Quantitative proteomics and bioinformatics analyses of human coronary artery endothelial cell injury induced by Kawasaki disease / 中国当代儿科杂志

OBJECTIVE@#To study the biomarkers for human coronary artery endothelial cell (HCAEC) injury induced by Kawasaki disease (KD) using isobaric tags for relative and absolute quantitation (iTRAQ) proteomics.@*METHODS@#HCAECs cultured with the serum of children with KD were used as the KD group, and those cultured with the serum of healthy children was used as the healthy control group. The iTRAQ technique was used to measure the expression of proteins in two groups. The data on proteins were analyzed by bioinformatics. Western blot was used for the validation of protein markers.@*RESULTS@#A total of 518 significantly differentially expressed proteins were identified (with an absolute value of difference fold of >1.2, P<0.05). The gene ontology analysis showed that the differentially expressed proteins were significantly enriched in biological processes (including cellular processes, metabolic processes, and biological regulation), cellular components (including cell parts, cells, and organelles), and molecular functions (including binding, catalytic activity, and molecular function regulators). The KEGG analysis showed that the proteins were significantly enriched in the signaling pathways of ribosomes, PI3K-Akt signaling pathway, and transcriptional dysregulation in cancer. The PPI network showed that the top 9 protein markers in relation density were PWP2, MCM4, MCM7, MCM5, MCM3, MCM2, SLD5, HDAC2, and MCM6, which were selected as the protein markers for coronary endothelial injury in KD. Western blot showed that the KD group had significantly lower expression levels of the protein markers HDAC2, PWP2, and MCM2 than the healthy control group (P<0.05).@*CONCLUSIONS@#The serum of children with KD significantly changes the protein expression pattern of HCAECs and affects the signaling pathways associated with the cardiovascular system, which provides a new basis for the pathophysiological mechanism and therapeutic targets of KD.

Effects of intravenous immunoglobulin and aspirin treatment on the functions of circulating endothelial progenitor cells in children with Kawasaki disease / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the effects of intravenous immunoglobulin (IVIG) and aspirin treatment on the functions of circulating endothelial progenitor cells (EPCs) in children with Kawasaki disease (KD) and possible mechanisms.</p><p><b>METHODS</b>Blood samples were obtained in 10 children with KD before and 7 days after the treatment by IVIG and aspirin. MTT method, modified Boyden chamber method and cell culture plate adhesion method were used to assess the functions of EPCs, including proliferation, adhension and migration activities. The plasma levels of tumor necrosis factor-α (TNF-α) and high-sensitivity C reactive protein (hs-CRP) were also measured.</p><p><b>RESULTS</b>The functions of circulating EPCs 7 days after IVIG and aspirin treatment were significantly improved. IVIG and aspirin treatment significantly reduced plasma TNF-α and hs-CRP concentrations. There was a significant linear regression relationship between the reduced plasma TNF-α and hs-CRP levels and the increased functions of circulating EPCs.</p><p><b>CONCLUSIONS</b>IVIG and aspirin treatment can improve the functions of circulating EPCs, possibly through reducing plasma concentrations of TNF-α and hs-CRP.</p>

Decreased circulating endothelial progenitor cell function: relationship with serum concentrations of high-sensitivity C-reactive protein in children with Kawasaki disease / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the function of circulating endothelial progenitor cells and its relationship with serum concentrations of high-sensitivity C-reactive protein (Hs-CRP) in children with Kawasaki disease.</p><p><b>METHODS</b>Ten children with Kawasaki disease and ten healthy children as a control group were enrolled. The peripheral mononuclear cells were induced into endothelial progenitor cells using Dulbecco's Modified Eagle Medium containing vascular endothelial growth factor and basic fibroblast growth factor. The proliferative ability, migratory ability and adhesive ability of endothelial progenitor cells were assessed by MTT methods, modified Boyden chamber methods and cell culture plate adhesion method, respectively. The concentrations of serum Hs-CRP were measured by latex enhanced turbidimetric immunoassay.</p><p><b>RESULTS</b>The proliferative ability, migratory ability and adhesive ability of endothelial progenitor cells in the Kawasaki disease group were significantly lower than those in the control group (P<0.01). The serum concentrations of Hs-CRP in the Kawasaki disease group were significantly higher than those in the control group (87.1+/-30.2 mg/L vs 5.3+/-3.4 mg/L; P<0.01). The function of circulating endothelial progenitor cells was negatively correlated with serum concentrations of Hs-CRP in the Kawasaki disease group.</p><p><b>CONCLUSIONS</b>The function of circulating endothelial progenitor cells is decreased in children with Kawasaki disease, which may be associated with the abnormal expression of inflammatory mediators.</p>

