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Monosialic ganglioside GM3 specifically suppresses the monocyte adhesion to endothelial cells for inflammation
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Short communication
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Monosialic ganglioside GM3 specifically suppresses the monocyte adhesion to endothelial cells for inflammation
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Seok-Jo Kim a,b,1 , Tae-Wook Chung a,1 , Hee-Jung Choi a,c , Un-Ho Jin a , Ki-Tae Ha c , Young-Choon Lee b,∗∗ , Cheorl-Ho Kim a,∗ a Molecular and Cellular Glycobiology Unit, Department of Biological Science, SungKyunKwan University, 300 Chunchun-Dong, Jangan-Gu, Suwon City, Kyunggi-Do 440-746, Republic of Korea b Department of Medicinal Biotechnology, Dong-A University, Saha-Gu, Busan 604-714, Republic of Korea c Division of Applied Medicine, School of Korean Medicine, Pusan National University, Yangsan City, Gyeongsangnam-Do 626-770, Republic of Korea
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Article history: Received 3 June 2013 Received in revised form 10 September 2013 Accepted 28 September 2013 Available online xxx
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Keywords: Cell adhesion molecule Monosialic ganglioside GM3 Vascular endothelial growth factor (VEGF) Q2 Inflammation Monocyte-endothelial adhesion Proinflammatory cytokine
Vascular endothelial growth factor (VEGF) is well known as a significant angiogenic factor, and also functions as a proinflammatory cytokine, which induces adhesion of leukocyte to endothelial cells in inflammation reaction. In this study, we show that ganglioside GM3 inhibits the VEGF-induced expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) through activation of nuclear factor-B (NF-B) via protein kinase B (AKT) signaling in human umbilical vein endothelial cells (HUVECs), relating with leukocyte recruitment to endothelial cells under inflammatory conditions. In addition, ganglioside GM3 significantly reduced the monocyte adhesion to HUVECs as determined by the monolayer cell adhesion assay. Furthermore, in VEGF-injected mice for the inflammatory condition, ganglioside GM3 markedly decreased the expression of ICAM-1 and VCAM-1 in vein tissues. These results suggest that ganglioside GM3 has an anti-inflammatory role by suppressing the expression of inflammatory-related molecules during in vitro and in vivo inflammation. © 2013 Published by Elsevier Ltd.
1. Introduction Inflammatory cytokines including interleukin (IL)-1, IL-6, IL8, transforming growth factor (TGF)- and tumor necrosis factor (TNF)-␣ are released from various inflammatory cells through complex network signaling in inflammation responses (Angelo and Kurzrock, 2007; Wang et al., 2006). These molecules lead to inflammation-related diseases such as cancer progression, cardiovascular disease, diabetic retinopathy, rheumatoid arthritis, retinal ischemia and inflammatory psoriasis by inducing VEGF expression, which is closely linked to angiogenesis (Angelo and Kurzrock, 2007). VEGF upregulated during inflammatory reaction activates endothelial cells to induce expression of cell adhesion molecules including intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), resulting in recruitment of leukocytes at inflammation site to further activate inflammation response (Angelo and Kurzrock, 2007; Kim et al., 2001a,b; Zittermann and Issekutz, 2006). The expression of VCAM-1 and
ICAM-1 is regulated by NF-B activation via PI3K/Akt signal transduction in endothelial cells during inflammatory reaction (De Martin et al., 2000; Kim et al., 2001a). Gangliosides are sialic acid-complex glycosphingolipids located on mammalian cell membranes (Hakomori, 1990). Gangliosides also participate in regulation for various cellular events of several biologic processes on cell proliferation, adhesion, cell–cell or cell–matrix interactions, differentiation, tumorigenesis and metastasis (Hakomori, 2002; Hakomori and Igarashi, 1995; Varki, 1993). However, the role of gangliosides on leukocyte recruitment to endothelial cells for inflammation remains unclear. In this study, we found that ganglioside GM3 inhibits the expression of ICAM-1 and VCAM-1 in VEGF-exposure endothelial cells and VEGF-injected mice, thereby resulting in reduction of monocyte adhesion to endothelial cells stimulated by VEGF. Here we show the antiinflammatory role of ganglioside GM3 during in vitro and in vivo inflammation.
2. Results and discussion ∗ Corresponding author. Tel.: +82 31 290 7002; fax: +82 31 290 7015. ∗ ∗ Corresponding author. Tel.: +82 51 200 7591. E-mail addresses: [email protected] (Y.-C. Lee), [email protected] (C.-H. Kim). 1 These authors contributed equally to this study.
Gangliosides are composed of various types in the regulation of carbohydrate biosynthesis such as a-series: gangliosides GM3, GM2, GM1 and GD1a; b-series: gangliosides GD3, GD2 and
1357-2725/$ – see front matter © 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.biocel.2013.09.015
Please cite this article in press as: Kim S-J, et al. Monosialic ganglioside GM3 specifically suppresses the monocyte adhesion to endothelial cells for inflammation. Int J Biochem Cell Biol (2013), http://dx.doi.org/10.1016/j.biocel.2013.09.015
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Fig. 1. Inhibition of ICAM-1 and VCAM-1 expression by ganglioside GM3 in VEGF-induced HUVECs. (A) After starvation for 6 h, HUVECs were incubated with the various gangliosides and lactosylceramide (30 M) for 1 h and treated with VEGF (50 ng/mL) for 6 h. To investigate mRNA expression for ICAM-1 and VCAM-1, RT-PCR was performed using specific primers of ICAM-1 and VCAM-1. Data obtained by densitometric analyses are shown as bar graph. Relative ratio of each molecule was calculated with -actin level. # P < 0.05 vs. the untreated control. *P < 0.05 vs. the VEGF-treated control. (B and C) After starvation for 6 h, HUVECs were incubated with the indicated concentration of ganglioside GM3 for 1 h and induced with VEGF (50 ng/mL) for 6 h. To investigate mRNA expression for ICAM-1 and VCAM-1, RT-PCR was performed using specific primers of ICAM-1 and VCAM-1 (B) and Western blot was performed with the specific antibodies for ICAM-1 and VCAM-1 (C). -Actin (B) and GAPDH (C) were used as a control. (D) For immunofluorescence analysis of ICAM-1 and VCAM-1 expression, HUVECs were pre-incubated with GM3 (30 M) for 1 h and induced with VEGF (50 ng/mL) for 6 h. The cells were immunostained as described in Section 3.
