Lysoplasmenylcholine increases neutrophil adherence to human coronary artery endothelial cells

We demonstrated previously that thrombin stimulation of human coronary artery endothelial cells (HCAEC) results in release of choline lysophospholipids [lysophosphatidylcholine (lysoPtdCho) and lysoplasmenylcholine (lysoPlsCho)]. These amphiphilic metabolites have been implicated in arrhythmogenesis following the onset of myocardial ischemia, but studies examining their direct effects on the vasculature remain limited. We and others have shown that thrombin and lysoPtdCho can increase cell surface adhesion molecules and adherence of circulating inflammatory cells to the endothelium. This study supports our hypothesis that these changes may be mediated, at least in part, by lysoPlsCho, thus implicating this metabolite as an inflammatory mediator in the coronary vasculature and a modulator of the progression of atherosclerosis. Apical stimulation of HCAEC with thrombin resulted in the production and release of choline lysophospholipids from the apical surface of the HCAEC monolayer. Basolateral stimulation had no effect on choline lysophospholipid production or release from either the apical or basolateral surface of the HCAEC monolayer. Incubation of HCAEC with lysoPlsCho or lysoPtdCho resulted in similar increases in HCAEC surface expression of P-selectin and E-selectin. Furthermore, lysoPlsCho increased cell surface expression of P-selectin, E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 with a time course similar to that of thrombin stimulation. Increased presence of cell surface adhesion molecules may contribute to the significant increase in adherence of neutrophils to either thrombin- or lysoPlsCho-stimulated HCAEC. These results demonstrate that the presence of thrombin at sites of vascular injury in the coronary circulation, resulting in increased choline lysophospholipid release from the HCAEC apical surface, has the potential to propagate vascular inflammation by upregulation of adhesion molecules and recruitment of circulating inflammatory cells to the endothelium. endothelium; arrhythmogenesis; inflammation; lysophospholipids     

Wound closure in sheared endothelial cells is enhanced by modulation of vascular endothelial-cadherin expression and localization

We previously demonstrated that laminar shear stress enhances human coronary artery endothelial cell (HCAEC) wound closure via the mechanisms of cell spreading and migration. Because cell-cell junctional proteins such as vascular endothelial cell cadherin (VE-cadherin) are critical to cell-cell adhesion and motility, we tested the hypothesis that modulation of VE-cadherin expression under shear stress may be linked to this enhancement in wound closure. HCAEC monolayers were preconditioned to attain cellular alignment by shearing at 12 dynes/cm(2) for 18 hr in a parallel-plate flow chamber. Subsequently, they were divided into the following three groups: (i) control; (ii) treated with anti-cadherin-5 antibody; or (iii) treated with the calcium chelating agent EGTA. Next, the monolayers were wounded with a metal spatula and resheared at 20 dynes/cm(2) or left static. Time-lapse imaging was performed during the first 3 hr after imposition of these conditions. Immunocytochemistry or Western blot analyses for VE-cadherin expression were performed on all wounded monolayers. Deconvolution microscopy, three-dimensional cell-cell junctional reconstruction images, and histogram analyses of interendothelial junction signal intensities were performed on cells at the wound edge of a monolayer. Under shear, HCAEC demonstrated increased VE-cadherin immunofluorescence and protein expression despite an enhancement in wound closure compared with static conditions. In separate experiments, application with anti-cadherin-5 antibody or treatment with EGTA attenuated VE-cadherin expression and further enhanced wound closure compared with control shear and all static conditions. In addition, the pattern of VE-cadherin localization with these treatments became more intracellular and nuclear in appearance. These findings of changes in this junctional adhesion protein expression and localization may further our understanding of laminar shear stress-induced endothelial repair in the coronary circulation.     

