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Upregulation of thromboxane synthase mediates visfatin-induced interleukin-8 expression and angiogenic activity in endothelial cells
Biochemical and Biophysical Research Communications 418 (2012) 662–668
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Upregulation of thromboxane synthase mediates visfatin-induced interleukin-8 expression and angiogenic activity in endothelial cells Su-Ryun Kim a,1, Yun-Hoa Jung b,1, Hyun-Joo Park a,c, Mi-Kyoung Kim a, Joo-Won Jeong d, Hye-Ock Jang c, Il Yun c, Soo-Kyung Bae c, Moon-Kyoung Bae a,⇑ a
Department of Oral Physiology, School of Dentistry, Pusan National University, Yangsan 626-870, South Korea Department of Oral & Maxillofacial Radiology, School of Dentistry, Pusan National University, Yangsan 626-870, South Korea Department of Dental Pharmacology, School of Dentistry, Pusan National University, Yangsan 626-870, South Korea d School of Medicine, Kyung Hee University, Seoul 130-701, South Korea b c
a r t i c l e
i n f o
Article history: Received 7 January 2012 Available online 24 January 2012 Keywords: Visfatin Thromboxane synthase Thromboxane A2 Interleukin-8 Vascular endothelial cells Angiogenesis
a b s t r a c t Thromboxane synthase (TXAS) is an enzyme that catalyzes the synthesis of thromboxane A2 (TXA2). Overexpression of TXAS is associated with a variety of vascular diseases. Recently, we reported that visfatin, a novel adipokine, exhibits angiogenic actions. In this study, we showed that visfatin increased mRNA and protein levels of TXAS and stimulated TXA2 biosynthesis in vascular endothelial cells. In addition, visfatin induced the expression and secretion of interleukin-8 (IL-8), which is blocked by a TXAS inhibitor and by the transfection of siRNA specific for TXAS. Furthermore, the inhibition of TXAS activity and blockade of the IL-8 receptor attenuated visfatin-induced endothelial angiogenesis. Together, these results showed that visfatin promoted IL-8 production by upregulation of TXAS, leading to angiogenic activation in endothelial cells. Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved.
1. Introduction Visfatin is a novel adipokine predominantly produced by visceral adipose tissue [1]. Visfatin was originally identified as a growth factor, called pre-B cell colony enhancing factor (PBEF), for the maturation of B-cell precursors [2]. Both intracellular and extracellular forms of visfatin were known as nicotinamide phosphoribosyltransferase (NAMPT), an enzyme involved in mammalian nicotinamide adenine dinucleotide biosynthesis [3]. Recently, we and others showed that visfatin has proinflammatory activity (e.g., induction of inflammatory cytokines and cell adhesion molecules) in human monocytes and vascular endothelial cells [4,5]. Visfatin also has proangiogenic activity mediated by enhancing the production of soluble factors, such as vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6), in endothelial cells [6–10]. Thromboxane synthase (TXAS) is a downstream enzyme of arachidonic acid metabolism and catalyzes the synthesis of thromboxane A2 (TXA2) [11]. TXA2 was originally known to control platelet aggregation and smooth muscle contraction; more recently, it has been reported to act as a pathophysiological modulator of ⇑ Corresponding author. Fax: +82 51 510 8239. 1
E-mail address: [email protected] (M.-K. Bae). Denotes co-first authors.
endothelial function [12,13]. TXA2 stimulates vascular inflammatory responses by increasing the expression of the endothelial cell adhesion molecules, such as ICAM-1 and VCAM-1, and regulates endothelial angiogenesis and permeability [14–16]. TXAS is highly overexpressed in various types of vascularized solid tumors, and its expressional level is associated with increased tumor microvessel density [17–20]. However, the molecular mechanisms underlying the induction of TXAS expression as well as the functional significance of TXAS signaling in vascular endothelium remain poorly defined. In the present study, we examined the effect of visfatin on TXAS gene expression in vascular endothelial cells and investigated the underlying mechanisms. Our data demonstrate for the first time that visfatin enhances TXAS and its metabolite, TXA2, thus inducing endothelial interleukin-8 (IL-8) production and angiogenesis.
2. Materials and methods 2.1. Reagents and recombinant proteins SB203580, LY294002, and U0126 were purchased from BIOMOL. Carbethoxyhexyl imidazole (CI) was acquired from Enzo Life Sciences. Rabbit polyclonal anti-TXAS and mouse monoclonal anti-PECAM-1 antibodies were obtained from Cayman and BD Biosciences, respectively. Mouse monoclonal anti-a-tubulin
0006-291X/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.01.072
S.-R. Kim et al. / Biochemical and Biophysical Research Communications 418 (2012) 662–668
antibodies were supplied by Biogenex. IL-8 neutralizing antibodies were purchased from R&D systems. Recombinant human visfatin protein was prepared in our laboratory as previously described [4]. Polymyxin B (PMB) and repertaxin were obtained from Sigma.
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with siRNAs using oligofectamine and Amaxa Nucleofector, respectively, according to the manufacturer’s instructions.
