IL-6 and IL-8 content of KLF2-transduced BOECs.

<p>(A) Immunofluorescence image showing co-localization of IL-6 (green) and VWF (red) in IL-1β-treated KLF2- and mock-transduced BOECs. Nuclei were visualized with DAPI (blue). Scale bars: 10 µm. (B) Western blot analysis for VWF, KLF2, IL-8 and IL-6 expression in lysates of mock- and KLF2-transduced BOECs; α-tubulin was shown as a loading control. (C) Immunofluorescence image showing co-localization of IL-8 (green) and VWF (red) in IL-1β-treated KLF2- and mock-transduced BOECs. Nuclei were visualized with DAPI (blue). Scale bars: 10 µm. (D) Release of VWF from PMA-stimulated KLF2 (black bars)- and mock (white bars)-transduced cells (IL-1β-treated), measured by determining the concentration of VWF in the conditioned medium by ELISA. **P<0.001; ***P<0.0001 by Students t-test (E-F) Release of IL-6 and IL-8 from PMA-stimulated KLF2 (black bars)- and mock (white bars)-transduced cells (IL-1β-treated), measured by determining the concentration of IL-6 and IL-8 in the conditioned medium by ELISA. The amount of IL-6 released without stimulation was slightly reduced in KLF2 expressing cells when compared to mock-transduced cells. NS: non-significant; *P<0.01; ***P<0.0001 by Students t-test.</p

OPG content of mock- or KLF2-transduced BOECs.

<p>(A) Immunofluorescence image showing co-localization of OPG (green) and VWF (red) in both mock- and KLF2-tranduced BOECs. Nuclei were stained using DAPI (blue). Scale bars: 10 µm.(B) Western blot analysis for VWF, KLF2, IL-8 and IL-6 expression in lysates of mock- and KLF2-transduced BOECs; α-tubulin was shown as a loading control.</p

AQP1 immunohistochemistry in vascular tissue specimen showing different stages of atherosclerosis.

<p>Overviews, showing immunohistochemistry for macrophages (HAM56, left column), are given for arteries without lesions (A, external iliac artery), with focal lesions of the initial stage (B, abdominal aorta, intimal xanthoma/fatty streak) or the advanced stage (C, common iliac artery, fibro-calcific plaque with signs of rupture). Rectangles within the HAM56 overview indicate the position of areas that are shown as magnification of serial sections stained for ICAM-1 (middle column) and AQP1 (right column). In addition, these sections/areas were further characterized with regard to their cellular composition by staining for macrophages (HAM56) and smooth muscle cells (anti α-actin) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145777#pone.0145777.s003" target="_blank">S3</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145777#pone.0145777.s005" target="_blank">S5</a> Figs).</p

KLF2 dependency of AQP1 expression.

<p>(A, B) Expression of KLF2 was induced in HUVEC by laminar flow (shear), lentiviral transduction (KLF2) or incubation with atorvastatin (statin). mRNA levels were determined by semi-quantitative RT-PCR for AQP1 (A) and eNOS (B), normalized to P0 and shown as mean and SEM of the fold induction (grey) relative to the corresponding control (white). The following conditions are depicted: shear; exposure to laminar shear stress for ≥ 4 days at an average of 18 dyne/cm², induction relative to static control (N = 6), KLF2; transduction with a lentiviral vector carrying KLF2 under control of the PGK promoter and subsequent growth for ≥ 4 days, induction relative to mock transduced cells (N = 7), statin; incubation with atorvastatin at a final concentration of 10 μM during 24 hours, induction relative to vehicle control (N = 5). *P<0.05, **P<0.01. (C) HUVECs were transduced with a lentiviral vector encoding an shRNA targeting KLF2. 24 hours later, expression of KLF2 was induced by incubation with atorvastatin at a final concentration of 10 μM during 24 hours. mRNA levels were determined for KLF2, AQP1 and eNOS, normalized to P0 and shown as mean and SEM mRNA level (grey) relative to control cells transduced with a non-targeting construct (white)(N = 3). *P<0.05, **P<0.01.</p

Proteomic Screen Identifies IGFBP7 as a Novel Component of Endothelial Cell-Specific Weibel-Palade Bodies

Vascular endothelial cells contain unique storage organelles, designated Weibel-Palade bodies (WPBs), that deliver inflammatory and hemostatic mediators to the vascular lumen in response to agonists like thrombin and vasopressin. The main component of WPBs is von Willebrand factor (VWF), a multimeric glycoprotein crucial for platelet plug formation. In addition to VWF, several other components are known to be stored in WPBs, like osteoprotegerin, monocyte chemoattractant protein-1 and angiopoetin-2 (Ang-2). Here, we used an unbiased proteomics approach to identify additional residents of WPBs. Mass spectrometry analysis of purified WPBs revealed the presence of several known components such as VWF, Ang-2, and P-selectin. Thirty-five novel candidate WPB residents were identified that included insulin-like growth factor binding protein-7 (IGFBP7), which has been proposed to regulate angiogenesis. Immunocytochemistry revealed that IGFBP7 is a bona fide WPB component. Cotransfection studies showed that IGFBP7 trafficked to pseudo-WPB in HEK293 cells. Using a series of deletion variants of VWF, we showed that targeting of IGFBP7 to pseudo-WPBs was dependent on the carboxy-terminal D4-C1-C2-C3-CK domains of VWF. IGFBP7 remained attached to ultralarge VWF strings released upon exocytosis of WPBs under flow. The presence of IGFBP7 in WPBs highlights the role of this subcellular compartment in regulation of angiogenesis

