Activation Of Vascular Endothelial Cells Research Articles

Sirt1 Inhibits Oxidative Stress in Vascular Endothelial Cells.

The vascular endothelium is a layer of cells lining the inner surface of vessels, serving as a barrier that mediates microenvironment homeostasis. Deterioration of either the structure or function of endothelial cells (ECs) results in a variety of cardiovascular diseases. Previous studies have shown that reactive oxygen species (ROS) is a key factor that contributes to the impairment of ECs and the subsequent endothelial dysfunction. The longevity regulator Sirt1 is a NAD+-dependent deacetylase that has a potential antioxidative stress activity in vascular ECs. The mechanisms underlying the protective effects involve Sirt1/FOXOs, Sirt1/NF-κB, Sirt1/NOX, Sirt1/SOD, and Sirt1/eNOs pathways. In this review, we summarize the most recent reports in this field to recapitulate the potent mechanisms involving the protective role of Sirt1 in oxidative stress and to highlight the beneficial effects of Sirt1 on cardiovascular functions.

RHO-KINASE AS A KEY PARTICIPANT IN THE REGULATION OF VASCULAR TONE IN NORMAL CIRCULATION AND VASCULAR DISORDERS

Rho-kinase was shown to regulate the functions of almost all cells of our body. The key activator of Rhokinase is the small guanosine triphosphate (GTP)-binding protein RhoA, but RhoA-independent mechanisms of Rho-kinase regulation exist as well. In this review we describe the mechanisms affecting Rho-kinase activity in vascular smooth muscle and endothelial cells, Rho-kinase regulatory influences on fundamental physiological processes in these cells, as well as its role in the pathogenesis of vascular disorders in systemic and pulmonary arterial hypertension and diabetes mellitus.

β-arrestin is critical for early shear stress-induced Akt/eNOS activation in human vascular endothelial cells

Recent evidence suggests that β-arrestins, which are involved in G protein-coupled receptors desensitization, may influence mechanotransduction. Here, we observed that nitric oxide (NO) production was abrogated in human saphenous vein endothelial cells (SVECs) transfected with siRNA against β-arrestin 1 and 2 subjected to shear stress (SS, 15 dynes/cm2, 10 min). The downregulation of β-arrestins 1/2 in SVECs cells also prevented the SS-induced rise in levels of phosphorylation of Akt and endothelial nitric oxide synthase (eNOS, Serine 1177). Interestingly, immunoprecipitation revealed that β-arrestin interacts with Akt, eNOS and caveolin-1 and these interactions are not influenced by SS. Our data indicate that β-arrestins and Akt/eNOS downstream signaling are required for early SS-induced NO production in SVECs, which is consistent with the idea that β-arrestins and caveolin-1 are part of a pre-assembled complex associated with the cellular mechanotransduction machinery.

Endothelin-1 Regulates Molecules of the Major Histocompatibility Complex: Role in Sickle Cell Disease

Molecules of the Major Histocompatibility Complex (MHC), and in particular specific human leukocyte antigen (HLA) alleles, have been proposed in the pathophysiology of immune and vascular alterations leading to vasoocclusive crises (VOC) and stroke in Sickle Cell Disease (SCD). Endothelial cells express MHC molecules following exposure to cytokines. SCD is characterized, in part, by vascular endothelial cell activation, increased oxidative stress, sickle cell adhesion and excess levels of the potent mitogen, endothelin-1 (ET-1). ET-1 activates endothelial cells, induces oxidative stress and inflammation in the vascular wall and regulates erythrocyte homeostasis. However, the role of ET-1 on MHC regulation in SCD is not clear. We first characterized the effect of ET-1 on MHC expression in the human endothelial cell line, EA.hy926. We observed dose-dependent increases in the expression of MHC class I (HLA-A2 4.8 ± 2.1 folds p<0.01 n=4), MHC class II (HLA-DR 4.4 ± 1.7 folds p<0.01 n=4) and MHC transcription factor (CIITA 3.5 ± 1.8 folds p<0.05 n=4) in EA.hy926 cells. ET-1-stimulated expression of HLA-A2, HLA-DR and CIITA were significantly blocked by pre-incubation of cells with 10 µM BQ788, a selective blocker of ET-1 type B receptors (p<0.01, n=4). Chromatin immunoprecipitation (ChIP) studies in EA.hy926 cells showed that ET-1 increased Histone H3 acetylation of the promoter region within MHC molecules (HLA-A2 62% ± 5%, HLA-DRB 33% ± 18%, p<0.01, n=4); an event that was likewise blocked by BQ788. We then studied two sickle transgenic knockout mouse models of moderate to severe disease phenotype, βSAntilles and Berkeley (BERK) mice, respectively. We observed significant increases in MHC molecule, H2-Aa mRNA levels (n=7; p<0.01) in spleens from sickle transgenic mice when compared to transgenic knockout mice expressing human hemoglobin A (HbA). We then treated BERK, βSAntilles and HbA mice for 14 days with ET-1 receptor antagonists and observed significant reductions in H2-Aa mRNA levels in spleen tissue from sickle transgenic mice but not in HbA mice (n=7; p<0.05). These results implicate ET-1 as a novel regulator of MHC molecules and suggest that ET-1 receptor blockade represents a promising therapeutic approach to regulate both immune and vascular responses in SCD. DisclosuresNo relevant conflicts of interest to declare.

