Inhibition of CXCL12-mediated chemotaxis of Jurkat cells by direct immunotoxicants.

Directional migration of cells to specific locations is required in tissue development, wound healing, and immune responses. Immune cell migration plays a crucial role in both innate and adaptive immunity. Chemokines are small pro-inflammatory chemoattractants that control the migration of leukocytes. In addition, they are also involved in other immune processes such as lymphocyte development and immune pathology. In a previous toxicogenomics study using the Jurkat T cell line, we have shown that the model immunotoxicant TBTO inhibited chemotaxis toward the chemokine CXCL12. In the present work, we aimed at assessing a novel approach to detecting chemicals that affect the process of cell migration. For this, we first evaluated the effects of 31 chemicals on mRNA expression of genes that are known to be related to cell migration. With this analysis, seven immunotoxicants were identified as potential chemotaxis modulators, of which five (CoCl2 80 µM, MeHg 1 µM, ochratoxin A 10 µM, S9-treated ochratoxin A 10 µM, and TBTO 100 nM) were confirmed as chemotaxis inhibitor in an in vitro trans-well chemotaxis assay using the chemokine CXCL12. The transcriptome data of the five compounds together with previously obtained protein phosphorylation profiles for two out of five compounds (i.e., ochratoxin A and TBTO) revealed that the mechanisms behind the chemotaxis inhibition are different for these immunotoxicants. Moreover, the mTOR inhibitor rapamycin had no effect on the chemotaxis of Jurkat cells, indicating that the mTOR pathway is not involved in CXCL12-mediated chemotaxis of Jurkat cells, which is opposite to the findings on human primary T cells (Munk et al. in PLoS One 6(9):e24667, 2011). Thus, the results obtained from the chemotaxis assay conducted with Jurkat cells might not fully represent the results obtained with human primary T cells. Despite this difference, the present study indicated that some compounds may exert their immunotoxic effects through inhibition of CXCL12-mediated chemotaxis.

Protein phosphorylation profiling identifies potential mechanisms for direct immunotoxicity.

Signaling networks are essential elements that are involved in diverse cellular processes. One group of fundamental components in various signaling pathways concerns protein tyrosine kinases (PTK). Various toxicants have been demonstrated to exert their toxicity via modulation of tyrosine kinase activity. The present study aimed to identify common cellular signaling pathways that are involved in chemical-induced direct immunotoxicity. To this end, an antibody array-based profiling approach was applied to assess effects of five immunotoxicants, two immunosuppressive drugs and two non-immunotoxic control chemicals on the phosphorylation of 28 receptor tyrosine kinases and 11 crucial signaling nodes in Jurkat T-cells. The phosphorylation of ribosomal protein S6 (RPS6) and of kinases Akt, Src and p44/42 were found to be commonly regulated by immunotoxicants and/or immunosuppressive drugs (at least three compounds), with the largest effect observed upon RPS6. Flow cytometry and Western blotting were used to further examine the effect of the model immunotoxicant TBTO on the components of the mTOR-p70S6K-RPS6 pathway. These analyses revealed that both TBTO and the mTOR inhibitor rapamycin inactivate RPS6, but via different mechanisms. Finally, a comparison of the protein phosphorylation data to previously obtained transcriptome data of TBTO-treated Jurkat cells resulted in a good correlation at the pathway level and indicated that TBTO affects ribosome biogenesis and leukocyte migration. The effect of TBTO on the latter process was confirmed using a CXCL12 chemotaxis assay.

Expression of Nitric Oxide-Transporting Aquaporin-1 Is Controlled by KLF2 and Marks Non-Activated Endothelium In Vivo.

