Induction Of Endothelial Dysfunction Research Articles

The vicious circle of UHRF1 down-regulation and KEAP1/NRF2/HO-1 pathway impairment promotes oxidative stress-induced endothelial cell apoptosis in diabetes.

Oxidative stress is recognized as a key factor in the induction of endothelial dysfunction in diabetes. However, the specific mechanisms have not been fully elucidated. We herein hypothesized that ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) might have a role in oxidative stress-induced endothelial cell (EC) apoptosis in diabetes. Western blot, qPCR, wound healing assay, apoptosis assay, reactive oxygen species (ROS) detection, dual-luciferase reporter assay, methylation-specific PCR, bisulfite sequencing PCR and chromatin immunoprecipitation assay were performed. UHRF1 expression levels were significantly decreased in endothelial colony-forming cells derived from peripheral blood of participants with type 2 diabetes compared with individuals without diabetes. ECs treated with high glucose, palmitate or hydrogen peroxide in vitro also exhibited decreased UHRF1 protein levels. Silencing of UHRF1 led to decreased migration ability and increased apoptosis and ROS production in ECs, which might be related to impaired Kelch-like ECH-associated protein 1 (KEAP1)/nuclear factor erythroid 2-related factor 2 (NRF2)/haeme oxygenase-1 pathway. Mechanistically, UHRF1 is closely implicated in epigenetic regulation of chromatin modification status at KEAP1 genomic locus via histone acetylation. NRF2 down-regulation in turn inhibits UHRF1 protein level, which might be due to increased ROS generation. Diabetes-induced oxidative stress can mediate down-regulation of UHRF1, which enhances ROS production by regulating KEAP1/p-NRF2 pathway through histone acetylation and might also form a self-perpetuating feedback loop with KEAP1/p-NRF2 to further promote oxidative stress-induced apoptosis of ECs in diabetes.

Abstract 11270: SGLT2 Expression in the Coronary Microvessel Endothelium and Cardiomyocytes of Cardiac Patients: Determinant Role of Low-Grade Inflammation and Induction of Endothelial Dysfunction

Introduction: Sodium-glucose co-transporter2 inhibitors (SGLT2i) showed benefit in major cardiovascular diseases characterized by low-grade inflammation. However, the role and function of SGLT2 in the heart remain unclear. Hypothesis: This study evaluated whether SGLT1/2 are expressed in the heart of patients with cardiac diseases, and determined the role of low-grade inflammation and the functional consequences. Methods: Human left ventricle (LV) biopsies were collected from 17 patients with aortic and mitral valves stenosis or hypertrophic cardiomyopathy at Strasbourg Hospital. Cultured endothelial cells (EC) were from pig coronary arteries. Expression levels were assessed by RT-qPCR, Western blot analysis and immunofluorescence staining, and the level of oxidative stress and nitric oxide (NO) using fluorescent probes. Results: SGLT1/2 protein levels were observed in the LV of cardiac patients and correlated p-p65 NFκB levels. SGLT2, VCAM-1 and TNF-α staining was observed in the endothelium of coronary microvessels and, to some extent, cardiomyocytes. TNF-α upregulated SGLT1 and 2, VCAM-1, AT1R and ACE expression and decreased that of eNOS and the bradykinin-stimulated NO formation in EC. The stimulatory effect of TNF-α was inhibited by an NF-kB inhibitor and SGLT2 siRNA but not by SGLT1 siRNA. Oxidative stress in LV sections and TNF-α-treated EC was inhibited by VAS-2870 (NADPH oxidase inhibitor), losartan (AT1R antagonist) and empagliflozin (SGLT2i), and in LV by infliximab (TNF-α receptor inhibitor). Conclusions: The findings indicate that SGLT2 is expressed in the LV of cardiac patients in the coronary endothelium and cardiomyocytes, and associated with their low-grade inflammatory status. Moreover, TNF-α upregulated the AT1R/NAPDH oxidase/SGLT2 crosstalk to sustain oxidative stress promoting endothelial dysfunction. Thus, SGLT2 appears as an interesting target to protect the coronary microcirculation.

Mechanisms of Oxidized LDL-Mediated Endothelial Dysfunction and Its Consequences for the Development of Atherosclerosis.