Defective Bone Repair in C57Bl6 Mice With Acute Systemic Inflammation.

Bone repair is initiated with a local inflammatory response to injury. The presence of systemic inflammation impairs bone healing and often leads to malunion, although the underlying mechanisms remain poorly defined. Our research objective was to use a mouse model of cortical bone repair to determine the effect of systemic inflammation on cells in the bone healing microenvironment. QUESTION/PURPOSES: (1) Does systemic inflammation, induced by lipopolysaccharide (LPS) administration affect the quantity and quality of regenerating bone in primary bone healing? (2) Does systemic inflammation alter vascularization and the number or activity of inflammatory cells, osteoblasts, and osteoclasts in the bone healing microenvironment? Cortical defects were drilled in the femoral diaphysis of female and male C57BL/6 mice aged 5 to 9 months that were treated with daily systemic injections of LPS or physiologic saline as control for 7 days. Mice were euthanized at 1 week (Control, n = 7; LPS, n = 8), 2 weeks (Control, n = 7; LPS, n = 8), and 6 weeks (Control, n = 9; LPS, n = 8) after surgery. The quantity (bone volume per tissue volume [BV/TV]) and microarchitecture (trabecular separation and thickness, porosity) of bone in the defect were quantified with time using microCT. The presence or activity of vascular endothelial cells (CD34), macrophages (F4/80), osteoblasts (alkaline phosphatase [ALP]), and osteoclasts (tartrate-resistant acid phosphatase [TRAP]) were evaluated using histochemical analyses. Only one of eight defects was bridged completely 6 weeks after surgery in LPS-injected mouse bones compared with seven of nine defects in the control mouse bones (odds ratio [OR], 0.04; 95% CI, 0.003-0.560; p = 0.007). The decrease in cortical bone in LPS-treated mice was reflected in reduced BV/TV (21% ± 4% vs 39% ± 10%; p < 0.01), increased trabecular separation (240 ± 36 μm vs 171 ± 29 μm; p < 0.01), decreased trabecular thickness (81 ± 18 μm vs 110 ± 22 μm; p = 0.02), and porosity (79% ± 4% vs 60% ± 10%; p < 0.01) at 6 weeks postoperative. Defective healing was accompanied by decreased CD34 (1.1 ± 0.6 vs 3.4 ± 0.9; p < 0.01), ALP (1.9 ± 0.9 vs 6.1 ± 3.2; p = 0.03), and TRAP (3.3 ± 4.7 vs 7.2 ± 4.0; p = 0.01) activity, and increased F4/80 (13 ± 2.6 vs 6.8 ± 1.7; p < 0.01) activity at 2 weeks postoperative. The results indicate that LPS-induced systemic inflammation reduced the amount and impaired the quality of bone regenerated in mouse femurs. The effects were associated with impaired revascularization, decreased bone turnover by osteoblasts and osteoclasts, and by increased catabolic activity by macrophages. Results from this preclinical study support clinical observations of impaired primary bone healing in patients with systemic inflammation. Based on our data, local administration of VEGF in the callus to stimulate revascularization, or transplantation of stem cells to enhance bone turnover represent potentially feasible approaches to improve outcomes in clinical practice.