The flow-responsive transcription factor Krüppel-like factor 2 (KLF2) maintains an anti-coagulant, anti-inflammatory endothelium with sufficient nitric oxide (NO)-bioavailability. In this study, we aimed to explore, both in vitro and in human vascular tissue, expression of the NO-transporting transmembrane pore aquaporin-1 (AQP1) and its regulation by atheroprotective KLF2 and atherogenic inflammatory stimuli. In silico analysis of gene expression profiles from studies that assessed the effects of KLF2 overexpression in vitro and atherosclerosis in vivo on endothelial cells, identifies AQP1 as KLF2 downstream gene with elevated expression in the plaque-free vessel wall. Biomechanical and pharmaceutical induction of KLF2 in vitro is accompanied by induction of AQP1. Chromosome immunoprecipitation (CHIP) confirms binding of KLF2 to the AQP1 promoter. Inflammatory stimulation of endothelial cells leads to repression of AQP1 transcription, which is restrained by KLF2 overexpression. Immunohistochemistry reveals expression of aquaporin-1 in non-activated endothelium overlying macrophage-poor intimae, irrespective whether these intimae are characterized as being plaque-free or as containing advanced plaque. We conclude that AQP1 expression is subject to KLF2-mediated positive regulation by atheroprotective shear stress and is downregulated under inflammatory conditions both in vitro and in vivo. Thus, endothelial expression of AQP1 characterizes the atheroprotected, non-inflamed vessel wall. Our data provide support for a continuous role of KLF2 in stabilizing the vessel wall via co-temporal expression of eNOS and AQP1 both preceding and during the pathogenesis of atherosclerosis.

Successful validation of genomic biomarkers for human immunotoxicity in Jurkat T cells in vitro.

Previously, we identified 25 classifier genes that were able to assess immunotoxicity using human Jurkat T cells. The present study aimed to validate these classifiers. For that purpose, Jurkat cells were exposed for 6 h to subcytotoxic doses of nine immunotoxicants, five non-immunotoxicants and four compounds for which human immunotoxicity has not yet been fully established. RNA was isolated and subjected to Fluidigm quantitative real time (qRT)-PCR analysis. The sensitivity, specificity and accuracy of the screening assay as based on the nine immunotoxicants and five non-immunotoxicants used in this study were 100%, 80% and 93%, respectively, which is better than the performance in our previous study. Only one compound was classified as false positive (benzo-e-pyrene). Of the four potential (non-)immunotoxicants, chlorantraniliprole and Hidrasec were classified immunotoxic and Sunset yellow and imidacloprid as non-immunotoxic. ToxPi analysis of the PCR data provided insight in the molecular pathways that were affected by the compounds. The immunotoxicants 2,3-dichloro-propanol and cypermethrin, although structurally different, affected protein metabolism and cholesterol biosynthesis and transport. In addition, four compounds, i.e. chlorpyrifos, aldicarb, benzo-e-pyrene and anti-CD3, affected genes in cholesterol metabolism and transport, protein metabolism and transcription regulation. qRT-PCR on eight additional genes coding for similar processes as defined in ToxPi analyzes, supported these results. In conclusion, the 25 immunotoxic classifiers performed very well in a screening with new non-immunotoxic and immunotoxic compounds. Therefore, the Jurkat screening assay has great promise to be applied within a tiered approach for animal free testing of human immunotoxicity.

Heme oxygenase-1 is required for angiogenic function of bone marrow-derived progenitor cells: role in therapeutic revascularization.

AIMS: Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that can be down-regulated in diabetes. Its importance for mature endothelium has been described, but its role in proangiogenic progenitors is not well known. We investigated the effect of HO-1 on the angiogenic potential of bone marrow-derived cells (BMDCs) and on blood flow recovery in ischemic muscle of diabetic mice. RESULTS: Lack of HO-1 decreased the number of endothelial progenitor cells (Lin(-)CD45(-)cKit(-)Sca-1(+)VEGFR-2(+)) in murine bone marrow, and inhibited the angiogenic potential of cultured BMDCs, affecting their survival under oxidative stress, proliferation, migration, formation of capillaries, and paracrine proangiogenic potential. Transcriptome analysis of HO-1(-/-) BMDCs revealed the attenuated up-regulation of proangiogenic genes in response to hypoxia. Heterozygous HO-1(+/-) diabetic mice subjected to hind limb ischemia exhibited reduced local expression of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), stromal cell-derived factor 1 (SDF-1), VEGFR-1, VEGFR-2, and CXCR-4. This was accompanied by impaired revascularization of ischemic muscle, despite a strong mobilization of bone marrow-derived proangiogenic progenitors (Sca-1(+)CXCR-4(+)) into peripheral blood. Blood flow recovery could be rescued by local injections of conditioned media harvested from BMDCs, but not by an injection of cultured BMDCs. INNOVATION: This is the first report showing that HO-1 haploinsufficiency impairs tissue revascularization in diabetes and that proangiogenic in situ response, not progenitor cell mobilization, is important for blood flow recovery. CONCLUSIONS: HO-1 is necessary for a proper proangiogenic function of BMDCs. A low level of HO-1 in hyperglycemic mice decreases restoration of perfusion in ischemic muscle, which can be rescued by a local injection of conditioned media from cultured BMDCs.

