The effects of coenzyme Q10 supplementation on gene expression related to insulin, lipid and inflammation in patients with polycystic ovary syndrome.

OBJECTIVE: This research was conducted to assess the effects of coenzyme Q10 (CoQ10) intake on gene expression related to insulin, lipid and inflammation in subjects with polycystic ovary syndrome (PCOS). METHODS: This randomized double-blind, placebo-controlled trial was conducted on 40 subjects diagnosed with PCOS. Subjects were randomly allocated into two groups to intake either 100 mg CoQ10 (n = 20) or placebo (n = 20) per day for 12 weeks. Gene expression related to insulin, lipid and inflammation were quantified in blood samples of PCOS women with RT-PCR method. RESULTS: Results of RT-PCR shown that compared with the placebo, CoQ10 intake downregulated gene expression of oxidized low-density lipoprotein receptor 1 (LDLR) (p < 0.001) and upregulated gene expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) (p = 0.01) in peripheral blood mononuclear cells of subjects with PCOS. In addition, compared to the placebo group, CoQ10 supplementation downregulated gene expression of interleukin-1 (IL-1) (p = 0.03), interleukin-8 (IL-8) (p = 0.001) and tumor necrosis factor alpha (TNF-α) (p < 0.001) in peripheral blood mononuclear cells of subjects with PCOS. CONCLUSIONS: Overall, CoQ10 intake for 12 weeks in PCOS women significantly improved gene expression of LDLR, PPAR-γ, IL-1, IL-8 and TNF-α.

Molecular hydrogen stabilizes atherosclerotic plaque in low-density lipoprotein receptor-knockout mice.

Hydrogen (H(2)) attenuates the development of atherosclerosis in mouse models. We aimed to examine the effects of H(2) on atherosclerotic plaque stability. Low-density lipoprotein receptor-knockout (LDLR(-/-)) mice fed an atherogenic diet were dosed daily with H(2) and/or simvastatin. In vitro studies were carried out in an oxidized-LDL (ox-LDL)-stimulated macrophage-derived foam cell model treated with or without H(2). H(2) or simvastatin significantly enhanced plaque stability by increasing levels of collagen, as well as reducing macrophage and lipid levels in plaques. The decreased numbers of dendritic cells and increased numbers of regulatory T cells in plaques further supported the stabilizing effect of H(2) or simvastatin. Moreover, H(2) treatment decreased serum ox-LDL level and apoptosis in plaques with concomitant inhibition of endoplasmic reticulum stress (ERS) and reduction of reactive oxygen species (ROS) accumulation in the aorta. In vitro, like the ERS inhibitor 4-phenylbutyric acid, H(2) inhibited ox-LDL- or tunicamycin (an ERS inducer)-induced ERS response and cell apoptosis. In addition, like the ROS scavenger N-acetylcysteine, H(2) inhibited ox-LDL- or Cu(2+) (an ROS inducer)-induced reduction in cell viability and increase in cellular ROS. Also, H(2) increased Nrf2 (NF-E2-related factor-2, an important factor in antioxidant signaling) activation and Nrf2 small interfering RNA abolished the protective effect of H(2) on ox-LDL-induced cellular ROS production. The inhibitory effects of H(2) on the apoptosis of macrophage-derived foam cells, which take effect by suppressing the activation of the ERS pathway and by activating the Nrf2 antioxidant pathway, might lead to an improvement in atherosclerotic plaque stability.

Atorvastatina, anticuerpos LDL oxidada y su relación con la edad / Atorvastatin and oxidized low density lipoprotein antibody. Relationship to age

