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The adipokine visfatin induces tissue factor expression in human coronary artery endothelial cells
Thrombosis Research 130 (2012) 403–408
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Regular Article
The adipokine visfatin induces tissue factor expression in human coronary artery endothelial cells Another piece in the adipokines puzzle Plinio Cirillo a,⁎, Vito Di Palma a, Fabio Maresca a, Francesco Pacifico b, Francesca Ziviello a, Michele Bevilacqua a, Bruno Trimarco a, Antonio Leonardi c, Massimo Chiariello a a b c
Department of Clinical Medicine, Cardiovascular and Immunological Science (Division of Cardiology) Istituto di Endocrinologia e Oncologia Sperimentale, CNR Dipartimento di Biologia e Patologia Cellulare e Molecolare, University of Naples “Federico II”
a r t i c l e
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Article history: Received 8 March 2012 Accepted 4 June 2012 Available online 21 June 2012 Keywords: Adipokines Atherothrombosis Tissue Factor Visfatin
a b s t r a c t Introduction: Adipocytes are nowadays recognized as cells able to produce and secrete a large variety of active substances with direct effects on vascular cells, known as adipokines. Visfatin is a recently identified adipokine not yet completely characterized for its pathophysiological role in cardiovascular disease. Increased levels of visfatin are measurable in the plasma of patients with coronary artery disease and specifically in those with acute coronary syndromes (ACS). Several studies have indicated that Tissue Factor (TF) plays a pivotal role in the pathophysiology of ACS by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates the effects of visfatin on TF in human coronary endothelial cells (HCAECs). Methods: HCAECs were stimulated with visfatin in a concentration range usually measurable in plasma of patients with ACS and than processed to evaluate TF-mRNA levels as well as TF expression/activity. Finally, the role of NF-κB pathway was investigated. Results: We demonstrate that visfatin induces transcription of mRNA for TF by Real Time PCR. In addition, we show that this adipokine promotes surface expression of TF that is functionally active since we measured increased procoagulant activity. Visfatin effects on TF appear modulated by the activation of the transcription factor, NF-κB, since NF-κB inhibitors suppressed TF expression. Finally, we show that the nicotinamide phopsphoribosyltransferase enzymatic activity of visfatin seems to play a pivotal role in modulating the NF-κB driven regulation of TF. Discussion: Data of the present study, although in vitro, indicate that visfatin, at doses measurable in ACS patient plasma, induces a procoagulant phenotype in human coronary endothelial cells by promoting TF expression. These observations support the hypothesis that this adipokine might play a relevant role as an active partaker in athero-thrombotic disease. © 2012 Elsevier Ltd. All rights reserved.
Introduction Obesity, a disease in which adipose tissue is largely represented, is actually considered as strong cardiovascular risk factor [1,2]. Thus, although it is established that increase of visceral fat is causally involved in the development of cardiovascular disease, the possible link between these entities is not completely understood yet. Indeed, the current point of view about the adipose tissue is actually changed, indicating that adipocytes have no longer to be considered only storage cells for fat, but they have to be now recognized as cells able to produce and secrete a large variety of active substances named adipokines. Several ⁎ Corresponding author at: Division of Cardiology, University of Naples “Federico II”, Via Sergio Pansini 5, 80131 Naples, Italy. Tel./fax: + 39 081 7462235. E-mail address: [email protected] (P. Cirillo). 0049-3848/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2012.06.007
adipokines have been extensively characterized and are considered of particular interest in cardiovascular pathophysiology [3]. Conversely, other novel adipokines, such as visfatin, have been poorly investigated. Interestingly, elevated plasma levels of visfatin have been measured in plasma of patients with acute coronary syndromes compared with controls [4]. Thus, it has been proposed that this adipokine, might be a marker of increased cardiovascular risk, specially in patients with metabolic syndrome [5]. Increasing evidences have assigned a central role to thrombosis in the progression and complication of atherosclerosis and have linked it to the clinical occurrence of acute coronary syndromes [6-8]. Several studies have clearly indicated that tissue factor (TF), the key initiator of extrinsic coagulation pathway, plays a pivotal role in the pathophysiology of acute coronary syndromes by triggering the formation of intracoronary thrombi following endothelial injury [9-12].
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In the present study, we provide support, in vitro, for the hypothesis that visfatin is not only a marker of cardiovascular risk, but it might effectively play an active role in pathophysiology of coronary acute events by promoting a pro-thrombotic state in human coronary artery endothelial cells (HCAECs) through induction of TF. Furthermore, we investigated the potential involvement of the NF-κB intracellular pathway in this phenomenon. Methods HCAECs purchased from Lonza (Basel, Switzerland) were grown in EGM 2 medium with 10% FBS and used at passages 2 to 5. To investigate the visfatin effects on TF expression and activity, cells were enzimatically harvested and counted in a haemocytometer and sub cultured in 24-well plates at an initial density of about 5 × 10 4 cell/ well, while at confluence, cell density was of about 8.5 × 10 4 cell/ well. For other set of experiments, cells were grown in 100 mm cell plates and, at confluence, cell density was of about 2 × 10 6 cells per plate. At confluence, cell were starved in serum-free medium for 24 hours and then used in the different set of experiments. Visfatin (Sigma Chemical Co., St Louis, USA) used in all studies was analyzed founding endotoxin level to be b0.125 EU/mL (b12.5 pg/mL) by Limulus assay (Bio Whittaker, Walkersville, USA). All media, and water were also tested and endotoxin level found to be b0.125 EU/mL. In addition, in order to avoid a possible bias from contaminating LPS, all experiments were then repeated in the presence of Polymyxin B (100 μg/ml). Effects of Visfatin on TF-mRNA Levels The dose–response effects of visfatin on TF-mRNA transcription were preliminary investigated by semi-quantitative polymerase chain reaction. Then, the effect of visfatin on TF-mRNA was investigated by real-time reverse transcription analysis as previously described [13]. Briefly, HCAECs were incubated with visfatin (20 ng/ml, concentration chosen on the basis of semi-quantitative results) for 30 minutes. Then, cells were washed with PBS and then fresh medium (EGM 2 containing 0,1% serum) was added. Total mRNA was extracted by cell cultures using TRIzol reagent (GIBCO, Carlsbad, USA), at baseline, 30, 60 and 120 min after visfatin stimulation and TF mRNA levels were examined by real-time reverse transcription (RT) and polymerase chain reaction (PCR) by LightCycler (Roche Diagnostics, Basel, Switzerland). In positive control experiments, HCAECs were incubated with LPS (50 μg/ml, Sigma Chemical Co., St Louis, USA), for 30 min and then mRNA was extracted at 60 min. Total mRNA was extracted from cell cultures using TRIzol reagent (GIBCO, Carlsbad, USA), according to the manufacturer's instructions. Reverse transcription was performed using mMLV (GIBCO, Carlsbad, USA) and 100 ng of the RNA samples from each culture condition. Samples were run in triplicate in 50 μl reactions by using an ABI PRISM 5700 sequence detector system (Applied Bio systems, Foster City, USA). Samples were incubated at 50 °C for 2 min, 95 °C for 10 min and then underwent 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Specific oligonucleotides for human GAPDH and human TF were designed on the basis of published sequences using PRIMER EXPRESS Software (Applied Bio systems) and validated for their specificity. SYBR-green chemistry was used to detect fluorescence and an internal standard (Applied Bio systems, Foster City, USA) was used for quantization of the message. The results were analyzed using a comparative method, and the values were normalized to the GAPDH expression and converted into percentage change. Three different experiments were performed for each experimental condition. Effects of Visfatin on TF surface Expression and Activity HCAECs cultivated as above were washed with PBS and then incubated with increasing concentrations of visfatin (5, 10, 20 or 40 ng/ml) for 6 hours.