Transcriptome-based functional classifiers for direct immunotoxicity.

Current screening methods for direct immunotoxic chemicals are mainly based on general toxicity studies with rodents. The present study aimed to identify transcriptome-based functional classifiers that can eventually be exploited for the development of in vitro screening assays for direct immunotoxicity. To this end, a toxicogenomics approach was applied in which gene expression changes in human Jurkat lymphoblastic T cells were investigated in response to a wide range of compounds, including direct immunotoxicants, immunosuppressive drugs, and non-immunotoxic control chemicals. On the basis of DNA microarray data previously obtained by the exposure of Jurkat cells to 31 test compounds (Shao et al. in Toxicol Sci 135(2):328-346, 2013), we identified a set of 93 genes, of which 80 were significantly regulated (|numerical ratio| ≥1.62) by at least three compounds and the other 13 genes were significantly regulated by either one single compound or compound class. A total of 28 most differentially regulated genes were selected for qRT-PCR verification using a training set of 44 compounds consisting of the above-mentioned 31 compounds (23 immunotoxic and 8 non-immunotoxic) and 13 additional immunotoxicants. Good correlation between the results of microarray and qRT-PCR (Pearson's correlation, R ≥ 0.69) was found for 27 out of the 28 genes. Redundancy analysis of these 27 potential classifiers led to a final set of 25 genes. To assess the performance of these genes, Jurkat cells were exposed to 20 additional compounds (external verification set) followed by qRT-PCR. The classifier set of 25 genes gave a good performance in the external verification: accuracy 85 %, true positive rate (sensitivity) 88 %, and true negative rate (specificity) 67 %. Furthermore, on the basis of the gene ontology annotation of the 25 classifier genes, the immunotoxicants examined in this study could be categorized into distinct functional subclasses. In conclusion, we have identified and validated classifier genes that can be used for the development of an in vitro assay for the identification and initial characterization of hazards for direct immunotoxicity of chemicals and drugs. This assay promises to complement animal-free toxicity testing approaches within the field of direct immunotoxicity.

Toxicogenomics-based identification of mechanisms for direct immunotoxicity.

Compounds with direct immunotoxic properties, including metals, mycotoxins, agricultural pesticides, and industrial chemicals, form potential human health risks due to exposure through food, drinking water, and the environment. Insights into the mechanisms of action are currently lacking for the majority of these direct immunotoxicants. Therefore, the present work aimed to gain insights into the molecular mechanisms underlying direct immunotoxicity. To this end, we assessed in vitro the effects of 31 test compounds on the transcriptome of the human Jurkat T-cell line. These compounds included direct immunotoxicants, immunosuppressive drugs with different mode of actions, and nonimmunotoxic control chemicals. Pathway analysis of the microarray data allowed us to identify canonical pathways and Gene Ontology processes that were transcriptionally regulated in common by immunotoxicants (1) with structural similarities, such as tributyltin chloride and tributyltin oxide that activated the retinoic acid/X receptor signaling pathway and (2) without structural similarities, such as As2O3, dibutyltin chloride, diazinon, MeHg, ochratoxin A (OTA), S9-treated OTA, S9-treated cyclophosphamide, and S9-treated benzo[a]pyrene, which activated unfolded protein response, and FTY720, lindane, and propanil, which activated the cholesterol biosynthesis pathway. In addition, processes uniquely affected by individual immunotoxicants were identified, such as the induction of Notch receptor signaling and the downregulation of acute-phase response genes by OTA. These findings were validated by quantitative real-time PCR analysis of genes involved in these processes. Our study indicated that diverse modes of action are involved in direct immunotoxicity and that a set of pathways or genes, rather than one single gene, can be used to screen compounds for direct immunotoxicity.