Abstract A36: Resistance to IGF-1R targeted therapy by insulin-like growth factor 2-mediated tumor microenvironment network

Abstract Drug resistance is a major impediment in medical oncology. Recent studies have emphasized the importance of the tumor microenvironment (TME) to innate resistance to molecularly targeted therapies. In this study, we investigate the role of TME in resistance to cixutumumab, an anti-IGF-1R mAb that has shown limited clinical efficacy. We show that treatment with cixutumumab accelerates tumor infiltration of stromal cells and metastatic tumor growth and, ultimately, decreases overall survival of mice. Cixutumumab treatment stimulates recruitment of macrophages and fibroblasts via STAT3-dependent transcriptional up-regulation of IGF2 in cancer cells and paracrine IGF2/IGF-2R activation, resulting in the stroma-derived CXCL8 production and thus angiogenic and metastatic environment. Silencing IGF2 or STAT3 expression in cancer cells or IGF-2R or CXCL8 expression in stromal cells significantly inhibits the cancer-stroma communication and angiogenic activities of vascular endothelial cells. These findings suggest that blocking the STAT3/IGF-2/IGF-2R intercellular signaling network may overcome the adverse consequences of anti-IGF-1R mAb-based therapies. Citation Format: Ji-Sun Lee, Ho-Young Lee. Resistance to IGF-1R targeted therapy by insulin-like growth factor 2-mediated tumor microenvironment network. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A36.

Histidine-Rich Glycoprotein Prevents Septic Lethality through Regulation of Immunothrombosis and Inflammation

Sepsis is a major cause of death worldwide. We show that a plasma protein histidine-rich glycoprotein (HRG) was decreased significantly in septic mice with cecal ligation and puncture (CLP) and supplementary treatment of septic mice with exogenous HRG improved survival, with strong inhibition of tight attachment of neutrophils to pulmonary vasculatures, subsequent immunothrombosis, DIC state, lung inflammation, hypercytokinemia, and activation of vascular endothelial cells (VECs). In contrast, knockdown of HRG by siRNA exacerbated lethality. Purified human HRG reversibly induced morphological changes in human neutrophils in vitro; induction of spherical shape with reduced microvilli and adhesiveness to VECs. HRG maintained the passage of neutrophils through microcapillaries and abolished production of reactive oxygen species. These results suggested that the supplementary therapy with HRG may provide a novel strategy for the treatment of sepsis through suppression of excessive systemic inflammation and immunothrombosis by keeping circulating neutrophils quiescent and preventing uncontrolled activation of VECs.

Human iPSC-derived endothelial cell sprouting assay in synthetic hydrogel arrays

Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events during angiogenesis in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can recapitulate one or more aspects of angiogenesis in vitro, they are often limited by undefined substrates and lack of dependence on key angiogenic signaling axes. Here, we designed and characterized a chemically-defined model of endothelial sprouting behavior in vitro using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs). We rapidly encapsulated iPSC-ECs at high density in poly(ethylene glycol) (PEG) hydrogel spheres using thiol-ene chemistry and subsequently encapsulated cell-dense hydrogel spheres in a cell-free hydrogel layer. The hydrogel sprouting array supported pro-angiogenic phenotype of iPSC-ECs and supported growth factor-dependent proliferation and sprouting behavior. iPSC-ECs in the sprouting model responded appropriately to several reference pharmacological angiogenesis inhibitors of vascular endothelial growth factor, NF-κB, matrix metalloproteinase-2/9, protein kinase activity, and β-tubulin, which confirms their functional role in endothelial sprouting. A blinded screen of 38 putative vascular disrupting compounds from the US Environmental Protection Agency’s ToxCast library identified six compounds that inhibited iPSC-EC sprouting and five compounds that were overtly cytotoxic to iPSC-ECs at a single concentration. The chemically-defined iPSC-EC sprouting model (iSM) is thus amenable to enhanced-throughput screening of small molecular libraries for effects on angiogenic sprouting and iPSC-EC toxicity assessment. Statement of SignificanceAngiogenesis assays that are commonly used for drug screening and toxicity assessment applications typically utilize natural substrates like MatrigelTM that are difficult to spatially pattern, costly, ill-defined, and may exhibit lot-to-lot variability. Herein, we describe a novel angiogenic sprouting assay using chemically-defined, bioinert poly(ethylene glycol) hydrogels functionalized with biomimetic peptides to promote cell attachment and degradation in a reproducible format that may mitigate the need for natural substrates. The quantitative assay of angiogenic sprouting here enables precise control over the initial conditions and can be formulated into arrays for screening. The sprouting assay here was dependent on key angiogenic signaling axes in a screen of angiogenesis inhibitors and a blinded screen of putative vascular disrupting compounds from the US-EPA.

