Endothelium : journal of endothelial cell research最新文献

During the inflammatory response, endothelial cell (EC) functions and mechanics change dramatically. To understand these responses, the authors analyzed changes in EC gene expression in an in vitro model of inflammation using cDNA microarrays. After interleukin-1 beta (IL1beta) stimulation, over 2500 genes were differentially expressed, of which approximately 2000 had not been previously identified by microarray studies of IL1beta stimulation in human umbilical vein endothelial cells (HUVECs). Functional grouping of these genes according to gene ontologies revealed genes associated with apoptosis, cell cycle, nuclear factor (NF)-kappa B cascade, chemotaxis, and immune response. Interestingly, claudin-1, known to exist in endothelial cell-cell junctions was up-regulated, but claudin-5 and occludin, which also exist in EC junctions, were down-regulated. Pre-b-cell colony enhancing factor (PBEF), a cytokine which may play a role in regulating endothelial permeability, was also up-regulated following IL1beta stimulation. Neutrophil transmigration across IL1beta-stimulated ECs did not induce changes in EC gene expression as strongly as IL1beta stimulation alone. Nineteen genes after 1 h and 22 genes after 3 h of neutrophil application were differentially expressed. These results indicate that, in terms of transcriptional effects on ECs, neutrophil transmigration is a relatively small perturbation in comparison to the background of large scale changes induced in ECs by cytokine stimulation. Supplementary materials are available for this article. Go to the publisher's online edition of Endothelium for the following free supplementary resources: supplementary figures and tables.

Receptor desensitization, or decreased responsiveness of a receptor to agonist stimulation, represents a regulatory process with the potential to have a significant impact on cell behavior. P2Y(2), a G-protein-coupled receptor activated by extracellular nucleotides, undergoes desensitization at many tissues, including the vascular endothelium. Endothelial cells from a variety of vascular beds are normally exposed to extracellular nucleotides released from damaged cells and activated platelets. The purpose of the present study was to compare P2Y(2) receptor desensitization observed in endothelial cells derived from bovine retina, a model of microvascular endothelium, and human umbilical vein endothelial cells (HUVECs), a model of a large blood vessel endothelium. P2Y(2) receptor desensitization was monitored by following changes in UTP-stimulated intracellular free Ca(2 +) in single cells using fura-2 microfluorometry. Both endothelial cell models exhibited desensitization of the P2Y(2) receptor after stimulation with UTP. However, the cells differed in the rate, dependence on agonist concentration, and percentage of maximal desensitization. These results suggest differential mechanisms of P2Y(2) receptor desensitization and favors heterogeneity in extracellular nucleotide activity in endothelial cells according to its vascular bed origin.

High altitude and hypoxia are known to induce polycythemia, pulmonary hypertension, and vascular remodeling. The authors investigated a number of blood cell populations in 15 mountain trekkers before and after 12 days spent at >3000 m. Red blood cell and platelet count increased, whereas circulating hematopoietic stem cell (enumerated as CD34bright cells), circulating endothelial cell (CEC) and circulating endothelial progenitor (CEP) count significantly decreased. In particular, the authors observed a decrease in the count of viable CECs, and a decrease in the circulating levels of RNA of the endothelial-specific gene VE-cadherin, whereas the fraction of apoptotic/necrotic CECs was stable. These data suggest a unique pattern of modulation of surrogate markers of vascular remodeling induced by exposure to hypobaric hypoxia.

Shear stress is thought to be important in maintaining vascular homeostasis and regulating vascular remodeling. The authors have previously shown that low shear stress increases interleukin (IL)-8 gene expression in endothelial cells. However, the detailed events that contribute to the regulation of this expression remain to be identified. In this study, the authors examined whether the shear stress-induced IL-8 mRNA expression in endothelial cells is mechanotransduced by integrins and the cytoskeleton. Exposure of endothelial cells to low shear stress (4.2 dyne/cm(2)) rapidly increased the expression of IL-8 mRNA. The induced IL-8 mRNA expression was inhibited by GRGDNP, which blocked the binding of integrins to the extracellular matrix. Additionally, this increase was attenuated by both anti-alpha(v)beta(3) integrin and anti-beta(1) integrin antibodies. Treatment of endothelial cells with either low shear stress or cytochalasin D resulted in the disruption of the cytoskeleton. Following the disruption of filamentous F-actin, the IL-8 mRNA expression in endothelial cells also increased, indicating that the shear stress-induced IL-8 mRNA expression may be mediated by the disruption of actin fibers. Taken together, these data suggest that integrins and the actin cytoskeleton play important roles in regulating the shear stress-induced IL-8 gene expression. Supplementary materials are available for the Materials and Methods section of this article. Go to the publisher's online of edition of Endothelium for this free supplemental resource.