TF expression on cell surface was investigated with FACS analysis. After incubation, endothelial cells were detached with 10 mmol/L EDTA in PBS (without trypsin) and stained with FITClabelled monoclonal antibody (Pharmingen, Franklin Lakes, USA) against TF, or with the appropriate isotype IgG (phycoerythrin or FITC) as control. Fluorescence intensity of 9000 cells for each sample was quantified by a FACSCalibur analyzer (Becton-Dickinson, Franklin Lakes, USA). To evaluate whether visfatin-induced TF was functionally active, TF procoagulant activity was determined by a two-step colorimetric assay, based on the ability of TF to promote generation of coagulation FXa, as previously described [13]. Briefly, after stimulation with visfatin, cells were incubated with 1 nM of recombinant human FVIIa (Novo Nordisk A/S Gentofte, Denmark), followed by 100 nM of purified human factor X (Calbiochem-Novobiochem, La Jolla, CA, USA) and 5 mM CaCl2 for 15 min at 37 °C. A chromogenic substrate, specific for factor X (Cromozym X, Roche Diagnostics, Mannheim, Germany, 0.5 mmol/l) was then added and incubated for 30 min at 37 °C. The reaction was stopped by adding 200 μl/ml of sample of a 30% solution of acetic acid and the change in optical density at 405 nm was quantified with a spectrophotometer. Purified factor Xa of known concentration (Sigma Chemical Co., St Louis, USA) allowed generation of calibration curves. To evaluate whether visfatin-induced expression of TF resulted from de novo synthesis, in another set of experiments, HCAECs were pre-incubated for 120 minutes with cycloheximide (10 μg/ ml), an inhibitor of protein synthesis, or with 5, 6-dichloro-1-β-dribofuranosylbenzimidazole (DRB, 10 μg/ml), an inhibitor of DNA transcription, before adding visfatin (20 ng/ml). In additional control experiments cells were pre-incubated for 10 minutes with a mouse monoclonal antibody against human TF (5 μg/ml, American Diagnostica Inc, Greenwich, CT, USA). Positive control experiments included cells incubated for 6 hours with LPS (50 μg/ml). Six different experiments were performed for each experimental condition. Since it has been demonstrated that effects of visfatin may be mediated by its intrinsic nicotinamide phosphoribosyltransferase (NAMPT) enzymatic activity [14], in an additional set of experiments we have investigated whether nicotine-amide mononucleotide (NMN), the product of NAMPT activity, was able to mimic the effects of this adipokine on TF, in HCAECs. Thus, cells were stimulated with NMN (100 μM), and then TF expression as well as TF procoagulant activity were determined as described above. Moreover, in control experiments, HCAECs were incubated with the NAMPT inhibitor FK866 (10 nmol/L) for 1 hour before stimulating cells with visfatin (20 ng/ml).
Effects of Visfatin on NF-κB Activation To investigate whether expression of TF induced by visfatin involved activation of the NF-κB pathway, we performed luciferase assays. HCAEC cells (3 × 10 5) were transfected with 1.5 μg of the Ig-κB-luciferase reporter gene plasmid, containing NF-κB-binding sites of immunoglobulin promoter region, and, 24 h after transfection, were treated with increasing amounts of visfatin (5, 10, 20 or 40 ng/ml). After 3 h of incubation at 37 °C, cell extracts were prepared and reporter gene activity was determined by the luciferase system (Promega). A pRSV-β-galactosidase vector (0.5 μg) was used to normalize for transfection efficiencies.
Statistical Analysis Data are presented as mean ± SD. Differences between groups were determined by a one-way ANOVA followed by a Student's t test with Bonferroni's correction. A p value b 0.05 was considered statistically significant.
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Results Effects of Visfatin on TF MRNA Levels Visfatin effects of TF-mRNA transcription were investigated by real-time reverse transcription analysis. TF mRNA levels were very low in unstimulated HCAECs, as expected [12]. Visfatin (20 ng/ml), caused a time-dependent increase in TF mRNA levels, as compared to unstimulated cells. The peak of TF mRNA levels was observed at 60 min, while at 120 min, TF mRNA levels started decreasing (Fig. 1). In experiments repeated in the presence of Polymyxin B, visfatin had similar effects on mRNA transcription (data not shown). Effects of Visfatin on TF Surface Expression and Activity HCAECs were incubated with increasing concentrations of visfatin (5, 10, 20, or 40 ng/ml) for 6 hours, and then processed to evaluate TF expression on cell surface with FACS analysis. TF expression was almost undetectable on unstimulated HCAECs, at baseline. Visfatin caused progressive increase of TF expression, in a dose-dependent fashion (Fig. 2, A). Similarly, TF procoagulant activity, determined by a two-step colorimetric assay, based on the ability of TF to promote generation of coagulation FXa, was almost undetectable at baseline, and progressively increased after stimulation with increasing visfatin doses (Fig. 3, A). In experiments repeated in the presence of Polymyxin B, visfatin had similar effects on TF expression and activity (Fig. 4). Pre-incubation with cycloheximide (10 μg/ml), an inhibitor of protein synthesis, or with DRB (10 μg/ml), an inhibitor of DNA transcription, significantly inhibited TF expression as well as TF procoagulant activity (Figs. 2 and 3, B), suggesting that visfatin is able to induce de novo synthesis of TF and these new TF molecules are then expressed
Fig. 2. Panel A: Dose–response effects of visfatin (5, 10, 20 and 40 ng/ml for 6 hours) on TF expression in HCAECs determined by FACS analysis. Visfatin induced a dose–response increase in TF expression. LPS represents positive control experiments. Panel B: Control experiments performed by pre-incubating endothelial cells with cycloheximide (CE), DRB, with a mouse monoclonal antibody directed against human TF (Anti-TF), with pyrrolidine dithio carbamate ammonium (PDTC), or with Bay 11–7082. Each bar represents the mean ± SD of 6 different experiments. *= p b 0.005 vs unstimulated, control cells. **= p b 0.005 visfatin (Visf).