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GD1b; neutral glycosphingolipid: lactosylceramide (van Echten and Sandhoff, 1993). Some gangliosides including GM3, GM1, GD1a and GD3, and lactosylceramide are known to regulate the receptor functions on membrane (Chung et al., 2009; Liu et al., 2006). VEGF, as a potent pro-inflammatory cytokine as well as angiogenic stimulator, plays a key role during inflammation by stimulating the expression of cellular adhesion molecules such as ICAM-1 and VCAM-1 through activation of NF-B in HUVECs (Kim et al., 2001a; Min et al., 2005; Yancopoulos et al., 2000). However, explanation on relationship between gangliosides and expression of ICAM-1 and VCAM-1 for anti-inflammatory function remains unclear. In order to examine whether the gangliosides regulate the VEGFinduced inflammation or not, the expression levels of ICAM-1 and
VCAM-1 induced by VEGF have been assessed in HUVECs. Interestingly, the expression level of ICAM-1 and VCAM-1 was specifically down-regulated by GM3 only when various gangliosides including GM3, GM2, GM1, GD1a, GD3, GD2 and GD1b and neutral glycosphingolipid were treated in VEGF-induced HUVECs. The fact that GM3 is specific for the down-regulation of ICAM-1 and VCAM-1 expressions was very remarkable. Then, the effect of ganglioside GM3 on expression of ICAM-1 and VCAM-1 has been further checked in VEGF-induced HUVECs. The increased mRNA and protein levels of ICAM-1 and VCAM-1 by VEGF were significantly suppressed by co-treatment of VEGF with GM3 in a dose-dependent manner (Fig. 1B and C). When the immunofluorescence levels of ICAM-1 and VCAM-1 expression in the HUVECs were examined, the data
Please cite this article in press as: Kim S-J, et al. Monosialic ganglioside GM3 specifically suppresses the monocyte adhesion to endothelial cells for inflammation. Int J Biochem Cell Biol (2013), http://dx.doi.org/10.1016/j.biocel.2013.09.015
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has clearly shown that ganglioside GM3 apparently blocked the expression of ICAM-1 and VCAM-1 in the VEGF-stimulated HUVECs (Fig. 1D). It has been reported that ganglioside GD1a enhances proliferation and migration of HUVECs induced at low concentration of VEGF, and lowers the threshold for VEGF-mediated signaling (Lang et al., 2001; Liu et al., 2006). Thus, we have also examined whether gangliosides have an effect on ICAM-1 and VCAM-1 expression in HUVEC stimulated at low concentration (5 ng/mL) of VEGF. After starvation for 6 h, HUVECs were incubated with the various gangliosides and lactosylceramide (30 M) for 1 h and treated with VEGF (5 ng/mL) for 6 h. RT-PCR was performed to check the levels of mRNA expression for ICAM-1 and VCAM-1. Just we could get the same data like Fig. 1A. On the other hand, usually, Ladish group treated HUVECs with GD1a in the presence or absence of VEGF for 24 h to check whether GD1a has any effect on the enhancement of proliferation or migration in HUVECs with or without VEGF (Lang et al., 2001; Liu et al., 2006). Thus, we also rechecked whether ganglioside GM3 and GD1a have effect on ICAM-1 and VCAM-1 expression in HUVEC stimulated at low concentration (5 ng/mL) of VEGF. After starvation for 6 h, HUVECs were incubated with ganglioside GM3 or GD1a (30 M) for 1 h and treated with VEGF (5 ng/mL) for 24 h. Ganglioside GM3 clearly decreased expression of ICAM-1 and VCAM-1 induced by 5 ng/mL VEGF in HUVEC. In contrast, ganglioside GD1a affected the enhanced expression of ICAM-1 and VCAM-1 in 5 ng/mL VEGF-treated HUVECs (data not shown). These results indicate that ganglioside GM3 significantly inhibits the expression of ICAM-1 and VCAM-1 in endothelial cells, which are known to directly relate to inflammatory reaction, in in vitro inflammatory system using VEGF as a mediator. As ganglioside GM3 inhibits the mRNA level of ICAM-1 and VCAM-1 in VEGF-mediated endothelial cells (Fig. 1A and B), the transcription activities of the ICAM-1 and VCAM-1 promoters (Min et al., 2005) using luciferase reporter assay system has been analyzed. As shown in Fig. 2A and B, the transcriptional activities of the ICAM-I and VCAM-1 promoters including NF-B binding sites were significantly increased when HUVECs were treated with VEGF as compared with control. Upon GM3 treatment, the activities of the ICAM-1 promoters including NF-B binding sites were significantly inhibited in VEGF-treated HUVECs, as compared with only VEGF (Fig. 2A). As expected similarly, GM3 inhibited the transcriptional activities of the VCAM-1 promoters including NF-B binding sites in VEGF-treated HUVECs too (Fig. 2B). During inflammation, the increased expressions of ICAM-1 and VCAM-1 mRNAs are mainly regulated by activation of the transcriptional factor NF-B through AKT/IB-␣ signal pathway in HUVECs (Kim et al., 2001a,b; Marumo et al., 1999; Meng et al., 2002). Therefore, we checked whether ganglioside GM3 is involved in the expression of ICAM-1 and VCAM-1 via AKT/IB-␣/NF-B pathway in VEGF-induced inflammatory condition using Western blot analysis and EMSA. As shown in Fig. 2C, the increased levels of the VEGF-induced AKT and IB-␣ phosphorylation were significantly suppressed by GM3 in HUVECs, while normal forms of AKT and IB-␣ were not changed. We next checked whether the activated NF-B as a transcription factor is inhibited by GM3 in VEGF-induced HUVECs using Western blot analysis and EMSA. VEGF-stimulated translocation of NF-B to nucleus was clearly inhibited by GM3 in HUVECs as evidenced by Western blot analysis (Fig. 2D). Furthermore, electromobility shift data showed that GM3 clearly inhibited electromobility shift complex between double-stranded 32 P-labeled oligo of NF-B and the nuclear lysates from VEGF-treated HUVECs (Fig. 2E). These results clearly suggest that ganglioside GM3 inhibits the transcriptional regulation of the ICAM-1 and VCAM-1 genes by blocking NF-B activation and the related phosphorylation factors such as AKT and IB-␣ in HUVECs. In vascular inflammation, the induction of adhesion molecules including ICAM-1 and VCAM-1 brings out leukocyte adhesion to
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endothelial cells as a critical step in in vivo and in vitro models (Goda et al., 2000; Kim et al., 2001b; Min et al., 2005; Thomas-Ecker et al., 2007). Therefore, we examined whether ganglioside GM3 inhibits monocyte adhesion to endothelial cells using a monolayer cell adhesion assay. As shown in Fig. 3, VEGF highly induced the adhesiveness of human THP-1 monocyte to confluent HUVECs induced by VEGF, compared with control or ganglioside GM3 (30 M) alone. The monocyte adhesion to VEGF-induced HUVECs, however, was reduced by GM3 (Fig. 3). These results clearly indicate that ganglioside GM3 suppresses the monocyte adhesion to endothelial cells during VEGF-mediated inflammation. Miklavcic et al. (2012) have reported that low level of GM3 was associated with higher production of proinflammatory signals. It has been also shown that GM3 deficiency exacerbated inflammatory arthritis in the mouse model of rheumatoid arthritis (Tsukuda et al., 2012). Furthermore, our data showed that ganglioside GM3 suppressed VEGF-mediated vascular inflammation through the reduction of the monocyte adhesion to HUVECs by decreasing expression of ICAM-1 and VCAM-1 induced by VEGF in HUVECs. These results suggest that some inflammatory conditions may be negatively regulated by ganglioside GM3. On the other hand, Gracheva et al. have showed that activity of GM3 synthase and ganglioside GM3 product in mononuclear cells and monocytes from atherosclerotic patients was higher than those from healthy donors. The adhesion of ganglioside GM3-treated monocytes to the HUVEC cells was also increased compared to ganglioside GM3untreated monocytes through the induction of CD11b, known as one of ligands for ICAM-1, on ganglioside GM3-stimulated monocytes (Gracheva et al., 2007). Based on these results and reports, difference in amount of ganglioside GM3 content on the several inflammatory conditions or on inflammatory microenvironment with inflammatory cytokine such as VEGF might be represented as a positive or negative regulator for the inflammatory homeostasis. Inflammation exhibits the expression of various molecules related to angiogenic and inflammatory cytokines such as of IL1, IL-6, IL-8, TGF-, TNF-␣ and VEGF (Angelo and Kurzrock, 2007; Wang et al., 2006). These cytokines induce the tumor growth, angiogenesis, inflammation and metastasis (Angelo and Kurzrock, 2007). Among these cytokines, VEGF acts as a growth factor regulating the physiology and pathology of the vascular system such as angiogenesis, inflammation and vascular injury (Detmar et al., 1998; Ferrara and Davis-Smyth, 1997). In angiogenesis, the VEGFmediated activation of the vascular endothelial cell regulates the proliferation, migration and tube formation which need the formation of a new tiny capillaries from blood vessels (Ferrara and Davis-Smyth, 1997). During inflammatory reaction, the activation of VEGF-stimulated endothelial cells induces leukocyte adhesion and infiltration in vivo and in vitro through the increased expression of ICAM-1 and VCAM-1 (Kim et al., 2001a,b; Min et al., 2005). Furthermore, TNF-␣ also induces leukocyte recruitment to endothelial cells by inducing expression of ICAM-1 and VCAM-1 on endothelial cells like VEGF (Chen et al., 1999; Mackay et al., 1993). From the above results, our in vitro experiments have clearly shown that ganglioside GM3 suppresses the VEGF-mediated expression of ICAM-1 and VCAM-1 and leukocyte recruitment in HUVECs. Therefore, to further investigate whether the ganglioside GM3 regulates the expression of pro-inflammatory mediators in vivo, we injected the VEGF or VEGF/TNF-␣ into mice using a method of low-pressure tail vein (LTV) injection into the BALB/c mice (Sebestyen et al., 2006; Zittermann and Issekutz, 2006). As shown in Fig. 4A and B, the mRNA levels of ICAM-1 and VCAM-1 were induced in mice vein tissues of the VEGF-treated group. In addition, the co-treatment with VEGF and TNF-␣ showed the increased expression levels of the molecules, compared with VEGF treatment group only (Fig. 4A and B). However, interestingly, the expression levels of the CAMs when induced by VEGF and VEGF/TNF-␣ were significantly decreased
Please cite this article in press as: Kim S-J, et al. Monosialic ganglioside GM3 specifically suppresses the monocyte adhesion to endothelial cells for inflammation. Int J Biochem Cell Biol (2013), http://dx.doi.org/10.1016/j.biocel.2013.09.015
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Fig. 2. Inhibition of transcriptional activity of the CAMs genes and NF-B activation via AKT/IB-␣ pathway by ganglioside GM3 in VEGF-induced HUVECs. (A and B) After transfection with each promoter, HUVECs were pre-incubated with GM3 (30 M) for 1 h and incubated with VEGF (50 ng/mL) for 6 h. Luciferase activity from the cells was analyzed as described in Section 3. Relative luciferase activity was normalized with the activity of pCMV-gal plasmid. The data represents the mean ± SD for triplicate independent experiments in control untreated cells. # P < 0.05 vs. the untreated control. *P < 0.05 vs. the VEGF-treated control. HUVECs were pre-incubated with GM3 (30 M) for 1 h and incubated with VEGF (50 ng/mL) for 6 h. (C) Western blot was determinated with the specific antibodies for phospho-AKT, AKT, phospho-IB-␣ and IB-␣. GAPDH was used as a control. (D) The nuclear extracts of the HUVECs were prepared as described in Section 3. After separation of the nuclear extracts, Western blot was performed with the specific antibodies for NF-B p65 and hnRNP1 used as a control. (E) The NF-B binding activity from nuclear extracts was assayed by EMSA as described in Section 3. N.C. indicated negative control; S.C, specific competitor used as 100-fold of non-labeled NF-B oligonucleotide.
