Xiao Lei Moore

Structure/Function Relationships of Apolipoprotein A-I Mimetic Peptides: Implications for Antiatherogenic Activities of High-Density Lipoprotein

Rationale: Apolipoprotein (apoA)-I mimetic peptides are a promising type of antiatherosclerosis therapy, but how the structural features of these peptides relate to the multiple antiatherogenic functions of HDL is poorly understood. Objective: To establish structure/function relationships of apoA-I mimetic peptides with their antiatherogenic functions. Methods and Results: Twenty-two bihelical apoA-I mimetic peptides were investigated in vitro for the capacity and specificity of cholesterol efflux, inhibition of inflammatory response of monocytes and endothelial cells, and inhibition of low-density lipoprotein (LDL) oxidation. It was found that mean hydrophobicity, charge, size of hydrophobic face, and angle of the link between the helices are the major factors determining the efficiency and specificity of cholesterol efflux. The peptide with optimal parameters was more effective and specific toward cholesterol efflux than human apoA-I. Charge and size of hydrophobic face were also the major factors affecting antiinflammatory properties, and the presence of cysteine and histidine residues was the main factor determining antioxidant properties. There was no significant correlation between capacities of the peptides to support individual functions; each function had its own optimal set of features. Conclusions: None of the peptides was equally effective in all the antiatherogenic functions tested, suggesting that different functions of HDL may have different mechanisms and different structural requirements. The results do suggest, however, that rationalizing the design of apoA-I mimetic peptides may improve their therapeutic value and may lead to a better understanding of mechanisms of various antiatherogenic functions of HDL.

Caveolin-1 Plays a Critical Role in the Differentiation of Monocytes into Macrophages

Objective—Monocyte to macrophage differentiation is an essential step in atherogenesis. The structure protein of caveolae, caveolin-1, is increased in primary monocytes after its adhesion to endothelium. We explore the hypothesis that caveolin-1 plays a role in monocyte differentiation to macrophages. Methods and Results—Both phorbol myristate acetate–induced THP-1 and colony-stimulating factor–induced primary monocyte differentiation was associated with an increase in cellular caveolin-1 expression. Overexpression of caveolin-1 by transfection increased macrophage surface markers and inflammatory genes, whereas caveolin-1 knockdown by small interfering RNA or knockout reduced these. Also, caveolin-1 knockdown inhibited the differentiation–induced nuclear translocation of early growth response 1 (EGR-1) through extracellular signal-regulated kinase phosphorylation, further decreased the binding of EGR-1 to CD115 promoter, thus decreasing EGR-1 transcriptional activity. In functional assays, caveolin-1 inhibited transmigration but promoted phagocytosis in the monocyte–macrophage lineage. Decreasing caveolin-1 inhibited the uptake of modified low-density lipoprotein and reduced cellular lipid content. Finally, we showed that caveolin-1 knockout mice displayed less monocyte differentiation than wild-type mice and that EGR-1 transcription activity was also decreased in these mice because of the inhibition of extracellular signal-regulated kinase phosphorylation. Conclusion—Caveolin-1 promotes monocyte to macrophage differentiation through the regulation of EGR-1 transcriptional activity, suggesting that phagocytic caveolin-1 may be critical for atherogenesis.

Selective Endothelial Overexpression of Arginase II Induces Endothelial Dysfunction and Hypertension and Enhances Atherosclerosis in Mice

Background Cardiovascular disorders associated with endothelial dysfunction, such as atherosclerosis, have decreased nitric oxide (NO) bioavailability. Arginase in the vasculature can compete with eNOS for L-arginine and has been implicated in atherosclerosis. The aim of this study was to evaluate the effect of endothelial-specific elevation of arginase II expression on endothelial function and the development of atherosclerosis. Methodology/Principal Findings Transgenic mice on a C57BL/6 background with endothelial-specific overexpression of human arginase II (hArgII) gene under the control of the Tie2 promoter were produced. The hArgII mice had elevated tissue arginase activity except in liver and in resident peritoneal macrophages, confirming endothelial specificity of the transgene. Using small-vessel myography, aorta from these mice exhibited endothelial dysfunction when compared to their non-transgenic littermate controls. The blood pressure of the hArgII mice was 17% higher than their littermate controls and, when crossed with apoE −/− mice, hArgII mice had increased aortic atherosclerotic lesions. Conclusion We conclude that overexpression of arginase II in the endothelium is detrimental to the cardiovascular system.