Assessment of the usefulness of the murine cytotoxic T cell line CTLL-2 for immunotoxicity screening by transcriptomics.

A toxicogenomics approach was applied to assess the usefulness of the mouse cytotoxic T cell line CTLL-2 for in vitro immunotoxicity testing. CTLL-2 cells were exposed for 6 h to two model immunotoxic compounds: (1) the mycotoxin deoxynivalenol (DON, 1 and 2 µM), a ribotoxic stress inducer, and (2) the organotin compound tributyltin oxide (TBTO, 100 and 200 nM), an endoplasmic reticulum (ER) stress inducer. Effects on whole-genome mRNA expression were assessed by microarray analysis. The biological interpretation of the microarray data indicated that TBTO (200 nM) induced genes involved in T cell activation, ER stress, NFκB activation and apoptosis, which agreed very well with results obtained before on TBTO exposed Jurkat cells and mouse primary thymocytes. Remarkably, DON (2 µM) downregulated genes involved in T cell activation, ER stress and apoptosis, which is opposite to results obtained before for DON-exposed Jurkat cells and mouse primary thymocytes. Furthermore, the results for DON in CTLL-2 cells are also opposite to the results obtained for TBTO in CTLL-2 cells. In agreement with the lack of induction of ER stress and apoptosis, viability assays showed that CTLL-2 cells are much more resistant to the toxicity of DON than Jurkat cells and primary thymocytes. We propose that CTLL-2 cells lack the signal transduction that induces ER stress and apoptosis in response to ribotoxic stress. Based on the results for TBTO and DON, the CTLL-2 cell line does not yield an added value for immunotoxicity compared to the human Jurkat T cell line.

Galectin-2 expression is dependent on the rs7291467 polymorphism and acts as an inhibitor of arteriogenesis.

AIMS: In patients with obstructive coronary artery disease (CAD), the growth of collateral arteries, i.e. arteriogenesis, can preserve myocardial tissue perfusion and function. Monocytes modulate this process, supplying locally the necessary growth factors and degrading enzymes. Knowledge on factors involved in human arteriogenesis is scarce. Thus, the aim of the present study is to identify targets in monocytes that are critical for arteriogenesis in patients with CAD. METHODS AND RESULTS: A total of 50 patients with a chronic total coronary occlusion were dichotomized according to their collateral flow index. From each patient, RNA was isolated from unstimulated peripheral blood monocytes, monocytes stimulated by lipopolysaccharide (LPS) or interleukin (IL)-4, and from macrophages. Increased mRNA expression of galectin-2 was found in three out of four monocytic cell types of patients with a low capacity of the collateral circulation (P= 0.03 for unstimulated monocytes; P= 0.02 for LPS-stimulated monocytes; P= 0.20 for IL-4-stimulated monocytes; P= 0.02 for macrophages). Additionally, galectin-2 mRNA expression was significantly associated with the rs7291467 polymorphism in LGALS2 encoding galectin-2 in all four monocytic cell types. Patient with the rs7291467 CC genotype displayed highest galectin-2 expression, and also tended to have a lower arteriogenic response. To evaluate the effect of galectin-2 on arteriogenesis in vivo, we used a murine hindlimb model. Treatment with galectin-2 markedly impaired the perfusion restoration at Day 7. CONCLUSION: Collectively, these results identify galectin-2 as a novel inhibitor of arteriogenesis. Modulation of galectin-2 may constitute a new therapeutic strategy for the stimulation of arteriogenesis in patients with CAD.