Atherosclerosis is an immuno-metabolic disease involving chronic inflammation, oxidative stress, epigenetics, and metabolic dysfunction. There is compelling evidence suggesting numerous modifications including the change of the size, density, and biochemical properties in the low-density lipoprotein (LDL) within the vascular wall. These modifications of LDL, in addition to LDL transcytosis and retention, contribute to the initiation, development and clinical consequences of atherosclerosis. Among different atherogenic modifications of LDL, oxidation represents a primary modification. A series of pathophysiological changes caused by oxidized LDL (oxLDL) enhance the formation of foam cells and atherosclerotic plaques. OxLDL also promotes the development of fatty streaks and atherogenesis through induction of endothelial dysfunction, formation of foam cells, monocyte chemotaxis, proliferation and migration of SMCs, and platelet activation, which culminate in plaque instability and ultimately rupture. This article provides a concise review of the formation of oxLDL, enzymes mediating LDL oxidation, and the receptors and pro-atherogenic signaling pathways of oxLDL in vascular cells. The review also explores how oxLDL functions in different stages of endothelial dysfunction and atherosclerosis. Future targeted pathways and therapies aiming at reducing LDL oxidation and/or lowering oxLDL levels and oxLDL-mediated pro-inflammatory responses are also discussed.

Hyperuricemia-induced endothelial insulin resistance: the nitric oxide connection.

Hyperuricemia, defined as elevated serum concentrations of uric acid (UA) above 416µmol L-1, is related to the development of cardiometabolic disorders, probably via induction of endothelial dysfunction. Hyperuricemia causes endothelial dysfunction via induction of cell apoptosis, oxidative stress, and inflammation; however, it's interfering with insulin signaling and decreased endothelial nitric oxide (NO) availability, resulting in the development of endothelial insulin resistance, which seems to be a major underlying mechanism for hyperuricemia-induced endothelial dysfunction. Here, we elaborate on how hyperuricemia induces endothelial insulin resistance through the disruption of insulin-stimulated endothelial NO synthesis. High UA concentrations decrease insulin-induced NO synthesis within the endothelial cells by interfering with insulin signaling at either the receptor or post-receptor levels (i.e., proximal and distal steps). At the proximal post-receptor level, UA impairs the function of the insulin receptor substrate (IRS) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) in the insulin signaling pathway. At the distal level, high UA concentrations impair endothelial NO synthase (eNOS)-NO system by decreasing eNOS expression and activity as well as by direct inactivation of NO. Clinically, UA-induced endothelial insulin resistance is translated into impaired endothelial function, impaired NO-dependent vasodilation, and the development of systemic insulin resistance. UA-lowering drugs may improve endothelial function in subjects with hyperuricemia.

Roles and Therapeutic Implications of Endoplasmic Reticulum Stress and Oxidative Stress in Cardiovascular Diseases

In different pathological states that cause endoplasmic reticulum (ER) calcium depletion, altered glycosylation, nutrient deprivation, oxidative stress, DNA damage or energy perturbation/fluctuations, the protein folding process is disrupted and the ER becomes stressed. Studies in the past decade have demonstrated that ER stress is closely associated with pathogenesis of obesity, insulin resistance and type 2 diabetes. Excess nutrients and inflammatory cytokines associated with metabolic diseases can trigger or worsen ER stress. ER stress plays a critical role in the induction of endothelial dysfunction and atherosclerosis. Signaling pathways including AMP-activated protein kinase and peroxisome proliferator-activated receptor have been identified to regulate ER stress, whilst ER stress contributes to the imbalanced production between nitric oxide (NO) and reactive oxygen species (ROS) causing oxidative stress. Several drugs or herbs have been proved to protect against cardiovascular diseases (CVD) through inhibition of ER stress and oxidative stress. The present article reviews the involvement of ER stress and oxidative stress in cardiovascular dysfunction and the potential therapeutic implications.

The Role of Inflammation and Myeloperoxidase-Related Oxidative Stress in the Pathogenesis of Genetically Triggered Thoracic Aortic Aneurysms.