Fundamento y objetivos: En pacientes hipercolesterolémicos, estudiamos las relaciones de los valores plasmáticos de anticuerpos anti lipoproteínas de baja densidad (LDL) oxidadas con diferentes variables de interés cardiovascular y sus cambios tras el tratamiento con atorvastatina. Pacientes y método: En 48 pacientes se determinaron los valores de anticuerpos anti-LDL oxidadas, biomarcadores lipídicos, de estrés oxidativo e inflamatorios, a la entrada del estudio y tras 24 semanas de tratamiento con 20mg de atorvastatina. Resultados: Los valores basales de anticuerpos anti-LDL oxidadas se correlacionaron con la edad (r=0,41, p=0,03), cintura (r=0,38, p=0,04) y la proteína C reactiva ultrasensible (PCRs) (r=0,46, p=0,02), pero no con el resto de variables. El tratamiento con atorvastatina no disminuyó los valores de anticuerpos anti-LDL oxidadas (valor basal medio [intervalo de confianza del 95%] de 413 mUI/ml [187-1196] y a las 24 semanas de 349 mUI/ml [101-1559]). El porcentaje de cambio de anticuerpos anti-LDL oxidadas en la semana 24 se correlacionó negativamente con la edad (r=−0,37, p=0,03), pero no con cambios en el resto de las variables. Conclusiones: En sujetos con hipercolesterolemia, los valores plasmáticos de anticuerpos anti-LDL oxidadas se relacionaron positivamente con la edad, cintura y PCRs. No se observaron cambios de los valores plasmáticos de anticuerpos anti-LDL oxidadas tras el tratamiento con atorvastatina, pero su variación se relacionó con la edad, lo que sugiere que las acciones inmunomoduladoras de las estatinas pueden depender de ésta (AU)

Background and objectives: In hypercholesterolemic patients, we studied the relationships of plasma levels of LDLoxab with cardiovascular variables and its changes after treatment with atorvastatin. Patients and methods: We studied, in 48 patients, the levels of LDLoxab, as well as lipid, oxidative stress and inflammatory biomarkers, at baseline and 24 weeks after treatment with 20mg of atorvastatin. Results: Baseline: a correlation was observed between LDLoxab and age (r= 0.41, P=.03), waist (r=0.38, P=.04) and C reactive protein (r= 0.46, P=.02), but not with other variables. Atorvastatin treatment did not decrease LDLoxab;(mU/mL, median [CI 95%]: baseline: 413 [187-1,196] and 24 weeks: 349 [101-1559]). The percentage change at week 24, was negatively correlated with age (r=−0.37, P=.03) but not with other variables. Conclusion: In hypercholesterolemic subjects plasma LDLoxab levels were positively corelated with age, waist and C reactive protein. There were no changes in plasma levels of LDLoxab after treatment with atorvastatin, but the variation was associated with age, suggesting that the immunomodulatory actions may depend of this (AU)

Caloric restriction, aerobic exercise training and soluble lectin-like oxidized LDL receptor-1 levels in overweight and obese post-menopausal women.

BACKGROUND: Elevated circulating levels of soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) have been observed in obese persons and are reduced by weight loss. However, it is not known whether combining caloric restriction (CR) with exercise training is better in reducing sLOX-1 levels than CR alone. OBJECTIVE: We examined whether the addition of aerobic exercise to a weight loss intervention differentially affects sLOX-1 levels in 61 abdominally obese post-menopausal women randomly assigned to a CR only (n = 22), CR+moderate-intensity exercise (n = 22) or CR+vigorous-intensity exercise (n = 17) intervention for 20 weeks. The caloric deficit was ~2800 kcal per week for all groups. RESULTS: The intervention groups were similar at baseline with respect to body weight, body composition, lipids and blood pressure. However, plasma sLOX-1 levels were higher in the CR-only group (99.90 ± 8.23 pg ml(-1)) compared with both the CR+moderate-intensity exercise (69.39 ± 8.23 pg ml(-1), P = 0.01) and the CR+vigorous-intensity exercise (72.83 ± 9.36 pg ml(-1), P = 0.03) groups. All three interventions significantly reduced body weight (~14%), body fat and waist and hip circumferences to a similar degree. These changes were accompanied by a 23% reduction in sLOX-1 levels overall (-19.00 ± 30.08 pg ml(-1), P < 0.0001), which did not differ among intervention groups (P = 0.13). Changes in body weight, body fat and maximal oxygen consumption (VO(2) max) were not correlated with changes in sLOX-1 levels. In multiple regression analyses in all women combined, baseline sLOX-1 levels (ß = -0.70 ± 0.06, P < 0.0001), age (ß = 0.92 ± 0.43, P = 0.03) and baseline body mass index (BMI) (ß = 1.88 ± 0.66, P = 0.006) were independent predictors of the change in sLOX-1 with weight loss. CONCLUSIONS: Weight loss interventions of equal energy deficit have similar effects on sLOX-1 levels in overweight and obese post-menopausal women, with the addition of aerobic exercise having no added benefit when performed in conjunction with CR.