2.7. Tube formation assay 2.2. Cell culture Human microvascular endothelial cells (HMECs) were obtained from the CDC (Atlanta, GA). The cells were maintained in MCDB supplemented with 10% FBS (Invitrogen), 1% antibiotics, 10 lM/ ml of l-glutamine, 1 lg/ml of hydrocortisone, and 10 ng/ml of hEGF at 37 °C under a humidified atmosphere containing (vol/vol) 95% air and 5% CO2. Primary human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cord veins by collagenase treatment as described previously [21]. The HUVECs were plated onto a 0.2% gelatin-coated dish and grown in sterile endothelial growth medium (EGM-2, CLONTECTICS). EGM-2 consisted of an endothelial basal medium (EBM-2, CLONTECTICS), trace elements, growth factors, and antibiotics. For cells treated with visfatin, the culture medium was supplemented with an endotoxinneutralizing agent (10 lg/ml polymyxin B). 2.3. RT-PCR Total RNA was isolated from the HMECs using a TRIzol reagent kit (Invitrogen). cDNA synthesis was performed using 3 lg of total RNA with a reverse transcription kit (Promega). The oligonucleotide primers for PCR were designed as follows: b-actin, 50 GACTACCTCATGAAGATC-30 and 50 -GATCCACATCTGCTGGAA-30 ; human TXAS, 50 -AGATGGTTCCCCTCATCAGC-30 and 50 -GCCCACAATCTCAT CCACAG-30 ; human IL-8, 50 -GAAGGTGCAGTTTTGCCAAG-30 and 50 -ACCCTCTGCACCCAGTTTTC-30 . 2.4. Western blot analysis The harvested cells were lysed in a lysis buffer (40 mM Tris-Cl, 10 mM EDTA, 120 mM NaCl, and 0.1% NP-40 with protease inhibitor cocktail [Sigma]). A constant protein concentration (30 lg/lane) was used. The proteins were separated by sodium dodecyl sulfate– polyacrylamide gel electrophoresis and were transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech). The membrane was blocked with 5% skim milk in phosphate-buffered saline (PBS) containing 0.1% Tween-20 for 1 h at room temperature and probed with the appropriate antibodies. This signal was developed using an enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech). 2.5. Enzyme-linked immunosorbent assays The cells were treated with visfatin in the presence or absence of pretreatment with CI or signaling inhibitors. At the end of the treatment, culture media were collected and centrifuged at 14,000 rpm for 5 min. The supernatants were stored in aliquots at 70 °C. Secreted IL-8 protein and TXB2 levels were determined using enzyme-linked immunosorbent assays (ELISAs) with human IL-8 ELISA Max™ Set Deluxe Kits (BioLegend) and thromboxane B2 EIA kit (Cayman Chemical), respectively, according to the manufacturer’s instructions. 2.6. Transient transfection of small interfering RNA Small interfering RNA (siRNA) oligonucleotides against human TXAS (50 - GUUGAGAACUUCAGUAACUG-30 , 50 -CAGUUACUGAAGUU CUCAAC-30 ) were synthesized [22], and negative control siRNA was purchased from Bioneer. HMECs and HUVECs were transfected
Growth factor-reduced (GFR) Matrigel (300 lL) (BD Bioscience) was pipetted into 24-well culture plates and solidified for 30 min at 37 °C. HUVECs were seeded onto the surface of the Matrigel and then treated with visfatin for indicated times at 37 °C. Morphological changes of the endothelial cells were observed under a phase contrast microscope and photographed.
2.8. Rat aortic ring sprouting assay The aorta was excised from 6-week-old male Sprague–Dawley rats, and the fibroadipose tissue was removed. The aorta was sectioned into 1-mm-thick slices; the aortic rings were placed on Matrigel-coated wells, covered with an additional 50 lL of Matrigel, and allowed to gel for 30 min at 37 °C. The aortic rings were cultured with visfatin in the presence or absence of CI. After 3 days, the outgrowth was photographed under a phase contrast microscope.
2.9. In vivo mouse Matrigel plug assay C57/BL6 mice were subcutaneously injected with 0.5 mL of Matrigel containing the indicated amounts of visfatin and heparin (10 units/lL) in presence or absence of CI. After 7 days, the mice were killed, and the Matrigel plugs were recovered, fixed with 4% paraformaldehyde in PBS, and embedded in paraffin. Each plug was fixed, sectioned, and stained with hematoxylin-eosin or immunostained with antibody for PECAM-1, a marker for endothelial cells. The hemoglobin level was measured using the Drabkin method for quantifying blood vessel formation.
3. Results 3.1. Visfatin increases TXAS expression and TXA2 production in human endothelial cells We first investigated whether visfatin could induce the expression of TXAS in endothelial cells. HMECs were incubated with visfatin, and TXAS mRNA levels were analyzed by RT-PCR. As shown in Fig. 1A, visfatin increased the expression of TXAS mRNA in a timedependent manner. The levels of TXAS protein in visfatin-treated HMECs were then analyzed by western blotting. Consistent with the RT-PCR data, TXAS protein levels were time-dependently increased in cell lysates after visfatin treatment (Fig. 1B). Next, we determined the release of TXB2, a stable metabolite product of TXA2, in the culture medium after treatment with visfatin. As shown in Fig. 1C, the incubation of HMECs with visfatin induced the release of TXB2 in a time-dependent fashion. Recently, we and others have shown that visfatin stimulates ERK1/2 or PI3 K/ Akt signaling in human endothelial cells, thereby promoting angiogenic events [6,7]. Therefore, to determine signal transduction mechanisms involved in visfatin-induced TXA2 production, we examined the effects of several specific pharmacologic inhibitors on visfatin-induced TXB2 secretion. As shown in Fig. 1D, the ERK1/2 inhibitor U0126 significantly reduced the visfatin-induced elevation of TXB2 levels in endothelial cells. Thus, visfatin induces the production of TXA2 through activating ERK1/2 in endothelial cells.
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Fig. 1. Effects of visfatin on the expression of TXAS and the biosynthesis of TXA2. HMECs were treated with visfatin (500 ng/ml) for the indicated times. (A) Total RNA was isolated and then analyzed by RT-PCR by using specific primers to human TXAS. b-Actin served as an internal control. (B) Western blots were probed with anti-TXAS antibodies. a-Tubulin served as the loading control. (C) TXA2 synthesis was quantified by the amount of TXB2, which was measured using ELISA. (D) HMECs were preincubated with LY294002 (2 lM), SB203580 (2 lM), and U0126 (2 lM) for 1 h prior to stimulation with visfatin (500 ng/ml). The TXB2 levels in cell supernatants were measured by ELISA. Data are the mean ± SE values relative to the control in triplicate experiments. ⁄P < 0.01, compared to the control; #P < 0.01, compared to visfatin.