Associations Between Fibrocytes and Postcontrast Myocardial T1 Times in Hypertrophic Cardiomyopathy

Background Fibrocytes are bone marrow‐derived mesenchymal progenitors that have been linked to various fibrotic disorders. This study was undertaken to investigate whether fibrocytes are increased in diffuse myocardial fibrosis in humans. Methods and Results Thirty‐seven patients with hypertrophic cardiomyopathy (HCM) and 20 healthy controls were recruited. Cardiac magnetic resonance imaging with postcontrast T1 mapping was performed to non‐invasively quantify diffuse myocardial fibrosis and these patients were classified into 2 groups (T1<470 ms or T1≥470 ms, as likely or unlikely to have diffuse fibrosis, respectively). Circulating fibrocytes (CD45+/CD34+/collagen I+) were measured by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were cultured for 13 days and fibrocytes were quantitated by flow cytometry (CD45+/collagen I+) and real‐time PCR (gene expression of matrix proteins). Plasma cytokines/chemokines mediating fibrocyte trafficking and differentiation were measured by multiplex assays. Circulating fibrocytes were decreased in HCM patients compared to controls. The proportion of fibrocytes derived from PBMCs was increased in patients with diffuse fibrosis compared with those without or controls (31.1±4.1% versus 18.9±3.9% and 10.9±2.0%, P<0.05 and P<0.001, respectively), and the proportion of fibrocytes was inversely correlated with T1 time (r=−0.37, P=0.03). Plasma levels of stromal cell‐derived factor‐1 were elevated in patients with diffuse fibrosis compared with those without or controls (5131±271 pg/mL versus 3893±356 pg/mL and 4172±185 pg/mL, respectively, both P<0.05). Conclusions HCM patients with diffuse fibrosis as assessed by postcontrast T1 mapping have elevated plasma SDF and an enhanced ability of PBMCs to differentiate into fibrocytes, suggesting that fibrocytes may contribute to the pathogenesis of myocardial fibrosis.

Anti-atherogenic effects of high-density lipoprotein on nitric oxide synthesis in the endothelium

1. The endothelium is critical in the control of vascular haemodynamics and haemostasis. Endothelial dysfunction, typically characterized by decreased nitric oxide bioavailability and response to endothelium‐dependent agonists, is well accepted as a defining characteristic of early atherosclerosis.

Lipidomic profiling in inflammatory bowel disease: Comparison between ulcerative colitis and Crohn's disease

Background:Inflammatory bowel disease (IBD), which encompasses ulcerative colitis (UC) and Crohns disease (CD), is believed to be caused by abnormal host immune responses to the intestinal microbiome. However, the precise etiology of IBD remains unknown. Lipid metabolism and signaling are suggested to play important roles in inflammation with significant implications for IBD. In this study, we aimed to characterize lipidomic profiles in IBD with comparison between healthy controls, UC, and CD. Methods:Patients with IBD (n = 40, UC: 16 and CD: 24) and age- and gender-matched healthy volunteers (n = 84) were recruited. Plasma lipid profiles containing 333 lipid species were measured using electrospray ionization–tandem mass spectrometry. Results:A total of 86 individual lipid species were significantly changed in CD compared with controls (78 decreased while 8 increased), with the majority belonging to the ether lipids including the alkylphospholipids (alkylphosphatidylcholine and alkylphosphatidylethanolamine) and plasmalogens (alkenylphosphatidylcholine and alkenylphosphatidylethanolamine). Of these 86 lipid species, 33 remained significantly and negatively associated with CD after adjusting for age, sex, waist circumference, current smoking, and diastolic blood pressure in logistic regression. In contrast, only 5 lipid species significantly differed between UC and controls. Conclusions:We demonstrate that a number of ether lipids (alkylphospholipid and plasmalogens) are significantly and negatively associated with CD. These alterations of lipid profiles particularly plasmalogens may contribute to the pathogenesis of IBD.

Endothelial dysfunction in patients with type 2 diabetes post acute coronary syndrome

This single visit study examined whether endothelial function, in addition to cardiovascular (CV) risk factors and plasma microparticle content, was normalised in 15 patients with type 2 diabetes + acute coronary syndrome (ACS) (6 weeks–6 months post cardiac event) undergoing standard clinical care compared to 16 sex- and age-matched healthy controls. Results: While total and low-density lipoprotein (LDL) cholesterol levels were well controlled in the patients with type 2 diabetes + ACS, residual CV risk profiles such as increased body mass index (BMI), systolic blood pressure, glucose levels and triglycerides and lower high-density lipoprotein (HDL) levels were still apparent. Endothelium-dependent responses to acetylcholine (ACh) were significantly lower in type 2 diabetes + ACS patients compared to controls. Correspondingly, the reactive hyperaemic index (RHI) was lower in the patient cohort. Endothelial microparticle (EMP) levels (CD31+, CD41−) were 40% lower in the patient cohort. Simultaneous analysis of platelet microparticle (PMP) levels (CD41+) showed no difference between cohorts. Conclusions: Patients with type 2 diabetes suffering from recent ACS exhibit residual CV risk factors despite being on standard clinical care. In addition, these patients continue to present with endothelial dysfunction despite having lower levels of EMPs.