Effects of berberine on expression of LOX-1 and SR-BI in human macrophage-derived foam cells induced by ox-LDL.

This study investigates the effects of beriberine on the expression of lectin-like ox-LDL receptor-1 (LOX-1), scavenger receptor A (SR-A), SR class B type I (SR-BI) and ATP-binding cassette transporter A1 (ABCA1) in human macrophage-derived foam cells induced by ox-LDL. Different concentrations of Berberine were co-cultured with THP-1 derived foam cells. The mRNA and protein expressions of LOX-1, SR-A, SR-BI and ABCA1 were determined by RT-PCR and Western blot analysis, respectively. Ox-LDL significantly increased the expression of LOX-1 and inhibited the expression of SR-BI in a dose- and time-dependent manner. Berberine significantly inhibited the effects of ox-LDL in a dose- and time-dependent manner. Moreover, ox-LDL significantly promoted ABCA1 expression. However, berberine had no effect on SR-A or ABCA1 expression. Berberine can inhibit the expression of LOX-1 and promote the expression of SR-BI in macrophage-derived foam cells. Therefore, berberine could be used to treat atherosclerotic diseases.

Curcumin eliminates oxidized LDL roles in activating hepatic stellate cells by suppressing gene expression of lectin-like oxidized LDL receptor-1.

Type II diabetes mellitus (T2DM) is often accompanied by non-alcoholic steatohepatitis (NASH) and associated with hypercholesterolemia, that is, increased levels of plasma low-density lipoprotein (LDL) and oxidized LDL (ox-LDL). Approximately one-third of NASH develops hepatic fibrosis. The role of hypercholesterolemia in T2DM and NASH-associated hepatic fibrogenesis remains obscure. We previously reported that the phytochemical curcumin inhibited the activation of hepatic stellate cells (HSCs), the major effector cells during hepatic fibrogenesis, and protected the liver from fibrogenesis in vitro and in vivo. The aims of this study are to evaluate the role of ox-LDL in activation of HSCs, to assess curcumin effects on eliminating the role of ox-LDL, and to further explore the underlying mechanisms. In this report, we observe that ox-LDL alters the expression of genes closely relevant to HSC activation, which is eliminated by curcumin. Curcumin suppresses gene expression of lectin-like oxidized LDL receptor-1 (LOX-1), leading to the blockade of the transport of extracellular ox-LDL into cells. This suppressive effect of curcumin results from the interruption of Wnt signaling and the activation of peroxisome proliferator-activated receptor-gamma (PPARgamma). In conclusion, these results support our initial hypothesis and demonstrate that ox-LDL stimulates HSC activation, which is eliminated by curcumin by suppressing lox-1 expression by interrupting Wnt signaling and stimulating PPARgamma activity. These results provide novel insights into the role of ox-LDL in T2DM and NASH-associated hepatic fibrogenesis and mechanisms by which curcumin suppresses ox-LDL-induced HSC activation, as well as the implication of curcumin in the treatment of T2DM and NASH-associated hepatic fibrosis.

Effect of ABCG2, PPARGC1A, OLR1 and SCD1 gene polymorphism on estimated breeding values for functional and production traits in Polish Holstein-Friesian bulls.

This study investigated the impact of 6 polymorphisms located in the ABCG2, PPARGC1A, OLR1 and SCD1 genes on estimated breeding values for milk production, longevity, somatic cell count and reproductive traits. The analysis was conducted on 453 Polish Holstein-Friesian bulls. Genotypes were identified using PCR-RFLP, and haplotype inferences were performed for 3 linked mutations of PPARGC1A. The most significant associations were found between the A/C polymorphism located in exon 14 of ABCG2 and milk fat production traits as well as calving-to-first insemination interval, and between the T/C substitution in intron 9 of the PPARGC1A and non-return rate in heifers.

Effects of red wine polyphenolic compounds on paraoxonase-1 and lectin-like oxidized low-density lipoprotein receptor-1 in hyperhomocysteinemic mice.