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in the mice injected with ganglioside GM3 (Fig. 4A and B). These results suggest that ganglioside GM3 suppresses the expression of inflammatory-related molecules during the in vivo inflammation. As described, the present study is representative of the artificial treatment of the ganglioside GM3 to the proinflammatory conditions constructed with the inflammatory cells such as endothelial cells and monocytes, and the biologically regulating factors such as VEGF or TNF-␣. Then the present finding raises if the GM3 is indeed
one of the positive regulators for the in vivo anti-inflammatory homeostasis, how the GM3 is produced for the homeostasis during the in vivo inflammatory conditions such as infection, cancer, atherosclerosis, or any other diseases status. For the GM3 biosynthesis in vertebrates, the microenvironmental distribution of the GM3-expressing cells such as immune cells is suggested to be crucial (Kiguchi et al., 1990). Therefore, our further study will be focused on the issue in animals.
Please cite this article in press as: Kim S-J, et al. Monosialic ganglioside GM3 specifically suppresses the monocyte adhesion to endothelial cells for inflammation. Int J Biochem Cell Biol (2013), http://dx.doi.org/10.1016/j.biocel.2013.09.015
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Fig. 3. Inhibition of leukocyte (THP-1) adhesiveness on VEGF-induced HUVECs by ganglioside GM3. (A) After starvation for 6 h, the confluent monolayers of HUVECs were pre-incubated with ganglioside GM3 (30 M) for 1 h and induced with VEGF (50 ng/mL) for 6 h. Then, leukocyte (THP-1) adhesion to HUVECs as described in Section 3. Phase-contrast microscopic pictures of representative experiments were shown. (B) Quantification of the leukocyte adhesion to HUVECs was shown. The ratio of THP-1 cells adhering to the HUVEC was counted and calculated. Represent data was mean ± SD relative to adhesion ratio of control cells from the results of triplicate experiments. # P < 0.05 vs. the untreated control. *P < 0.05 vs. the VEGF-treated control.
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In conclusion, we show that GM3 significantly suppresses the leukocyte adhesion to the endothelial cells by regulating expression of ICAM-1 and VCAM-1 in endothelium induced by inflammatory cytokines, and might also be advantageous in therapy of various inflammation linked diseases.
3. Materials and methods
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Ganglioside GM3, GM2 and GD2 were purchased from Alexsis (San Diego, CA). Lactosylceramide, GM1, GD3, GD1a and GD1b were purchased from Calbiochem (San Diego, CA). VEGF and TNF-␣ were obtained from R&D Systems (Minneapolis, MN). The antibodies to ICAM-1, VCAM-1, p-AKT, AKT, p-IB␣, COX-2, p65 subunit of NF-B and hnRNP A1, and anti-rabbit, anti-goat and anti-mouse IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz Biotechnology, CA). Anti-GAPDH antibody was obtained from Chemicon, Inc. (Temecula, CA). Alexa Flour 488-conjugated goat anti-rabbit IgG were obtained from Molecular Probes (Carlsbad, CA). 3.2. Cell culture HUVECs, obtained from Cambrex Bio Science (MD, USA), were cultured in sterile endothelial cell growth medium (EGM-2, Cambrex Bio Science) and were maintained as described previously (Chung et al., 2009). 3.3. Reverse transcription polymerase chain reaction (RT-PCR) analysis
Fig. 4. Inhibition of the VEGF and VEGF/TNF-␣-induced expression of CAMs by ganglioside GM3 in in vivo experiments. After pre-injection of ganglioside GM3 (0.1 mg/kg or 1 mg/kg) for 30 min, the BALB/c mice (n = 6) were injected with VEGF (50 ng/mL) or VEGF/TNF-␣ for 4 h as described in Section 3. (A and B) To investigate mRNA expression for ICAM-1 and VCAM-1, RT-PCR was performed using specific primers of mICAM-1 and mVCAM-1. -Actin was used as a control. Data obtained from densitometric analyses are shown in (B). Relative ratio of each molecule is calculated with GAPDH level. # P < 0.05 and ## P < 0.05 vs. the untreated control. *P < 0.05 vs. the VEGF-treated control. **P < 0.05 vs. the VEGF and TNF-␣-treated control.
Total RNAs from cells and tissues were extracted using the Corezol reagent (Corebio Co., Seoul, Republic of Korea). cDNAs were synthesized by RT-PCR kit (Bioneer Co., Daejon, Republic of Korea) according to the manufacturer’s RT-PCR protocol and amplified by specific primers: human VCAM-1, 5-GATACAACCGTCTTGGTCAGCCC3 (sense), and 5-CGCATCCTTCAACTGGCCTT-3 (antisense); human ICAM-1, 5-CAGTGACCATCTACAGCTTTCCGG-3 (sense), 5-GCTGCTACCACAGTGATGAT-GACAA-3 (antisense); and human -actin, 5 -CA AGAGATGGCCACGGCTGCT-3 (sense)
Please cite this article in press as: Kim S-J, et al. Monosialic ganglioside GM3 specifically suppresses the monocyte adhesion to endothelial cells for inflammation. Int J Biochem Cell Biol (2013), http://dx.doi.org/10.1016/j.biocel.2013.09.015
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and 5 -TCCTTCTGCATCCTGTCGGCA-3 (antisense), mouse VCAM-1, 5-GGCTCTGGGAAGCTGGAACGAAG-3 (sense), and 5-ACTCCAGAGTCTTCCATCCTCATAGC-3 (antisense); mouse 5-CACCCCAAGGACCCCAAGGAGAT-3 (sense), and ICAM-1, 5-CGACGCCGCTCAGAAGAACCAC-3 (antisense); mouse GAPDH, 5 -GCCAAGGTCATCCATGACAAC-3 (sense) and 5 GTCCACCACCCTGTTGCTGTA-3 (antisense). For the internal standard of each mRNA in the RT-PCR assay, the expression levels of -actin and GAPDH were measured (Kang et al., 2008). The PCR products were visualized by electrophoresis on 1.5% agarose containing ethidium bromide with 1× Tris–acetate–EDTA (TAE) buffer.
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3.4. Western blot analysis
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Western blot analysis was performed as described previously (Kim et al., 2006a; Song et al., 2010). Briefly, 30 g of total proteins from HUVECs exposed to various concentrations of ganglioside GM3 with or without VEGF were separated by SDS-PAGE and electro-transferred to POTRAN nitrocellulose transfer membranes (Whatman GmbH, Germany). The nitrocellulose transfer membranes blocked by blocking buffer were incubated with ICAM-1, VCAM-1, p-AKT, AKT, p-IB␣, p65 subunit of NF-B, hnRNP A1 and GAPDH antibodies, respectively. The signals were visualized using the ECL chemiluminescence system (Amersham Biosciences, UK) with anti-rabbit, anti-goat and anti-mouse IgG antibodies linked with the horseradish peroxidase.