in an active form on the cell surface. Control experiments, performed by pre-incubating cells with a mouse monoclonal antibody directed against human TF, confirmed that only TF molecules have been detected by FACS analysis, and that procoagulant activity measured was really due to TF expression on cell surface after visfatin induction (Figs. 2 and 3, B). Pre-treatment of cells with pyrrolidine di-thio carbamate ammonium (PDTC, 100 μmol/L), or with Bay 11–7082 (5 μmol/L), two inhibitors of NF-κB activation with a different mechanism of action, for 60 min before stimulation with visfatin (20 ng/ml), significantly reduced TF expression and activity. (Figs. 2 and 3, B). In cells incubated with PDTC, Bay 11–782 and with the monoclonal anti-TF antibody but not stimulated with visfatin, TF was almost undetectable (Fig. 4). Interestingly, inhibition of the visfatin intrinsic enzymatic activity by FK588 (10 nmol/L) resulted in the significant reduction of TF expression as well as activity. Conversely, NMN (100 μM) product of visfatin enzymatic activity, was able to mimic the visfatin effects observed on TF (Fig. 5, A and B). Effects of Visfatin on NF-κB Activation
Fig. 1. Effects of visfatin on TF-mRNA levels in HCAECs assessed by Real Time quantitative PCR. TF mRNA was undetectable at baseline (Base) in unstimulated cells. Visfatin (20 ng/ml), caused the time-dependent increase in levels of TF mRNA that peaked at 60 min and started decreasing at 120 min. LPS (50 μg/ml) represents positive control. Each bar represents the mean ± SD of 3 different experiments. Data are expressed as % change versus control gene represented by GAPDH. (* = p b 0.005 vs Base).
Since we have previously demonstrated that different stimuli are able to up-regulate TF expression via NF-κB in HCAEC cells [13,15] and given the property of visfatin to induce gene target expression through NF-κB activation [16], we investigated if visfatin-mediated TF upregulation was determined by NF-κB activation. Thus, we analyzed NF-κB activity in HCAEC cells transfected with a plasmid containing a luciferase reporter gene downstream the promoter region of immunoglobulin κ light chain responsive to NF-κB activity and incubated them with different concentrations of visfatin. Visfatin induced NF-κB
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Fig. 3. Panel A and panel B: Effects of visfatin on TF activity in HCAECs, determined by a two-step colorimetric assay based on the ability of TF/FVIIa to promote generation of coagulation FXa. TF activity reflects results observed for TF expression, confirming that TF was functionally active. Each bar represents the mean ± SD of 6 different experiments. * = p b 0.005 vs unstimulated, control cells. ** = p b 0.005 visfatin (Visf).
activation in a dose-dependent manner with the luciferase activity that started to be significantly higher at 20 ng/ml concentration, indicating that it up-regulates TF expression in HCAEC cells via NF-κB activation (Fig. 6, A). Interestingly, FK588 (10 nmol/L), inhibitor of the visfatin intrinsic enzymatic activity, caused the significant reduction of NF-κB activity, expressed as luciferase activation (Fig. 6, B). Conversely, NMN was able to mimic the NF-κB effects observed after visfatin stimulation (Fig. 6, B). Discussion The main findings of the present study are: a) Visfatin induces TFmRNA transcription and de novo synthesis of functionally active TF in
Fig. 4. Control experiments. Visfatin effects on TF expression in presence of Polymyxin B. * = p b 0.005 vs unstimulated, control cells. In HCAECs stimulated with a mouse monoclonal antibody directed against human TF (Anti-TF), with pyrrolidine dithio carbamate ammonium (PDTC), or with Bay 11–7082, but not stimulated with Visfatin, TF was undetectable. Each bar represents the mean ± SD of 6 different experiments.
Fig. 5. NAMPT enzymatic activity mediates visfatin effects on TF. Panel A. Inhibition of the visfatin (NAMPT) intrinsic enzymatic activity by FK588 (10 nmol/L) caused the significant reduction of TF activity determined by a two-step colorimetric assay based on the ability of TF/FVIIa to promote generation of coagulation FXa. * = p b 0.005 vs Visfatin stimulated cells (Visf). In control experiments, NMN (100 μM), product of visfatin enzymatic activity, mimicked visfatin effects on TF activity. Panel B. Similar results were observed when TF expression was evaluated. * = p b 0.005 vs Visfatin stimulated cells (Visf).
human coronary artery endothelial cells in culture; b) these phenomena appear to be regulated through activation of NF-κB pathway; c) the visfatin enzymatic activity as nicotinamide phosphoribosyltransferase seems to play a pivotal role in modulating the effects of this adipokine. Several epidemiological studies have clearly demonstrated that human obesity, a disease in which adipose tissue is largely represented, is a strong cardiovascular risk factor causally involved in the development of cardiovascular disease [1,17]. Starting from these epidemiological observations, many efforts have been recently done to investigate the adipose tissue and its relationship with cardiovascular disease. Emerging evidence have transformed the current point of view about this tissue, indicating that adipocytes should no longer be considered only for their role in fat storage, as they are being recognized as cells able to secrete cytokines [17,18]. Specifically, some of these adiposederived cytokines, known as adipokines, such as leptin, and resistin, contribute to a proinflammatory environment and exert pro-atherogenic effects on vascular cells [19,20]. Visfatin belongs to the family of the most recently identified adipokines [21]. It is produced by visceral adipose tissue and it seems to mimic the effects of insulin [21]. Interestingly, this adipokine has been shown to be produced by immune cells such as neutrophils and macrophages usually involved in the pathophysiology of acute coronary events too [22,23]. Specifically, it has been demonstrated that visfatin contributes in promoting inflammation because it induces the expression of proinflammatory molecules such as TNF-α, IL1-β and IL -6 in
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Fig. 6. Visfatin mediates TF up-regulation through NF-κB activation. Panel A. HCAEC cells were transfected with the Ig-κB-luciferase reporter gene plasmid, containing NF-kBbinding sites of immunoglobulin promoter region, and, 24 h after transfection, were treated with increasing visfatin concentrations. Results are the means (± S.D.) of at least three independent experiments, normalized for b-galactosidase activity of a co-transfected Rous sarcoma virus b-galactosidase plasmid. * = p b 0.005 vs unstimulated, control cells (Base). Panel B. NAMPT enzymatic activity mediates visfatin effects on NF-κB. Inhibition of the visfatin (NAMPT) intrinsic enzymatic activity by FK588 caused the significant reduction of Luciferase NF-κB-driven expression. NMN, product of visfatin enzymatic activity, mimicked visfatin effects on this transcription factor. * = p b 0.005 vs visfatin stimulated cells (Visf).