Lamellipodial motility in wounded endothelial cells exposed to physiologic flow is associated with different patterns of beta1-integrin and vinculin localization

Integrins- and cytoskeletal-associated focal adhesion proteins may participate in the process of endothelial wound closure, but their relationship in these wounds and in the presence of shear forces has not been defined. The goal in this study was to test the hypotheses that (1) modulation of beta(1)-integrin in human coronary artery endothelial cells (HCAEC) would alter endothelial wound closure under shear stress, and (2) beta(1)-integrin association with vinculin would be necessary for mediating this closure. HCAEC monolayers were pre-conditioned to attain alignment by shearing at 12 dynes/cm(2) for 18 h in a parallel-plate flow chamber. Subsequently, they were divided into three groups: (a) control, (b) treated with anti-beta(1)-integrin adhesion blocking antibody, or (c) treated with anti-beta(1)-integrin adhesion promoting antibody. Next, the monolayers were wounded with a metal spatula, and re-sheared at 20 dynes/cm(2) or left static. Time-lapse imaging and deconvolution microscopy were then performed for 3 h. Immunocytochemistry for beta(1)-integrin expression and vinculin was performed on all wounded monolayers. Under shear stress, vinculin localized to the ends of stress fibers, while beta(1)-integrin took on an intracellular macroaggregate appearance. Treatment with anti-beta(1)-integrin adhesion blocking antibody enhanced wound closure, left the vinculin staining at the lamellipodial tips unchanged, but was associated with beta(1)-integrin staining at the lateral cell edges. Treatment with the anti-beta(1)-integrin adhesion promoting antibody retarded wound closure, increased vinculin staining at cell-cell junctions, and was associated with a fibrillar pattern of beta(1)-integrin staining. Modulation of beta(1)-integrin and changes in beta(1)-integrin and vinculin localization may further our understanding of laminar shear stress-induced endothelial repair in the coronary circulation.     

Decreased circulating endothelial progenitor cell levels and function in patients with nonalcoholic fatty liver disease

Objectives

Nonalcoholic fatty liver disease (NAFLD) is associated with advanced atherosclerosis and a higher risk of cardiovascular disease. Increasing evidence suggests that injured endothelial monolayer is regenerated by circulating bone marrow derived-endothelial progenitor cells (EPCs), and levels of circulating EPCs reflect vascular repair capacity. However, the relation between NAFLD and EPC remains unclear. Here, we tested the hypothesis that patients with nonalcoholic fatty liver disease (NAFLD) might have decreased endothelial progenitor cell (EPC) levels and attenuated EPC function.

Methods and Results

A total of 312 consecutive patients undergoing elective coronary angiography because of suspected coronary artery disease were screened and received examinations of abdominal ultrasonography between July 2009 and November 2010. Finally, 34 patients with an ultrasonographic diagnosis of NAFLD, and 68 age- and sex-matched controls without NAFLD were enrolled. Flow cytometry with quantification of EPC markers (defined as CD34⁺, CD34⁺KDR⁺, and CD34⁺KDR⁺CD133⁺) in peripheral blood samples was used to assess circulating EPC numbers. The adhesive function, and migration, and tube formation capacities of EPCs were also determined in NAFLD patients and controls. Patients with NAFLD had a significantly higher incidence of metabolic syndrome, previous myocardial infarction, hyperuricemia, and higher waist circumference, body mass index, fasting glucose and triglyceride levels. In addition, patients with NAFLD had significantly decreased circulating EPC levels (all P<0.05), attenuated EPC functions, and enhanced systemic inflammation compared to controls. Multivariate logistic regression analysis showed that circulating EPC level (CD34⁺KDR⁺ [cells/10⁵ events]) was an independent reverse predictor of NAFLD (Odds ratio: 0.78; 95% confidence interval: 0.69–0.89, P<0.001).

Conclusions

NAFLD patients have decreased circulating EPC numbers and functions than those without NAFLD, which may be one of the mechanisms to explain atherosclerotic disease progression and enhanced cardiovascular risk in patients with NAFLD.     