Galectin-2 Induces a Proinflammatory, Anti-Arteriogenic Phenotype in Monocytes and Macrophages

<div><p>Galectin-2 is a monocyte-expressed carbohydrate-binding lectin, for which increased expression is genetically determined and associated with decreased collateral arteriogenesis in obstructive coronary artery disease patients. The inhibiting effect of galectin-2 on arteriogenesis was confirmed <i>in vivo</i>, but the mechanism is largely unknown. In this study we aimed to explore the effects of galectin-2 on monocyte/macrophage phenotype <i>in vitro</i> and <i>vivo</i>, and to identify the receptor by which galectin-2 exerts these effects. We now show that the binding of galectin-2 to different circulating human monocyte subsets is dependent on monocyte surface expression levels of CD14. The high affinity binding is blocked by an anti-CD14 antibody but not by carbohydrates, indicating a specific protein-protein interaction. Galectin-2 binding to human monocytes modulated their transcriptome by inducing proinflammatory cytokines and inhibiting pro-arteriogenic factors, while attenuating monocyte migration. Using specific knock-out mice, we show that galectin-2 acts through the CD14/toll-like receptor (TLR)-4 pathway. Furthermore, galectin-2 skews human macrophages to a M1-like proinflammatory phenotype, characterized by a reduced motility and expression of an anti-arteriogenic cytokine/growth factor repertoire. This is accompanied by a switch in surface protein expression to CD40-high and CD206-low (M1). In a murine model we show that galectin-2 administration, known to attenuate arteriogenesis, leads to increased numbers of CD40-positive (M1) and reduced numbers of CD206-positive (M2) macrophages surrounding actively remodeling collateral arteries. In conclusion galectin-2 is the first endogenous CD14/TLR4 ligand that induces a proinflammatory, non-arteriogenic phenotype in monocytes/macrophages. Interference with CD14-Galectin-2 interaction may provide a new intervention strategy to stimulate growth of collateral arteries in genetically compromised cardiovascular patients.</p></div

Human galectin-2 affects migration of monocytes and drives M1-type polarization of macrophages.

<p>(A) Spontaneous migration of human monocytes across fibronectin-coated Transwell inserts was assessed in the absence or presence of rh-gal-2. Representative pictures are shown of migrated cells after 24h. Quantified migrated cell counts represent the mean ± SEM from at least 3 independent experiments. *<i>P</i> < 0.05. (B) Human monocyte-derived differentiated macrophage subtypes (M0, M1 and M2) were stimulated at day 7 with either vehicle (control) or rh-gal-2 for 24 hours followed by actin staining (TRITC-labeled phalloidin). Representative images from three independent experiments are shown. A scale bar (25um) indicates the size of the cells, which become elongated (M1-like) in the presence of rh-gal-2. (C) Human monocyte-derived macrophage subtypes were stimulated at day 7 with either vehicle (control) or rh-gal-2. Motility is presented as mean traveled distance ± SEM, from at least 3 independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Galectin-2 binds CD14 and mimics LPS.

<p>(A) Human monocytes were incubated with biotinylated rh-gal-2 or—rh-gal-1. Binding of galectins (expressed as MFI) was analyzed by flow cytometry in the presence or absence of neutralizing anti-human CD14 antibody (100ug/ml). (B) IFN-β gene expression was measured in human monocytes by real-time PCR after treatment with TLR4 ligand LPS (10 ng/ml), TLR2 ligand PGN (1 μg/ml), rh-gal-2 (10 μg/ml), or combinations for three hours. Expression levels were compared of untreated vs. h-gal-2, LPS, and PGN. LPS vs. LPS + h-gal-2, and PGN vs. PGN + h-gal-2. Gene expression is expressed relative to untreated sample (set at 1). *<i>P</i> <0.05, **<i>P</i> < 0.01. Results in panel A and B are presented as mean ± SEM from at least 3 independent experiments. (C) Whole blood from WT, TLR4-/-, or CD14-/- mice was stimulated with rm-gal-2,or LPS in the absence or presence of polymyxin B (PMB). TNF-α protein levels were measured in supernatants and compared between wt and CD14- or TLR4-deficient cells stimulated as indicated. Within the mouse strains the effect of PMB was evaluated. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001, wt vs. knockout. '''<i>P</i> < 0.001, LPS vs. LPS + PMB. (D) Human monocytes were incubated with rh-gal-2 (10 μg/ml) or LPS (as indicated) for three hours in the presence or absence of neutralizing anti-human CD14 antibody (20 μg/ml).IFN-β gene expression was determined by real-time PCR. Results represent the mean expression ± SD of one experiment measured in triplicate. ***<i>P</i> < 0.001, ****<i>P</i> < 0.0001</p

Binding analysis of recombinant human galectin-2 to monocytes and macrophages.

<p>Cells were incubated with 10 μg/ml biotinylated recombinant human galectin-2 (rh-gal-2). (A) The effect of lactose (20mM) and thiodigalactoside (20mM) on rh-gal-2 binding to monocytes. (B) Binding (expressed as MFI) of human galectin-2 to human monocyte subsets i.e. non-classical (gate R1; CD14+CD16+), intermediate (gate R2; CD14++/CD16+), and classical (gate R3; CD14++CD16-). Results are presented as mean ± SEM from at least 3 independent experiments. * <i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001, untreated vs. h-gal-2. (C) Correlation of rh-gal-2 binding with CD14 expression. (D) Binding of rh-gal-2 to human monocyte-derived M0 macrophages and mouse macrophages (RAW264.7). Representative histograms from three independent experiments are depicted in all three panels.</p