Fluid shear stress upregulated endothelial nitric oxide synthase gene expression and nitric oxide formation in human endothelial progenitor cells / 中华心血管病杂志

<p><b>OBJECTIVE</b>To observe the effect of fluid shear stress on the eNOS gene expression and NO production in endothelial progenitor cells (EPCs).</p><p><b>METHODS</b>The peripheral blood mononuclear cells from healthy volunteers were inducted into EPCs and divided into stationary group (0 dyn/cm(2), 1 dyn/cm(2) = 0.1 Pa), low-flow shear stress group (5 dyn/cm(2)), medium-flow shear stress group (15 dyn/cm(2)) and high-flow shear stress group (25 dyn/cm(2)). The effects of shear stress on the endothelial nitric oxide synthase (eNOS) gene expression and nitric oxide (NO) production in human EPCs were measured.</p><p><b>RESULTS</b>Typical "spindle-shaped" appearance was shown in EPCs derived from peripheral blood mononuclear cells and were positively labeled by acetylated-LDL, lectin, FLK-1 and vWF. After 4 hours treatment with various shear stresses, the ratio of eNOS/beta-actin mRNA expression by human EPCs in low, medium and high-flow shear stress group was 0.364, 0.505 and 0.548 respectively, which was significantly higher than that in stationary group (0.183, all P < 0.05) and the NO secretion in human EPCs in low, medium and high-flow shear stress group was also significantly higher than that in stationary group (all P < 0.05).</p><p><b>CONCLUSION</b>Fluid shear stress enhances the eNOS mRNA expression and NO secretion in human EPCs, therefore, shear stress could potentiate the repair efficacy of EPCs for endothelial injury.</p>

Increased brachial-ankle pulse wave velocity is associated with impaired endothelial function in patients with coronary artery disease / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Pulse wave velocity and flow-mediated vasodilation (FMD) are widely used as noninvasive modalities for evaluating atherosclerosis. However, it is not known whether pulse wave velocity is related to FMD in patients with coronary artery disease (CAD). Therefore, the present study was designed to investigate the alteration in brachial-ankle pulse wave velocity (baPWV) and endothelial function in CAD patients.</p><p><b>METHODS</b>Thirty-three patients with CAD and thirty control subjects were recruited for this study. baPWV was measured non-invasively using a VP 1000 automated PWV/ABI analyzer (PWV/ABI, Colin Co. Ltd., Komaki, Japan). Endothelial function as reflected by FMD in the brachial artery was assessed with a high-resolution ultrasound device.</p><p><b>RESULTS</b>baPWV was increased in CAD patients compared with control subjects [(1756.1 +/- 253.1) cm/s vs (1495.3 +/- 202.3) cm/s, P < 0.01]. FMD was significantly reduced in CAD patients compared with control subjects [(5.2 +/- 2.1)% vs (11.1 +/- 4.4)%, P < 0.01]. baPWV correlated with FMD (r = -0.68, P < 0.001). The endothelium-independent vasodilation induced by sublingual nitroglycerin in the brachial artery was similar in the CAD group compared with the control group.</p><p><b>CONCLUSIONS</b>CAD is associated with increased baPWV and endothelial dysfunction. Increased baPWV parallels diminished endothelial function. Our data therefore suggest that baPWV can be used as a noninvasive surrogate index in clinical evaluation of endothelial function.</p>