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3.5. Immunofluorescence microscopy
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Immunostaining with fluorescence was performed as described previously (Chung et al., 2009). Briefly, the treated HUVECs spreaded on gelatin-coated 12 mm coverslips in 24-well tissue culture plates were fixed in 3.7% formaldehyde and washed with PBS. Non-specific sites of the slide were blocked with 1% bovine serum albumin (BSA) for 1 h and incubated with ICAM-1 and VCAM-1 antibodies diluted 1:100 in phosphate-buffered saline (PBS) containing 1% BSA at 4 ◦ C for overnight. Then, the cells were washed and incubated with Alexa Flour 488-conjugated goat anti-rabbit IgG (1:500) at room temperature. After 1 h, the cells were washed with PBS, and then analyzed using a fluorescence microscope (Nikon).
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3.6. Transfection and luciferase assay
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The constructed plasmid for each promoter of ICAM-1 and VCAM-1 was provided from Dr. Kwon laboratory (Min et al., 2005). Luciferase assay was performed as described previously (Kim et al., 2006b). Cells were co-transfected with 0.5 pmol of each promoterluciferase reporter constructs and 1 g of pCMV--galactosidase plasmid by the Welfect method (Welgene Co., Daegu, Republic of Korea) for 24 h. After transfection, cells were treated with various concentrations of ganglioside GM3 with or without VEGF (50 ng/mL). Luciferase activity and -galactosidase activity were performed by using the luciferase and -galactosidase enzyme assay system (Promega, MD, USA). Luciferase activity was normalized with the -galactosidase activity.
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3.7. Electrophoretic gel mobility shift analysis (EMSA)
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The nuclear proteins of each cell were extracted as described previously (Choi et al., 2008). NF-B–DNA binding activities were measured in a gel shift assay system kit (Promega). Briefly, doublestranded oligonucleotides containing the consensus sequence for NF-B (5-AGTTGAGGGGACTTTCCCAGGC-3) were end-labeled with [␥-32 P]ATP using T4 polynucleotide kinase and used as probes for EMSA. Nuclear extract proteins (2 g) of each sample were
pre-incubated with the gel shift-binding buffer [4% glycerol, 1 mM MgCl2 , 0.5 mM ethylenediamine tetra-acetic acid (EDTA), 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris–HCl (pH 7.5), and 0.05 mg/mL poly(deoxyinosine-deoxycytosine)]. After 10 min, each sample was incubated with the labeled probe for 20 min at room temperature. Each sample was separated on a 4% nondenaturing polyacrylamide gel in a 0.5× TBE buffer. After running, the gel was dried and detected by autoradiography. 3.8. Monocyte adhesion assay HUVECs were spread on 2% gelatin-coated 24-well plates and treated various concentration of ganglioside GM3 with or without VEGF (50 ng/mL) for 6 h. Then, human THP-1 cells (5 × 104 cells/200 L/well) were added onto the confluent monolayers of HUVECs and incubated for 20 min at 37 ◦ C with rotation at 120 rpm, and washed out 3 times with PBS removing the unattached cells. Under a microscope, the number of attached cells was counted. 3.9. Mice and VEGF-induced inflammatory reaction in mice using low-pressure tail vein injections Female BALB/c mice (8 weeks old) were used in the experiment. 0.1 mg/kg or 1 mg/kg of ganglioside GM3 dissolved in 50 L of PBS with 5 g heparin was pre-injected into the mice tail vein using a 30 G 1/2 needle (Becton Dickinson, Franklin lakes, NJ). After 30 min, 50 ng/50 L VEGF or VEGF and TNF-␣ dissolved in PBS with 5 g heparin were injected into the mice tail vein for 4 h. Each reagent was injected by a method for low-pressure tail vein injections (Sebestyen et al., 2006). Then, the vein tissues were surgically isolated from the mice. 3.10. Statistical analyses Data were statistically presented as mean ± SD and all experiments were repeated at least three times. The statistical comparison between experimental groups was compared using a standard ANOVA followed by the Student’s t-test. Authors’ contribution S.J.K., T.W.C., Y.C.L. and C.H.K. designed research; S.J.K., T.W.C., H.J.C. and U.H.J. performed research; S.J.K., T.W.C., H.J.C., U.H.J., K.T.H., Y.C.L. and C.H.K. analyzed data; S.J.K., T.W.C., Y.C.L. and C.H.K. wrote paper. Acknowledgments This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST), Korea (Grant No. 2011-0008546). References Angelo LS, Kurzrock R. Vascular endothelial growth factor and its relationship to inflammatory mediators. Clin Cancer Res 2007;13:2825–30. Chen KH, Reece LM, Leary JF. Mitochondrial glutathione modulates TNF-alphainduced endothelial cell dysfunction. Free Radic Biol Med 1999;27:100–9. Choi HJ, Chung TW, Kim SJ, Cho SY, Lee YS, Lee YC, et al. The AP-2alpha transcription factor is required for the ganglioside GM3-stimulated transcriptional regulation of a PTEN gene. Glycobiology 2008;18:395–407. Chung TW, Kim SJ, Choi HJ, Kim KJ, Kim MJ, Kim SH, et al. Ganglioside GM3 inhibits VEGF/VEGFR-2-mediated angiogenesis: direct interaction of GM3 with VEGFR2. Glycobiology 2009;19:229–39. De Martin R, Hoeth M, Hofer-Warbinek R, Schmid JA. The transcription factor NFkappa B and the regulation of vascular cell function. Arterioscler Thromb Vasc Biol 2000;20:E83–8.
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Detmar M, Brown LF, Schon MP, Elicker BM, Velasco P, Richard L, et al. Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice. J Invest Dermatol 1998;111:1–6. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997;18:4–25. Goda S, Imai T, Yoshie O, Yoneda O, Inoue H, Nagano Y, et al. CX3Cchemokine, fractalkine-enhanced adhesion of THP-1 cells to endothelial cells through integrin-dependent and -independent mechanisms. J Immunol 2000;164:4313–20. Gracheva EV, Samovilova NN, Golovanova NK, Andreeva ER, Andrianova IV, Tararak EM, et al. Activation of ganglioside GM3 biosynthesis in human monocyte/macrophages during culturing in vitro. Biochemistry (Mosc) 2007;72:772–7. Hakomori S. Bifunctional role of glycosphingolipids. Modulators for transmembrane signaling and mediators for cellular interactions. J Biol Chem 1990;265:18713–6. Hakomori S. Glycosylation defining cancer malignancy: new wine in an old bottle. Proc Natl Acad Sci U S A 2002;99:10231–3. Hakomori S, Igarashi Y. Functional role of glycosphingolipids in cell recognition and signaling. J Biochem 1995;118:1091–103. Kang SK, Kim YS, Kong YJ, Song KH, Chang YC, Park YG, et al. Disialoganglioside GD3 synthase expression recruits membrane transglutaminase 2 during erythroid differentiation of the human chronic myelogenous leukemia K562 cells. Proteomics 2008;8:3317–28. Kiguchi K, Henning-Chubb CB, Huberman E. Glycosphingolipid patterns of peripheral blood lymphocytes, monocytes, and granulocytes are cell specific. J Biochem 1990;107:8–14. Kim I, Moon SO, Kim SH, Kim HJ, Koh YS, Koh GY. Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-kappa B activation in endothelial cells. J Biol Chem 2001a;276:7614–20. Kim I, Moon SO, Park SK, Chae SW, Koh GY. Angiopoietin-1 reduces VEGF-stimulated leukocyte adhesion to endothelial cells by reducing ICAM-1, VCAM-1, and Eselectin expression. Circ Res 2001b;89:477–9. Kim SJ, Choi HJ, Jin UH, Lee YC, Kim CH. Transcriptional regulation of the human Sia-alpha2,3-Gal-beta1,4-GlcNAc-R:alpha2,8-sialyltransferase (hST8Sia III) by retinoic acid in human glioblastoma tumor cell line. Biochim Biophys Acta 2006a;1759:451–7. Kim SJ, Chung TW, Jin UH, Suh SJ, Lee YC, Kim CH. Molecular mechanisms involved in transcriptional activation of the human Sia-alpha2,3-Gal-beta1,4GlcNAc-R:alpha2,8-sialyltransferase (hST8Sia III) gene induced by KCl in human glioblastoma cells. Biochem Biophys Res Commun 2006b;344:1057–64. Lang Z, Guerrera M, Li R, Ladisch S. Ganglioside GD1a enhances VEGF-induced endothelial cell proliferation and migration. Biochem Biophys Res Commun 2001;282:1031–7.