Regulation of endotoxin-induced proinflammatory activation in human coronary artery cells: expression of functional membrane-bound CD14 by human coronary artery smooth muscle cells

Low-level endotoxemia has been identified as a powerful risk factor for atherosclerosis. However, little is known about the mechanisms that regulate endotoxin responsiveness in vascular cells. We conducted experiments to compare the relative responses of human coronary artery endothelial cells (HCAEC) and smooth muscle cells (HCASMC) to very low levels of endotoxin, and to elucidate the mechanisms that regulate endotoxin responsiveness in vascular cells. Endotoxin (10-fold higher in magnitude at >10-fold lower threshold concentrations (10-30 pg/ml) compared with HCAEC. This remarkable sensitivity of HCASMC to very low endotoxin concentrations, comparable to that found in circulating monocytes, was not due to differential expression of TLR4, which was detected in HCAEC, HCASMC, and intact coronary arteries. Surprisingly, membrane-bound CD14 was detected in seven different lines of HCASMC, conferring responsiveness to endotoxin and to lipoteichoic acid, a product of Gram-positive bacteria, in these cells. These results suggest that the low levels of endotoxin associated with increased risk for atherosclerosis are sufficient to produce inflammatory responses in coronary artery cells. Because CD14 recognizes a diverse array of inflammatory mediators and functions as a pattern recognition molecule in inflammatory cells, expression of membrane-bound CD14 in HCASMC implies a potentially broader role for these cells in transducing innate immune responses in the vasculature.     

Peroxynitrite induces integrin-dependent adhesion of human neutrophils to endothelial cells via activation of the Raf-1/MEK/Erk pathway.

Accumulating evidence suggests that enhanced peroxynitrite (ONOO-) formation occurs during inflammation. We have studied the impact and the mechanisms of ONOO- action on expression of adhesion molecules on human neutrophils and coronary artery endothelial cells (HCAEC) and binding of neutrophils to HCAEC. Addition of ONOO- (0.1 to 200 5M) to isolated neutrophils resulted in a concentration-dependent down-regulation of L-selectin expression, and up-regulation of CD11b/CD18 expression. ONOO- stimulation of Erk activity was accompanied by activation of Ras, Raf-1 and MEK (mitogen-activated protein kinase kinase), and was sensitive to the MEK inhibitor PD 98059. We have observed a tight association between Erk activation and changes in CD11b/CD18 expression. ONOO- also evoked activation of neutrophil p38 MAPK. Neither ONOO--induced up-regulation of CD11b/CD18 expression nor Erk activation was affected by SB 203580, a selective inhibitor of p38 MAPK. ONOO- by itself had little effect on expression of ICAM-1 and E-selectin on HCAEC, whereas it markedly enhanced attachment of neutrophils to lipopolysaccharide-activated HCAEC only when it was added together with neutrophils. Increases in neutrophil adhesion evoked by ONOO- were blocked by an anti-CD18 monoclonal antibody. These data suggest that ONOO- activates Erk in neutrophils via the Ras/Raf-1/MEK signal transduction pathway, leading to up-regulation of surface expression of CD11b/CD18 and consequently to increased neutrophil adhesion to endothelial cells.     

The anti-inflammatory peptides, antiflammins, regulate the expression of adhesion molecules on human leukocytes and prevent neutrophil adhesion to endothelial cells.