3.2. TXA2 production by visfatin mediates IL-8 production in human endothelial cells We recently reported that TXA2 upregulates IL-8 in human vascular endothelial cells [16]. Therefore, we examined whether visfatin could affect TXA2-dependent IL-8 expression in endothelial cells. Treatment of HMECs with visfatin increased IL-8 mRNA expression and protein secretion in a time-dependent manner (Fig. 2A and B). To determine the role of IL-8 in visfatin-induced angiogenesis, we treated endothelial cells with an IL-8 neutralizing antibody or repertaxin, a specific inhibitor of IL-8 receptors. Pretreatment with IL-8 neutralizing antibody decreased visfatininduced tubular formation of endothelial cells (Fig. 2C). Repertaxin
treatment reduced microvessel sprouting in response to visfatin (Fig. 2D). Next, to elucidate the role of TXAS in mediating visfatin-induced IL-8 expression, we examined the effect of CI, an inhibitor of TXAS, on visfatin-induced expression and production of IL-8. As shown in Fig. 3A, CI suppressed visfatin-induced IL-8 gene expression. In addition, preincubation with CI abolished visfatin-induced IL-8 protein secretion (Fig. 3B). To further confirm that TXAS mediates visfatin-induced increase in IL-8 in endothelial cells, we investigated the effect of specific knockdown of TXAS by siRNA in endothelial cells. TXAS siRNA reduced endothelial IL-8 mRNA expression and protein production induced by visfatin (Fig. 3C and D). Taken together, these results indicate that TXAS mediates visfatin-induced production of IL-8 in endothelial cells.
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Fig. 2. The role of IL-8 in visfatin-induced angiogenesis. (A) HMECs were treated with visfatin for the indicated times. Total RNA was isolated and then analyzed by RT-PCR by using primers specific to human IL-8. b-Actin served as an internal control. (B) The amount of IL-8 protein secreted was measured by ELISA. (C) HUVECs were plated onto the surface of the GFR-Matrigel and treated without or with visfatin (500 ng/mL), visfatin + IL-8 neutralizing antibody (1 lg/ml), or IL-8 neutralizing antibody alone. Capillary-like tube formation was observed, and photographs were taken after 8 h (left). The newly formed tube branches and area were quantified from at least 3 individual experiments (right). ⁄P < 0.05, compared to the control; #P < 0.05, compared to visfatin. (D) Rat aortic rings were embedded into Matrigel in a 48-well plate and then treated with visfatin (5 lg/mL) in the presence or absence of Repertaxin. On day 4, microvessel outgrowths were photographed using a phase contrast microscope (left). The degree of microvessel formation was quantified by counting the newly formed microvessels extending from the cultured aortic rings (right). The results shown represent at least 3 independent experiments. ⁄⁄P < 0.01 vs. control; #P < 0.05 vs. visfatin alone.
3.3. TXAS-dependent IL-8 production is required for visfatin- induced angiogenesis To investigate the function of TXAS-dependent IL-8 production in visfatin-induced angiogenesis, we examined the effect of TXAS siRNA on visfatin-induced endothelial angiogenesis in vitro. Knockdown of TXAS with specific siRNA significantly suppressed visfatin-induced endothelial tube-like formation on Matrigel (Fig. 4A). Rat aortic rings were placed in Matrigel and incubated with visfatin, in the presence or absence of CI. As shown in Fig. 4B, CI diminished vessel-sprout formation in rat aortic rings stimulated by visfatin. In addition, total RNA was isolated from microvessels of rat aortic explants; IL-8 mRNA expression was then
measured using RT-PCR. As shown in Fig. 4C, IL-8 mRNA was increased in visfatin-stimulated sprouting vessels in the rat aorta, and this effect was blocked by treatment with CI. We also performed a mouse Matrigel plug assay, an in vivo angiogenesis model. Visfatin-treated Matrigel plugs produced more neovessels than control-treated plugs, but Matrigel plugs containing visfatin plus CI showed less neovascularization (Fig. 4D). The amount of functional blood vessels was quantified by measuring the hemoglobin content in the Matrigel matrixes. Co-treatment of visfatin with CI significantly decreased hemoglobin concentrations in Matrigel implants as compared with plugs treated with visfatin alone (Fig. 4E). For histological analysis, each section of the Matrigel plug was stained with hematoxylin-eosin and anti-CD31 (PECAM-1)
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Fig. 3. Effect of TXAS inhibition on visfatin-induced IL-8 production. (A) HMECs were treated with visfatin (500 ng/ml) for 16 h in the presence or absence of CI (20 lM). Total RNA was isolated and then analyzed by RT-PCR using specific primers to human IL-8. b-Actin served as an internal control. (B) HMECs were incubated with visfatin (500 ng/ ml) for 16 h in the presence or absence of CI (20 lM), and secreted IL-8 protein was analyzed by ELISA. Data are the mean ± SE relative to control (set at 100%) in triplicate experiments. ⁄P < 0.01 compared to control; #P < 0.01 compared to visfatin. (C) HMECs were transfected with TXAS siRNA (200 nM) or control siRNA (200 nM) according to the manufacturer’s instructions. After transfection, cells were treated with visfatin (500 ng/ml) for 16 h before cell harvesting. Total RNA was extracted and subjected to RTPCR analysis using specific primers for human IL-8. TXAS protein level was examined by western blotting by using an anti-TXAS antibody. (D) After siRNA transfection, cell supernatants were collected and quantified using human IL-8 ELISA. Data are the mean ± SE values relative to control (set at 100%) in triplicate experiments. ⁄P < 0.01, compared to visfatin-treated control siRNA.
antibody. The visfatin-induced increase in the vessel staining area was strongly suppressed by CI (Fig. S1). Overall, these results suggest that visfatin promotes angiogenesis through TXAS-dependent production of IL-8 in endothelial cells.