Genetically triggered thoracic aortic aneurysms (TAAs) are usually considered to exhibit minimal levels of inflammation. However, emerging data demonstrate that specific features of an inflammatory response can be observed in TAA, and that the extent of the inflammatory response can be correlated with the severity, in both mouse models and in human studies. Myeloperoxidase (MPO) is a key mediator of the inflammatory response, via production of specific oxidative species, e.g., the hypohalous acids. Specific tissue modifications, mediated by hypohalous acids, have been documented in multiple cardiovascular pathologies, including atherosclerosis associated with coronary artery disease, abdominal aortic, and cerebral aneurysms. Similarly, data are now emerging that show the capacity of MPO-derived oxidative species to regulate mechanisms important in TAA pathogenesis, including alterations in extracellular matrix homeostasis, activation of matrix metalloproteinases, induction of endothelial dysfunction and vascular smooth muscle cell phenotypic switching, and activation of ERK1/2 signaling. The weight of evidence supports a role for inflammation in exacerbating the severity of TAA progression, expanding our understanding of the pathogenesis of TAA, identifying potential biomarkers for early detection of TAA, monitoring severity and progression, and for defining potential novel therapeutic targets.

Evaluation of the Correlation between Serum Concentrations of Asymmetric Dimethylarginine and Corrected TIMI Frame Count in Patients with Slow Coronary Flow.

Coronary slow flow (CSF) is an important angiographic entity that is characterized by delayed opacification of coronary arteries in the absence of epicardial occlusive disease. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase. Elevated levels of ADMA cause the induction of endothelial dysfunction and thus promote atherosclerosis. This study was aimed at determining the role of ADMA in the development of CSF. One hundred twenty-nine subjects who fulfilled the inclusion criteria were enrolled in this study. According to coronary angiography results, these subjects were divided into five groups. The serum concentration of ADMA was measured in these subjects. In this study, there was no significant correlation between serum concentrations of ADMA and mean corrected TIMI frame count (CTFC) (P > 0.05). However, the ADMA level was significantly correlated with CTFC in the left anterior descending (LAD) coronary artery in patients with CSF (r = −0.381, P = 0.045). Also, plasma ADMA levels were significantly higher in patients with CSF and without CAD compared to patients without CSF and with CAD (50-90%) (P = 0.034). Besides, serum concentrations of ADMA were significantly higher in subjects with BMI < 25 kg/m2 compared with those having BMI > 30 kg/m2 (P = 0.003). It was also shown that the levels of ADMA were significantly higher in subjects with age as a cardiovascular risk factor compared with those without this risk factor (P = 0.024). Further studies with larger population sizes are needed to confirm the present findings on the association between the serum concentrations of ADMA and CSF.

Time‐dependent induction of vascular oxidative stress, inflammation, endothelial dysfunction and high blood pressure by aircraft noise exposure in mice

BackgroundTransportation noise is recognized as an important cardiovascular risk factor. It was shown that traffic noise exposure is associated with cerebro/cardiovascular diseases such as hypertension, myocardial infarction, and stroke. Aircraft noise causes endothelial dysfunction, inflammation and increases cerebral/cardiovascular oxidative stress in mice and traffic noise in general increased stroke mortality in large population studies. Chronic aircraft noise also causes learning and memory impairment in children. We recently studied the effects of different time exposure protocols (sleep versus awake phase) of aircraft noise exposure on C57Bl/6JRj mice and demonstrated that sleep phase noise exposure impairs the circadian clock and causes oxidative stress, endothelial dysfunction and high blood pressure.Methods and ResultsBefore the main study, we ensured that the longest noise exposure did not cause hearing loss in the mice by using audiometry. C57Bl/6JRj mice were exposed to aircraft noise for 4, 7, 14 and 28 days at a maximum sound level of 85 dB(A) and a mean sound pressure level of 72 dB(A) and compared with unexposed control mice. The induction of endothelial dysfunction and elevation of blood pressure was comparable in all exposure groups. Oxidative burst in the whole blood, stimulated with phorbol 12,13‐dibutyrate (PDBu) or zymosan A, was increased in all exposed groups with a maximum at 4 or 7 days. Vascular ROS formation as envisaged by dihydroethidium (DHE) staining was increased in noise‐exposed mice without a significant time response. Noise‐induced aortic, cardiac and cerebral superoxide formation as measured by DHE HPLC analysis increased in dependence of noise exposure duration. Similarly, levels of interleukin‐6 (IL‐6) were time dependently increased by trend in plasma and brain tissue in all noise exposed mice. We also established increased NOX‐2 expression in aortic tissue of noise exposed mice, while levels of P‐VASP were decreased after exposure.ConclusionWe here present novel data on the effects of different aircraft noise exposure protocols (4–28 days) indicating that most adverse vascular effects are established already after 4 days. Importantly, our present data prove that the applied moderate noise levels do not cause induction of hearing loss, even not after long‐term exposures. Thus our present data provide important technical insights in noise exposure protocols for future translational studies in mice with relevance for the human situation.Support or Funding InformationOur experimental studies on noise effects in mice were supported by vascular biology research grants from the Boehringer Ingelheim Foundation for the collaborative research group „Novel and neglected cardiovascular risk factors: molecular mechanisms and therapeutic implications” and Foundation Heart of Mainz