leucocytes. These inflammatory molecules, in turn, are able to upregulate visfatin expression [24], creating a vicious circle operating in inflammatory cells within atherosclerotic lesions and potentially responsible of plaque destabilization. Clinical reports have highlighted the relationship existing between visfatin and atherosclerosis [5,25]. In addition, it has been demonstrated that patients with coronary artery disease have increased plasma levels of visfatin and that these levels were significantly elevated in patients with acute coronary syndromes [4]. Interestingly, it has been recently demonstrated that circulating visfatin levels at admission in patients with acute myocardial infarction are associated with thrombotic occlusion of infarct related artery [26]. Literature about mechanisms by which visfatin might have a role in acute coronary syndromes is still scanty. It has been suggested that this adipokine might trigger plaque rupture by accumulating in atherosclerotic plaques where it stimulates monocyte metalloproteinase activity [25]. Then, it has been demonstrated that this molecule could promote another cause of plaque expansion and vulnerability such as angiogenesis [27]. In the present study we have demonstrated, using a cell culture model, that visfatin induces TF expression in Human Coronary Endothelial Cells in a dose-dependent fashion and that this phenomenon appears to be mainly related to the synthesis of new TF molecules, since
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cycloheximide and DRB, an inhibitor of protein synthesis and mRNA transcription, respectively, completely inhibited the visfatin effects on TF expression. Of particular pathophysiological interest was the finding that these newly formed TF molecules were functionally active, as demonstrated by the parallel increase in TF-procoagulant activity, which was detectable on the surface of stimulated cells. This observation might have important pathophysiological consequences, considering that endothelial cells are at the interface between the vessel wall and circulating blood coagulation factors; thus, visfatin seems to be able to induce a “procoagulant” phenotype in coronary endothelial cells. Taken together, these data might explain, at least in part, why patients with acute myocardial infarction and increased circulating visfatin levels at admission had higher rate of infarct related artery thrombotic occlusion [26]. This hypothesis appears strongly supported by the observation that patients with occluded artery had median plasma visfatin levels of about 20 ng/ml, in line with concentrations tested in the present study. However, it should be kept in mind that it cannot be known if, in vivo, there is a compartmentalization of the effects or how the observed effects are influenced by other conditions. Thus, future research should address this issue. Interestingly, we have demonstrated that visfatin modulates TF by the transcription factor NF-κB. Luciferase reporter gene downstream the promoter region responsive to NF-κB was expressed after visfatin stimulation and TF expression/activity was abolished by pre-treatment of cells with PDTC, or with Bay 11–7082, two inhibitors of NF-κB activation with a different mechanism of action. NF-κB is present but inactive in the cytoplasm of many cells such as lymphocytes, monocytes, endothelial and smooth muscle cells. It seems to be activated by several stimuli during the atherosclerotic process and it is responsible of the expression of inflammatory proteins that actively participate in this process, leading to plaque disruption and acute coronary events [28]. Specifically, binding sites for this transcription factor are contained in the promoter for TF [29]. Moreover, NF-κB has been demonstrated to be activated in the unstable plaques of patients with acute coronary syndromes [30,31]. Another important finding in this study is that the enzymatic activity of visfatin is required for its action. It has been recently demonstrated that visfatin has intrinsic nicotinamide phosphoribosyltransferase (NAMPT) enzymatic activity. Specifically, Revollo et al. have demonstrated that this activity is fundamental in pancreatic cells to regulate insulin secretion [14]. Moreover, it has been recently shown that the NAMPT activity is involved in visfatin's effects on smooth muscle cells [32,33]. Results of the present report appear to be in line with these previous data since the visfatin effects on TF expression as well as activity were abolished by the NAMPT inhibitor, FK866. Conversely, NMN, the product of NAMPT activity, was able to mimic the visfatin effects on TF. Of note, the NAMPT enzymatic activity seems to be pivotal in modulating the nuclear transcription factor NF-κB, since FK866 pretreatment abolished the Luciferase reporter gene downstream the promoter region responsive to NF-κB, that was expressed after visfatin stimulation. This observation is in line with previous observation by Fan et al. who have been demonstrated that Visfatin/Nampt exerts its effects on macrophages via this transcription factor [34] Previous studies from our group have investigated the role of other adipocitokynes in the pathophysiology of acute coronary syndromes indicating their potential involvement in these phenomena [13,16]. The present study gives a newer point of view on the potential role of visfatin in the pathophysiology of ACS. It could be speculated that this adipocytokine, besides promoting plaque disruption [25,27], might be actively involved in triggering TF-mediated thrombus formation, finally leading to the occurrence of acute coronary events. In conclusion, the present study, although in vitro, permits to shed a brighter light on the shadows of the complex puzzle depicting the relationship existing between the adipose tissue, the adipocytokines and the cardiovascular disease.
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[18] Ahima RS, Flier JS. Adipose tissue as an endocrine organ. Trends Endocrinol Metab 2000;11:327–32. [19] Yudkin JS, Stehouwer CD, Emeis JJ, Coppack SW. C-reactive protein in healthy subjects: associations with obesity, insulin resistance, and endothelial dysfunction: a potential role for cytokines originating from adipose tissue? Arterioscler Thromb Vasc Biol 1999;19:972–8. [20] Calabro P, Yeh ET. Obesity, inflammation, and vascular disease: the role of the adipose tissue as an endocrine organ. Subcell Biochem 2007;42:63–91. [21] Fukuhara A, Matsuda M, Nishizawa M, Segawa K, Tanaka M, Kishimoto K, Matsuki Y, Murakami M, Ichisaka T, Murakami H, Watanabe E, Takagi T, Akiyoshi M, Ohtsubo T, Kihara S, Yamashita S, Makishima M, Funahashi T, Yamanaka S, Hiramatsu R, Matsuzawa Y, Shimomura I. Visfatin: a protein secreted by visceral fat that mimics the effects of insulin. Science 2005;307:426–30. [22] Samal B, Sun Y, Stearns G, Xie C, Suggs S, McNiece I. Cloning and characterization of the cDNA encoding a novel human pre-B-cell colony-enhancing factor. Mol Cell Biol Feb 1994;14(2):1431–7. [23] McGlothlin JR, Gao L, Lavoie T, Simon BA, Easley RB, Ma SF, Rumala BB, Garcia JG, Ye SQ. Molecular cloning and characterization of canine pre-B-cell colony-enhancing factor. Biochem Genet Apr 2005;43(3–4):127–41. [24] Moschen AR, Kaser A, Enrich B, Mosheimer