Activin A induces a non-fibrotic phenotype in smooth muscle cells in contrast to TGF-ß.

AIMS: Activin A and transforming growth factor-ß1 (TGF-ß1) belong to the same family of growth and differentiation factors that modulate vascular lesion formation in distinct ways, which we wish to understand mechanistically. METHODS AND RESULTS: We investigated the expression of cell-surface receptors and activation of Smads in human vascular smooth muscle cells (SMCs) and demonstrated that activin receptor-like kinase-1 (ALK-1), ALK-4, ALK-5 and endoglin are expressed in human SMCs. As expected, TGF-ß1 activates Smad1 and Smad2 in these cells. Interestingly, activin A also induces phosphorylation of both Smads, which has not been reported for Smad1 before. Transcriptome analyses of activin A and TGF-ß1 treated SMCs with subsequent Gene-Set Enrichment Analyses revealed that many downstream gene networks are induced by both factors. However, the effect of activin A on expression kinetics of individual genes is less pronounced than for TGF-ß1, which is explained by a more rapid dephosphorylation of Smads and p38-MAPK in response to activin A. Substantial differences in expression of fibronectin, alpha-V integrin and total extracellular collagen synthesis were observed. CONCLUSIONS: Genome-wide mRNA expression analyses clarify the distinct modulation of vascular lesion formation by activin A and TGF-ß1, most significantly because activin A is non-fibrotic.

Blocking interferon {beta} stimulates vascular smooth muscle cell proliferation and arteriogenesis.

Increased interferon (IFN)-ß signaling in patients with insufficient coronary collateralization and an inhibitory effect of IFNß on collateral artery growth in mice have been reported. The mechanisms of IFNß-induced inhibition of arteriogenesis are unknown. In stimulated monocytes from patients with chronic total coronary artery occlusion and decreased arteriogenic response, whole genome expression analysis showed increased expression of IFNß-regulated genes. Immunohistochemically, the IFNß receptor was localized in the vascular media of murine collateral arteries. Treatment of vascular smooth muscle cells (VSMC) with IFNß resulted in an attenuated proliferation, cell-cycle arrest, and increased expression of cyclin-dependent kinase inhibitor-1A (p21). The growth inhibitory effect of IFNß was attenuated by inhibition of p21 by RNA interference. IFNß-treated THP1 monocytes showed enhanced apoptosis. Subsequently, we tested if collateral artery growth can be stimulated by inhibition of IFNß-signaling. RNA interference of the IFNß receptor-1 (IFNAR1) increased VSMC proliferation, cell cycle progression, and reduced p21 gene expression. IFNß signaling and FAS and TRAIL expression were attenuated in monocytes from IFNAR1(-/-) mice, indicating reduced monocyte apoptosis. Hindlimb perfusion restoration 1 week after femoral artery ligation was improved in IFNAR1(-/-) mice compared with wild-type mice as assessed by infusion of fluorescent microspheres. These results demonstrate that IFNß inhibits collateral artery growth and VSMC proliferation through p21-dependent cell cycle arrest and induction of monocyte apoptosis. Inhibition of IFNß stimulates VSMC proliferation and collateral artery growth.

Mycophenolate mofetil attenuates plaque inflammation in patients with symptomatic carotid artery stenosis.

Atherosclerosis as well as the subsequent progression towards cardiovascular events are considered to, at least partially, be a consequence of chronic inflammatory activity. Therefore, we decided to evaluate the impact of short-term immunosuppressive treatment on plaque characteristics in patients with symptomatic carotid artery stenosis. Twenty-one patients were randomized to receive either 1000 mg. Mycophenolate mofetil (MMF) BD or placebo for at least 2 weeks prior to undergoing carotid endarterectomy (CEA). The serial sections of the CEA specimens were immunostained for activated T-cells (CD3+CD69+), regulatory T-cells (CD3+FOXP3+) and macrophages (CD68). In addition, gene expression profiling was performed by Illumina gene-array. Immunostaining revealed a reduction of activated T-cells in nine MMF-treated patients compared to 11 placebo-treated control patients (19.7% vs. 28.1%; p<0.05) as well as an increase of regulatory T-cells (3.8% vs. 1.8%; p=0.05). Microarray analyses confirmed beneficial changes to plaque phenotype, showing reduced expression of pro-inflammatory genes. Significantly reduced expression of metalloproteinases and osteopontin was observed in three out of nine MMF-treated patients compared to nil out of 11 in the placebo group. In the present study we show that immunosuppressive treatment for two-and-a-half weeks prior to CEA elicits changes in the plaque phenotype of symptomatic patients. These changes include reduced inflammatory cell presence with a concomitant decrease in pro-inflammatory gene expression.