The uptake mechanism and intracellular fate of Paraoxonase-1 in endothelial cells

Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-associated lactonase that plays a significant role in the anti-atherosclerotic activity of HDL. However, several studies have shown that PON1 localizes in cells, where it operates independently of HDL. Previously, we showed that PON1 localizes in endothelial cells (ECs), and impairs vasodilation mediated by the endothelium-derived hyperpolarizing factor (EDHF) 5,6-δ-DHTL. However, the internalization pathway of PON1 into ECs, and the intracellular fate of PON1 are unknown. Therefore, the present study aimed to elucidate the uptake mechanism, intracellular trafficking and the function of PON1 in ECs. ‏We conducted a series of inhibition experiments of fluorescently labeled recombinant PON1 (rePON1) in ECs, followed by FACS analyses. We found that rePON1 binds the EC membrane via specific binding sites located in lipid-rafts/caveolae microdomains that are shared with HDL, and internalized through dynamin-dependent endocytosis. Qualitative assessments of the intracellular trafficking of rePON1, using confocal z-stack images, showed colocalization of the labeled rePON1 with early and late endosome/lysosome markers. Accordingly, a “pulse-chase” incubation of rePON1, followed by lactonase activity measurement in EC lysate, revealed that rePON1 retains its lactonase activity after binding to the cells. However, this activity decreases over time. Finally, induction of endothelial dysfunction with high glucose, angiotensin II, or palmitic acid increased rePON1 uptake by ECs. In conclusion, these results indicate that free PON1 interacts with ECs via binding sites located in lipid-rafts/caveolae, where it is enzymatically active and regulates endothelial functions. However, once internalized, PON1 is degraded. Additionally, alteration in endothelial function affects PON1 uptake by ECs.

P6266Circulating microparticules of patients with coronary artery disease up-regulate the expression of sodium-glucose cotransporters 1 and 2 in coronary artery endothelial cells: role of angiotensin II

Abstract Introduction Circulating microparticles (MPs) from patients with coronary artery diseases (CAD) have been shown to promote endothelial senescence and dysfunction involving the pro-oxidant local angiotensin system. Sodium-glucose cotransporters (SGLTs)2 inhibitors decreased the risk of cardiovascular disease in patients with type 2 diabetes and this effect appears to be independent of glycemic control. Moreover, high glucose and H2O2 have been shown to cause a redox-sensitive upregulation of SGLT1 and 2 in coronary artery endothelial cells (ECs). Aim Therefore, this study examined whether angiotensin II (Ang II, a potent NADPH oxidase-dependent inducer of oxidative stress) and CAD MPs stimulate SGLT1 and 2 expression in ECs, and assessed their role in the induction of endothelial dysfunction. Methods ECs were isolated from porcine coronary arteries. The protein expression level was assessed by Western blot analysis and immunocytochemical staining, oxidative stress using dihydroethidium staining, and senescence by senescence-associated beta-galactosidase activity (SA-beta-gal activity). Circulating CAD MPs were collected from blood samples of patients (61–79 year) with established cardiovascular disease. Results Control ECs expressed low levels of SGLT1 and SGLT2 proteins. Exposure of ECs to Ang II caused a time- and concentration-dependent increase in the protein level of SGLT1 and SGLT2 with a significant increase observed at concentrations as low as 10 nM. Exposure of ECs to CAD MPs (10 nM PhtdSer eq) from 3/5 patients increased the SGLT1 and SGLT2 protein level. An increased SGLT1 and SGLT2 immunofluorescence signal was also observed in response to Ang II and H2O2. Ang II increased the level of oxidative stress, SA-beta-gal activity, senescence markers (p53, p21 and p16), VCAM-1, MCP-1, tissue factor (TF) and the local angiotensin system (ACE, AT1R), and down-regulated that of eNOS. CAD MPs from 4/5 patients decreased eNOS level and from 5/5 patients increased VCAM-1 level. All the Ang II-induced effects were prevented by the dual SGLT1/2 inhibitor LX-4211 and the selective SGLT2 inhibitor, empagliflozin. Conclusions The present findings indicate that CAD MPs and Ang II upregulate the expression of SGLT1 and SGLT2 protein levels in ECs, and that they promote endothelial dysfunction. They further suggest that inhibition of SGLT1 and/or SGLT2 might be an attractive strategy to protect the arterial wall and, hence, the development of cardiovascular diseases. Acknowledgement/Funding Unrestricted research grant from Boehringer Ingelheim Pharma GmbH &amp; Co. KG