Hyperhomocysteinemia, or abnormally high plasma homocysteine (Hcy) concentration, has often been associated with vascular thrombosis and the development of premature atherosclerosis. Many studies have shown that moderate wine consumption has potential beneficial effects related to the prevention of atherosclerosis, in part attributed to the biological properties of polyphenolic components, mainly flavonoids. The aim of the present study is to determine the effects of a red wine polyphenolic extract (PE) administration on hyperhomocysteinemia due to cystathionine beta-synthase (CBS) deficiency and on the associated biochemical markers of hepatic and endothelial dysfunctions in mice. Red wine PE was added for 4 weeks to the drinking water of heterozygous CBS-deficient mice fed a high-methionine diet, a murine model of hyperhomocysteinemia. Red wine PE supplementation at low dose significantly reduced plasma Hcy levels and restored the hepatic and plasma-decreased paraoxonase-1 activity induced by chronic hyperhomocysteinemia. Moreover, aortic expression of proinflammatory cytokines and adhesion molecules and levels of soluble lectin-like oxidized low-density lipoprotein receptor-1 were reduced in hyperhomocysteinemic mice fed the red wine PE supplementation. These findings suggest that red wine PE administration in low quantities has beneficial effects on biochemical markers of endothelial dysfunction due to hyperhomocysteinemia.

Impact of plasma oxidized low-density lipoprotein removal on atherosclerosis.

BACKGROUND: Several clinical studies of statin therapy have demonstrated that lowering low-density lipoprotein (LDL) cholesterol prevents atherosclerotic progression and decreases cardiovascular mortality. In addition, oxidized LDL (oxLDL) is suggested to play roles in the formation and progression of atherosclerosis. However, whether lowering oxLDL alone, rather than total LDL, affects atherogenesis remains unclear. METHODS AND RESULTS: To clarify the atherogenic impact of oxLDL, lectin-like oxLDL receptor 1 (LOX-1), an oxLDL receptor, was expressed ectopically in the liver with adenovirus administration in apolipoprotein E-deficient mice at 46 weeks of age. Hepatic LOX-1 expression enhanced hepatic oxLDL uptake, indicating functional expression of LOX-1 in the liver. Although plasma total cholesterol, triglyceride, and LDL cholesterol levels were unaffected, plasma oxLDL was markedly and transiently decreased in LOX-1 mice. In controls, atherosclerotic lesions, detected by Oil Red O staining, were markedly increased (by 38%) during the 4-week period after adenoviral administration. In contrast, atherosclerotic progression was almost completely inhibited by hepatic LOX-1 expression. In addition, plasma monocyte chemotactic protein-1 and lipid peroxide levels were decreased, whereas adiponectin was increased, suggesting decreased systemic oxidative stress. Thus, LOX1 expressed in the livers of apolipoprotein E-deficient mice transiently removes oxLDL from circulating blood and possibly decreases systemic oxidative stress, resulting in complete prevention of atherosclerotic progression despite the persistence of severe LDL hypercholesterolemia and hypertriglyceridemia. CONCLUSIONS: OxLDL has a major atherogenic impact, and oxLDL removal is a promising therapeutic strategy against atherosclerosis.

Isorhamnetin prevent endothelial cell injuries from oxidized LDL via activation of p38MAPK.