4. Discussion Obesity is a risk factor for cardiovascular diseases and tumor growth, which is accelerated by dysregulated angiogenesis [23]. Fat-derived adipokines, such as leptin, resistin, and adiponectin, are upregulated in obesity; these adipokines regulate multiple angiogenic processes in vascular endothelial cells [24,25]. We and others have demonstrated the angiogenic properties of the adipokine visfatin and its ability to modulate the secretion of angiogenic factors [7–10]. In the present study, our data showed a role for TXA2 in mediating visfatin-induced endothelial production of IL-8 and angiogenesis. Specifically, visfatin upregulated the expression of TXAS and biosynthesis of TXA2 in endothelial cells. Further, visfatin enhanced the expression and secretion of IL-8, and this increase was diminished by blocking TXAS activity. Finally, inhibition of TXAS or IL-8 suppressed visfatin-induced angiogenesis in vitro and in vivo. Prostanoids, including thromboxanes and prostaglandins, are bioactive lipid mediators called eicosanoids, which are metabolic products of arachidonic acid [26]. TXA2 is synthesized from prostaglandin H2 (PGH2) by thromboxane synthase, which is expressed
in different cell types in the blood and vascular wall, such as platelets, vascular smooth muscle cells, and vascular endothelial cells, in response to various physiological and pathological stimuli [11,12,27]. In particular, potent angiogenic factors, VEGF and bFGF, augment the biosynthesis of TXA2 in endothelial cells, and enhanced TXA2 production is involved in VEGF- or bFGF-induced endothelial cell migration [15]. In vascular smooth muscle cells, incubation with TNF-a or inducing hypoxia increases TXAS expression and TXA2 formation by upregulating NADPH oxidase [28]. We previously reported that visfatin induces the generation of reactive oxygen species (ROS) though NADPH oxidase activation in endothelial cells [4], suggesting that ROS derived from visfatin-stimulated NADPH oxidase may influence TXAS expression as well as TXA2 biosynthesis. Overexpression of TXAS and high levels of TXA2 have been observed in a variety of cancers, including prostate cancer, lung cancer, colorectal cancer, glioma, and breast carcinoma [18,19,29–31]. TXAS and its metabolite, TXA2, have diverse roles in regulating tumor-promoting properties, such as tumor cell growth, migration, invasion, metastasis, and angiogenesis [15,32–34]. Considering the stimulatory effect of visfatin on TXAS expression, it is likely that TXA2 acts as a mediator of visfatin-dependent tumor angiogenesis, tumor cell motility, and growth, which leads to the development and progression of TXAS-overexpressing cancers, including breast cancer. It has been reported that an elevation in serum visfatin is associated with postmenopausal breast tumors [35], and overexpression of visfatin was observed in breast cancer tissues [36,37]. Further investigations should be performed to determine the
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Fig. 4. Visfatin induces angiogenesis through TXAS-dependent IL-8 production. (A) HUVECs were transiently transfected with control siRNA or with TXAS siRNA. Following transfection and incubation for 24 h, HUVECs were plated onto the surface of the GFR-Matrigel and treated with or without visfatin (500 ng/mL). Capillary-like tube formation was observed, and photomicrographs were taken under a phase contrast microscope (left). The newly formed tube branches and area were quantified (right). Three independent experiments were performed in duplicate. ⁄P < 0.05, compared to visfatin-treated control siRNA. (B) Rat aortic rings were embedded into Matrigel in 48-well plate and then treated with visfatin (5 lg/ml), in the presence or absence of CI (20 lM) in human endothelial growth medium (200 ll). On day 3, microvessel outgrowth was photographed under a phase contrast microscope (left). The newly formed microvessel formation was quantified by counting the numbers of microvessels extending from the cultured aortic rings (right). (C) Total RNA was isolated from microvessels of rat aortic rings and then analyzed for rat IL-8 mRNA levels by RT-PCR. GAPDH served as an internal control. (D) Matrigel plugs mixed with visfatin (5 lg/ml) in the presence or absence of CI (20 lM) were photographed. (E) The concentration of hemoglobin within the plug was quantified from a parallel assay with a known amount of hemoglobin (right panel). ⁄P < 0.01, compared to control; #P < 0.01, compared to visfatin.
correlation between the expression of visfatin and TXAS and the role of visfatin-TXA2 signaling in breast tumorigenesis. The Human TXAS gene is transcriptionally regulated by binding of p45 NF-E2 or NF-E2 related factor 2 (NRF2) to the NF-E2 site within the human TXAS promoter region [38–40]. In our preliminary study, we found that treatment with visfatin induces nuclear translocation of NRF2 in endothelial cells. Hence, it appears that
NRF2 is involved in visfatin-induced transcriptional activation of the TXAS gene in endothelial cells. It will be interesting to study whether the TXAS promoter contains an NRF2 binding site to which NRF2 binds in response to visfatin. This possibility is under investigation. In conclusion, our findings provide the first evidence of TXASdependent IL-8 upregulation by visfatin in endothelial cells and
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of IL-8’s role in mediating visfatin-induced angiogenesis. Therefore, these results suggest that TXAS expression by visfatin in endothelial cells and the downstream effectors could be promising therapeutic targets for various vascular pathologies. Acknowledgments This work was supported by the research grant from Korea Research Foundation Grant funded by Korea Government (MOEHRD, Basic Research Promotion Fund) (KRF-2008-531-C00050) and Medical Research Institute Grant (2010–21), Pusan National University Hospital. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2012.01.072. References [1] C. Hug, H.F. Lodish, Medicine. Visfatin: a new adipokine, Science 307 (2005) 366–367. [2] B. Samal, Y. Sun, G. Stearns, C. Xie, S. Suggs, I. McNiece, Cloning and characterization of the cDNA encoding a novel human pre-B-cell colonyenhancing factor, Molecular and Cellular Biology 14 (1994) 1431–1437. [3] J.R. Revollo, A. Korner, K.F. Mills, A. Satoh, T. Wang, A. Garten, B. Dasgupta, Y. Sasaki, C. Wolberger, R.R. Townsend, J. Milbrandt, W. Kiess, S. Imai, Nampt/ PBEF/visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme, Cell Metabolism 6 (2007) 363–375. [4] S.R. Kim, Y.H. Bae, S.K. Bae, K.S. Choi, K.H. Yoon, T.H. Koo, H.O. Jang, I. Yun, K.W. Kim, Y.G. Kwon, M.A. Yoo, M.K. Bae, Visfatin enhances ICAM-1 and VCAM-1 expression through ROS-dependent NF-kappaB activation in endothelial cells, Biochimica et Biophysica Acta 1783 (2008) 886–895. [5] A.R. Moschen, A. Kaser, B. Enrich, B. Mosheimer, M. Theurl, H. Niederegger, H. Tilg, Visfatin, an adipocytokine with proinflammatory and immunomodulating properties, Journal of Immunology 178 (2007) 1748–1758. [6] S.R. Kim, S.K. Bae, K.S. Choi, S.Y. Park, H.O. Jun, J.Y. Lee, H.O. Jang, I. Yun, K.H. Yoon, Y.J. Kim, M.A. Yoo, K.W. Kim, M.K. Bae, Visfatin promotes angiogenesis by activation of extracellular signal-regulated kinase 1/2, Biochemical and Biophysical Research Communications 357 (2007) 150–156. [7] R. Adya, B.K. Tan, A. Punn, J. Chen, H.S. Randeva, Visfatin induces human endothelial VEGF and MMP-2/9 production via MAPK and PI3K/Akt signalling pathways: novel insights into visfatin-induced angiogenesis, Cardiovascular Research 78 (2008) 356–365. [8] R. Adya, B.K. Tan, J. Chen, H.S. Randeva, Pre-B cell colony enhancing factor (PBEF)/visfatin induces secretion of MCP-1 in human endothelial cells: role in visfatin-induced angiogenesis, Atherosclerosis 205 (2009) 113–119. [9] Y.H. Bae, M.K. Bae, S.R. Kim, J.H. Lee, H.J. Wee, S.K. Bae, Upregulation of fibroblast growth factor-2 by visfatin that promotes endothelial angiogenesis, Biochemical and Biophysical Research Communications 379 (2009) 206–211. [10] J.Y. Kim, Y.H. Bae, M.K. Bae, S.R. Kim, H.J. Park, H.J. Wee, S.K. Bae, Visfatin through STAT3 activation enhances IL-6 expression that promotes endothelial angiogenesis, Biochimica et Biophysica Acta 1793 (2009) 1759–1767. [11] M.A. Iniguez, C. Cacheiro-Llaguno, N. Cuesta, M.D. Diaz-Munoz, M. Fresno, Prostanoid function and cardiovascular disease, Archives of Physiology and Biochemistry 114 (2008) 201–209. [12] A. Alfranca, M.A. Iniguez, M. Fresno, J.M. Redondo, Prostanoid signal transduction and gene expression in the endothelium: role in cardiovascular diseases, Cardiovascular Research 70 (2006) 446–456. [13] N. Nakahata, Thromboxane A2: physiology/pathophysiology, cellular signal transduction and pharmacology, Pharmacology and Therapeutics 118 (2008) 18–35. [14] T. Ishizuka, M. Kawakami, T. Hidaka, Y. Matsuki, M. Takamizawa, K. Suzuki, A. Kurita, H. Nakamura, Stimulation with thromboxane A2 (TXA2) receptor agonist enhances ICAM-1, VCAM-1 or ELAM-1 expression by human vascular endothelial cells, Clinical and Experimental Immunology 112 (1998) 464–470. [15] D. Nie, M. Lamberti, A. Zacharek, L. Li, K. Szekeres, K. Tang, Y. Chen, K.V. Honn, Thromboxane A(2) regulation of endothelial cell migration, angiogenesis, and tumor metastasis, Biochemical and Biophysical Research Communications 267 (2000) 245–251. [16] S.R. Kim, S.K. Bae, H.J. Park, M.K. Kim, K. Kim, S.Y. Park, H.O. Jang, I. Yun, Y.J. Kim, M.A. Yoo, M.K. Bae, Thromboxane A(2) increases endothelial permeability through upregulation of interleukin-8, Biochemical and Biophysical Research Communications 397 (2010) 413–419.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Upregulation of thromboxane synthase mediates visfatin-induced interleukin-8 expression and angiogenic activity in endothelial cells Su-Ryun Kim a,1, Yun-Hoa Jung b,1, Hyun-Joo Park a,c, Mi-Kyoung Kim a, Joo-Won Jeong d, Hye-Ock Jang c, Il Yun c, Soo-Kyung Bae c, Moon-Kyoung Bae a,⇑ a
Department of Oral Physiology, School of Dentistry, Pusan National University, Yangsan 626-870, South Korea Department of Oral & Maxillofacial Radiology, School of Dentistry, Pusan National University, Yangsan 626-870, South Korea Department of Dental Pharmacology, School of Dentistry, Pusan National University, Yangsan 626-870, South Korea d School of Medicine, Kyung Hee University, Seoul 130-701, South Korea b c
a r t i c l e
i n f o
Article history: Received 7 January 2012 Available online 24 January 2012 Keywords: Visfatin Thromboxane synthase Thromboxane A2 Interleukin-8 Vascular endothelial cells Angiogenesis
a b s t r a c t Thromboxane synthase (TXAS) is an enzyme that catalyzes the synthesis of thromboxane A2 (TXA2). Overexpression of TXAS is associated with a variety of vascular diseases. Recently, we reported that visfatin, a novel adipokine, exhibits angiogenic actions. In this study, we showed that visfatin increased mRNA and protein levels of TXAS and stimulated TXA2 biosynthesis in vascular endothelial cells. In addition, visfatin induced the expression and secretion of interleukin-8 (IL-8), which is blocked by a TXAS inhibitor and by the transfection of siRNA specific for TXAS. Furthermore, the inhibition of TXAS activity and blockade of the IL-8 receptor attenuated visfatin-induced endothelial angiogenesis. Together, these results showed that visfatin promoted IL-8 production by upregulation of TXAS, leading to angiogenic activation in endothelial cells. Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved.
1. Introduction Visfatin is a novel adipokine predominantly produced by visceral adipose tissue [1]. Visfatin was originally identified as a growth factor, called pre-B cell colony enhancing factor (PBEF), for the maturation of B-cell precursors [2]. Both intracellular and extracellular forms of visfatin were known as nicotinamide phosphoribosyltransferase (NAMPT), an enzyme involved in mammalian nicotinamide adenine dinucleotide biosynthesis [3]. Recently, we and others showed that visfatin has proinflammatory activity (e.g., induction of inflammatory cytokines and cell adhesion molecules) in human monocytes and vascular endothelial cells [4,5]. Visfatin also has proangiogenic activity mediated by enhancing the production of soluble factors, such as vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6), in endothelial cells [6–10]. Thromboxane synthase (TXAS) is a downstream enzyme of arachidonic acid metabolism and catalyzes the synthesis of thromboxane A2 (TXA2) [11]. TXA2 was originally known to control platelet aggregation and smooth muscle contraction; more recently, it has been reported to act as a pathophysiological modulator of ⇑ Corresponding author. Fax: +82 51 510 8239. 1
E-mail address: [email protected] (M.-K. Bae). Denotes co-first authors.
endothelial function [12,13]. TXA2 stimulates vascular inflammatory responses by increasing the expression of the endothelial cell adhesion molecules, such as ICAM-1 and VCAM-1, and regulates endothelial angiogenesis and permeability [14–16]. TXAS is highly overexpressed in various types of vascularized solid tumors, and its expressional level is associated with increased tumor microvessel density [17–20]. However, the molecular mechanisms underlying the induction of TXAS expression as well as the functional significance of TXAS signaling in vascular endothelium remain poorly defined. In the present study, we examined the effect of visfatin on TXAS gene expression in vascular endothelial cells and investigated the underlying mechanisms. Our data demonstrate for the first time that visfatin enhances TXAS and its metabolite, TXA2, thus inducing endothelial interleukin-8 (IL-8) production and angiogenesis.