Induction of endothelial dysfunction by oxidized low-density lipoproteins via downregulation of Erk-5/Mef2c/KLF2 signaling: Amelioration by fisetin

Extra-cellular signal regulated kinase-5 (Erk-5), a transcriptional activator and regulator of endothelial cells (ECs) homeostasis, has been implicated in shear stress-induced endothelial dysfunction (ED), however its role in oxidized low-density lipoprotein (oxLDL)- induced ED during metabolic stress is not known. Herein, regulation and function of Erk-5 in oxLDL-induced EC death, inflammation and dysfunction has been investigated. Primary Human Umbilical Vein Endothelial Cells (pHUVECs) were stimulated with oxLDL. MTT and Trypan blue exclusion assays to assess cell viability, RT-qPCR and Western blotting assays to determine expression of endothelial and inflammatory markers and ED mediators at mRNA and protein levels, respectively were performed. Monocyte adhesion assay was performed to examine monocytes adherence to oxLDL-stimulated pHUVECs. The exposure of oxLDL induced a dose- and time-dependent decrease in pHUVECs viability, which concurred with decreased Erk-5 expression. Further, oxLDL (100 μg/ml) decreased the expression of endothelial markers eNOS and vWF, and increased the expression of ICAM-1, at both mRNA and protein levels. SiRNA-mediated silencing of Erk-5 or its inhibition showed that changes in eNOS, vWF and ICAM-1 expression could be mediated through Erk-5. Furthermore, oxLDL decreased the levels of Erk-5's upstream regulator MEK5 and downstream regulators Mef2c and KLF2, which were similar to their expressions in Erk-5 silenced cells. Fisetin, a phytochemical and bioflavonoid, could reduce the effect of oxLDL in ECs by upregulating the expression of endothelial markers including Erk-5, and downregulating the expression of inflammation markers. These results suggest that Erk-5 could be a critical regulator of oxLDL-induced EC death, inflammation and dysfunction via downregulation of Erk-5/Mef2c-KLF2 signaling pathway, which can be ameliorated by a bioflavonoid, fisetin.

Acute exposure to air pollution is associated with novel changes in blood levels of endothelin-1 and circulating angiogenic cells in young, healthy adults