Antiflammin-1 and antiflammin-2 are nonapeptides corresponding to the region of highest similarity between glucocorticoid-inducible proteins lipocortin-1 and uteroglobin. We have studied whether antiflammins could affect expression of adhesion molecules on human leukocytes and coronary artery endothelial cells (HCAEC) and binding of neutrophils (PMNs) to HCAEC. Although neither antiflammin-1 nor antiflammin-2 affected expression of adhesion molecules on resting PMNs, monocytes, and lymphocytes in whole blood, they attenuated changes in L-selectin and CD11/CD18 expression evoked by platelet-activating factor or interleukin-8 with IC(50) values of 4-20 micromol/l. The maximum inhibition was similar to those seen with human recombinant lipocortin-1 (100 microgram/ml). Unlike dexamethasone (100 nmol/l), the antiflammins had little effect on LPS-stimulated expression of E-selectin and ICAM-1 on HCAEC. Consistently, culture of HCAEC with dexamethasone, but not with antiflammins, decreased PMN binding to endothelial cells. Preincubation of PMNs with antiflammins markedly decreased their adhesion to LPS-activated HCAEC. Inhibition of adhesion was additive with function blocking anti-E-selectin and anti-L-selectin antibodies, but was not additive with anti-CD18 antibody. These results show that antiflammins inhibit PMN adhesion to HCAEC by attenuating activation-induced up-regulation of CD11/CD18 expression on leukocytes, and suggest that antiflammins may represent a novel therapeutic approach in blocking leukocyte trafficking in host defense and inflammation.     

The influence of garlic (Allium sativum) extract on interleukin 1alpha-induced expression of endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1.

Inflammation plays an important role in both the initiation of atherosclerosis and development of atherothrombotic events. The adherence of leukocytes/monocytes to the endothelium is an early event in atherogenesis. Phytotherapeutica as garlic and garlic extracts were shown to have beneficial modulating effects in patients with atherosclerotic disease. The aim of this study was to evaluate in vitro the influence of water-soluble garlic (Allium sativum) extract on the cytokine-induced expression of endothelial leukocyte adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1, CD54) and vascular cell adhesion molecule-1 (VCAM-1, CD106). Cytokine-induced expression of cellular adhesion molecules was measured on primary human coronary artery endothelial cell (HCAEC) cultures. HCAEC were cultured in microvascular endothelial cell growth medium and preincubated with garlic extract at various concentrations (0.25-4.0 mg/ml), after which human interleukin-1alpha (IL-1alpha, 10 ng/ml) was added for 1 day. Fluorescein isothiocyanate (FITC)-labeled anti-ICAM-1 and FITC-labeled anti-VCAM-1 were used to analyze the IL-1alpha-induced expression of ICAM-1 and VCAM-1 by flow cytometry. Incubation of HCAEC with garlic extract significantly decreased ICAM-1 and VCAM-1 expression induced by IL-1alpha. In addition, we examined the effects of garlic extract on the adhesion of monocytes to endothelial cells, using the monocytic U937 cell line. The presence of garlic extract significantly inhibited the adhesion of monocytes to IL-1alpha-stimulated endothelial cells. These results indicate that garlic extract modulates the expression of ICAM-1 and VCAM-1, thus potentially contributing to the beneficial effects traditionally attributed to garlic.     

Androgens inhibit tumor necrosis factor-α-induced cell adhesion and promote tube formation of human coronary artery endothelial cells

Endothelial cells contribute to the function and integrity of the vascular wall, and a functional aberration may lead to atherogenesis. There is increasing evidence on the atheroprotective role of androgens. Therefore, we studied the effect of the androgens-testosterone and dihydrotestosterone-and estradiol on human coronary artery endothelial cell (HCAEC) function. We found by MTT assay that testosterone is not cytotoxic and enhances HCAEC proliferation. The effect of testosterone (10-50 nM), dihydrotestosterone (5-50 nM), and estradiol (0.1-0.4 nM) on the adhesion of tumor necrosis factor-α (TNF-α)-stimulated HCAECs was determined at different time points (12-96 h) by assessing their binding with human monocytic THP-1 cells. In addition, the expression of adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1), was determined by ELISA and Western blot analysis. Both testosterone and dihydrotestosterone attenuated cell adhesion and the expression of VCAM-1 and ICAM-1 in a dose- and time-dependent manner. Furthermore, androgen treatment for a longer duration inhibited cell migration, as demonstrated by wound-healing assay, and promoted tube formation on a Matrigel. Western blot analysis demonstrated that the expression of phosphorylated endothelial nitric oxide synthase (eNOS) increased, whereas that of inducible nitric oxide synthase (iNOS) decreased following the 96-h steroid treatment of TNF-α-stimulated HCAECs. Our findings suggest that androgens modulate endothelial cell functions by suppressing the inflammatory process and enhancing wound-healing and regenerative angiogenesis, possibly through an androgen receptor (AR)-dependent mechanism.     