Impaired endothelium-dependent vasodilation and arterial elasticity in patients with coronary artery disease / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the change in endothelium-dependent vasodilation and arterial elasticity and the association between them in patients with coronary artery disease (CAD).</p><p><b>METHODS</b>Thirty patients with CAD and thirty control subjects were recruited for this study. Flow-mediated dilation (FMD) in the brachial artery was evaluated by ultrasound Doppler flow method. They also underwent a non-invasive assessment of C(1) large artery and C(2) small artery indices by using pulse wave analysis.</p><p><b>RESULTS</b>FMD was significantly reduced in CAD group compared with that in control group [(5.17 +/- 2.13)% vs (11.1 +/- 4.36)%, P < 0.05], C(1) large artery elasticity index was similar between the two groups [(11.59 +/- 4.56) ml/mm Hg x 10 vs (12.11 +/- 3.82) ml/mm Hg x 10, P > 0.05]. However, C(2) small artery elasticity index was significantly reduced in CAD group compared with that in control group [(4.20 +/- 1.80) ml/mm Hg x 100 vs (6.26 +/- 2.36) ml/mm Hg x 100, P < 0.05]. There was a positive association between reduced C(2) small artery elasticity index and impaired FMD (r = 0.53, P < 0.05).</p><p><b>CONCLUSIONS</b>There were impaired endothelium-dependent vasodilation and reduced C(2) small artery elasticity index in the patients with CAD, which were closely correlated with each other. The present study suggested that the measurement of C(2) small artery elasticity might be used as a novel index for the determination of endothelial function.</p>

A study of association between age-related circulating endothelial progenitor cells and arterial elasticity / 中华心血管病杂志

<p><b>OBJECTIVE</b>Reduced arterial elasticity is a hallmark of aging in healthy humans independently of diseases and endothelial-cell injury and dysfunction may be responsible for this fall in arterial elasticity. We hypothesized that circulating endothelial progenitor cells (EPCs) are involved in endothelial repair and that lack of EPCs contributes to impaired arterial elasticity.</p><p><b>METHODS</b>A total of 56 healthy male volunteers were divided into young (n = 26) and elderly (n = 30) groups. Large and small artery elasticity indices were non-invasively assessed by using pulse wave analysis. Flow cytometer was used to count the number of circulating CD34(+) mononuclear cells (MNCs), which were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. EPCs were characterized as adherent cells double positive staining for DiI-acLDL uptake and lectin binding with using fluorescent microscope.</p><p><b>RESULTS</b>C(1) (large artery elasticity index) and C(2) (small artery elasticity index) were significantly reduced in the elderly group compared with those in the young group (11.73 +/- 1.45 vs 16.89 +/- 1.69 ml/mm Hg x 10, P < 0.001; 8.40 +/- 1.45 vs 10.58 +/- 1.18 ml/mm Hg x 100, P < 0.001 respectively). In parallel, the number of circulating EPCs was significantly reduced in the elderly group compared with the young group (0.13 +/- 0.02 vs 0.17 +/- 0.04%, P < 0.05). The number of circulating EPCs correlated with C(1) large and C(2) small artery elasticity indices (r = 0.47, P < 0.01; r = 0.4, P < 0.01). Fluorescent microscope was used to identify EPCs, which were double positive staining for DiI-acLDL uptake and lectin binding.</p><p><b>CONCLUSION</b>The present findings suggested that the fall in circulating EPCs with subsequently impaired endothelial-cell repair and function might contribute to reduced arterial elasticity in humans with aging. The decrease in circulating EPCs could serve as a surrogate biologic measure of vascular function and human age.</p>

Fluid shear stress regulates secretion of tissue-type plasminogen activator in human endothelial progenitor cells / 中华心血管病杂志