Acute and chronic exposures to particulate matter (PM2.5) air pollution increase the risk for cardiovascular disease (CVD). A hypothesized mechanism linking PM2.5 exposure and CVD is the induction of endothelial dysfunction - a key step to increased CVD risk. Although PM2.5 exposure is associated with endothelial dysfunction and the vasoconstrictor peptide endothelin-1 (ET-1) is upregulated in endothelial dysfunction, the effects of PM2.5 on ET-1 and whether or not ET-1 mediates the downstream effects of PM2.5 are unclear. In addition to examining associations between acute changes in ambient PM2.5 and circulating levels of ET-1, we also looked at whether changes in ET-1 were associated with changes in markers of vascular health and systemic injury. For example, endothelial function is maintained in part by circulating angiogenic cell (CAC)-mediated repair, and our recent studies show that CACs in humans and mice are decreased by ambient PM2.5 exposure. In the current study, we recruited young, healthy adults who were exposed to natural variations in PM2.5, and we analyzed associations between PM2.5 and circulating levels of ET-1, between ET-1 and CACs, and between ET-1 and other biomarkers of injury using linear regression analyses. Surprisingly, ET-1 levels were negatively associated with PM2.5 levels (β = −0.773, P = 0.0005), yet, in contrast, positively associated with two CACs: CAC-2 (CD31+/CD34+/CD45+) and CAC-4 (CD31+/CD34+/CD45+/CD133+). Interestingly, ET-1 levels were negatively associated with some biomarkers (platelet factor 4, β = −0.148, P = 0.0003; triglycerides, β = −0.095, P = 0.041) and positively with other biomarkers: albumin (β = 0.035, P = 0.006) and IL-lβ (β = 0.082, P = 0.012). These findings further reveal the insidious nature of PI2.5’s anti-angiogenic effect including a novel relationship between ET-1 and CACs in young adults exposed to acute elevations of air pollution.

Tryptophan-Derived Uremic Toxins and Thrombosis in Chronic Kidney Disease.

Patients with chronic kidney disease (CKD) display an elevated risk of thrombosis. Thrombosis occurs in cardiovascular events, such as venous thromboembolism, stroke, and acute coronary syndrome, and is a cause of hemodialysis vascular access dysfunction. CKD leads to the accumulation of uremic toxins, which exerts toxic effects on blood and the vessel wall. Some uremic toxins result from tryptophan metabolization in the gut through the indolic and the kynurenine pathways. An increasing number of studies are highlighting the link between such uremic toxins and thrombosis in CKD. In this review, we describe the thrombotic mechanisms induced by tryptophan-derived uremic toxins (TDUT). These mechanisms include an increase in plasma levels of procoagulant factors, induction of platelet hyperactivity, induction of endothelial dysfunction/ impairment of endothelial healing, decrease in nitric oxide (NO) bioavailability, and production of procoagulant microparticles. We focus on one important prothrombotic mechanism: The induction of tissue factor (TF), the initiator of the extrinsic pathway of the blood coagulation. This induction occurs via a new pathway, dependent on the transcription factor Aryl hydrocarbon receptor (AhR), the receptor of TDUT in cells. A better understanding of the prothrombotic mechanisms of uremic toxins could help to find novel therapeutic targets to prevent thrombosis in CKD.

Concept and connotation of oxidative stress in preeclampsia.

BACKGROUND:Preeclampsia (PE) is a systemic pregnancy-related disorder characterized by hypertension, proteinuria, and edema. Free radicals seem to play an important role in the induction of endothelial dysfunction in PE.AIM:The aim of the present study was to investigate serum levels of nitric oxide (NO), peroxynitrite (ONOO−), paraoxonase (PON-1), malondialdehyde (MDA), and lipid profile in preeclamptic patients compared to the women with normal pregnancy.MATERIALS AND METHODS:A total of 68 pregnant women were recruited. They were divided into two groups - Group A, 40 women were a newly diagnosed with PE and Group B, 28 women with normal pregnancy. Anthropometric measurements including body mass index and blood pressure in accordance with biochemical measurements including NO, ONOO−, PON-1, MDA, and lipid profile were done for preeclamptic pregnant women compared to the controls.RESULTS:Pregnant women with pre-eclampsia illustrated insignificant differences in the age (31.22±2.87) compared to the age of control P > 0.05. There were significant changes in the body mass index (BMI), type of delivery and smoking status of pregnant women with pre-eclampsia compared to the control P < 0.05. Both systolic and diastolic blood pressures were high in pregnant women with pre-eclampsia compared to the control P < 0.01. PON-1 and NO serum levels were significantly decreased (P < 0.01) while ONOO− and MDA serum levels were significantly increased in PE compared to the women with normal pregnancy.CONCLUSIONS:This study concluded that PE is associated with the augmentation of oxidative stress and reduction of endogenous antioxidant capacity regarding PON-1.