The present investigation was undertaken to determine the protective effects of isorhamnetin on endothelial cell line EA.hy926 injuries induced by oxidized low-density lipoprotein (ox-LDL) and to uncover some of the underlying mechanisms of these effects. Indices such as cell viability, lactate dehydrogenase (LDH), and nitric oxide (NO) release were measured to evaluate the protective effects of isorhamnetin. 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, superoxide dismutase (SOD), superoxide and reactive oxygen species (ROS) generation were also detected to evaluate the antioxidant effects of isorhamnetin. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to confirm the expression of endothelial nitric oxide synthase (eNOS) mRNA and lectin-like ox-LDL receptor-1 mRNA. Western blotting was used to evaluate the protein expression of this receptor and eNOS, as well as p38-mitogen-activated protein kinase (p38MAPK) phosphorylation and NF-kappaB p65 translocation. As a result, cell viability decreased significantly (P<0.01) after 24 h treatment with ox-LDL, accompanied with apparent secretion disorders such as NO reduction and LDH increase. Pretreatment with isorhamnetin resulted in remarkable increase of cell viability (P<0.05) and modulation of secretion disorders mediated by ox-LDL in a concentration-dependent manner. Besides, ox-LDL led to upregulation of lectin-like ox-LDL receptor-1, phosphorylation of p38MAPK, translocation of NF-kappaB, and downregulation of the eNOS expression in endothelial cells. Isorhamnetin pretreatment inhibited the ox-LDL-induced downregulation of eNOS, upregulation of lectin-like ox-LDL receptor-1, phosphorylation of the p38MAPK and translocation of NF-kappaB. Moreover, isorhamnetin exhibited strong antioxidant activity, which was shown by its inhibition effects on ox-LDL-induced superoxide, ROS overproduction and significant SOD reduction. The data indicated the protective effects of isorhamnetin on endothelial cell line EA.hy926 from ox-LDL-induced cell injuries. These effects were obtained via inhibition of lectin-like ox-LDL receptor-1 upregulation, interference of ox-LDL-mediated intracellular signaling pathway (p38MAPK activation, NF-kappaB nuclear translocation, eNOS expression) and the antioxidant activity of isorhamnetin.

[Effects of lecithin-like oxidized low-density lipoprotein receptor-1 on p38 mitogen-activated protein kinase induced by oxidized low-density lipoprotein in endothelial cells].

OBJECTIVE: To investigate the effects of lecithin-like oxidized low-density lipoprotein receptor-1 (LOX-1) on the p38 mitogen-activated protein kinase (p38MAPK) induced by oxidized low-density lipoprotein (ox-LDL) in endothelial cells. METHODS: Human umbilical vein endothelial cells of the line ECV304 were cultured and treated with LDL 25 microg/ml and ox-LDL of 3 different concentrations (25 microg/ml, 50 microg/ml, and 100 microg/ml) for 24 hours. ECV304 cells without treatment of ox-LDL were used as control group. MTT method was used to detect the optical density (OD) of different groups. RT-PCR was used to detect the mRNA expression of LOX-1. Western blotting was used to detect the protein expression of LOX-1. Another ECV304 cells were randomly divided into 7 groups to be treated with LDL (25 microg/ml), ox-LDL of the concentrations of 25 microg/ml, 50 microg/ml, and 100 microg/ml, ox-LDL 100 microg/ml + JTX92 (LOX-1 blocking antibody) 10 microg/ml, or JTX92 10 microg/ml, or not to be treated with LOX-1 and/or JTX92 (as control group). The p38MAPK protein expression and phosphorylated p38MAPK (p-p38MAPK) protein expression were detected by Western blotting. RESULTS: The A values of the ECV304 cells treated with ox-LDL were significantly lower than that of the control group dose-dependently (all P < 0.05), however, the A value of the LDL group was not significantly different from that of the control group (P > 0.05). The LOX-1 mRNA expression and protein expression were significantly increased in the ox-LDL groups dose-dependently (both P < 0.01). However, the LOX-1 mRNA expression and protein expression of the LDL group were not significantly different from those of the control group (both P > 0.05). The p38MAPK protein expression level was not significantly different among different groups (all P > 0.05). The p-p38MAPK protein expression was up-regulated by ox-LDL dose-dependently (P < 0.05). The p-p38MAPK protein level of the ox-LDL + JTX92 group was significantly lower than that of the ox-LDL group (P < 0.05), However, the p-p38MAPK protein expression of the JTX92 group was not significantly different from that of the control group. CONCLUSION: ox-LDL activates the p38MAPK signal pathway through its receptor LOX-1. Upregulation of LOX-1 is an important ring causing atherosclerosis.

Cyclic tensile stretch load and oxidized low density lipoprotein synergistically induce lectin-like oxidized ldl receptor-1 in cultured bovine chondrocytes, resulting in decreased cell viability and proteoglycan synthesis.

