Pharmacogenomics of Interferon-ß Therapy in Multiple Sclerosis: Baseline IFN Signature Determines Pharmacological Differences between Patients

Multiple sclerosis (MS) is a heterogeneous disease. In order to understand the partial responsiveness to IFNbeta in Relapsing Remitting MS (RRMS) we studied the pharmacological effects of IFNbeta therapy. Large scale gene expression profiling was performed on peripheral blood of 16 RRMS patients at baseline and one month after the start of IFNbeta therapy. Differential gene expression was analyzed by Significance Analysis of Microarrays. Subsequent expression analyses on specific genes were performed after three and six months of treatment. Peripheral blood mononuclear cells (PBMC) were isolated and stimulated in vitro with IFNbeta. Genes of interest were measured and validated by quantitative realtime PCR. An independent group of 30 RRMS patients was used for validation. Pharmacogenomics revealed a marked variation in the pharmacological response to IFNbeta between patients. A total of 126 genes were upregulated in a subset of patients whereas in other patients these genes were downregulated or unchanged after one month of IFNbeta therapy. Most interestingly, we observed that the extent of the pharmacological response correlates negatively with the baseline expression of a specific set of 15 IFN response genes (R = -0.7208; p = 0.0016). The negative correlation was maintained after three (R = -0.7363; p = 0.0027) and six (R = -0.8154; p = 0.0004) months of treatment, as determined by gene expression levels of the most significant correlating gene. Similar results were obtained in an independent group of patients (n = 30; R = -0.4719; p = 0.0085). Moreover, the ex vivo results could be confirmed by in vitro stimulation of purified PBMCs at baseline with IFNbeta indicating that differential responsiveness to IFNbeta is an intrinsic feature of peripheral blood cells at baseline. These data imply that the expression levels of IFN response genes in the peripheral blood of MS patients prior to treatment could serve a role as biomarker for the differential clinical response to IFNbet

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

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

The shear stress-induced transcription factor Kruppel-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 NH2-terminal kinase (JNK) and its downstream targetsATF2/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 thrombinor 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. (Blood. 2010; 115: 2533-2542

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 Kruppel-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 Tcell 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. (C) 2008 Elsevier Inc. All rights reserve

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

AQP1 is preferentially expressed in endothelium overlying plaque-free intimae and is induced by KLF2.

<p>(A, B) Heatmaps of the top 25 ranking genes as determined by GSEA. (A) Gene expression in endothelium overlying plaque-free (PF) intimae from human large arteries is compared to gene expression in endothelium from early- and advanced (ADV) lesions. Data set from Volger et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145777#pone.0145777.ref002" target="_blank">2</a>]. Aquaporin 1 (AQP1) is identified as the highest ranking membrane-expressed gene in plaque-free lesions (green rectangle). (B) Time courses (24, 48 and 72 hours) of gene expression in mock-transduced HUVEC (control, c) and KLF2-transduced HUVEC are compared. Data set from Boon et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145777#pone.0145777.ref007" target="_blank">7</a>]. The position of AQP1 is indicated by a green rectangle. (C) Correlation of KLF2—and AQP1 transcript levels during time courses (24, 48 and 72 hours) of gene expression in mock- and KLF2- transduced HUVECs (same data set as used for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145777#pone.0145777.g001" target="_blank">Fig 1B</a>).</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

Suppression of AQP1 transcription by TNF-α is restrained by KLF2.

<p>HUVECs were transfected with either empty lentiviral vector (mock) or with a lentiviral vector carrying KLF2 under control of the PGK promoter (KLF2) and two days after transfection incubated with vehicle (ctr) or TNF-α at a concentration of 20 ng/ml during 24 hours (TNF). mRNA levels were determined for AQP1, normalized to P0 and expressed as mean and SEM (N = 4). *P<0.05, **P<0.01.</p

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