Association of ICAM-1 K469E polymorphism with dengue infection in North Indian population

Dengue infection is caused by flavivirus is one of the leading cause of mortality. There are certain factors which play role in the transformation of a mild form of the disease (DF) into a severe form (DHF) but the most important ones are: viral strain virulence, host genetics, and host immune status. In severe dengue infection, plasma leakage occurs due to vascular endothelial cell activation through expression of adhesion molecule like intercellular cell adhesion molecule-1 (ICAM-1). A total of 100 dengue patients (DF; n = 53 and DHF/DSS; n = 47) and 200 healthy controls were included in the study. ICAM-1 K469E genotyping was done by polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP). Expression of ICAM-1 mRNA was done by Real time reverse transcription- PCR (rRT-PCR). Patients with homozygous genotype (EE) have 3.22 fold risk (P = 0.008) of developing severe form of disease (DHF/DSS) as compared to other genotypes. Patients with DHF/DSS exhibit higher expression of ICAM-1 mRNA as compared to dengue fever and controls (P = 0.001 and < 0.001). Patients (DHF/DSS) with homozygous (EE) genotype exhibit higher expression of ICAM-1 mRNA when compared with wild type (KK) genotype (P = 0.005). This study suggests a possible association between the ICAM-1 polymorphism and the disease severity.

Abstract 347: Endothelin-1 Regulates Molecules of the Major Histocompatibility Complex: Role in Sickle Cell Disease

Sickle Cell Disease (SCD) is characterized, in part, by vascular endothelial cell activation, increased oxidative stress and sickle cell adhesion. Excess levels of the vasoconstrictor, endothelin-1 (ET-1), activate endothelial cells, induce oxidative stress and inflammation in the vascular wall and regulate erythrocyte homeostasis, in part, via activation of protein disulfide isomerase (PDI). We measured circulating ET-1, TGFβ1 and PDI activity in two sickle transgenic knockout mouse models expressing human sickle hemoglobin (Hb), BERK and βS Antilles . We observed significant increases in all three markers (n=6; p&lt;0.01) when compared to either C57BL/J6 mice or knockout mice expressing normal human Hb A. We then intraperitoneally injected BERK mice for 14 days with ET-1 receptor antagonists and observed significant reductions in these circulating markers (n=6; p&lt;0.05). These studies are important as PDI is proposed to control hemostasis, thrombosis and is reported to regulate the major histocompatibility complex (MHC). In addition, activated endothelial cells express PDI and MHC. However, the relationship between ET-1 and MHC in SCD remains unclear. We characterized the role of ET-1 on MHC expression in the endothelial cell line, EA.hy926. We observed dose-dependent increases in the expression of MHC class I (HLA-A2 4.8 ± 2.1 folds p&lt;0.01 n=4), MHC class II (HLA-DR 4.4 ± 1.7 folds p&lt;0.01 n=4) and MHC transcription factor (CIITA 3.5 ± 1.8 folds p&lt;0.05 n=4) in EA.hy926 cells. Increased expression of MHC was significantly blocked by co-incubation of cells with 10μM BQ788, a selective blocker of ET-1 type B receptors. Chromatin immunoprecipitation studies showed that ET-1 incubation produced an increase in Histone H3 acetylation of the promoter in MHC molecules (HLA-A2 4.6 ± 1.4 folds, HLA-DRB 6.0 ± 0.7 folds, p&lt;0.01, n=3) in these cells; an event that was likewise blocked by BQ788. In addition, ET-1 stimulated recruitment of CIITA to the promoter of HLA-A2 (1.9 ± 0.7 folds, p&lt;0.05, n=3) and HLA-DRB (3.0 ± 0.6 folds, p&lt;0.01, n=3). These results implicate ET-1 as a novel regulator of MHC promoter activity and suggest that ET-1 receptor blockade represents an important therapeutic approach to improve both the inflammatory and vascular complications of SCD.

Essential Role of Pituitary Tumor Transforming Gene 1 (PTTG1) in Vascular Endothelial Cell Activation and Remodeling