B, Theurl M, Niederegger H, Tilg H. Visfatin, an adipocytokine with proinflammatory and immunomodulating properties. J Immunol Feb 1 2007;178(3):1748–58. [25] Dahl TB, Yndestad A, Skjelland M, Øie E, Dahl A, Michelsen A, Damås JK, Tunheim SH, Ueland T, Smith C, Bendz B, Tonstad S, Gullestad L, Frøland SS, Krohg-Sørensen K, Russell D, Aukrust P, Halvorsen B. Increased expression of visfatin in macrophages of human unstable carotid and coronary atherosclerosis: possible role in inflammation and plaque destabilization. Circulation 2007;115:972–80. [26] Yu TH, Lu LF, Hung WC, Chiu CA, Liu YT, Yang CY, Tsai IT, Yen YC, Chen CY, Wang CP. Circulating visfatin level at admission is associated with occlusion of infarct-related artery in patients with acute ST-segment elevation myocardial infarction. Acta Cardiol Sin 2011;27:77–85. [27] Adya R, Tan BK, Punn A, Chen J, Randeva HS. Visfatin induces human endothelial VEGF and MMP/2/9 production via MAPK and PI3K/Akt signalling pathway: novel insights into visfatin-induced angiogenesis. Cardiovasc Res 2008;78:356–65. [28] Barnes PJ, Karin M. Nuclear factor-kappa B: a pivotal transcription factor in chronic inflammatory disease. N Engl J Med 1997;336:1066–71. [29] Oeth P, Parry GC, Mackman N. Regulation of the tissue factor gene in human monocytic cells. Arterioscler Thromb Vasc Biol 1997 Feb;17(2):365–74. [30] Ritchie ME. Nuclear Factor- kB is selectively and markedly activated in humans with unstable angina pectoris. Circulation 1998;98:1707–13. [31] Wilson SH, Best PJ, Edwards WD, Holmes Jr DR, Carlson PJ, Celermajer DS, Lerman A. Nuclear factor-kappaB immunoreactivity is present in human coronary plaque and enhanced in patients with unstable angina pectoris. Atherosclerosis 2002;160: 147–53. [32] Wang P, Xu TY, Guan YF, Su DF, Fan GR, Miao CY. Perivascular adipose tissue-derived visfatin is a vascular smooth muscle cell growth factor: role of nicotinamide dicluneotide. Cardiovasc Res 2009;81:370–80. [33] Romacho T, Azcutia V, Vázquez-Bella M, Matesanz N, Cercas E, Nevado J, Carraro R, Rodríguez-Mañas L, Sánchez-Ferrer CF, Peiró C. Extracellular PBEF/NAMPT/visfatin activates pro-inflammatory signalling in human vascular smooth muscle cells through nicotinamide phosphoribosyltransferase activity. Diabetologia 2009;51: 2455–63. [34] Fan Y, Meng S, Wang Y, Cao J, Wang C. Visfatin/PBEF/Nampt induces EMMPRIN and MMP-9 production in macrophages via the NAMPT-MAPK (p38, ERK1/2)-NF-κB signaling pathway. Int J Mol Med Apr 2011;27(4):607–15.
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Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Regular Article
The adipokine visfatin induces tissue factor expression in human coronary artery endothelial cells Another piece in the adipokines puzzle Plinio Cirillo a,⁎, Vito Di Palma a, Fabio Maresca a, Francesco Pacifico b, Francesca Ziviello a, Michele Bevilacqua a, Bruno Trimarco a, Antonio Leonardi c, Massimo Chiariello a a b c
Department of Clinical Medicine, Cardiovascular and Immunological Science (Division of Cardiology) Istituto di Endocrinologia e Oncologia Sperimentale, CNR Dipartimento di Biologia e Patologia Cellulare e Molecolare, University of Naples “Federico II”
a r t i c l e
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Article history: Received 8 March 2012 Accepted 4 June 2012 Available online 21 June 2012 Keywords: Adipokines Atherothrombosis Tissue Factor Visfatin
a b s t r a c t Introduction: Adipocytes are nowadays recognized as cells able to produce and secrete a large variety of active substances with direct effects on vascular cells, known as adipokines. Visfatin is a recently identified adipokine not yet completely characterized for its pathophysiological role in cardiovascular disease. Increased levels of visfatin are measurable in the plasma of patients with coronary artery disease and specifically in those with acute coronary syndromes (ACS). Several studies have indicated that Tissue Factor (TF) plays a pivotal role in the pathophysiology of ACS by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates the effects of visfatin on TF in human coronary endothelial cells (HCAECs). Methods: HCAECs were stimulated with visfatin in a concentration range usually measurable in plasma of patients with ACS and than processed to evaluate TF-mRNA levels as well as TF expression/activity. Finally, the role of NF-κB pathway was investigated. Results: We demonstrate that visfatin induces transcription of mRNA for TF by Real Time PCR. In addition, we show that this adipokine promotes surface expression of TF that is functionally active since we measured increased procoagulant activity. Visfatin effects on TF appear modulated by the activation of the transcription factor, NF-κB, since NF-κB inhibitors suppressed TF expression. Finally, we show that the nicotinamide phopsphoribosyltransferase enzymatic activity of visfatin seems to play a pivotal role in modulating the NF-κB driven regulation of TF. Discussion: Data of the present study, although in vitro, indicate that visfatin, at doses measurable in ACS patient plasma, induces a procoagulant phenotype in human coronary endothelial cells by promoting TF expression. These observations support the hypothesis that this adipokine might play a relevant role as an active partaker in athero-thrombotic disease. © 2012 Elsevier Ltd. All rights reserved.
Introduction Obesity, a disease in which adipose tissue is largely represented, is actually considered as strong cardiovascular risk factor [1,2]. Thus, although it is established that increase of visceral fat is causally involved in the development of cardiovascular disease, the possible link between these entities is not completely understood yet. Indeed, the current point of view about the adipose tissue is actually changed, indicating that adipocytes have no longer to be considered only storage cells for fat, but they have to be now recognized as cells able to produce and secrete a large variety of active substances named adipokines. Several ⁎ Corresponding author at: Division of Cardiology, University of Naples “Federico II”, Via Sergio Pansini 5, 80131 Naples, Italy. Tel./fax: + 39 081 7462235. E-mail address: [email protected] (P. Cirillo). 0049-3848/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2012.06.007
adipokines have been extensively characterized and are considered of particular interest in cardiovascular pathophysiology [3]. Conversely, other novel adipokines, such as visfatin, have been poorly investigated. Interestingly, elevated plasma levels of visfatin have been measured in plasma of patients with acute coronary syndromes compared with controls [4]. Thus, it has been proposed that this adipokine, might be a marker of increased cardiovascular risk, specially in patients with metabolic syndrome [5]. Increasing evidences have assigned a central role to thrombosis in the progression and complication of atherosclerosis and have linked it to the clinical occurrence of acute coronary syndromes [6-8]. Several studies have clearly indicated that tissue factor (TF), the key initiator of extrinsic coagulation pathway, plays a pivotal role in the pathophysiology of acute coronary syndromes by triggering the formation of intracoronary thrombi following endothelial injury [9-12].