2. Materials and methods 2.1. Reagents and recombinant proteins SB203580, LY294002, and U0126 were purchased from BIOMOL. Carbethoxyhexyl imidazole (CI) was acquired from Enzo Life Sciences. Rabbit polyclonal anti-TXAS and mouse monoclonal anti-PECAM-1 antibodies were obtained from Cayman and BD Biosciences, respectively. Mouse monoclonal anti-a-tubulin
0006-291X/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.01.072
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antibodies were supplied by Biogenex. IL-8 neutralizing antibodies were purchased from R&D systems. Recombinant human visfatin protein was prepared in our laboratory as previously described [4]. Polymyxin B (PMB) and repertaxin were obtained from Sigma.
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with siRNAs using oligofectamine and Amaxa Nucleofector, respectively, according to the manufacturer’s instructions.
2.7. Tube formation assay 2.2. Cell culture Human microvascular endothelial cells (HMECs) were obtained from the CDC (Atlanta, GA). The cells were maintained in MCDB supplemented with 10% FBS (Invitrogen), 1% antibiotics, 10 lM/ ml of l-glutamine, 1 lg/ml of hydrocortisone, and 10 ng/ml of hEGF at 37 °C under a humidified atmosphere containing (vol/vol) 95% air and 5% CO2. Primary human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cord veins by collagenase treatment as described previously [21]. The HUVECs were plated onto a 0.2% gelatin-coated dish and grown in sterile endothelial growth medium (EGM-2, CLONTECTICS). EGM-2 consisted of an endothelial basal medium (EBM-2, CLONTECTICS), trace elements, growth factors, and antibiotics. For cells treated with visfatin, the culture medium was supplemented with an endotoxinneutralizing agent (10 lg/ml polymyxin B). 2.3. RT-PCR Total RNA was isolated from the HMECs using a TRIzol reagent kit (Invitrogen). cDNA synthesis was performed using 3 lg of total RNA with a reverse transcription kit (Promega). The oligonucleotide primers for PCR were designed as follows: b-actin, 50 GACTACCTCATGAAGATC-30 and 50 -GATCCACATCTGCTGGAA-30 ; human TXAS, 50 -AGATGGTTCCCCTCATCAGC-30 and 50 -GCCCACAATCTCAT CCACAG-30 ; human IL-8, 50 -GAAGGTGCAGTTTTGCCAAG-30 and 50 -ACCCTCTGCACCCAGTTTTC-30 . 2.4. Western blot analysis The harvested cells were lysed in a lysis buffer (40 mM Tris-Cl, 10 mM EDTA, 120 mM NaCl, and 0.1% NP-40 with protease inhibitor cocktail [Sigma]). A constant protein concentration (30 lg/lane) was used. The proteins were separated by sodium dodecyl sulfate– polyacrylamide gel electrophoresis and were transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech). The membrane was blocked with 5% skim milk in phosphate-buffered saline (PBS) containing 0.1% Tween-20 for 1 h at room temperature and probed with the appropriate antibodies. This signal was developed using an enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech). 2.5. Enzyme-linked immunosorbent assays The cells were treated with visfatin in the presence or absence of pretreatment with CI or signaling inhibitors. At the end of the treatment, culture media were collected and centrifuged at 14,000 rpm for 5 min. The supernatants were stored in aliquots at 70 °C. Secreted IL-8 protein and TXB2 levels were determined using enzyme-linked immunosorbent assays (ELISAs) with human IL-8 ELISA Max™ Set Deluxe Kits (BioLegend) and thromboxane B2 EIA kit (Cayman Chemical), respectively, according to the manufacturer’s instructions. 2.6. Transient transfection of small interfering RNA Small interfering RNA (siRNA) oligonucleotides against human TXAS (50 - GUUGAGAACUUCAGUAACUG-30 , 50 -CAGUUACUGAAGUU CUCAAC-30 ) were synthesized [22], and negative control siRNA was purchased from Bioneer. HMECs and HUVECs were transfected
Growth factor-reduced (GFR) Matrigel (300 lL) (BD Bioscience) was pipetted into 24-well culture plates and solidified for 30 min at 37 °C. HUVECs were seeded onto the surface of the Matrigel and then treated with visfatin for indicated times at 37 °C. Morphological changes of the endothelial cells were observed under a phase contrast microscope and photographed.
2.8. Rat aortic ring sprouting assay The aorta was excised from 6-week-old male Sprague–Dawley rats, and the fibroadipose tissue was removed. The aorta was sectioned into 1-mm-thick slices; the aortic rings were placed on Matrigel-coated wells, covered with an additional 50 lL of Matrigel, and allowed to gel for 30 min at 37 °C. The aortic rings were cultured with visfatin in the presence or absence of CI. After 3 days, the outgrowth was photographed under a phase contrast microscope.
2.9. In vivo mouse Matrigel plug assay C57/BL6 mice were subcutaneously injected with 0.5 mL of Matrigel containing the indicated amounts of visfatin and heparin (10 units/lL) in presence or absence of CI. After 7 days, the mice were killed, and the Matrigel plugs were recovered, fixed with 4% paraformaldehyde in PBS, and embedded in paraffin. Each plug was fixed, sectioned, and stained with hematoxylin-eosin or immunostained with antibody for PECAM-1, a marker for endothelial cells. The hemoglobin level was measured using the Drabkin method for quantifying blood vessel formation.