Angiopoietin 1 (Ang-1) is the main ligand for endothelial cell-specific tyrosine kinase (Tie-2) receptors and it promotes migration and proliferation and inhibits apoptosis and vascular leakage. The exact mechanisms through which the Ang-1 exerts these effects remain unclear. The authors exposed human umbilical vein endothelial cells (HUVECs) to Ang-1 (300 ng/mL) for 4 h and conducted gene expression profiling using oligonucleotide microarrays. Real-time polymerase chain reaction (PCR) was also conducted to verify several of the genes that were regulated by Ang-1. Exposure to Ang-1 resulted in induction of 86 genes that are involved in endothelial cell (EC) proliferation, differentiation, migration, and survival. Thirty-six of these genes, including stanniocalcin, cyclin D1, vascular endothelial growth factor C, fms-related tyrosine kinase 1, interleukin 8, and CXCR4 have previously been shown to be induced by vascular endothelial growth factor (VEGF), suggesting significant similarities between VEGF and Ang-1 pathways. Ang-1 exposure also inhibited mRNA expressions of 49 genes, most of which are involved in cell cycle arrest, apoptosis, and suppression of transcription. These results indicate that Ang-1 triggers coordinated responses in endothelial cells designed to inhibit the expression of proapoptotic and antiproliferative genes and up-regulate proproliferative, proangiogenic, and antiapoptotic pathways. Moreover, we also found that the Erk1/2, phosphatidylinositol (PI) 3-kinase, and the mTOR pathways are involved in Ang-1-induced gene expression in HUVECs.

Endothelial dysfunction is a major feature of vascular diseases. A practical, minimally invasive method to effectively "probe" gene transcription for an individual patient's endothelium has potential to "customize" assessment for an individual at risk of vascular disease as well as pathophysiologic insight in an in vivo human, clinical context. Published literature lacks a methodology to identify endothelial differential gene expression in individuals with vascular disease. We describe a methodology to do so. The aim of this study was to specifically utilize (a) cutaneous microvascular biopsy, (b) laser capture microdissection, (c) cDNA amplification, (d) suppression subtractive hybridization, (e) high-throughput sequencing techniques, (f) real-time polymerase chain reaction (PCR), and (g) in combination of these methods, to profile differential gene expression in the context of cardiovascular and cerebrovascular disease. Endothelial cells were obtained by laser capture microdissection from a patient and a healthy sibling's microvascular biopsy tissues. Endothelial RNA was extracted, reverse transcribed, and amplified to ds cDNA. Suppression subtractive hybridization was used to establish an endothelial differential gene expression library. Real-time PCR confirmed SERP1, caspase 8, IGFBP7, S100A4, F85, and F147 up-regulation between 1.4- and 3.47-fold. The authors have successfully established a methodology to profile endothelial differential gene expression and identified six differentially expressed genes. This minimally invasive novel method has potential wide application in the customized assessment of many patients suffering vascular diseases.

The second half of the 20th century has witnessed the birth and growth of biological ultrastructural research in which electron microscopy plays a crucial role. The electron microscope has contributed greatly to biology and medicine, as is clearly indicated by the large number of publications devoted to reporting observations on the fine structure of biological specimens. Ultrastructural studies, initially concerned with macromolecular complexes, had by the mid-1950s revealed a previous unimaginable complexity in the organization of the eukaryotic cell. This review article summarizes the literature data concerning the important role played by the electron microscope in the study and characterization of capillary endothelial cells.