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Liu Y, McCarthy J, Ladisch S. Membrane ganglioside enrichment lowers the threshold for vascular endothelial cell angiogenic signaling. Cancer Res 2006;66:10408–14. Mackay F, Loetscher H, Stueber D, Gehr G, Lesslauer W. Tumor necrosis factor alpha (TNF-alpha)-induced cell adhesion to human endothelial cells is under dominant control of one TNF receptor type, TNF-R55. J Exp Med 1993;177:1277–86. Marumo T, Schini-Kerth VB, Busse R. Vascular endothelial growth factor activates nuclear factor-kappaB and induces monocyte chemoattractant protein-1 in bovine retinal endothelial cells. Diabetes 1999;48:1131–7. Meng F, Liu L, Chin PC, D‘Mello SR. Akt is a downstream target of NF-kappa B. J Biol Chem 2002;277:29674–80. Miklavcic JJ, Schnabl KL, Mazurak VC, Thomson AB, Clandinin MT. Dietary ganglioside reduces proinflammatory signaling in the intestine. J Nutr Metab 2012;2012:280286. Min JK, Lee YM, Kim JH, Kim YM, Kim SW, Lee SY, et al. Hepatocyte growth factor suppresses vascular endothelial growth factor-induced expression of endothelial ICAM-1 and VCAM-1 by inhibiting the nuclear factor-kappaB pathway. Circ Res 2005;96:300–7. Sebestyen MG, Budker VG, Budker T, Subbotin VM, Zhang G, Monahan SD, et al. Mechanism of plasmid delivery by hydrodynamic tail vein injection. I. Hepatocyte uptake of various molecules. J Gene Med 2006;8:852–73. Song KH, Kang YJ, Jin UH, Park YI, Kim SM, Seong HH, et al. Cloning and functional characterization of pig CMP-N-acetylneuraminic acid hydroxylase(pCMAH) for the synthesis of N-glycolylneuraminic acid as the xenoantigenic determinant in pig-to-human xenotransplantation. Biochem J 2010;427:179–88. Thomas-Ecker S, Lindecke A, Hatzmann W, Kaltschmidt C, Zanker KS, Dittmar T. Alteration in the gene expression pattern of primary monocytes after adhesion to endothelial cells. Proc Natl Acad Sci U S A 2007;104:5539–44. Tsukuda Y, Iwasaki N, Seito N, Kanayama M, Fujitani N, Shinohara Y, et al. Ganglioside GM3 has an essential role in the pathogenesis and progression of rheumatoid arthritis. PLoS One 2012;7:e40136. van Echten G, Sandhoff K. Ganglioside metabolism. Enzymology, topology, and regulation. J Biol Chem 1993;268:5341–4. Varki A. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 1993;3:97–130. Wang XJ, Han G, Owens P, Siddiqui Y, Li AG. Role of TGF beta-mediated inflammation in cutaneous wound healing. J Investig Dermatol Symp Proc 2006;11:112–7. Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ, Holash J. Vascular-specific growth factors and blood vessel formation. Nature 2000;407:242–8. Zittermann SI, Issekutz AC. Endothelial growth factors VEGF and bFGF differentially enhance monocyte and neutrophil recruitment to inflammation. J Leukoc Biol 2006;80:247–57.
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Short communication
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Monosialic ganglioside GM3 specifically suppresses the monocyte adhesion to endothelial cells for inflammation
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Seok-Jo Kim a,b,1 , Tae-Wook Chung a,1 , Hee-Jung Choi a,c , Un-Ho Jin a , Ki-Tae Ha c , Young-Choon Lee b,∗∗ , Cheorl-Ho Kim a,∗ a Molecular and Cellular Glycobiology Unit, Department of Biological Science, SungKyunKwan University, 300 Chunchun-Dong, Jangan-Gu, Suwon City, Kyunggi-Do 440-746, Republic of Korea b Department of Medicinal Biotechnology, Dong-A University, Saha-Gu, Busan 604-714, Republic of Korea c Division of Applied Medicine, School of Korean Medicine, Pusan National University, Yangsan City, Gyeongsangnam-Do 626-770, Republic of Korea
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Article history: Received 3 June 2013 Received in revised form 10 September 2013 Accepted 28 September 2013 Available online xxx
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Keywords: Cell adhesion molecule Monosialic ganglioside GM3 Vascular endothelial growth factor (VEGF) Q2 Inflammation Monocyte-endothelial adhesion Proinflammatory cytokine
Vascular endothelial growth factor (VEGF) is well known as a significant angiogenic factor, and also functions as a proinflammatory cytokine, which induces adhesion of leukocyte to endothelial cells in inflammation reaction. In this study, we show that ganglioside GM3 inhibits the VEGF-induced expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) through activation of nuclear factor-B (NF-B) via protein kinase B (AKT) signaling in human umbilical vein endothelial cells (HUVECs), relating with leukocyte recruitment to endothelial cells under inflammatory conditions. In addition, ganglioside GM3 significantly reduced the monocyte adhesion to HUVECs as determined by the monolayer cell adhesion assay. Furthermore, in VEGF-injected mice for the inflammatory condition, ganglioside GM3 markedly decreased the expression of ICAM-1 and VCAM-1 in vein tissues. These results suggest that ganglioside GM3 has an anti-inflammatory role by suppressing the expression of inflammatory-related molecules during in vitro and in vivo inflammation. © 2013 Published by Elsevier Ltd.
1. Introduction Inflammatory cytokines including interleukin (IL)-1, IL-6, IL8, transforming growth factor (TGF)- and tumor necrosis factor (TNF)-␣ are released from various inflammatory cells through complex network signaling in inflammation responses (Angelo and Kurzrock, 2007; Wang et al., 2006). These molecules lead to inflammation-related diseases such as cancer progression, cardiovascular disease, diabetic retinopathy, rheumatoid arthritis, retinal ischemia and inflammatory psoriasis by inducing VEGF expression, which is closely linked to angiogenesis (Angelo and Kurzrock, 2007). VEGF upregulated during inflammatory reaction activates endothelial cells to induce expression of cell adhesion molecules including intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), resulting in recruitment of leukocytes at inflammation site to further activate inflammation response (Angelo and Kurzrock, 2007; Kim et al., 2001a,b; Zittermann and Issekutz, 2006). The expression of VCAM-1 and
ICAM-1 is regulated by NF-B activation via PI3K/Akt signal transduction in endothelial cells during inflammatory reaction (De Martin et al., 2000; Kim et al., 2001a). Gangliosides are sialic acid-complex glycosphingolipids located on mammalian cell membranes (Hakomori, 1990). Gangliosides also participate in regulation for various cellular events of several biologic processes on cell proliferation, adhesion, cell–cell or cell–matrix interactions, differentiation, tumorigenesis and metastasis (Hakomori, 2002; Hakomori and Igarashi, 1995; Varki, 1993). However, the role of gangliosides on leukocyte recruitment to endothelial cells for inflammation remains unclear. In this study, we found that ganglioside GM3 inhibits the expression of ICAM-1 and VCAM-1 in VEGF-exposure endothelial cells and VEGF-injected mice, thereby resulting in reduction of monocyte adhesion to endothelial cells stimulated by VEGF. Here we show the antiinflammatory role of ganglioside GM3 during in vitro and in vivo inflammation.
2. Results and discussion ∗ Corresponding author. Tel.: +82 31 290 7002; fax: +82 31 290 7015. ∗ ∗ Corresponding author. Tel.: +82 51 200 7591. E-mail addresses: [email protected] (Y.-C. Lee), [email protected] (C.-H. Kim). 1 These authors contributed equally to this study.