High density lipoproteins (HDL) interrupt the sphingosine kinase signaling pathway. A possible mechanism for protection against atherosclerosis by HDL.

The ability of high density lipoproteins (HDL) to inhibit cytokine-induced adhesion molecule expression has been demonstrated in their protective function against the development of atherosclerosis and associated coronary heart disease. A key event in atherogenesis is endothelial activation induced by a variety of stimuli such as tumor necrosis factor-alpha (TNF), resulting in the expression of various adhesion proteins. We have recently reported that sphingosine 1-phosphate, generated by sphingosine kinase activation, is a key molecule in mediating TNF-induced adhesion protein expression. We now show that HDL profoundly inhibit TNF-stimulated sphingosine kinase activity in endothelial cells resulting in a decrease in sphingosine 1-phosphate production and adhesion protein expression. HDL also reduced TNF-mediated activation of extracellular signal-regulated kinases and NF-kappaB signaling cascades. Furthermore, HDL enhanced the cellular levels of ceramide which in turn inhibits endothelial activation. Thus, the regulation of sphingolipid signaling in endothelial cells by HDL provides a novel insight into the mechanism of protection against atherosclerosis.     

Inhibition of cholesteryl ester transfer protein by anacetrapib does not impair the anti-inflammatory properties of high density lipoprotein

Cholesteryl ester transfer protein (CETP) is a target of therapeutic intervention for coronary heart disease. Anacetrapib, a potent inhibitor of CETP, has been shown to reduce LDL-cholesterol by 40% and increase HDL-cholesterol by 140% in patients, and is currently being evaluated in a phase III cardiovascular outcomes trial. HDL is known to possess anti-inflammatory properties, however with such large increases in HDL-cholesterol, it is unclear whether CETP inhibition perturbs HDL functionality such as anti-inflammatory effects on endothelial cells. The purpose of the present study was to determine whether CETP inhibition by anacetrapib affects the anti-inflammatory properties of HDL. HDL was isolated from either hamsters treated with vehicle or anacetrapib for 2 weeks, or from normal human subjects treated either placebo, 20 mg, or 150 mg anacetrapib daily for 2 weeks. Anacetrapib treatment increased plasma HDL cholesterol levels by 65% and between 48 and 82% in hamsters and humans, respectively. Pre-incubation of human aortic endothelial cells with HDL isolated from both control and anacetrapib treated hamsters suppressed TNFα induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin. Similar results were obtained with human HDL samples pre and post treatment with placebo or anacetrapib. Further, HDL inhibited TNFα-induced MCP-1 secretion, monocyte adhesion and NF-κB activation in endothelial cells, and the inhibition was similar between control and anacetrapib treated groups. These studies demonstrate that anacetrapib treatment does not impair the ability of HDL to suppress an inflammatory response in endothelial cells.     

Both the unique and repeat regions of the Porphyromonas gingivalis hemagglutin A are involved in adhesion and invasion of host cells