RHEUMATIC DISEASES AND ENDOTHELIAL FUNCTION

The review analyzes the role of chronic inflammatory diseases in the induction of endothelial dysfunction as a factor in the acceleration of atherosclerotic changes in the cardiovascular system. The importance of non-invasive techniques for evaluating endothelial dysfunction to detect preclinical atherosclerotic changes in children and adolescents with rheumatic was discussed. It is postulated that the detection of early signs of endothelial dysfunction in children and adolescents with rheumatic diseases, along with traditional risk factors, should become an integral part of the strategy for preventing adverse cardiovascular events in subsequent age periods.

143 - Modification of Low-Density Lipoprotein by Myeloperoxidase Oxidants – a Pathway to Endothelial Dysfunction in Atherosclerosis?

There is strong evidence that low-density lipoprotein (LDL) modified by hypochlorous acid (HOCl) produced by myeloperoxidase is present in atherosclerotic lesions and can propagate inflammation. Less is known about the biological reactivity of LDL modified by hypothiocyanous acid (HOSCN), the other major oxidant produced by myeloperoxidase in vivo . We examined the role of HOCl- and HOSCN-modified LDL in the induction of endothelial dysfunction, which is characterised by decreased nitric oxide ( ● NO) formation and is a key pathway to atherosclerotic lesion development. Exposure of human coronary artery endothelial cells (HCAEC) to both HOCl- and HOSCN-LDL resulted in a greater extent of endothelial nitric oxide synthase (eNOS) uncoupling compared to control LDL, which correlated with a decrease in eNOS activity. A significant decrease in ● NO production was also seen on exposure of HCAEC to each modified LDL for 24 h, though no elevation in superoxide (O 2 ●– ) production from uncoupled eNOS was observed. We also performed studies to see if the reduced eNOS functionality is consistent with the caveolin-1-mediated down-regulation of eNOS activity. While eNOS and caveolin-1 remained colocalised in the cell, as shown by fluorescence microscopy, we did not observe any binding of eNOS and caveolin-1 in HCAEC exposed to each modified LDL. The changes in ● NO production were also independent of mRNA and protein expression of cellular adhesion molecules; ICAM-1, VCAM-1 and E-selectin, and cytokines; MCP-1 and IL-6, which remained unchanged. Together, these data provide new insights regarding the role of LDL modified by myeloperoxidase-derived oxidants, in altering endothelial function within the setting of atherosclerosis, and may have particular significance for smokers, who have elevated plasma thiocyanate, the precursor to HOSCN.

High Levels of Soluble Endoglin Induce a Proinflammatory and Oxidative-Stress Phenotype Associated with Preserved NO-Dependent Vasodilatation in Aortas from Mice Fed a High-Fat Diet

Aims: A soluble form of endoglin (sEng) was proposed to participate in the induction of endothelial dysfunction in small blood vessels. Here, we tested the hypothesis that high levels of sEng combined with a high-fat diet induce endothelial dysfunction in an atherosclerosis-prone aorta. Methods and Results: Six-month-old female and male transgenic mice overexpressing human sEng (Sol-Eng⁺) with low (Sol-Eng⁺low) or high (Sol-Eng⁺high) levels of plasma sEng were fed a high-fat rodent diet containing 1.25% cholesterol and 40% fat for 3 months. The plasma cholesterol and mouse sEng levels did not differ in the Sol-Eng⁺high and Sol-Eng⁺low mice. The expression of proinflammatory (P-selectin, ICAM-1, pNFκB and COX-2) and oxidative-stress-related markers (HO-1, NOX-1 and NOX-2) in the aortas of Sol-Eng⁺high female mice was significantly higher than in Sol-Eng⁺low female mice. Endothelium-dependent vasodilatation induced by acetylcholine was preserved better in the Sol-Eng⁺high female mice than in the Sol-Eng⁺low female mice. Conclusion: These results suggest that high concentrations of sEng in plasma in combination with a high-fat diet induce the simultaneous activation of proinflammatory, pro-oxidative and vasoprotective mechanisms in mice aorta and the balance of these biological processes determines whether the final endothelial phenotype is adaptive or maladaptive.

