endothelial cells causes enhancement of the acute response to estradiol (E 2 ) that is blocked by ER antagonism, specific to E 2 versus other agonists, and dependent on the ER␣ hormone binding domain. In addition, the acute stimulation of eNOS by E 2 can be reconstituted in COS-7 cells cotransfected with wild-type ER␣ and eNOS. 11 Thus, the short-term ef...