Porphyromonas gingivalis is one of the major etiologic agents of adult periodontitis and has been associated with cardiovascular diseases. It expresses multiple hemagglutinins that are significant virulence factors and play an important role in bacterial attachment and invasion of host cells. The objective of this study was to determine the impact of P. gingivalis hemagglutinin A (HagA) on the attachment to and invasion of human coronary artery endothelial cells (HCAEC) and gingival epithelial cells (GEC). Bacterial strains expressing the HagA protein (or subunits), including Escherichia coli carrying plasmid pEKS5, E. coli carrying plasmid ST2, and Salmonella enterica serovar Typhimurium with plasmid pNM1.1 were used in this study. The strains were tested for their ability to attach to and invade HCAEC and GEC using antibiotic protection assays. In addition, the unique 5' N-terminal non-repeated segment of HagA was purified in recombinant form and a monoclonal antibody was created against the polypeptide. The monoclonal antibody against the unique portion of HagA was tested for inhibitory activity in these assays. The attachment of both E. coli strains expressing HagA fragment to host cells was significantly increased compared to their respective controls. However, they did not invade GEC or HCAEC. Interestingly, HagA expression in the Salmonella strain increased both adherence to and invasion of HCAEC, which may be due to the presence of the entire hagA ORF. A monoclonal antibody against the unique 5' N-terminal portion of HagA reduced invasion. Further experiments are needed to determine the role of the unique and the repeat segments of P. gingivalis HagA.     

Oxidized LDL at low concentration promotes in-vitro angiogenesis and activates nitric oxide synthase through PI3K/Akt/eNOS pathway in human coronary artery endothelial cells

It has long been considered that oxidized low-density lipoprotein (oxLDL) causes endothelial dysfunction and is remarkably related to the development of atherosclerosis. However, the effect of oxLDL at very low concentration (<10 μg/ml) on the endothelial cells remains speculative. Nitric oxide (NO) has a crucial role in the endothelial cell function. In this study, we investigated the effect of oxLDL at low concentration on NO production and proliferation, migration, tube formation of the human coronary artery endothelial cells (HCAEC). Results showed that oxLDL at 5 μg/ml enhanced HCAEC proliferation, migration and tube formation. These phenomena were accompanied by an increased intracellular NO production. l-NAME (a NOS inhibitor), LY294002 and wortmannin (PI3K inhibitors) could abolish oxLDL-induced angiogenic effects and prevent NO production in the HCAEC. The phosphorylation of Akt, PI3K and eNOS were up-regulated by oxLDL, which was attenuated by LY294002. Our results suggested that oxLDL at low concentration could promote in-vitro angiogenesis and activate nitric oxide synthesis through PI3K/Akt/eNOS pathway in HCAEC.     

Lipid Lowering and HDL Raising Gene Transfer Increase Endothelial Progenitor Cells,Enhance Myocardial Vascularity,and Improve Diastolic Function

Background

Hypercholesterolemia and low high density lipoprotein (HDL) cholesterol contribute to coronary heart disease but little is known about their direct effects on myocardial function. Low HDL and raised non-HDL cholesterol levels carried increased risk for heart failure development in the Framingham study, independent of any association with myocardial infarction. The objective of this study was to test the hypothesis that increased endothelial progenitor cell (EPC) number and function after lipid lowering or HDL raising gene transfer in C57BL/6 low density lipoprotein receptor deficient (LDLr^−/−) mice may be associated with an enhanced relative vascularity in the myocardium and an improved cardiac function.

Methodology/principal findings

Lipid lowering and HDL raising gene transfer were performed using the E1E3E4-deleted LDLr expressing adenoviral vector AdLDLr and the human apolipoprotein A-I expressing vector AdA-I, respectively. AdLDLr transfer in C57BL/6 LDLr^−/− mice resulted in a 2.0-fold (p<0.05) increase of the circulating number of EPCs and in an improvement of EPC function as assessed by ex vivo EPC migration and EPC adhesion. Capillary density and relative vascularity in the myocardium were 28% (p<0.01) and 22% (p<0.05) higher, respectively, in AdLDLr mice compared to control mice. The peak rate of isovolumetric relaxation was increased by 12% (p<0.05) and the time constant of isovolumetric relaxation was decreased by 14% (p<0.05) after AdLDLr transfer. Similarly, HDL raising gene transfer increased EPC number and function and raised both capillary density and relative vascularity in the myocardium by 24% (p<0.05). The peak rate of isovolumetric relaxation was increased by 16% (p<0.05) in AdA-I mice compared to control mice.