Mechanical stimulation is known to be an essential factor in the regulation of cartilage metabolism. We tested the hypothesis that expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) can be modulated by cyclic tensile stretch load in chondrocytes. Cyclic loading of repeated stretch stress at 10 cycles per minute with 10 kPa of stress for 6 h induced expression of LOX-1 to 2.6 times control in cultured bovine articular chondrocytes, equivalent to the addition of 10 microg/mL oxidized low density lipoprotein (ox-LDL) (2.4 times control). Application of the cyclic load to the chondrocytes along with 10 microg/mL ox-LDL resulted in synergistically increased LOX-1 expression to 6.3 times control. Individual application of cyclic loading and 10 microg/mL ox-LDL significantly suppressed chondrocytes viability (84.6% +/- 3.4% and 80.9% +/- 3.2% of control at 24 h, respectively; n = 3; p < 0.05) and proteoglycan synthesis [81.0% +/- 7.1% and 85.7% +/- 5.2% of control at 24 h, respectively; p < 0.05 when compared with 94.6% +/- 4.6% for native-LDL (n = 3)]. Cyclic loading and 10 microg/mL ox-LDL synergistically affected cell viability and proteoglycan synthesis, which were significantly suppressed to 45.6% +/- 4.9% and 48.7% +/- 6.7% of control at 24 h, respectively (n = 3; p < 0.01 when compared with individual application of cyclic loading or 10 microg/mL ox-LDL). In this study, we demonstrated synergistic effects of cyclic tensile stretch load and ox-LDL on cell viability and proteoglycan synthesis in chondrocytes, which may be mediated through enhanced expression of LOX-1 and which has important implications in the progression of cartilage degeneration in osteoarthritis.

Association of the OLR1 gene with milk composition in Holstein dairy cattle.

Oxidized low-density lipoprotein receptor (OLR1) is the major protein that binds, internalizes, and degrades oxidized low-density lipoprotein. The role of OLR1 in lipid metabolism and the results of previous whole-genome scan studies prompted the investigation of OLR1 as a candidate gene affecting milk composition traits. Direct cDNA and genomic sequencing of OLR1 revealed 2 single nucleotide polymorphisms (SNP) in exon 4, 5 SNP in intron 4, and 1 in the 3' untranslated region (UTR). Four intragenic haplotypes comprising SNP positions 7,160, 7,161, 7,278, 7,381, 7,409, 7,438, 7,512, and 8,232 were inferred. Haplotype analysis showed that one of the haplotypes was associated with a significant increase in fat yield and fat percentage. Single SNP analysis showed that allele C of SNP 8,232 (in the 3'-UTR) had significant effects on fat yield and fat percentage, whereas SNP 7,160 and 7,161 (in exon 4) had no significant effects. Both single SNP and haplotype analyses indicate that SNP 8,232 in the 3'-UTR is associated with milk fat yield and percentage and it may be in linkage disequilibrium with the functional polymorphism. To provide support for the hypothesis that SNP 8,232 is responsible for OLR1 expression, OLR1 expression levels in individuals bearing different genotypes were assessed. It was found that OLR1 expression was reduced in genotype AA individuals compared with CC and AC individuals, suggesting that A at position 8,232 may be the nucleotide causing decreased OLR1 expression. The 3'-UTR polymorphism found in this study might control translation or stability of OLR1 mRNA.

The role of LOX-1, a novel lectin-like receptor for oxidized low density lipoprotein, in atherosclerosis.

There is strong evidence for the role of oxidative stress in all stages of atherosclerosis. Oxidized low density lipoprotein (ox-LDL), a marker of oxidative stress, is present in the plasma as well as in the atherosclerotic arteries of patients with atherosclerosis. Ox-LDL leads to endothelial activation, dysfunction and injury. Recently, a novel lectin-like receptor for ox-LDL (LOX-1) has been identified, primarily in the endothelial cells, that allows uptake of ox-LDL into endothelial cells. This receptor is transcriptionally upregulated by tumour necrosis factor-a, angiotensin II, shear stress and ox-LDL. The expression of this receptor activates a variety of intracellular processes that leads to expression of adhesion molecules and endothelial activation. Recent studies show that LOX-1 activation leads to the expression of CD40/40 L in endothelial cells and upregulation of matrix metalloproteinases. This receptor is highly expressed in blood vessels of animals and humans with hypertension, diabetes mellitus and atherosclerosis. Co-localization of LOX-1 along with ox-LDL in the rupture-prone plaque suggests that this receptor may be involved in the precipitation of acute myocardial ischemia. Identification and regulation of this receptor and understanding of signal transduction pathways may lead to new therapies in disease states characterized by endothelial dysfunction.