KLF2-induced actin shear fibers control both alignment to flow and JNK signaling in vascular endothelium.

The shear stress-induced transcription factor Krüppel-like factor 2 (KLF2) confers antiinflammatory properties to endothelial cells through the inhibition of activator protein 1, presumably by interfering with mitogen-activated protein kinase (MAPK) cascades. To gain insight into the regulation of these cascades by KLF2, we used antibody arrays in combination with time-course mRNA microarray analysis. No gross changes in MAPKs were detected; rather, phosphorylation of actin cytoskeleton-associated proteins, including focal adhesion kinase, was markedly repressed by KLF2. Furthermore, we demonstrate that KLF2-mediated inhibition of Jun NH(2)-terminal kinase (JNK) and its downstream targets ATF2/c-Jun is dependent on the cytoskeleton. Specifically, KLF2 directs the formation of typical short basal actin filaments, termed shear fibers by us, which are distinct from thrombin- or tumor necrosis factor-alpha-induced stress fibers. KLF2 is shown to be essential for shear stress-induced cell alignment, concomitant shear fiber assembly, and inhibition of JNK signaling. These findings link the specific effects of shear-induced KLF2 on endothelial morphology to the suppression of JNK MAPK signaling in vascular homeostasis via novel actin shear fibers.

Nrf2-dependent and -independent responses to nitro-fatty acids in human endothelial cells: identification of heat shock response as the major pathway activated by nitro-oleic acid.

Electrophilic fatty acid derivatives, including nitrolinoleic acid and nitro-oleic acid (OA-NO(2)), can mediate anti-inflammatory and pro-survival signaling reactions. The transcription factor Nrf2, activated by electrophilic fatty acids, suppresses redox-sensitive pro-inflammatory gene expression and protects against vascular endothelial oxidative injury. It was therefore postulated that activation of Nrf2 by OA-NO(2) accounts in part for its anti-inflammatory actions, motivating the characterization of Nrf2-dependent and -independent effects of OA-NO(2) on gene expression using genome-wide transcriptional profiling. Control and Nrf2-small interfering RNA-transfected human endothelial cells were treated with vehicle, oleic acid, or OA-NO(2), and differential gene expression profiles were determined. Although OA-NO(2) significantly induced the expression of Nrf2-dependent genes, including heme oxygenase-1 and glutamate-cysteine ligase modifier subunit, the majority of OA-NO(2)-regulated genes were regulated by Nrf2-independent pathways. Moreover, gene set enrichment analysis revealed that the heat shock response is the major pathway activated by OA-NO(2), with robust induction of a number of heat shock genes regulated by the heat shock transcription factor. Inasmuch as the heat shock response mediates anti-inflammatory and cytoprotective actions, this mechanism is proposed to contribute to the protective cell signaling functions of nitro-fatty acids and other electrophilic fatty acid derivatives.

Actin-binding rho activating protein (Abra) is essential for fluid shear stress-induced arteriogenesis.