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In the present study, we provide support, in vitro, for the hypothesis that visfatin is not only a marker of cardiovascular risk, but it might effectively play an active role in pathophysiology of coronary acute events by promoting a pro-thrombotic state in human coronary artery endothelial cells (HCAECs) through induction of TF. Furthermore, we investigated the potential involvement of the NF-κB intracellular pathway in this phenomenon. Methods HCAECs purchased from Lonza (Basel, Switzerland) were grown in EGM 2 medium with 10% FBS and used at passages 2 to 5. To investigate the visfatin effects on TF expression and activity, cells were enzimatically harvested and counted in a haemocytometer and sub cultured in 24-well plates at an initial density of about 5 × 10 4 cell/ well, while at confluence, cell density was of about 8.5 × 10 4 cell/ well. For other set of experiments, cells were grown in 100 mm cell plates and, at confluence, cell density was of about 2 × 10 6 cells per plate. At confluence, cell were starved in serum-free medium for 24 hours and then used in the different set of experiments. Visfatin (Sigma Chemical Co., St Louis, USA) used in all studies was analyzed founding endotoxin level to be b0.125 EU/mL (b12.5 pg/mL) by Limulus assay (Bio Whittaker, Walkersville, USA). All media, and water were also tested and endotoxin level found to be b0.125 EU/mL. In addition, in order to avoid a possible bias from contaminating LPS, all experiments were then repeated in the presence of Polymyxin B (100 μg/ml). Effects of Visfatin on TF-mRNA Levels The dose–response effects of visfatin on TF-mRNA transcription were preliminary investigated by semi-quantitative polymerase chain reaction. Then, the effect of visfatin on TF-mRNA was investigated by real-time reverse transcription analysis as previously described [13]. Briefly, HCAECs were incubated with visfatin (20 ng/ml, concentration chosen on the basis of semi-quantitative results) for 30 minutes. Then, cells were washed with PBS and then fresh medium (EGM 2 containing 0,1% serum) was added. Total mRNA was extracted by cell cultures using TRIzol reagent (GIBCO, Carlsbad, USA), at baseline, 30, 60 and 120 min after visfatin stimulation and TF mRNA levels were examined by real-time reverse transcription (RT) and polymerase chain reaction (PCR) by LightCycler (Roche Diagnostics, Basel, Switzerland). In positive control experiments, HCAECs were incubated with LPS (50 μg/ml, Sigma Chemical Co., St Louis, USA), for 30 min and then mRNA was extracted at 60 min. Total mRNA was extracted from cell cultures using TRIzol reagent (GIBCO, Carlsbad, USA), according to the manufacturer's instructions. Reverse transcription was performed using mMLV (GIBCO, Carlsbad, USA) and 100 ng of the RNA samples from each culture condition. Samples were run in triplicate in 50 μl reactions by using an ABI PRISM 5700 sequence detector system (Applied Bio systems, Foster City, USA). Samples were incubated at 50 °C for 2 min, 95 °C for 10 min and then underwent 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Specific oligonucleotides for human GAPDH and human TF were designed on the basis of published sequences using PRIMER EXPRESS Software (Applied Bio systems) and validated for their specificity. SYBR-green chemistry was used to detect fluorescence and an internal standard (Applied Bio systems, Foster City, USA) was used for quantization of the message. The results were analyzed using a comparative method, and the values were normalized to the GAPDH expression and converted into percentage change. Three different experiments were performed for each experimental condition. Effects of Visfatin on TF surface Expression and Activity HCAECs cultivated as above were washed with PBS and then incubated with increasing concentrations of visfatin (5, 10, 20 or 40 ng/ml) for 6 hours.
TF expression on cell surface was investigated with FACS analysis. After incubation, endothelial cells were detached with 10 mmol/L EDTA in PBS (without trypsin) and stained with FITClabelled monoclonal antibody (Pharmingen, Franklin Lakes, USA) against TF, or with the appropriate isotype IgG (phycoerythrin or FITC) as control. Fluorescence intensity of 9000 cells for each sample was quantified by a FACSCalibur analyzer (Becton-Dickinson, Franklin Lakes, USA). To evaluate whether visfatin-induced TF was functionally active, TF procoagulant activity was determined by a two-step colorimetric assay, based on the ability of TF to promote generation of coagulation FXa, as previously described [13]. Briefly, after stimulation with visfatin, cells were incubated with 1 nM of recombinant human FVIIa (Novo Nordisk A/S Gentofte, Denmark), followed by 100 nM of purified human factor X (Calbiochem-Novobiochem, La Jolla, CA, USA) and 5 mM CaCl2 for 15 min at 37 °C. A chromogenic substrate, specific for factor X (Cromozym X, Roche Diagnostics, Mannheim, Germany, 0.5 mmol/l) was then added and incubated for 30 min at 37 °C. The reaction was stopped by adding 200 μl/ml of sample of a 30% solution of acetic acid and the change in optical density at 405 nm was quantified with a spectrophotometer. Purified factor Xa of known concentration (Sigma Chemical Co., St Louis, USA) allowed generation of calibration curves. To evaluate whether visfatin-induced expression of TF resulted from de novo synthesis, in another set of experiments, HCAECs were pre-incubated for 120 minutes with cycloheximide (10 μg/ ml), an inhibitor of protein synthesis, or with 5, 6-dichloro-1-β-dribofuranosylbenzimidazole (DRB, 10 μg/ml), an inhibitor of DNA transcription, before adding visfatin (20 ng/ml). In additional control experiments cells were pre-incubated for 10 minutes with a mouse monoclonal antibody against human TF (5 μg/ml, American Diagnostica Inc, Greenwich, CT, USA). Positive control experiments included cells incubated for 6 hours with LPS (50 μg/ml). Six different experiments were performed for each experimental condition. Since it has been demonstrated that effects of visfatin may be mediated by its intrinsic nicotinamide phosphoribosyltransferase (NAMPT) enzymatic activity [14], in an additional set of experiments we have investigated whether nicotine-amide mononucleotide (NMN), the product of NAMPT activity, was able to mimic the effects of this adipokine on TF, in HCAECs. Thus, cells were stimulated with NMN (100 μM), and then TF expression as well as TF procoagulant activity were determined as described above. Moreover, in control experiments, HCAECs were incubated with the NAMPT inhibitor FK866 (10 nmol/L) for 1 hour before stimulating cells with visfatin (20 ng/ml).
Effects of Visfatin on NF-κB Activation To investigate whether expression of TF induced by visfatin involved activation of the NF-κB pathway, we performed luciferase assays. HCAEC cells (3 × 10 5) were transfected with 1.5 μg of the Ig-κB-luciferase reporter gene plasmid, containing NF-κB-binding sites of immunoglobulin promoter region, and, 24 h after transfection, were treated with increasing amounts of visfatin (5, 10, 20 or 40 ng/ml). After 3 h of incubation at 37 °C, cell extracts were prepared and reporter gene activity was determined by the luciferase system (Promega). A pRSV-β-galactosidase vector (0.5 μg) was used to normalize for transfection efficiencies.
Statistical Analysis Data are presented as mean ± SD. Differences between groups were determined by a one-way ANOVA followed by a Student's t test with Bonferroni's correction. A p value b 0.05 was considered statistically significant.