Gangliosides are composed of various types in the regulation of carbohydrate biosynthesis such as a-series: gangliosides GM3, GM2, GM1 and GD1a; b-series: gangliosides GD3, GD2 and
1357-2725/$ – see front matter © 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.biocel.2013.09.015
Please cite this article in press as: Kim S-J, et al. Monosialic ganglioside GM3 specifically suppresses the monocyte adhesion to endothelial cells for inflammation. Int J Biochem Cell Biol (2013), http://dx.doi.org/10.1016/j.biocel.2013.09.015
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Fig. 1. Inhibition of ICAM-1 and VCAM-1 expression by ganglioside GM3 in VEGF-induced HUVECs. (A) After starvation for 6 h, HUVECs were incubated with the various gangliosides and lactosylceramide (30 M) for 1 h and treated with VEGF (50 ng/mL) for 6 h. To investigate mRNA expression for ICAM-1 and VCAM-1, RT-PCR was performed using specific primers of ICAM-1 and VCAM-1. Data obtained by densitometric analyses are shown as bar graph. Relative ratio of each molecule was calculated with -actin level. # P < 0.05 vs. the untreated control. *P < 0.05 vs. the VEGF-treated control. (B and C) After starvation for 6 h, HUVECs were incubated with the indicated concentration of ganglioside GM3 for 1 h and induced with VEGF (50 ng/mL) for 6 h. To investigate mRNA expression for ICAM-1 and VCAM-1, RT-PCR was performed using specific primers of ICAM-1 and VCAM-1 (B) and Western blot was performed with the specific antibodies for ICAM-1 and VCAM-1 (C). -Actin (B) and GAPDH (C) were used as a control. (D) For immunofluorescence analysis of ICAM-1 and VCAM-1 expression, HUVECs were pre-incubated with GM3 (30 M) for 1 h and induced with VEGF (50 ng/mL) for 6 h. The cells were immunostained as described in Section 3.
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GD1b; neutral glycosphingolipid: lactosylceramide (van Echten and Sandhoff, 1993). Some gangliosides including GM3, GM1, GD1a and GD3, and lactosylceramide are known to regulate the receptor functions on membrane (Chung et al., 2009; Liu et al., 2006). VEGF, as a potent pro-inflammatory cytokine as well as angiogenic stimulator, plays a key role during inflammation by stimulating the expression of cellular adhesion molecules such as ICAM-1 and VCAM-1 through activation of NF-B in HUVECs (Kim et al., 2001a; Min et al., 2005; Yancopoulos et al., 2000). However, explanation on relationship between gangliosides and expression of ICAM-1 and VCAM-1 for anti-inflammatory function remains unclear. In order to examine whether the gangliosides regulate the VEGFinduced inflammation or not, the expression levels of ICAM-1 and
VCAM-1 induced by VEGF have been assessed in HUVECs. Interestingly, the expression level of ICAM-1 and VCAM-1 was specifically down-regulated by GM3 only when various gangliosides including GM3, GM2, GM1, GD1a, GD3, GD2 and GD1b and neutral glycosphingolipid were treated in VEGF-induced HUVECs. The fact that GM3 is specific for the down-regulation of ICAM-1 and VCAM-1 expressions was very remarkable. Then, the effect of ganglioside GM3 on expression of ICAM-1 and VCAM-1 has been further checked in VEGF-induced HUVECs. The increased mRNA and protein levels of ICAM-1 and VCAM-1 by VEGF were significantly suppressed by co-treatment of VEGF with GM3 in a dose-dependent manner (Fig. 1B and C). When the immunofluorescence levels of ICAM-1 and VCAM-1 expression in the HUVECs were examined, the data
Please cite this article in press as: Kim S-J, et al. Monosialic ganglioside GM3 specifically suppresses the monocyte adhesion to endothelial cells for inflammation. Int J Biochem Cell Biol (2013), http://dx.doi.org/10.1016/j.biocel.2013.09.015
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has clearly shown that ganglioside GM3 apparently blocked the expression of ICAM-1 and VCAM-1 in the VEGF-stimulated HUVECs (Fig. 1D). It has been reported that ganglioside GD1a enhances proliferation and migration of HUVECs induced at low concentration of VEGF, and lowers the threshold for VEGF-mediated signaling (Lang et al., 2001; Liu et al., 2006). Thus, we have also examined whether gangliosides have an effect on ICAM-1 and VCAM-1 expression in HUVEC stimulated at low concentration (5 ng/mL) of VEGF. After starvation for 6 h, HUVECs were incubated with the various gangliosides and lactosylceramide (30 M) for 1 h and treated with VEGF (5 ng/mL) for 6 h. RT-PCR was performed to check the levels of mRNA expression for ICAM-1 and VCAM-1. Just we could get the same data like Fig. 1A. On the other hand, usually, Ladish group treated HUVECs with GD1a in the presence or absence of VEGF for 24 h to check whether GD1a has any effect on the enhancement of proliferation or migration in HUVECs with or without VEGF (Lang et al., 2001; Liu et al., 2006). Thus, we also rechecked whether ganglioside GM3 and GD1a have effect on ICAM-1 and VCAM-1 expression in HUVEC stimulated at low concentration (5 ng/mL) of VEGF. After starvation for 6 h, HUVECs were incubated with ganglioside GM3 or GD1a (30 M) for 1 h and treated with VEGF (5 ng/mL) for 24 h. Ganglioside GM3 clearly decreased expression of ICAM-1 and VCAM-1 induced by 5 ng/mL VEGF in HUVEC. In contrast, ganglioside GD1a affected the enhanced expression of ICAM-1 and VCAM-1 in 5 ng/mL VEGF-treated HUVECs (data not shown). These results indicate that ganglioside GM3 significantly inhibits the expression of ICAM-1 and VCAM-1 in endothelial cells, which are known to directly relate to inflammatory reaction, in in vitro inflammatory system using VEGF as a mediator. As ganglioside GM3 inhibits the mRNA level of ICAM-1 and VCAM-1 in VEGF-mediated endothelial cells (Fig. 1A and B), the transcription activities of the ICAM-1 and VCAM-1 promoters (Min et al., 2005) using luciferase reporter assay system has been analyzed. As shown in Fig. 2A and B, the transcriptional activities of the ICAM-I and VCAM-1 promoters including NF-B binding sites were significantly increased when HUVECs were treated with VEGF as compared with control. Upon GM3 treatment, the activities of the ICAM-1 promoters including NF-B binding sites were significantly inhibited in VEGF-treated HUVECs, as compared with only VEGF (Fig. 2A). As expected similarly, GM3 inhibited the transcriptional activities of the VCAM-1 promoters including NF-B binding sites in VEGF-treated HUVECs too (Fig. 2B). During inflammation, the increased expressions of ICAM-1 and VCAM-1 mRNAs are mainly regulated by activation of the transcriptional factor NF-B through AKT/IB-␣ signal pathway in HUVECs (Kim et al., 2001a,b; Marumo et al., 1999; Meng et al., 2002). Therefore, we checked whether ganglioside GM3 is involved in the expression of ICAM-1 and VCAM-1 via AKT/IB-␣/NF-B pathway in VEGF-induced inflammatory condition using Western blot analysis and EMSA. As shown in Fig. 2C, the increased levels of the VEGF-induced AKT and IB-␣ phosphorylation were significantly suppressed by GM3 in HUVECs, while normal forms of AKT and IB-␣ were not changed. We next checked whether the activated NF-B as a transcription factor is inhibited by GM3 in VEGF-induced HUVECs using Western blot analysis and EMSA. VEGF-stimulated translocation of NF-B to nucleus was clearly inhibited by GM3 in HUVECs as evidenced by Western blot analysis (Fig. 2D). Furthermore, electromobility shift data showed that GM3 clearly inhibited electromobility shift complex between double-stranded 32 P-labeled oligo of NF-B and the nuclear lysates from VEGF-treated HUVECs (Fig. 2E). These results clearly suggest that ganglioside GM3 inhibits the transcriptional regulation of the ICAM-1 and VCAM-1 genes by blocking NF-B activation and the related phosphorylation factors such as AKT and IB-␣ in HUVECs. In vascular inflammation, the induction of adhesion molecules including ICAM-1 and VCAM-1 brings out leukocyte adhesion to