ObjectivePituitary tumor‐transforming gene‐1 (PTTG1) is a transforming gene that was first discovered in rat pituitary tumor cells. It possesses transcriptional activity and has securin functions. As a transcription factor, PTTG1 has been shown to mediate angiogenic process by activation of cell proliferation, migration, and tube formation of human endothelial cells (ECs). The purpose of this study is to determine whether PTTG1 is involved in inflammatory responses in vascular ECs, and if so, to elucidate the mechanism (s) involved.Methods and ResultsAnalyses of multiple microarray datasets derived indicated that mRNA levels of pituitary tumor transforming gene (PTTG1) are highly interrelated with shear stress‐induced vascular remodeling in vascular ECs. Our results show that the expression of PTTG1 is significantly down‐regulated in vascular ECs in response to laminar shear stress, indicating that PTTG1 may participate in the inflammatory process of endothelial cell activation. Indeed, treatment of ECs with inflammatory cytokines, such as interleukin‐1β (IL‐1β) and tumor necrosis factor‐alpha (TNF‐α), the expression of PTTG1 in vascular ECs was markedly increased in a dose and time‐dependent manner, as determined by both quantitative real‐time PCR (qRT‐PCR) and western blot analysis. Furthermore, we show that treatment of vascular ECs with either simvastatin or fluvastatin substantially decreased PTTG1 expression in a time and dose dependent manner. To determine the functional significance of PTTG1 in endothelial activation, we performed a loss‐of‐function study by using PTTG1 specific small interference RNA (siRNA). Our results demonstrate that knockdown of PTTG1 in vascular ECs substantially attenuates both IL‐1β and TNF‐α‐induced inflammatory responses. The expression of inflammatory cytokines and chemokines, such as IL‐6, IL‐8, and MCP‐1, was markedly inhibited in PTTG1 knockdown ECs. To further elucidate the functional significance of PTTG1 in vivo, we assessed the expression of PTTG1 in carotid arteries subjected to a carotid artery ligation‐induced injury. Our results demonstrate that the expression of PTTG1 was markedly decreased in the injured vessels. Furthermore, we found that in a mouse model of carotid artery ligation induced injury, PTTG1 gene deficiency substantially attenuated vascular remodeling, as manifested by decreased neointimal formation, microphage infiltration, and expression of inflammatory cytokines and adhesion molecules in the injured endothelium.ConclusionTogether, our results for the first time identify PTTG1 as a critical regulator implicated in vascular inflammation and remodeling, and suggest that targeted inhibition of PTTG1 in vasculature may represent a novel therapeutic strategy for preventing inflammatory cardiovascular diseases, such as atherosclerosis and restenosis.Support or Funding InformationSupported by the US National Institutes of Health (HL103869) to J. Sun

N‐myc downstream regulated gene‐1 promotes vascular inflammation through activating both NF‐κB and c‐Jun/AP‐1 pathways in endothelial cells

ObjectiveN‐myc downstream regulated gene‐1(NDRG‐1) is a member of NDRG family that has been shown to play essential roles not only in the regulation of cancer metastasis but also in the development of cardiovascular diseases, such as atherosclerosis and thrombus. The objective of this study is to determine whether NDRG1 is involved in vascular endothelial cell (EC) activation, and if so, to determine the molecular mechanism (s) involved.Methods and ResultsIn response to the stimulation of inflammatory cytokines, such as interleukin‐1β (IL‐1β) and tumor necrosis factor‐alpha (TNF‐α), we show that the expression of NDRG1 in vascular ECs was markedly increased in a dose and time‐dependent manner, as determined by both quantitative real‐time PCR (qRT‐PCR) and western blot analysis. To determine the role of NDRG1 in endothelial activation, we performed a loss‐of‐function study by using NDRG1 specific small interference RNA (siRNA). Our results demonstrate that knockdown of NDRG1 in vascular ECs by siRNA substantially attenuates both IL‐1β and TNF‐α‐induced inflammatory responses in human umbilical vein endothelial cells (HUVECs). The expression of inflammatory cytokines and chemokines, such as IL‐6, IL‐8, and MCP‐1, was markedly inhibited in NDRG1 knockdown ECs. Intriguingly, the IL‐1β‐induced expression of thrombotic markers, such as plasminogen activator inhibitor type 1 (PAI‐1) and tissue factor (TF), was also attenuated in NDRG1 knockdown cells. To further elucidate the functional significance of NDRG1 in vivo, we assessed the expression of NDRG1 in the vessels subjected to the carotid artery ligation‐induced injury. Our results demonstrate that the expression of NDRG1 was markedly increased in the injured vessels as compared with their un‐injured controls. Furthermore, we found that in a mouse model of carotid artery ligation induced injury, knockdown of NDRG1 expression substantially attenuates vascular remodeling, as manifested by decreased neointimal formation, microphage infiltration, and expression of adhesion molecules in injured arteries. Mechanistically, we found that inhibition of NDRG1 markedly inhibited the transcriptional activities of both NF‐κB and AP‐1 pathways in vascular ECs, as determined by both promoter‐driven luciferase and electrophoretic mobility shift assays.ConclusionTaken together, our results for the first time identify NDRG1 as a critical regulator implicated in vascular inflammation and remodeling, and suggest that targeted inhibition of NDRG1 in vasculature may represent a novel therapeutic strategy for preventing certain cardiovascular diseases, such as atherosclerosis and thrombosis.Support or Funding InformationNIH:RO1 HL103869‐01, NSCF: 81541003, SHSCF:15ZR1413400

Cardiac biomarkers in children with congenital heart disease.