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Results Effects of Visfatin on TF MRNA Levels Visfatin effects of TF-mRNA transcription were investigated by real-time reverse transcription analysis. TF mRNA levels were very low in unstimulated HCAECs, as expected [12]. Visfatin (20 ng/ml), caused a time-dependent increase in TF mRNA levels, as compared to unstimulated cells. The peak of TF mRNA levels was observed at 60 min, while at 120 min, TF mRNA levels started decreasing (Fig. 1). In experiments repeated in the presence of Polymyxin B, visfatin had similar effects on mRNA transcription (data not shown). Effects of Visfatin on TF Surface Expression and Activity HCAECs were incubated with increasing concentrations of visfatin (5, 10, 20, or 40 ng/ml) for 6 hours, and then processed to evaluate TF expression on cell surface with FACS analysis. TF expression was almost undetectable on unstimulated HCAECs, at baseline. Visfatin caused progressive increase of TF expression, in a dose-dependent fashion (Fig. 2, A). Similarly, TF procoagulant activity, determined by a two-step colorimetric assay, based on the ability of TF to promote generation of coagulation FXa, was almost undetectable at baseline, and progressively increased after stimulation with increasing visfatin doses (Fig. 3, A). In experiments repeated in the presence of Polymyxin B, visfatin had similar effects on TF expression and activity (Fig. 4). Pre-incubation with cycloheximide (10 μg/ml), an inhibitor of protein synthesis, or with DRB (10 μg/ml), an inhibitor of DNA transcription, significantly inhibited TF expression as well as TF procoagulant activity (Figs. 2 and 3, B), suggesting that visfatin is able to induce de novo synthesis of TF and these new TF molecules are then expressed
Fig. 2. Panel A: Dose–response effects of visfatin (5, 10, 20 and 40 ng/ml for 6 hours) on TF expression in HCAECs determined by FACS analysis. Visfatin induced a dose–response increase in TF expression. LPS represents positive control experiments. Panel B: Control experiments performed by pre-incubating endothelial cells with cycloheximide (CE), DRB, with a mouse monoclonal antibody directed against human TF (Anti-TF), with pyrrolidine dithio carbamate ammonium (PDTC), or with Bay 11–7082. Each bar represents the mean ± SD of 6 different experiments. *= p b 0.005 vs unstimulated, control cells. **= p b 0.005 visfatin (Visf).
in an active form on the cell surface. Control experiments, performed by pre-incubating cells with a mouse monoclonal antibody directed against human TF, confirmed that only TF molecules have been detected by FACS analysis, and that procoagulant activity measured was really due to TF expression on cell surface after visfatin induction (Figs. 2 and 3, B). Pre-treatment of cells with pyrrolidine di-thio carbamate ammonium (PDTC, 100 μmol/L), or with Bay 11–7082 (5 μmol/L), two inhibitors of NF-κB activation with a different mechanism of action, for 60 min before stimulation with visfatin (20 ng/ml), significantly reduced TF expression and activity. (Figs. 2 and 3, B). In cells incubated with PDTC, Bay 11–782 and with the monoclonal anti-TF antibody but not stimulated with visfatin, TF was almost undetectable (Fig. 4). Interestingly, inhibition of the visfatin intrinsic enzymatic activity by FK588 (10 nmol/L) resulted in the significant reduction of TF expression as well as activity. Conversely, NMN (100 μM) product of visfatin enzymatic activity, was able to mimic the visfatin effects observed on TF (Fig. 5, A and B). Effects of Visfatin on NF-κB Activation
Fig. 1. Effects of visfatin on TF-mRNA levels in HCAECs assessed by Real Time quantitative PCR. TF mRNA was undetectable at baseline (Base) in unstimulated cells. Visfatin (20 ng/ml), caused the time-dependent increase in levels of TF mRNA that peaked at 60 min and started decreasing at 120 min. LPS (50 μg/ml) represents positive control. Each bar represents the mean ± SD of 3 different experiments. Data are expressed as % change versus control gene represented by GAPDH. (* = p b 0.005 vs Base).
Since we have previously demonstrated that different stimuli are able to up-regulate TF expression via NF-κB in HCAEC cells [13,15] and given the property of visfatin to induce gene target expression through NF-κB activation [16], we investigated if visfatin-mediated TF upregulation was determined by NF-κB activation. Thus, we analyzed NF-κB activity in HCAEC cells transfected with a plasmid containing a luciferase reporter gene downstream the promoter region of immunoglobulin κ light chain responsive to NF-κB activity and incubated them with different concentrations of visfatin. Visfatin induced NF-κB
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Fig. 3. Panel A and panel B: Effects of visfatin on TF activity in HCAECs, determined by a two-step colorimetric assay based on the ability of TF/FVIIa to promote generation of coagulation FXa. TF activity reflects results observed for TF expression, confirming that TF was functionally active. Each bar represents the mean ± SD of 6 different experiments. * = p b 0.005 vs unstimulated, control cells. ** = p b 0.005 visfatin (Visf).
activation in a dose-dependent manner with the luciferase activity that started to be significantly higher at 20 ng/ml concentration, indicating that it up-regulates TF expression in HCAEC cells via NF-κB activation (Fig. 6, A). Interestingly, FK588 (10 nmol/L), inhibitor of the visfatin intrinsic enzymatic activity, caused the significant reduction of NF-κB activity, expressed as luciferase activation (Fig. 6, B). Conversely, NMN was able to mimic the NF-κB effects observed after visfatin stimulation (Fig. 6, B). Discussion The main findings of the present study are: a) Visfatin induces TFmRNA transcription and de novo synthesis of functionally active TF in
Fig. 4. Control experiments. Visfatin effects on TF expression in presence of Polymyxin B. * = p b 0.005 vs unstimulated, control cells. In HCAECs stimulated with a mouse monoclonal antibody directed against human TF (Anti-TF), with pyrrolidine dithio carbamate ammonium (PDTC), or with Bay 11–7082, but not stimulated with Visfatin, TF was undetectable. Each bar represents the mean ± SD of 6 different experiments.