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endothelial cells as a critical step in in vivo and in vitro models (Goda et al., 2000; Kim et al., 2001b; Min et al., 2005; Thomas-Ecker et al., 2007). Therefore, we examined whether ganglioside GM3 inhibits monocyte adhesion to endothelial cells using a monolayer cell adhesion assay. As shown in Fig. 3, VEGF highly induced the adhesiveness of human THP-1 monocyte to confluent HUVECs induced by VEGF, compared with control or ganglioside GM3 (30 M) alone. The monocyte adhesion to VEGF-induced HUVECs, however, was reduced by GM3 (Fig. 3). These results clearly indicate that ganglioside GM3 suppresses the monocyte adhesion to endothelial cells during VEGF-mediated inflammation. Miklavcic et al. (2012) have reported that low level of GM3 was associated with higher production of proinflammatory signals. It has been also shown that GM3 deficiency exacerbated inflammatory arthritis in the mouse model of rheumatoid arthritis (Tsukuda et al., 2012). Furthermore, our data showed that ganglioside GM3 suppressed VEGF-mediated vascular inflammation through the reduction of the monocyte adhesion to HUVECs by decreasing expression of ICAM-1 and VCAM-1 induced by VEGF in HUVECs. These results suggest that some inflammatory conditions may be negatively regulated by ganglioside GM3. On the other hand, Gracheva et al. have showed that activity of GM3 synthase and ganglioside GM3 product in mononuclear cells and monocytes from atherosclerotic patients was higher than those from healthy donors. The adhesion of ganglioside GM3-treated monocytes to the HUVEC cells was also increased compared to ganglioside GM3untreated monocytes through the induction of CD11b, known as one of ligands for ICAM-1, on ganglioside GM3-stimulated monocytes (Gracheva et al., 2007). Based on these results and reports, difference in amount of ganglioside GM3 content on the several inflammatory conditions or on inflammatory microenvironment with inflammatory cytokine such as VEGF might be represented as a positive or negative regulator for the inflammatory homeostasis. Inflammation exhibits the expression of various molecules related to angiogenic and inflammatory cytokines such as of IL1, IL-6, IL-8, TGF-, TNF-␣ and VEGF (Angelo and Kurzrock, 2007; Wang et al., 2006). These cytokines induce the tumor growth, angiogenesis, inflammation and metastasis (Angelo and Kurzrock, 2007). Among these cytokines, VEGF acts as a growth factor regulating the physiology and pathology of the vascular system such as angiogenesis, inflammation and vascular injury (Detmar et al., 1998; Ferrara and Davis-Smyth, 1997). In angiogenesis, the VEGFmediated activation of the vascular endothelial cell regulates the proliferation, migration and tube formation which need the formation of a new tiny capillaries from blood vessels (Ferrara and Davis-Smyth, 1997). During inflammatory reaction, the activation of VEGF-stimulated endothelial cells induces leukocyte adhesion and infiltration in vivo and in vitro through the increased expression of ICAM-1 and VCAM-1 (Kim et al., 2001a,b; Min et al., 2005). Furthermore, TNF-␣ also induces leukocyte recruitment to endothelial cells by inducing expression of ICAM-1 and VCAM-1 on endothelial cells like VEGF (Chen et al., 1999; Mackay et al., 1993). From the above results, our in vitro experiments have clearly shown that ganglioside GM3 suppresses the VEGF-mediated expression of ICAM-1 and VCAM-1 and leukocyte recruitment in HUVECs. Therefore, to further investigate whether the ganglioside GM3 regulates the expression of pro-inflammatory mediators in vivo, we injected the VEGF or VEGF/TNF-␣ into mice using a method of low-pressure tail vein (LTV) injection into the BALB/c mice (Sebestyen et al., 2006; Zittermann and Issekutz, 2006). As shown in Fig. 4A and B, the mRNA levels of ICAM-1 and VCAM-1 were induced in mice vein tissues of the VEGF-treated group. In addition, the co-treatment with VEGF and TNF-␣ showed the increased expression levels of the molecules, compared with VEGF treatment group only (Fig. 4A and B). However, interestingly, the expression levels of the CAMs when induced by VEGF and VEGF/TNF-␣ were significantly decreased
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Fig. 2. Inhibition of transcriptional activity of the CAMs genes and NF-B activation via AKT/IB-␣ pathway by ganglioside GM3 in VEGF-induced HUVECs. (A and B) After transfection with each promoter, HUVECs were pre-incubated with GM3 (30 M) for 1 h and incubated with VEGF (50 ng/mL) for 6 h. Luciferase activity from the cells was analyzed as described in Section 3. Relative luciferase activity was normalized with the activity of pCMV-gal plasmid. The data represents the mean ± SD for triplicate independent experiments in control untreated cells. # P < 0.05 vs. the untreated control. *P < 0.05 vs. the VEGF-treated control. HUVECs were pre-incubated with GM3 (30 M) for 1 h and incubated with VEGF (50 ng/mL) for 6 h. (C) Western blot was determinated with the specific antibodies for phospho-AKT, AKT, phospho-IB-␣ and IB-␣. GAPDH was used as a control. (D) The nuclear extracts of the HUVECs were prepared as described in Section 3. After separation of the nuclear extracts, Western blot was performed with the specific antibodies for NF-B p65 and hnRNP1 used as a control. (E) The NF-B binding activity from nuclear extracts was assayed by EMSA as described in Section 3. N.C. indicated negative control; S.C, specific competitor used as 100-fold of non-labeled NF-B oligonucleotide.
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in the mice injected with ganglioside GM3 (Fig. 4A and B). These results suggest that ganglioside GM3 suppresses the expression of inflammatory-related molecules during the in vivo inflammation. As described, the present study is representative of the artificial treatment of the ganglioside GM3 to the proinflammatory conditions constructed with the inflammatory cells such as endothelial cells and monocytes, and the biologically regulating factors such as VEGF or TNF-␣. Then the present finding raises if the GM3 is indeed
one of the positive regulators for the in vivo anti-inflammatory homeostasis, how the GM3 is produced for the homeostasis during the in vivo inflammatory conditions such as infection, cancer, atherosclerosis, or any other diseases status. For the GM3 biosynthesis in vertebrates, the microenvironmental distribution of the GM3-expressing cells such as immune cells is suggested to be crucial (Kiguchi et al., 1990). Therefore, our further study will be focused on the issue in animals.
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Fig. 3. Inhibition of leukocyte (THP-1) adhesiveness on VEGF-induced HUVECs by ganglioside GM3. (A) After starvation for 6 h, the confluent monolayers of HUVECs were pre-incubated with ganglioside GM3 (30 M) for 1 h and induced with VEGF (50 ng/mL) for 6 h. Then, leukocyte (THP-1) adhesion to HUVECs as described in Section 3. Phase-contrast microscopic pictures of representative experiments were shown. (B) Quantification of the leukocyte adhesion to HUVECs was shown. The ratio of THP-1 cells adhering to the HUVEC was counted and calculated. Represent data was mean ± SD relative to adhesion ratio of control cells from the results of triplicate experiments. # P < 0.05 vs. the untreated control. *P < 0.05 vs. the VEGF-treated control.
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In conclusion, we show that GM3 significantly suppresses the leukocyte adhesion to the endothelial cells by regulating expression of ICAM-1 and VCAM-1 in endothelium induced by inflammatory cytokines, and might also be advantageous in therapy of various inflammation linked diseases.
3. Materials and methods
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Ganglioside GM3, GM2 and GD2 were purchased from Alexsis (San Diego, CA). Lactosylceramide, GM1, GD3, GD1a and GD1b were purchased from Calbiochem (San Diego, CA). VEGF and TNF-␣ were obtained from R&D Systems (Minneapolis, MN). The antibodies to ICAM-1, VCAM-1, p-AKT, AKT, p-IB␣, COX-2, p65 subunit of NF-B and hnRNP A1, and anti-rabbit, anti-goat and anti-mouse IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz Biotechnology, CA). Anti-GAPDH antibody was obtained from Chemicon, Inc. (Temecula, CA). Alexa Flour 488-conjugated goat anti-rabbit IgG were obtained from Molecular Probes (Carlsbad, CA). 3.2. Cell culture HUVECs, obtained from Cambrex Bio Science (MD, USA), were cultured in sterile endothelial cell growth medium (EGM-2, Cambrex Bio Science) and were maintained as described previously (Chung et al., 2009). 3.3. Reverse transcription polymerase chain reaction (RT-PCR) analysis
Fig. 4. Inhibition of the VEGF and VEGF/TNF-␣-induced expression of CAMs by ganglioside GM3 in in vivo experiments. After pre-injection of ganglioside GM3 (0.1 mg/kg or 1 mg/kg) for 30 min, the BALB/c mice (n = 6) were injected with VEGF (50 ng/mL) or VEGF/TNF-␣ for 4 h as described in Section 3. (A and B) To investigate mRNA expression for ICAM-1 and VCAM-1, RT-PCR was performed using specific primers of mICAM-1 and mVCAM-1. -Actin was used as a control. Data obtained from densitometric analyses are shown in (B). Relative ratio of each molecule is calculated with GAPDH level. # P < 0.05 and ## P < 0.05 vs. the untreated control. *P < 0.05 vs. the VEGF-treated control. **P < 0.05 vs. the VEGF and TNF-␣-treated control.