<p><b>OBJECTIVE</b>In order to investigate the role of shear stress in the regulation of endothelial function, we assessed here effects of shear stress on tissue-type plasminogen activator in human endothelial progenitor cells (EPCs).</p><p><b>METHODS</b>The peripheral blood mononuclear cells were separated from healthy adult and inducted into EPCs, which were identified by double staining for the fluorescent labeled acetylated-LDL and lectin. EPCs were seeded on the small diameter artificial vessels, and then divided into four different experimental groups including stationary group, low-flow shear stress group (5 dyn/cm(2)), medium-flow shear stress group (15 dyn/cm(2)) and high-flow shear stress group (25 dyn/cm(2)). The levels of t-PA in EPC culture medium at 0 hour, 5 hours, 10 hours, 15 hours, 20 hours and 25 hours after culture were measured by enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>The peripheral blood mononuclear cells differentiated into EPCs after induction, which were positively labeled by fluorescent acetylated-LDL and lectin. Shear stress enhanced production of the t-PA by EPCs, which was paralleled to levels and times of shear stress.</p><p><b>CONCLUSIONS</b>Shear stress increases t-PA secretion by human EPCs, suggesting that shear stress not only regulates vascular endothelial function but also participates in the pathogenesis of arteriosclerosis.</p>

Change in endothelial progenitor cells and endothelial function in patients with unstable angina pectoris / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the relationship between endothelial progenitors cells (EPCs) and endothelium-dependent vasodilation in patients with unstable angina pectoris.</p><p><b>METHODS</b>Thirty patients with unstable angina pectoris (UAP) and thirty control subjects were recruited. Flow-mediated dilation (FMD) in the brachial artery was evaluated by using ultrasound Doppler flow method. The number of circulating EPCs was analyzed by flow cytometry analysis. Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. CD34 antigen of adherent cells was identified by immunohistochemical assay. EPCs were characterized as adherent cells double positive for FITC-UEA-I binding and DiI-acLDL uptake by direct fluorescent staining under inverted fluorescent microscope.</p><p><b>RESULTS</b>FMD was significantly impaired in the UAP group compared with the control group (5.93% +/- 2.67% vs 11.1% +/- 4.36%, P < 0.05). There was no significant difference in NMD between two groups (13.60% +/- 5.03% vs 14.18% +/- 4.50%, P > 0.05). The number of CD34(+) cells significantly increased in the UAP group compared with the control group (0.13% +/- 0.05% vs 0.09% +/- 0.04%, P < 0.05). There was a negative association between impaired FMD and increased CD34(+) cell (r = -0.385, P < 0.05). A positive antigen of CD34 of adhesion cells and double positive adhesion cells were found.</p><p><b>CONCLUSION</b>This study shows that the levels of peripheral CD34(+) cells in patients with UAP increase with an impaired endothelial function. The increase in EPCs may be an important compensatory response to acute coronary ischemia and impaired endothelium in patients with UAP.</p>

Tumor necrosis factor-alpha induces release of endothelial microparticles from human endothelial cells / 中华心血管病杂志

<p><b>OBJECTIVE</b>The present study was designed to investigate whether Tumor necrosis factor (TNF)-alpha stimulates release of endothelial microparticles (EMPs) by human endothelial cells, and whether EMPs may serve as a promising marker for endothelial injury and dysfunction.</p><p><b>METHODS</b>Human umbilical venous endothelial cells (HUVEC) were incubated with or without TNF-alpha for 24 hours at 37 degrees C. EMPs generated on the surface of HUVEC were observed with a scanning electron microscopy. The CD31 and CD51 positive EMPs in culture supernatants were measured by flow cytometer.</p><p><b>RESULTS</b>Fewer vesicles were observed on cell surface of control group, in TNF-alpha-stimulated one, however, cells manifested a blebby surface (eruption phenomenon) and more vesicles on surface were observed. The levels of EMPs were significantly increased in TNF-alpha stimulated cells compared with controls [CD31 + EMP, (164 +/- 63)/1000 cells vs. (42 +/- 10)/1000 cells, P < 0.05; CD51 + EMP, (260 +/- 108)/1000 cells vs. (19 +/- 4)/1000 cells, P < 0.05].</p><p><b>CONCLUSION</b>TNF-alpha can stimulate HUVEC to release EMPs which may serve as a surrogate marker for endothelial injury and dysfunction.</p>