OBJECTIVE: Arteriogenesis, the development of a collateral circulation, is important for tissue survival but remains functionally defective because of early normalization of fluid shear stress (FSS). Using a surgical model of chronically elevated FSS we showed that rabbits exhibited normal blood flow reserve after femoral artery ligature (FAL). Inhibition of the Rho pathway by Fasudil completely blocked the beneficial effect of FSS. In a genome-wide gene profiling we identified actin-binding Rho activating protein (Abra), which was highly upregulated in growing collaterals. METHODS AND RESULTS: qRT-PCR and Western blot confirmed highly increased FSS-dependent expression of Abra in growing collaterals. NO blockage by L-NAME abolished FSS-generated Abra expression as well as the whole arteriogenic process. Cell culture studies demonstrated an Abra-triggered proliferation of smooth muscle cells through a mechanism that requires Rho signaling. Local intracollateral adenoviral overexpression of Abra improved collateral conductance by 60% in rabbits compared to the natural response after FAL. In contrast, targeted deletion of Abra in CL57BL/6 mice led to impaired arteriogenesis. CONCLUSIONS: FSS-induced Abra expression during arteriogenesis is triggered by NO and leads to stimulation of collateral growth by smooth muscle cell proliferation.

Endothelial CD81 is a marker of early human atherosclerotic plaques and facilitates monocyte adhesion.

AIMS: In a recent report, we established at the genome-wide level those genes that are specifically upregulated in the endothelium of atherosclerotic plaques in human arteries. As the transcriptome data revealed that mRNA for the tetraspanin family member CD81 is significantly and specifically upregulated in the endothelium overlying early atheroma, we set out to validate these results on the protein level, and investigate the functional consequences of CD81 upregulation. METHODS AND RESULTS: Immunohistochemical analysis in an independent set of donor arteries verified in the endothelium of early human atherosclerotic lesions the enhanced expression of CD81, which appears oxidative stress-dependent. Using lentiviral overexpression and silencing in human umbilical endothelial cells, we established in an in vitro flow adhesion assay that elevated endothelial CD81 is associated with increased monocyte adhesion to non-activated CD81-transduced endothelial cells, approaching the levels normally only attained after tumour necrosis factor alpha stimulation. The CD81 effect was dependent on both intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as it was abolished in the presence of a mixture of anti-ICAM-1 and anti-VCAM-1 antibodies. Flow cytometry revealed that increased CD81 levels did not increase total ICAM-1 and VCAM-1 surface expression. Instead, it concentrated the available adhesion molecules into membrane clusters, as indicated by confocal and electron microscopy. CD81 also colocalized with ICAM-1 and VCAM-1 in the adhesion rings around bound monocytes. CONCLUSION: Endothelial CD81 upregulated in early human atheroma has the potential to play a crucial role in the initial stages of atherosclerotic plaque formation by increasing monocyte adhesion prior to the full-blown inflammatory response.

Suppression of inflammatory signaling in monocytes from patients with coronary artery disease.

Monocytes and T-cells play an important role in the development of atherosclerotic coronary artery disease (CAD). Transcriptome analysis of circulating mononuclear cells from carefully matched atherosclerotic and control patients will potentially provide insights into the pathophysiology of atherosclerosis and supply biomarkers for diagnostic purposes. From patients undergoing coronary angiography because of anginal symptoms, we carefully matched 18 patients with severe triple-vessel CAD to 13 control patients without angiographic signs of CAD. All patients were on statin and aspirin treatment. Elevated soluble-ICAM levels demonstrated increased vascular inflammation in atherosclerotic patients. RNA from circulating CD4+ T-cells, CD14+ monocytes, lipopolysaccharide-stimulated monocytes, and macrophages was subjected to genome-wide expression analysis. In CD14+ monocytes, few inflammatory genes were overexpressed in control patients, while atherosclerotic patients showed overexpression of a group of Krüppel-associated box - containing transcription factors involved in negative regulation of gene expression. These differences disappeared upon LPS-stimulation or differentiation towards macrophages. No consistent changes in T cell transcriptomes were detected. Large inter-individual variability prevented the use of single differentially expressed genes as biomarkers, while monocyte gene expression signature predicted patient status with an accuracy of 84%. In this comprehensive analysis of circulating cell transcriptomes in atherosclerotic CAD, cautious patient matching revealed only small differences in transcriptional activity in different mononuclear cell types. Only an indication of a negative feedback to inflammatory gene expression was detected in atherosclerotic patients. Transcriptome differences of circulating cells possibly play less of a role than hitherto thought in the individual patient's susceptibility to atherosclerotic CAD, when appropriately matched for clinical symptoms and medication taken.