Total RNAs from cells and tissues were extracted using the Corezol reagent (Corebio Co., Seoul, Republic of Korea). cDNAs were synthesized by RT-PCR kit (Bioneer Co., Daejon, Republic of Korea) according to the manufacturer’s RT-PCR protocol and amplified by specific primers: human VCAM-1, 5-GATACAACCGTCTTGGTCAGCCC3 (sense), and 5-CGCATCCTTCAACTGGCCTT-3 (antisense); human ICAM-1, 5-CAGTGACCATCTACAGCTTTCCGG-3 (sense), 5-GCTGCTACCACAGTGATGAT-GACAA-3 (antisense); and human -actin, 5 -CA AGAGATGGCCACGGCTGCT-3 (sense)
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and 5 -TCCTTCTGCATCCTGTCGGCA-3 (antisense), mouse VCAM-1, 5-GGCTCTGGGAAGCTGGAACGAAG-3 (sense), and 5-ACTCCAGAGTCTTCCATCCTCATAGC-3 (antisense); mouse 5-CACCCCAAGGACCCCAAGGAGAT-3 (sense), and ICAM-1, 5-CGACGCCGCTCAGAAGAACCAC-3 (antisense); mouse GAPDH, 5 -GCCAAGGTCATCCATGACAAC-3 (sense) and 5 GTCCACCACCCTGTTGCTGTA-3 (antisense). For the internal standard of each mRNA in the RT-PCR assay, the expression levels of -actin and GAPDH were measured (Kang et al., 2008). The PCR products were visualized by electrophoresis on 1.5% agarose containing ethidium bromide with 1× Tris–acetate–EDTA (TAE) buffer.
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3.4. Western blot analysis
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Western blot analysis was performed as described previously (Kim et al., 2006a; Song et al., 2010). Briefly, 30 g of total proteins from HUVECs exposed to various concentrations of ganglioside GM3 with or without VEGF were separated by SDS-PAGE and electro-transferred to POTRAN nitrocellulose transfer membranes (Whatman GmbH, Germany). The nitrocellulose transfer membranes blocked by blocking buffer were incubated with ICAM-1, VCAM-1, p-AKT, AKT, p-IB␣, p65 subunit of NF-B, hnRNP A1 and GAPDH antibodies, respectively. The signals were visualized using the ECL chemiluminescence system (Amersham Biosciences, UK) with anti-rabbit, anti-goat and anti-mouse IgG antibodies linked with the horseradish peroxidase.
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3.5. Immunofluorescence microscopy
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Immunostaining with fluorescence was performed as described previously (Chung et al., 2009). Briefly, the treated HUVECs spreaded on gelatin-coated 12 mm coverslips in 24-well tissue culture plates were fixed in 3.7% formaldehyde and washed with PBS. Non-specific sites of the slide were blocked with 1% bovine serum albumin (BSA) for 1 h and incubated with ICAM-1 and VCAM-1 antibodies diluted 1:100 in phosphate-buffered saline (PBS) containing 1% BSA at 4 ◦ C for overnight. Then, the cells were washed and incubated with Alexa Flour 488-conjugated goat anti-rabbit IgG (1:500) at room temperature. After 1 h, the cells were washed with PBS, and then analyzed using a fluorescence microscope (Nikon).
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3.6. Transfection and luciferase assay
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The constructed plasmid for each promoter of ICAM-1 and VCAM-1 was provided from Dr. Kwon laboratory (Min et al., 2005). Luciferase assay was performed as described previously (Kim et al., 2006b). Cells were co-transfected with 0.5 pmol of each promoterluciferase reporter constructs and 1 g of pCMV--galactosidase plasmid by the Welfect method (Welgene Co., Daegu, Republic of Korea) for 24 h. After transfection, cells were treated with various concentrations of ganglioside GM3 with or without VEGF (50 ng/mL). Luciferase activity and -galactosidase activity were performed by using the luciferase and -galactosidase enzyme assay system (Promega, MD, USA). Luciferase activity was normalized with the -galactosidase activity.
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3.7. Electrophoretic gel mobility shift analysis (EMSA)
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The nuclear proteins of each cell were extracted as described previously (Choi et al., 2008). NF-B–DNA binding activities were measured in a gel shift assay system kit (Promega). Briefly, doublestranded oligonucleotides containing the consensus sequence for NF-B (5-AGTTGAGGGGACTTTCCCAGGC-3) were end-labeled with [␥-32 P]ATP using T4 polynucleotide kinase and used as probes for EMSA. Nuclear extract proteins (2 g) of each sample were
pre-incubated with the gel shift-binding buffer [4% glycerol, 1 mM MgCl2 , 0.5 mM ethylenediamine tetra-acetic acid (EDTA), 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris–HCl (pH 7.5), and 0.05 mg/mL poly(deoxyinosine-deoxycytosine)]. After 10 min, each sample was incubated with the labeled probe for 20 min at room temperature. Each sample was separated on a 4% nondenaturing polyacrylamide gel in a 0.5× TBE buffer. After running, the gel was dried and detected by autoradiography. 3.8. Monocyte adhesion assay HUVECs were spread on 2% gelatin-coated 24-well plates and treated various concentration of ganglioside GM3 with or without VEGF (50 ng/mL) for 6 h. Then, human THP-1 cells (5 × 104 cells/200 L/well) were added onto the confluent monolayers of HUVECs and incubated for 20 min at 37 ◦ C with rotation at 120 rpm, and washed out 3 times with PBS removing the unattached cells. Under a microscope, the number of attached cells was counted. 3.9. Mice and VEGF-induced inflammatory reaction in mice using low-pressure tail vein injections Female BALB/c mice (8 weeks old) were used in the experiment. 0.1 mg/kg or 1 mg/kg of ganglioside GM3 dissolved in 50 L of PBS with 5 g heparin was pre-injected into the mice tail vein using a 30 G 1/2 needle (Becton Dickinson, Franklin lakes, NJ). After 30 min, 50 ng/50 L VEGF or VEGF and TNF-␣ dissolved in PBS with 5 g heparin were injected into the mice tail vein for 4 h. Each reagent was injected by a method for low-pressure tail vein injections (Sebestyen et al., 2006). Then, the vein tissues were surgically isolated from the mice. 3.10. Statistical analyses Data were statistically presented as mean ± SD and all experiments were repeated at least three times. The statistical comparison between experimental groups was compared using a standard ANOVA followed by the Student’s t-test. Authors’ contribution S.J.K., T.W.C., Y.C.L. and C.H.K. designed research; S.J.K., T.W.C., H.J.C. and U.H.J. performed research; S.J.K., T.W.C., H.J.C., U.H.J., K.T.H., Y.C.L. and C.H.K. analyzed data; S.J.K., T.W.C., Y.C.L. and C.H.K. wrote paper. Acknowledgments This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST), Korea (Grant No. 2011-0008546). References Angelo LS, Kurzrock R. Vascular endothelial growth factor and its relationship to inflammatory mediators. Clin Cancer Res 2007;13:2825–30. Chen KH, Reece LM, Leary JF. Mitochondrial glutathione modulates TNF-alphainduced endothelial cell dysfunction. Free Radic Biol Med 1999;27:100–9. Choi HJ, Chung TW, Kim SJ, Cho SY, Lee YS, Lee YC, et al. The AP-2alpha transcription factor is required for the ganglioside GM3-stimulated transcriptional regulation of a PTEN gene. Glycobiology 2008;18:395–407. Chung TW, Kim SJ, Choi HJ, Kim KJ, Kim MJ, Kim SH, et al. Ganglioside GM3 inhibits VEGF/VEGFR-2-mediated angiogenesis: direct interaction of GM3 with VEGFR2. Glycobiology 2009;19:229–39. De Martin R, Hoeth M, Hofer-Warbinek R, Schmid JA. The transcription factor NFkappa B and the regulation of vascular cell function. Arterioscler Thromb Vasc Biol 2000;20:E83–8.
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