Conclusions/Significance

Both lipid lowering and HDL raising gene transfer have beneficial effects on EPC biology, relative myocardial vascularity, and diastolic function. These findings raise concerns over the external validity of studies evaluating myocardial biology and cardiac repair in normocholesterolemic animals.     

High‐density lipoprotein cholesterol affects early endothelial progenitor cell number and endothelial function in obese women

Objective: In order to improve our understanding of high‐density lipoprotein cholesterol (HDL‐C) cardiovascular (CV) impact in obesity, the association of HDL‐C plasma level with circulating early endothelial progenitor cell (early‐EPC) number and endothelium‐dependent vasodilatation (EDV) in obese women with normal or high low‐density lipoprotein cholesterol (LDL‐C) plasma levels was evaluated. Design and Methods: One hundred thirteen obese female subjects and a control group of 78 healthy female subjects were recruited. Circulating early‐EPC were assessed by single‐ and two‐color flow cytometric analyses with a fluorescence activated cell sorting (FACScan) flow cytometer. EDV was evaluated as response to ischemia by strain gauge plethysmography. Results: Both early‐EPC number and EDV were significantly decreased in obese women compared with the control group. Obese women with low HDL‐C showed a further decrease of early‐EPC and EDV in the presence of both high or normal LDL‐C plasmatic levels. In the normal HDL‐C level subgroup, hypercholesterolemic and nonhypercholesterolemic subjects showed no difference in early‐EPC number, whereas slight EDV impairment was present in hypercholesterolemic subjects. Conclusion: In obese women, low HDL‐C is associated to decreased early‐EPC number and impaired EDV, suggesting the need to assess whether evaluation of early‐EPC and EDV may increase HDL‐C prognostic value in the stratification of CV risk.     

Wnt5A/Ryk signaling critically affects barrier function in human vascular endothelial cells

Satisfactory therapeutic strategies for septic shock are still missing. Previously we found elevated levels of Wnt5A in patients with severe sepsis and septic shock. Wnt5A is released by activated macrophages but knowledge of its effects in the vascular system remains scant. Here we investigate the response of human coronary artery endothelial cells (HCAEC) to Wnt5A. We used a genome-wide differential expression approach to define novel targets regulated by Wnt5A. Gene ontology analysis of expression profiles revealed clusters of genes involved in actin cytoskeleton remodeling as the predominant targets of Wnt5A. Wnt5A targeted Rho-associated protein serine/threonine kinase (ROCK), leading to phosphorylation of LIM kinase-2 (LIMK2) and inactivation of the actin depolymerization factor cofilin-1 (CFL1). Functional experiments recording cytoskeletal rearrangements in living cells showed that Wnt5A enhanced stress fiber formation as a consequence of reduced actin depolymerization. The antagonist Wnt inhibitory factor 1 (WIF1) that specifically interferes with the WIF domain of Ryk receptors prevented actin polymerization. Wnt5A disrupted β-catenin and VE-cadherin adherens junctions forming inter-endothelial gaps. Functional experiments targeting the endothelial monolayer integrity and live recording of trans-endothelial resistance revealed enhanced permeability of Wnt5A-treated HCAEC. Ryk silencing completely prevented Wnt5A-induced endothelial hyperpermeability. Wnt5A decreased wound healing capacity of HCAEC monolayers; this was restored by the ROCK inhibitor Y-27632. Here we show that Wnt5A acts on the vascular endothelium causing enhanced permeability through Ryk interaction and downstream ROCK/LIMK2/CFL1 signaling. Wnt5A/Ryk signaling might provide novel therapeutic strategies to prevent capillary leakage in systemic inflammation and septic shock.     