3. Results 3.1. Visfatin increases TXAS expression and TXA2 production in human endothelial cells We first investigated whether visfatin could induce the expression of TXAS in endothelial cells. HMECs were incubated with visfatin, and TXAS mRNA levels were analyzed by RT-PCR. As shown in Fig. 1A, visfatin increased the expression of TXAS mRNA in a timedependent manner. The levels of TXAS protein in visfatin-treated HMECs were then analyzed by western blotting. Consistent with the RT-PCR data, TXAS protein levels were time-dependently increased in cell lysates after visfatin treatment (Fig. 1B). Next, we determined the release of TXB2, a stable metabolite product of TXA2, in the culture medium after treatment with visfatin. As shown in Fig. 1C, the incubation of HMECs with visfatin induced the release of TXB2 in a time-dependent fashion. Recently, we and others have shown that visfatin stimulates ERK1/2 or PI3 K/ Akt signaling in human endothelial cells, thereby promoting angiogenic events [6,7]. Therefore, to determine signal transduction mechanisms involved in visfatin-induced TXA2 production, we examined the effects of several specific pharmacologic inhibitors on visfatin-induced TXB2 secretion. As shown in Fig. 1D, the ERK1/2 inhibitor U0126 significantly reduced the visfatin-induced elevation of TXB2 levels in endothelial cells. Thus, visfatin induces the production of TXA2 through activating ERK1/2 in endothelial cells.
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Fig. 1. Effects of visfatin on the expression of TXAS and the biosynthesis of TXA2. HMECs were treated with visfatin (500 ng/ml) for the indicated times. (A) Total RNA was isolated and then analyzed by RT-PCR by using specific primers to human TXAS. b-Actin served as an internal control. (B) Western blots were probed with anti-TXAS antibodies. a-Tubulin served as the loading control. (C) TXA2 synthesis was quantified by the amount of TXB2, which was measured using ELISA. (D) HMECs were preincubated with LY294002 (2 lM), SB203580 (2 lM), and U0126 (2 lM) for 1 h prior to stimulation with visfatin (500 ng/ml). The TXB2 levels in cell supernatants were measured by ELISA. Data are the mean ± SE values relative to the control in triplicate experiments. ⁄P < 0.01, compared to the control; #P < 0.01, compared to visfatin.
3.2. TXA2 production by visfatin mediates IL-8 production in human endothelial cells We recently reported that TXA2 upregulates IL-8 in human vascular endothelial cells [16]. Therefore, we examined whether visfatin could affect TXA2-dependent IL-8 expression in endothelial cells. Treatment of HMECs with visfatin increased IL-8 mRNA expression and protein secretion in a time-dependent manner (Fig. 2A and B). To determine the role of IL-8 in visfatin-induced angiogenesis, we treated endothelial cells with an IL-8 neutralizing antibody or repertaxin, a specific inhibitor of IL-8 receptors. Pretreatment with IL-8 neutralizing antibody decreased visfatininduced tubular formation of endothelial cells (Fig. 2C). Repertaxin
treatment reduced microvessel sprouting in response to visfatin (Fig. 2D). Next, to elucidate the role of TXAS in mediating visfatin-induced IL-8 expression, we examined the effect of CI, an inhibitor of TXAS, on visfatin-induced expression and production of IL-8. As shown in Fig. 3A, CI suppressed visfatin-induced IL-8 gene expression. In addition, preincubation with CI abolished visfatin-induced IL-8 protein secretion (Fig. 3B). To further confirm that TXAS mediates visfatin-induced increase in IL-8 in endothelial cells, we investigated the effect of specific knockdown of TXAS by siRNA in endothelial cells. TXAS siRNA reduced endothelial IL-8 mRNA expression and protein production induced by visfatin (Fig. 3C and D). Taken together, these results indicate that TXAS mediates visfatin-induced production of IL-8 in endothelial cells.
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Fig. 2. The role of IL-8 in visfatin-induced angiogenesis. (A) HMECs were treated with visfatin for the indicated times. Total RNA was isolated and then analyzed by RT-PCR by using primers specific to human IL-8. b-Actin served as an internal control. (B) The amount of IL-8 protein secreted was measured by ELISA. (C) HUVECs were plated onto the surface of the GFR-Matrigel and treated without or with visfatin (500 ng/mL), visfatin + IL-8 neutralizing antibody (1 lg/ml), or IL-8 neutralizing antibody alone. Capillary-like tube formation was observed, and photographs were taken after 8 h (left). The newly formed tube branches and area were quantified from at least 3 individual experiments (right). ⁄P < 0.05, compared to the control; #P < 0.05, compared to visfatin. (D) Rat aortic rings were embedded into Matrigel in a 48-well plate and then treated with visfatin (5 lg/mL) in the presence or absence of Repertaxin. On day 4, microvessel outgrowths were photographed using a phase contrast microscope (left). The degree of microvessel formation was quantified by counting the newly formed microvessels extending from the cultured aortic rings (right). The results shown represent at least 3 independent experiments. ⁄⁄P < 0.01 vs. control; #P < 0.05 vs. visfatin alone.
3.3. TXAS-dependent IL-8 production is required for visfatin- induced angiogenesis To investigate the function of TXAS-dependent IL-8 production in visfatin-induced angiogenesis, we examined the effect of TXAS siRNA on visfatin-induced endothelial angiogenesis in vitro. Knockdown of TXAS with specific siRNA significantly suppressed visfatin-induced endothelial tube-like formation on Matrigel (Fig. 4A). Rat aortic rings were placed in Matrigel and incubated with visfatin, in the presence or absence of CI. As shown in Fig. 4B, CI diminished vessel-sprout formation in rat aortic rings stimulated by visfatin. In addition, total RNA was isolated from microvessels of rat aortic explants; IL-8 mRNA expression was then
measured using RT-PCR. As shown in Fig. 4C, IL-8 mRNA was increased in visfatin-stimulated sprouting vessels in the rat aorta, and this effect was blocked by treatment with CI. We also performed a mouse Matrigel plug assay, an in vivo angiogenesis model. Visfatin-treated Matrigel plugs produced more neovessels than control-treated plugs, but Matrigel plugs containing visfatin plus CI showed less neovascularization (Fig. 4D). The amount of functional blood vessels was quantified by measuring the hemoglobin content in the Matrigel matrixes. Co-treatment of visfatin with CI significantly decreased hemoglobin concentrations in Matrigel implants as compared with plugs treated with visfatin alone (Fig. 4E). For histological analysis, each section of the Matrigel plug was stained with hematoxylin-eosin and anti-CD31 (PECAM-1)
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Fig. 3. Effect of TXAS inhibition on visfatin-induced IL-8 production. (A) HMECs were treated with visfatin (500 ng/ml) for 16 h in the presence or absence of CI (20 lM). Total RNA was isolated and then analyzed by RT-PCR using specific primers to human IL-8. b-Actin served as an internal control. (B) HMECs were incubated with visfatin (500 ng/ ml) for 16 h in the presence or absence of CI (20 lM), and secreted IL-8 protein was analyzed by ELISA. Data are the mean ± SE relative to control (set at 100%) in triplicate experiments. ⁄P < 0.01 compared to control; #P < 0.01 compared to visfatin. (C) HMECs were transfected with TXAS siRNA (200 nM) or control siRNA (200 nM) according to the manufacturer’s instructions. After transfection, cells were treated with visfatin (500 ng/ml) for 16 h before cell harvesting. Total RNA was extracted and subjected to RTPCR analysis using specific primers for human IL-8. TXAS protein level was examined by western blotting by using an anti-TXAS antibody. (D) After siRNA transfection, cell supernatants were collected and quantified using human IL-8 ELISA. Data are the mean ± SE values relative to control (set at 100%) in triplicate experiments. ⁄P < 0.01, compared to visfatin-treated control siRNA.