Abstract 559: Lectin-like Oxidised Low Density Lipoprotein Receptor-1 Expression in Atheroma of Patients with Coronary Artery Disease is Higher in Patients with Acute Coronary Syndrome but Decreased in Patients on Statins

Introduction: The lectin-like oxidised low density lipoprotein receptor-1, LOX-1, is expressed in endothelial cells and macrophages among others. Its expression can be induced by a variety of stimuli such as oxidised LDL, endothelin-1, angiotensin II and shear stress. LOX-1 seems to be important for the induction of endothelial dysfunction and is implicated in atherosclerotic plaque vulnerability. Methods: Atherectomy specimens were obtained from 37 patients (mean age 62.5±9.7 years, body mass index 28.6±5.3 kg/m2; n=30 with chest pain/angina pectoris), who underwent medially indicated directional coronary atherectomy in the Department of Internal Medicine B, University Medicine Greifswald, between July 2001 and April 2005, and were immediately frozen in liquid nitrogen. Human coronary atherectomy specimens were cut into 5 μm-slices using a kryotom and stained with haematoxylin and eosin (H&amp;E), Oil Red O (lipids) and Elastica-van Gieson (collagen). LOX-1 was detected by immunofluorescence using a FITC-labelled rabbit polyclonal antibody against amino acids 143 to 271 of human LOX-1. Digital images were analysed with Corel PHOTO-PAINT 12, SCION Image, and SigmaPlot 11. Correlations between signal intensities for LOX-1 expression and patients’ parameters were analysed by Spearman Rank Order testing. Patients gave informed consent. The study protocol complied with the Declaration of Helsinki and was approved by the local ethics committee. Results: LOX-1 expression correlated significantly with lipid content of the coronary atherectomy specimens. LOX-1 expression tended to be higher in specimens from patients with acute coronary syndrome (n=11) and to be lower in patients receiving statins (n=16). However, no correlations were found between LOX-1 expression and grade of stenosis, plaque localisation, age, sex, body mass index, smoking (n=15), hypertension (n=31), diabetes mellitus type II (n=9), and dyslipoproteinaemia (n=32). Conclusions: LOX-1 expression in plaques was positively correlated with lipid contents of plaque material and tended to be higher in patients with acute coronary syndrome and to be lower if patients had received statins. High LOX-1 expression in the atherosclerotic plaque may contribute to plaque instability.

Antiphospholipid Syndrome--Not a Noninflammatory Disease.

The autoimmune disease antiphospholipid syndrome (APS) is characterized by thrombosis or pregnancy morbidity in patients with persistent antiphospholipid antibodies (aPLs). Although inflammation is not a key feature of the clinical presentation of the syndrome, there are indications that the inflammatory response plays an important role in APS. The major antigen of aPLs, the plasma protein β2-glycoprotein I, is involved in clearance of microparticles and in the innate immune response. In light of these physiological functions, the formation of antibodies against the protein is easily understood, as antibodies might augment the clearance reaction. In addition, inflammatory mediators are thought to play a role in the activation of leukocytes and the induction of endothelial dysfunction in APS. Moreover, evidence for a role of complement activation in the pathogenesis of the syndrome is accumulating. This review will provide an overview of current knowledge on the physiological function of β2-glycoprotein I, the formation of autoantibodies against β2-glycoprotein I and will explore the contribution of inflammation to the clinical manifestations of APS.

Innervation of the arterial wall and its modification in atherosclerosis.

The autonomic nervous system (ANS) plays an essential role in the regulation of vascular tone. Sympathetic neurotransmitters epinephrine and norepinephrine are released from the terminals of perivascular nerves and suppress endothelial production of nitric oxide (NO), an important vasodilator. Sympathetic nerves also release neuropeptide Y, a co-transmitter that stimulates vasoconstriction and proliferation of vascular smooth muscle cells. Parasympathetic nerves release acetylcholine, which leads to vascular contraction when NO production is inhibited. The ANS produces a variety of other vasoactive substances including ATP, calcitonin gene-related peptide, dopamine, and serotonin. On the other hand, the vascular system can reciprocally influence ANS activity through the release of NO, reactive oxygen species (ROS), angiotensin II, and other mechanisms. In pathological conditions such as atherosclerosis, hyperactivation of sympathetic neural activity has pro-atherogenic effects on the vascular function by increasing vasoconstriction, accumulation of modified lipoproteins in the vascular wall, induction of endothelial dysfunction, and stimulation of oxidative stress and vascular remodeling. Indeed, suppression of the sympathetic ANS should be beneficial for the treatment of cardiovascular diseases.