Most congenital heart diseases (CHDs) have specific hemodynamics, including volume and pressure overload, as well as cyanosis and pulmonary hypertension, associated with anatomical abnormalities. Such hemodynamic abnormalities can cause activation of neurohormones, inflammatory cytokines, fibroblasts, and vascular endothelial cells, which in turn contribute to the development of pathologic conditions such as cardiac hypertrophy, fibrosis, and cardiac cell damages and death. Measuring biomarker levels facilitates the prediction of these pathological changes, and provides information about the stress placed on the myocardial cells, the severity of the damage, the responses of neurohumoral factors, and the remodeling of the ventricle. Compared to the ample information on cardiac biomarkers in adult heart diseases, data from children with CHD are still limited. We reviewed cardiac biomarkers-specifically focusing on troponin as a biomarker of myocardial damage, amino-terminal procollagen type III peptide (PIIIP) as a biomarker of myocardial fibrosis and stromal remodeling, and B-type natriuretic peptide (BNP)/N-terminal proBNP as biomarkers of cardiac load and heart failure, by introducing relevant publications, including our own, on pediatric CHD patients as well as adults. Levels of highly sensitive troponin I are elevated in patients with atrial septal defects (ASDs) and ventricular septal defects (VSDs). PIIIP levels are also elevated in patients with ASD, VSD, pulmonary stenosis, and Tetralogy of Fallot. Measurement of BNP and N-terminal proBNP levels shows good correlation with heart failure score in children. In the treatment of children with CHD requiring delicate care, it is vital to know the specific degree of myocardial damage and severity of heart failure. Cardiac biomarkers are useful tools for ascertaining the condition of CHDs with ease and are likely to be useful in determining the appropriate care of pediatric cardiology patients.

Gold nanoparticles induce heme oxygenase-1 expression through Nrf2 activation and Bach1 export in human vascular endothelial cells.

It has been reported that increased levels and activity of the heme oxygenase-1 (HO-1) protein ameliorate tissue injuries. In the present study, we investigated the effects and mechanisms of action of gold nanoparticles (AuNPs) on HO-1 protein expression in human vascular endothelial cells (ECs). The AuNPs induced HO-1 protein and mRNA expression in a concentration- and time-dependent manner. The induction was reduced by the thiol-containing antioxidants, including N-acetylcysteine and glutathione, but not by the non-thiol-containing antioxidants and inhibitors that block the enzymes for intracellular reactive oxygen species generation. The AuNPs enhanced Nrf2 protein levels but did not affect Nrf2 mRNA expression. In response to the AuNP treatment, the cytosolic Nrf2 translocated to the nucleus, and, concomitantly, Bach1 exited the nucleus and its tyrosine phosphorylation increased. The chromatin immunoprecipitation assay revealed that the translocated Nrf2 bound to the antioxidant-response element located in the E2 enhancer region of the HO-1 gene promoter and acted as a transcription factor. Although N-acetylcysteine inhibited the AuNP-induced Nrf2 nuclear translocation, the AuNPs did not promote intracellular reactive oxygen species production or endoplasmic reticulum stress in the ECs. Knockdown of Nrf2 expression by RNA interference significantly inhibited AuNP-induced HO-1 expression at the protein and mRNA levels. In summary, AuNPs enhance the levels and nuclear translocation of the Nrf2 protein and Bach1 export/tyrosine phosphorylation, leading to Nrf2 binding to the HO-1 E2 enhancer promoter region to drive HO-1 expression in ECs. This study, together with our parallel findings, demonstrates that AuNPs can act as an HO-1 inducer, which may partially contribute to their anti-inflammatory bioactivity in human vascular ECs.