809 RESIDUAL ENDOTHELIAL DYSFUNCTION IN PATIENTS WITH TYPE II DIABETES POST ACUTE CORONARY SYNDROME

Background: Patients with type II diabetes (T2D) with a previous history of acute coronary syndrome (ACS) are at increased risk for subsequent major cardiovascular events compared with patients without T2D. Objective: Since endothelial function plays a critical role in vascular health, we hypothesised that residual endothelial dysfunction in ACS+T2D, despite standard clinical care, may contribute to the increased risk. Methods and Results: Thirty-one patients (15 T2D+ACS 2-6 months post ACS; 16 sex and age-matched healthy controls) were recruited. T2D+ACS patients on standard clinical care exhibited residual cardiovascular risk profiles such as increased body mass index, glucose levels and triglycerides and lower HDL levels (P < 0.01). However, total cholesterol and LDL cholesterol levels were well controlled in this patient cohort. Endothelium-dependent responses to acetylcholine, measured using venous occlusion strain gauge plethysmography, were significantly decreased in T2D+ACS patients compared to controls (P = 0.046) while endothelium-independent responses to sodium nitroprusside were not (P = 0.142). Similarly, the reactive hyperemic index, a measure of endothelial dysfunction using reactive hyperemia peripheral arterial tonometry, was decreased in the T2D+ACS study group when compared to controls (1.75 ± 0.17 vs 2.43 ± 0.33 respectively; P = 0.04). Endothelial microparticle (EMP) numbers increase and correlate with endothelial dysfunction in T2D patients. EMP (CD34+, CD41-) numbers, measured by flow cytometric analysis, were reduced by 40% in the patient cohort (P = 0.019) while platelet-derived microparticles (PMPs) were unchanged (P > 0.05). This may be due to the medications received. Conclusions: Patients with T2D, less than 6 months post ACS, exhibit residual cardiovascular risk factors including endothelial dysfunction despite being on standard clinical care.

701 HIGH INTRALUMINAL PRESSURE INDUCES ENDOTHELIAL INFLAMMATION

Background and Aims: Hypertension is a potent risk factor for coronary artery disease. The contributing mechanism however, remains elusive. We hypothesise that high intraluminal pressure per se induces vascular inflammation and the adhesion cascade. Methods and Results: Using a specialised pressure vessel chamber we show that elevations in intraluminal pressure increases gene expression of adhesion molecules and functional leukocyte adhesion to the endothelium in harvested vessels (P < 0.001). We also show that endothelial microparticles are shed from pressurised HUVECs (P < 0.05). Low shear stress (LSS: 1.6 dynes/cm2) coupled with pressure (120 mmHg) increased leukocyte adhesion compared to HSS (16 dyne/cm2; 90 ± 23 vs 24 ± 9 leukocytes/field respectively; n = 5-6; P < 0.001). However, LSS on its own (with no pressure) had no influence (4 ± 3 leukocytes/field; n = 4) suggesting that circumferential stretch plays a critical role. Electron microscopy images of pressurised vessels demonstrate caveolae disassembly, resulting in reduced caveolae number. To investigate the signalling pathways involved, we demonstrate that this acute inflammatory response was reduced with the use of inhibitors of NADPH oxidase (P < 0.001) and mitochondrial permeability (P < 0.05). Increased reactive oxygen species production was also seen at 120 mmHg in HUVECs perfused with the global ROS dye DCFH (60 mmHg: 3 ± 6 vs 120 mmHg: 149 ± 65 intensity units; n = 5–7; P < 0.05). Conclusion: We conclude that high intraluminal pressure induces vascular inflammation via circumferential stretch acting on mechano-sensors on the endothelium triggering the release of ROS and consequential adhesion molecule expression. Inhibiting these pathways may prove to be therapeutically beneficial for hypertensive patients to help reduce the risk of atherosclerosis.

Anti-atherogenic effects of high-density lipoprotein on nitric oxide synthesis in the endothelium: Frontiers in research review: Physiological and pathological functions of high-density lipoprotein

1. The endothelium is critical in the control of vascular haemodynamics and haemostasis. Endothelial dysfunction, typically characterized by decreased nitric oxide bioavailability and response to endothelium‐dependent agonists, is well accepted as a defining characteristic of early atherosclerosis.