NAD(P)H oxidase-stimulating activity of serum from type 2 diabetic patients with retinopathy mediates enhanced endothelial expression of E-selectin

Endothelial expression of E-selectin is enhanced in diabetic patients with retinopathy, however, the underlying mechanisms are unclear. Therefore, this study was aimed to determine if endothelial expression of E-selectin is stimulated with serum from type 2 diabetic patients with retinopathy, and whether this process is related to NAD(P)H oxidase-derived oxidative stress. Serum was obtained from type 2 diabetic patients with (T2DR) or without (T2DM) retinopathy, and age-matched non-diabetic healthy person (Control). Serum was added to in vitro-grown human coronary artery endothelial cells (HCAEC), after which E-selectin expression, reactive oxygen species (ROS) production, and NAD(P)H oxidase activity were measured. Serum from T2DR induced a significantly higher expression of E-selectin than serum from T2DM and control in association with an enhanced production of ROS in HCAEC. T2DR serum enhanced E-selectin expression in a ROS-dependent manner since this process was significantly attenuated not only by tiron (1 mM), a superoxide scavenger, but also by DPI (10 micromol/L) and apocynin (100 micromol/L), inhibitors of NAD(P)H oxidase. Furthermore, the activity of NADH oxidase was markedly increased by T2DR serum, and this was accompanied by the enhanced membrane translocation of p47phox, a cytosolic subunit of NAD(P)H oxidase. These findings suggest that serum from T2DR induced up-regulation of E-selectin expression in HCAEC, and this process might be dependent on activation of endothelial NADH oxidase via an enhanced membrane translocation of p47phox.     

High-density lipoprotein nitration and chlorination catalyzed by myeloperoxidase impair its effect of promoting endothelial repair

High-density lipoprotein (HDL) plays a key role in protecting against atherosclerosis. In cardiovascular disease, HDL can be nitrated and chlorinated by myeloperoxidase (MPO). In this study, we discovered that MPO-oxidized HDL is dysfunctional in promoting endothelial repair compared to normal HDL. Proliferation assay, wound healing, and transwell migration experiments showed that MPO-oxidized HDL was associated with a reduced stimulation of endothelial cell (EC) proliferation and migration. In addition, we found that Akt and ERK1/2 phosphorylation in ECs was significantly lower when ECs were incubated with oxidized HDL compared with normal HDL. To further determine whether oxidized HDL diminished EC migration through the PI3K/Akt and MEK/ERK pathways, we performed experiments with inhibitors of both these pathways. The transwell experiments performed in the presence of these inhibitors showed that the migration capacity was reduced and the differences observed between normal HDL and oxidized HDL were diminished. Furthermore, to study the effects of oxidized HDL on endothelial cells in vivo, we performed a carotid artery electric injury model on nude mice injected with either normal or oxidized HDL. Oxidized HDL inhibited reendothelialization compared to normal HDL in vivo. These findings implicate a key role for MPO-oxidized HDL in the pathogenesis of cardiovascular disease.     

Hepatocyte growth factor is upregulated by low-density lipoproteins and inhibits endothelin-1 release

Low-density lipoproteins (LDL) are known to cause endothelial injury and to promote the development of atherosclerotic lesions. This study demonstrates a significant concentration-dependent stimulatory effect of LDL on hepatocyte growth factor (HGF) synthesis (maximum release: 423 +/- 16% of control) and HGF receptor mRNA expression in cultured human coronary artery endothelial cells (HCAEC). HGF is a potent mitogen for endothelial cells but does not affect smooth muscle cell proliferation. In contrast, endothelin-1 (ET-1) acts as a mitogen on vascular smooth muscle cells and seems to be upregulated in coronary atherosclerosis. In this study, the basal ET-1 synthesis in HCAEC was concentration-dependently reduced by HGF (minimum: 54 +/- 3% of control). This inhibitory effect seems to be mediated via the tyrosine kinase activity of the HGF receptor c-met, since it was antagonized by the tyrosine kinase inhibitor lavendustin A. In addition, HGF also significantly reduced the LDL-stimulated ET-1 release. The LDL-induced upregulation of HGF synthesis in HCAEC and the inhibitory effect of HGF on ET-1 synthesis suggest a protective role of HGF in coronary atherosclerosis.