antibody. The visfatin-induced increase in the vessel staining area was strongly suppressed by CI (Fig. S1). Overall, these results suggest that visfatin promotes angiogenesis through TXAS-dependent production of IL-8 in endothelial cells.
4. Discussion Obesity is a risk factor for cardiovascular diseases and tumor growth, which is accelerated by dysregulated angiogenesis [23]. Fat-derived adipokines, such as leptin, resistin, and adiponectin, are upregulated in obesity; these adipokines regulate multiple angiogenic processes in vascular endothelial cells [24,25]. We and others have demonstrated the angiogenic properties of the adipokine visfatin and its ability to modulate the secretion of angiogenic factors [7–10]. In the present study, our data showed a role for TXA2 in mediating visfatin-induced endothelial production of IL-8 and angiogenesis. Specifically, visfatin upregulated the expression of TXAS and biosynthesis of TXA2 in endothelial cells. Further, visfatin enhanced the expression and secretion of IL-8, and this increase was diminished by blocking TXAS activity. Finally, inhibition of TXAS or IL-8 suppressed visfatin-induced angiogenesis in vitro and in vivo. Prostanoids, including thromboxanes and prostaglandins, are bioactive lipid mediators called eicosanoids, which are metabolic products of arachidonic acid [26]. TXA2 is synthesized from prostaglandin H2 (PGH2) by thromboxane synthase, which is expressed
in different cell types in the blood and vascular wall, such as platelets, vascular smooth muscle cells, and vascular endothelial cells, in response to various physiological and pathological stimuli [11,12,27]. In particular, potent angiogenic factors, VEGF and bFGF, augment the biosynthesis of TXA2 in endothelial cells, and enhanced TXA2 production is involved in VEGF- or bFGF-induced endothelial cell migration [15]. In vascular smooth muscle cells, incubation with TNF-a or inducing hypoxia increases TXAS expression and TXA2 formation by upregulating NADPH oxidase [28]. We previously reported that visfatin induces the generation of reactive oxygen species (ROS) though NADPH oxidase activation in endothelial cells [4], suggesting that ROS derived from visfatin-stimulated NADPH oxidase may influence TXAS expression as well as TXA2 biosynthesis. Overexpression of TXAS and high levels of TXA2 have been observed in a variety of cancers, including prostate cancer, lung cancer, colorectal cancer, glioma, and breast carcinoma [18,19,29–31]. TXAS and its metabolite, TXA2, have diverse roles in regulating tumor-promoting properties, such as tumor cell growth, migration, invasion, metastasis, and angiogenesis [15,32–34]. Considering the stimulatory effect of visfatin on TXAS expression, it is likely that TXA2 acts as a mediator of visfatin-dependent tumor angiogenesis, tumor cell motility, and growth, which leads to the development and progression of TXAS-overexpressing cancers, including breast cancer. It has been reported that an elevation in serum visfatin is associated with postmenopausal breast tumors [35], and overexpression of visfatin was observed in breast cancer tissues [36,37]. Further investigations should be performed to determine the
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Fig. 4. Visfatin induces angiogenesis through TXAS-dependent IL-8 production. (A) HUVECs were transiently transfected with control siRNA or with TXAS siRNA. Following transfection and incubation for 24 h, HUVECs were plated onto the surface of the GFR-Matrigel and treated with or without visfatin (500 ng/mL). Capillary-like tube formation was observed, and photomicrographs were taken under a phase contrast microscope (left). The newly formed tube branches and area were quantified (right). Three independent experiments were performed in duplicate. ⁄P < 0.05, compared to visfatin-treated control siRNA. (B) Rat aortic rings were embedded into Matrigel in 48-well plate and then treated with visfatin (5 lg/ml), in the presence or absence of CI (20 lM) in human endothelial growth medium (200 ll). On day 3, microvessel outgrowth was photographed under a phase contrast microscope (left). The newly formed microvessel formation was quantified by counting the numbers of microvessels extending from the cultured aortic rings (right). (C) Total RNA was isolated from microvessels of rat aortic rings and then analyzed for rat IL-8 mRNA levels by RT-PCR. GAPDH served as an internal control. (D) Matrigel plugs mixed with visfatin (5 lg/ml) in the presence or absence of CI (20 lM) were photographed. (E) The concentration of hemoglobin within the plug was quantified from a parallel assay with a known amount of hemoglobin (right panel). ⁄P < 0.01, compared to control; #P < 0.01, compared to visfatin.
correlation between the expression of visfatin and TXAS and the role of visfatin-TXA2 signaling in breast tumorigenesis. The Human TXAS gene is transcriptionally regulated by binding of p45 NF-E2 or NF-E2 related factor 2 (NRF2) to the NF-E2 site within the human TXAS promoter region [38–40]. In our preliminary study, we found that treatment with visfatin induces nuclear translocation of NRF2 in endothelial cells. Hence, it appears that
NRF2 is involved in visfatin-induced transcriptional activation of the TXAS gene in endothelial cells. It will be interesting to study whether the TXAS promoter contains an NRF2 binding site to which NRF2 binds in response to visfatin. This possibility is under investigation. In conclusion, our findings provide the first evidence of TXASdependent IL-8 upregulation by visfatin in endothelial cells and
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