Preferential campesterol incorporation into various tissues in apolipoprotein E*3-Leiden mice consuming plant sterols or stanols.

Intestinal absorption of plant sterols and stanols is much lower as compared with that of cholesterol; and therefore, serum concentrations are low. Circulating plant sterols and stanols are incorporated into tissues. However, hardly any data are available about tissue distributions of individual plant sterols and stanols, particularly in relation to their serum concentrations. We therefore fed female apolipoprotein E*3-Leiden mice a control diet, a plant sterol-enriched diet (1g/100 g diet), or a plant stanol-enriched diet (1g/100 g diet) for 8 weeks. In the sterol group, serum cholesterol-standardized campesterol and sitosterol concentrations were, respectively, 8 and 7 times higher as compared with those in the control group. Consequently, the serum campesterol-sitosterol ratio remained essentially unchanged. Cholesterol-standardized plant sterol concentrations increased significantly in all analyzed tissues, except brain. However, the campesterol-sitosterol ratio also increased in all tissues (except in liver and spleen), suggesting that campesterol is preferentially incorporated over sitosterol in those tissues. For the stanol group, serum plant stanol concentrations also increased; but the increase was but less pronounced. We conclude that, in apolipoprotein E*3-Leiden mice, campesterol is preferentially incorporated into most tissues over sitosterol, which cannot be deduced from changes in serum concentrations.

KLF2 primes the antioxidant transcription factor Nrf2 for activation in endothelial cells.

OBJECTIVE: Atheroprotective blood flow induces expression of anti-inflammatory Krüppel-like factor 2 (KLF2) and activates antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in vascular endothelium. Previously, we obtained KLF2-induced gene expression profiles in ECs, containing several Nrf2 target genes. Our aim was to investigate the role of KLF2 in shear stress-mediated activation of Nrf2 in human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: Expression of Nrf2 and its targets NAD(P)H dehydrogenase quinone 1 (NQO1) and heme oxygenase (HO-1) was elevated by shear and KLF2. KLF2 knockdown showed that shear-induced expression of NQO1 but not Nrf2 was dependent on KLF2. KLF2 overexpression in absence of flow resulted in more efficient activation of Nrf2 by tert-butyl hydroquinone (tBHQ) through enhanced nuclear localization, and promoted expression of a large panel of Nrf2-dependent genes resulting in superior protection against oxidative stress. Comparison of shear-, KLF2-, and Nrf2-induced transcriptomes showed that the majority of shear-modulated gene sets is influenced by KLF2 or Nrf2. CONCLUSIONS: We report that KLF2 substantially enhances antioxidant activity of Nrf2 by increasing its nuclear localization and activation. The synergistic activity of these two transcription factors forms a major contribution to the shear stress-elicited transcriptome in endothelial cells.

SOX-18 controls endothelial-specific claudin-5 gene expression and barrier function.

Members of the claudin family constitute tight junction strands and are major determinants in specificity and selectivity of paracellular barriers. Transcriptional control of claudin gene expression is essential to establish individual claudin expression patterns and barrier properties. Using full genome expression profiling, we now identify sex-determining region Y-box (SOX)-18, a member of the SOX family of high-mobility group box transcription factors, as one of the most differentially induced genes during establishment of the endothelial barrier. We show that overexpression of SOX-18 and a dominant-negative mutant thereof, as well as SOX-18 silencing, greatly affect levels of claudin-5 (CLDN5). The relevance of an evolutionary conserved SOX-binding site in the CLDN5 promoter is shown using sequential promoter deletions, as well as point mutations. Furthermore, SOX-18 silencing abrogates endothelial barrier function, as measured by electric cell-substrate impedance sensing. Thus an obligatory role for SOX-18 in the regulation of CLDN5 gene expression in an endothelial-specific and cell density-dependent manner is established, as well as a crucial, nonredundant role for specifically SOX-18 in the formation of the endothelial barrier.