PP4 - Carbon monoxide releasing molecule-3 (CORM-3) modulates progression of M1/M2 phenotypes in alveolar macrophages

Background The outcome of the severe systemic disorders (e.g. sepsis, acute lung injury) largely depends on the efficacy of resolution of inflammation. We have reported that water-soluble carbon monoxide releasing molecule-3 (CORM-3) suppresses inflammatory activation in neutrophils and vascular endothelial cells and offers protection against sepsis-induced lung dysfunction in vivo. Alveolar macrophages are key contributors to both the promotion and resolution/remodeling of inflammation and are classified into pro-inflammatory (M1) and anti-inflammatory (M2) groups. The change in M1/M2 balance has been reported in various pulmonary diseases and is considered as a target for therapeutic intervention. The aim of this study was to assess the effects of CORM-3 in modulation of macrophage M1/M2 phenotype under “inflammatory” or “remodeling” conditions. Methods Rat alveolar macrophages (NR8383) in culture were stimulated with LPS (500ng/ml)/IFN-γ (100ng/ml) or IL-4 (1ng/ml)/IL-13(1ng/ml) to induce M1- and M2-phenotypes, respectively. Specific markers of M1-phenotype (iNOS expression) and M2-phenotype (mannose receptor; Man-R, expression) were assessed (western blotting) after 1, 3, 6, or 24 hours in the absence or presence of CORM-3 (0.1mM) treatment. Inactive CORM-3 was used as a control. Results M1-phenotype (iNOS expression) was markedly upregulated (10 to 24 –fold) in LPS/IFN-γ-stimulated macrophage after 6 and 24 hours, respectively. In parallel, IL-4/IL13 stimulation induced M2-phenotype as evidenced by increase in Man-R levels at 6 and 24 hrs. Administration of CORM-3 during macrophage stimulation effectively suppressed development of both, M1- and M2-phenotypes. Paradoxically, treatment of naive (unstimulated) macrophage with CORM-3 increased expression of Man-R after 1 hour (1.8 fold) and iNOS expression after 6 hours (2.5 fold). Conclusion These findings indicate that CORM-3 can modulate progression of M1- and M2-phenotypes in macrophages under inflammatory conditions in vitro.

Endothelial Angiogenesis and Barrier Function in Response to Thrombin Require Ca2+ Influx through the Na+/Ca2+ Exchanger

Thrombin acts on the endothelium by activating protease-activated receptors (PARs). The endothelial thrombin-PAR system becomes deregulated during pathological conditions resulting in loss of barrier function and a pro-inflammatory and pro-angiogenic endothelial phenotype. We reported recently that the ion transporter Na(+)/Ca(2+) exchanger (NCX) operating in the Ca(2+)-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells. Here, we investigated whether Ca(2+) influx through NCX was involved in ERK1/2 activation, angiogenesis, and endothelial barrier dysfunction in response to thrombin. Reverse-mode NCX inhibitors and RNAi-mediated NCX1 knockdown attenuated ERK1/2 phosphorylation in response to thrombin or an agonist of PAR-1, the main endothelial thrombin receptor. Conversely, promoting reverse-mode NCX by suppressing Na(+)-K(+)-ATPase activity enhanced ERK1/2 activation. Reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced primary human vascular endothelial cell angiogenesis, quantified as proliferation and tubular differentiation. Reverse-mode NCX inhibitors or NCX1 knockdown preserved barrier integrity upon thrombin stimulation in vitro. Moreover, the reverse-mode NCX inhibitor SEA0400 suppressed Evans' blue albumin extravasation to the lung and kidneys and attenuated edema formation and ERK1/2 activation in the lungs of mice challenged with a peptide activator of PAR-1. Mechanistically, thrombin-induced ERK1/2 activation required NADPH oxidase 2-mediated reactive oxygen species (ROS) production, and reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced ROS production. We propose that reverse-mode NCX is a novel mechanism contributing to thrombin-induced angiogenesis and hyperpermeability by mediating ERK1/2 activation in a ROS-dependent manner. Targeting reverse-mode NCX could be beneficial in pathological conditions involving unregulated thrombin signaling.

Pneumonia and venous thromboembolism: Is the evidence catching up with the guidelines?

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Insulin-like growth factor binding protein-3 inhibits cell adhesion via suppression of integrin β4 expression.