Fig. 5. NAMPT enzymatic activity mediates visfatin effects on TF. Panel A. Inhibition of the visfatin (NAMPT) intrinsic enzymatic activity by FK588 (10 nmol/L) caused the significant reduction of TF activity determined by a two-step colorimetric assay based on the ability of TF/FVIIa to promote generation of coagulation FXa. * = p b 0.005 vs Visfatin stimulated cells (Visf). In control experiments, NMN (100 μM), product of visfatin enzymatic activity, mimicked visfatin effects on TF activity. Panel B. Similar results were observed when TF expression was evaluated. * = p b 0.005 vs Visfatin stimulated cells (Visf).
human coronary artery endothelial cells in culture; b) these phenomena appear to be regulated through activation of NF-κB pathway; c) the visfatin enzymatic activity as nicotinamide phosphoribosyltransferase seems to play a pivotal role in modulating the effects of this adipokine. Several epidemiological studies have clearly demonstrated that human obesity, a disease in which adipose tissue is largely represented, is a strong cardiovascular risk factor causally involved in the development of cardiovascular disease [1,17]. Starting from these epidemiological observations, many efforts have been recently done to investigate the adipose tissue and its relationship with cardiovascular disease. Emerging evidence have transformed the current point of view about this tissue, indicating that adipocytes should no longer be considered only for their role in fat storage, as they are being recognized as cells able to secrete cytokines [17,18]. Specifically, some of these adiposederived cytokines, known as adipokines, such as leptin, and resistin, contribute to a proinflammatory environment and exert pro-atherogenic effects on vascular cells [19,20]. Visfatin belongs to the family of the most recently identified adipokines [21]. It is produced by visceral adipose tissue and it seems to mimic the effects of insulin [21]. Interestingly, this adipokine has been shown to be produced by immune cells such as neutrophils and macrophages usually involved in the pathophysiology of acute coronary events too [22,23]. Specifically, it has been demonstrated that visfatin contributes in promoting inflammation because it induces the expression of proinflammatory molecules such as TNF-α, IL1-β and IL -6 in
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Fig. 6. Visfatin mediates TF up-regulation through NF-κB activation. Panel A. HCAEC cells were transfected with the Ig-κB-luciferase reporter gene plasmid, containing NF-kBbinding sites of immunoglobulin promoter region, and, 24 h after transfection, were treated with increasing visfatin concentrations. Results are the means (± S.D.) of at least three independent experiments, normalized for b-galactosidase activity of a co-transfected Rous sarcoma virus b-galactosidase plasmid. * = p b 0.005 vs unstimulated, control cells (Base). Panel B. NAMPT enzymatic activity mediates visfatin effects on NF-κB. Inhibition of the visfatin (NAMPT) intrinsic enzymatic activity by FK588 caused the significant reduction of Luciferase NF-κB-driven expression. NMN, product of visfatin enzymatic activity, mimicked visfatin effects on this transcription factor. * = p b 0.005 vs visfatin stimulated cells (Visf).
leucocytes. These inflammatory molecules, in turn, are able to upregulate visfatin expression [24], creating a vicious circle operating in inflammatory cells within atherosclerotic lesions and potentially responsible of plaque destabilization. Clinical reports have highlighted the relationship existing between visfatin and atherosclerosis [5,25]. In addition, it has been demonstrated that patients with coronary artery disease have increased plasma levels of visfatin and that these levels were significantly elevated in patients with acute coronary syndromes [4]. Interestingly, it has been recently demonstrated that circulating visfatin levels at admission in patients with acute myocardial infarction are associated with thrombotic occlusion of infarct related artery [26]. Literature about mechanisms by which visfatin might have a role in acute coronary syndromes is still scanty. It has been suggested that this adipokine might trigger plaque rupture by accumulating in atherosclerotic plaques where it stimulates monocyte metalloproteinase activity [25]. Then, it has been demonstrated that this molecule could promote another cause of plaque expansion and vulnerability such as angiogenesis [27]. In the present study we have demonstrated, using a cell culture model, that visfatin induces TF expression in Human Coronary Endothelial Cells in a dose-dependent fashion and that this phenomenon appears to be mainly related to the synthesis of new TF molecules, since
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cycloheximide and DRB, an inhibitor of protein synthesis and mRNA transcription, respectively, completely inhibited the visfatin effects on TF expression. Of particular pathophysiological interest was the finding that these newly formed TF molecules were functionally active, as demonstrated by the parallel increase in TF-procoagulant activity, which was detectable on the surface of stimulated cells. This observation might have important pathophysiological consequences, considering that endothelial cells are at the interface between the vessel wall and circulating blood coagulation factors; thus, visfatin seems to be able to induce a “procoagulant” phenotype in coronary endothelial cells. Taken together, these data might explain, at least in part, why patients with acute myocardial infarction and increased circulating visfatin levels at admission had higher rate of infarct related artery thrombotic occlusion [26]. This hypothesis appears strongly supported by the observation that patients with occluded artery had median plasma visfatin levels of about 20 ng/ml, in line with concentrations tested in the present study. However, it should be kept in mind that it cannot be known if, in vivo, there is a compartmentalization of the effects or how the observed effects are influenced by other conditions. Thus, future research should address this issue. Interestingly, we have demonstrated that visfatin modulates TF by the transcription factor NF-κB. Luciferase reporter gene downstream the promoter region responsive to NF-κB was expressed after visfatin stimulation and TF expression/activity was abolished by pre-treatment of cells with PDTC, or with Bay 11–7082, two inhibitors of NF-κB activation with a different mechanism of action. NF-κB is present but inactive in the cytoplasm of many cells such as lymphocytes, monocytes, endothelial and smooth muscle cells. It seems to be activated by several stimuli during the atherosclerotic process and it is responsible of the expression of inflammatory proteins that actively participate in this process, leading to plaque disruption and acute coronary events [28]. Specifically, binding sites for this transcription factor are contained in the promoter for TF [29]. Moreover, NF-κB has been demonstrated to be activated in the unstable plaques of patients with acute coronary syndromes [30,31]. Another important finding in this study is that the enzymatic activity of visfatin is required for its action. It has been recently demonstrated that visfatin has intrinsic nicotinamide phosphoribosyltransferase (NAMPT) enzymatic activity. Specifically, Revollo et al. have demonstrated that this activity is fundamental in pancreatic cells to regulate insulin secretion [14]. Moreover, it has been recently shown that the NAMPT activity is involved in visfatin's effects on smooth muscle cells [32,33]. Results of the present report appear to be in line with these previous data since the visfatin effects on TF expression as well as activity were abolished by the NAMPT inhibitor, FK866. Conversely, NMN, the product of NAMPT activity, was able to mimic the visfatin effects on TF. Of note, the NAMPT enzymatic activity seems to be pivotal in modulating the nuclear transcription factor NF-κB, since FK866 pretreatment abolished the Luciferase reporter gene downstream the promoter region responsive to NF-κB, that was expressed after visfatin stimulation. This observation is in line with previous observation by Fan et al. who have been demonstrated that Visfatin/Nampt exerts its effects on macrophages via this transcription factor [34] Previous studies from our group have investigated the role of other adipocitokynes in the pathophysiology of acute coronary syndromes indicating their potential involvement in these phenomena [13,16]. The present study gives a newer point of view on the potential role of visfatin in the pathophysiology of ACS. It could be speculated that this adipocytokine, besides promoting plaque disruption [25,27], might be actively involved in triggering TF-mediated thrombus formation, finally leading to the occurrence of acute coronary events. In conclusion, the present study, although in vitro, permits to shed a brighter light on the shadows of the complex puzzle depicting the relationship existing between the adipose tissue, the adipocytokines and the cardiovascular disease.
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