Plasma levels of endothelin-1 (ET-1) and adrenomedullin (ADM), two opposingly acting peptides, correlate with mortality in endotoxemia, but their measurement is cumbersome. New sandwich assays have been introduced that measure more stable precursor fragments. The objective of this study was to investigate the counterplay of their precursor peptides in septic patients and to compare them with disease severity and other biomarkers. Blood samples of an observational study in 95 consecutive critically ill patients admitted to the intensive care unit (ICU) were analyzed. CT-proET-1 and MR-proADM concentrations on admission were measured using new sandwich immunoassays. Depending on the clinical severity of the infection, both CT-proET-1 and MR-proADM levels exhibited a gradual increase from Systemic Inflammatory Response Syndrome (SIRS) to sepsis and septic shock (p < .001). Compared to the group of survivors, the group of nonsurvivors had higher median values of MR-proADM (5.7 nmol/L [range 0.4 to 21.0] versus 1.9 nmol/L [range 0.3 to 17.1], p < .02) and similar CT-proET-1 levels (56.0pmol/L [range 0.5 to 271.0] versus 54.1pmol/L [range 1.0 to 506.0], p = .86). Receiver operating characteristics (ROC) curve analysis showed a higher prognostic accuracy of the calculated ratio of both counteracting substances as compared to CT-proET-1 (p = 0.001) and C-reactive protein (CRP) (p = .001) and in the range of MR-proADM (p = .51), procalcitonin (p = 0.22), and the APACHE II score (p = .61). Endothelin-1 and adrenomedullin precursor peptides gradually increase with increasing severities of infection in critically ill patients. The ratio of the two counteracting peptides correlates with mortality and shows a prognostic accuracy to predict adverse outcome comparable to the APACHE II score.

Low-birth-weight babies have an increased risk of cardiovascular disease (CVD) in later life. The authors hypothesize that fetal hypoxia alters the structure and function of the developing cardiovascular system resulting in CVD. They investigated the effects of chronic hypoxia on cardiac performance, hemodynamic control, and growth during the second half of embryonic chick development. Three stages of hemodynamic adaptations were identified in hypoxic chick embryos. At embryonic day 13 (E13), heart rate and blood pressure were higher in hypoxic embryos. At E17, this was followed by sympathetic hyperinnervation of peripheral arteries, resulting in increased vasoconstriction during a chemoreflex. This was accompanied by dilatation of the left ventricle and a 50% reduction in cardiac contractility. E19 hypoxic embryos had a 33% higher baseline vascular tone, but failed to maintain blood pressure during acute stress, indicating cardiac failure. Reduced body, heart, and liver weights followed the hemodynamic changes. Chronic hypoxia induces dilated cardiomyopathy and sympathetic hyperinnervation of the peripheral vasculature leading to aberrant fetal hemodynamics and fetal growth restriction. This study identifies that alterations in fetal hemodynamic regulation are in the causal pathway between disturbances in fetal environment, restricted fetal growth and CVD, and establishes fetal hypoxia as a novel risk factor for cardiovascular disease.

Many cerebrovascular disorders are accompanied by an increased homocysteine (Hcy) levels. We have previously shown that acute hyperhomocysteinemia (HHcy) leads to an increased microvascular permeability in the mouse brain. Hcy competitively binds to gamma -aminbuturic acid (GABA) receptors and may increase vascular permeability by acting as an excitatory neurotransmitter. However, the role of GABA-A (GABA(A)) receptor in Hcy-induced endothelial cell (EC) permeability remains unclear. In the present study we attempted to determine the role of GABA(A) receptor and the possible mechanisms involved in Hcy-induced EC layer permeability. Mouse aortic and brain ECs were grown in Transwells and treated with 50 mu M Hcy in the presence or absence of GABA(A)-specific agonist muscimol. Role of matrix metalloproteinase-9 (MMP-9) was determined using its activity inhibitor GM-6001. Involvement of extracellular signal-regulated kinase (ERK) signaling was assessed using its kinase activity inhibitors PD98059 or U0126. EC permeability to the known content of bovine serum albumin (BSA)-conjugated with Alexa Flour-488 was assessed by measuring fluorescence intensity of the solutes in the Transwell's lower chambers. It was found that Hcy induced the formation of filamentous actin (F-actin). Hcy-induced EC permeability to BSA was significantly decreased by GABA and muscimol treatments. Presence of MMP-9 or ERK kinase activity inhibitors restored the Hcy-induced EC permeability to its baseline level. The mediation BSA leakage through the ECs was further confirmed in the experiments where Hcy-induced alterations in transendothelial electrical resistance of confluent ECs were assessed. The data suggest that Hcy increases EC layer permeability through inhibition of GABA(A) receptor and F-actin formation, in part, by transducing ERK and MMP-9 activation.