We previously reported that IGF binding protein-3 (IGFBP-3), a major IGF-binding protein in human serum, regulates angiogenic activities of human head and neck squamous cell carcinoma (HNSCC) cells and human umbilical vein endothelial cells (HUVECs) through IGF-dependent and IGF-independent mechanisms. However, the role of IGFBP-3 in cell adhesion is largely unknown. We demonstrate here that IGFBP-3 inhibits the adhesion of HNSCC cells and HUVECs to the extracellular matrix (ECM). IGFBP-3 reduced transcription of a variety of integrins, especially integrin β4, and suppressed phosphorylation of focal adhesion kinase (FAK) and Src in these cells through both IGF-dependent and IGF-independent pathways. IGFBP-3 was found to suppress the transcription of c-fos and c-jun and the activity of AP1 transcription factor. The regulatory effect of IGFBP-3 on integrin β4 transcription was attenuated by blocking c-jun and c-fos gene expression via siRNA transfection. Taken together, our data show that IGFBP-3 has IGF-dependent and -independent inhibitory effects on intracellular adhesion signaling in HNSCC and HUVECs through its ability to block c-jun and c-fos transcription and thus AP-1-mediated integrin β4 transcription. Collectively, our data suggest that IGFPB-3 may be an effective cancer therapeutic agent by blocking integrin-mediated adhesive activity of tumor and vascular endothelial cells.

Roles of ROS and PKC-βII in ionizing radiation-induced eNOS activation in human vascular endothelial cells

Vascular endothelial cells can absorb higher radiation doses than any other tissue in the body, and post-radiation impaired endothelial nitric oxide synthase (eNOS) function may be developed as a potential contributor to the pathogenesis of vascular injury. In this study, we investigated early alterations of eNOS signaling in human umbilical venous endothelial cells (HUVECs) exposed to X-ray radiation. We found that ionizing radiation increased eNOS phosphorylation at Ser-1177 and dephosphorylation at Thr-495 in HUVECs in a dose-dependent (≤20Gy) and time-dependent (6–72h) manner. The total expression levels of eNOS were unchanged by radiation. Although a transient but significant increase in extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation and a biphasic decline in Akt phosphorylation were observed after irradiation, these inhibitors were without effect on the radiation-induced changes in eNOS phosphorylation. There was an increase in protein kinase C-βII (PKC-βII) expression and the ablation of PKC-βII by small interfering RNA (siRNA) negated the radiation effect on the two eNOS phosphorylation events. Furthermore, when the radiation-induced increase in reactive oxygen species (ROS) generation was prevented by the anti-oxidant N-acetyl-l-cysteine, eNOS Ser-1177 phosphorylation and Thr-495 dephosphorylation in irradiated HUVECs were significantly reduced. However, transfection of PKC-β siRNA did not alter ROS production after irradiation, and NAC failed to block the radiation-induced increase in PKC-βII expression. Taken together, our results suggest that ionizing radiation-induced eNOS activation in human vascular endothelial cells is attributed to both the up-regulation of PKC-βII and the increase in ROS generation which were independent of each other.

Investigation of proposed mechanisms of chemotherapy-induced venous thromboembolism: endothelial cell activation and procoagulant release due to apoptosis.

Venous thromboembolism (VTE) during chemotherapy is common, with 7% mortality in metastatic breast cancer (MBC). In a prospective cohort study of patients with breast cancer, we investigated whether vascular endothelial cell activation (VECA), and whether apoptosis, is the cause of chemotherapy-induced VTE. Serum markers of VECA, E-selectin (E-sel), vascular cell adhesion molecule 1 (VCAM-1) and d-dimer (fibrin degradation and hypercoagulability marker) were measured prechemotherapy and at 1, 4, and 8 days following chemotherapy. Clinical deep vein thrombosis (DVT) or pulmonary embolism and occult DVT detected by duplex ultrasound imaging were recorded as VTE-positive (VTE+). In patients with MBC, hypercoagulable response to chemotherapy was compared between patients with and without cancer progression. Development of VTE and cancer progression was assessed 3 months following starting chemotherapy. Of the 134 patients, 10 (7.5%) developed VTE (6 [17%] of 36 MBC receiving palliation, 0 of 11 receiving neoadjuvant to downsize tumor, and 4 [5%] of 87 early breast cancer receiving adjuvant chemotherapy, P = .06). Levels of E-sel and VCAM-1 decreased in response to chemotherapy (P < .001) in both VTE+ and patients not developing VTE (VTE-). However, decrease in VECA markers was similar in VTE+ and VTE- patients, implying this is not the cause of VTE. In patients with MBC following chemotherapy, d-dimer (geometric mean) increased by 36% in the 21 patients with MBC responding to chemotherapy but steadily decreased by 11% in the 15 who progressed (day 4, P < .01), implying patients with tumor response (apoptosis) had an early hypercoagulable response. During chemotherapy for breast cancer, VECA is induced; however, this is not the primary mechanism for VTE. Chemotherapy-induced apoptosis may enhance hypercoagulability and initiate VTE.