Up-Regulation of Cell Adhesion Molecule Genes in Human Endothelial Cells Stimulated by Lymphotoxin Alpha: DNA Microarray Analysis

160 Journal of Atherosclerosis and Thrombosis Vol.15, No.3 Gene Regulation by LTA in Endothelial Cells 161 Original Article

Shinichiro Suna1, Yasuhiko Sakata1, Hiroshi Sato1, Hiroya Mizuno1, Daisaku Nakatani1, Masahiko Shimizu1, Masaya Usami1, Seiji Takashima1, Hiroshi Takeda2, and Masatsugu Hori1

1Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Japan. 2Department of Medical Information Science, Osaka University Graduate School of Medicine, Suita, Japan.

Background: We recently reported that the A252G polymorphism of the Lymphotoxin-alpha (LTA) gene, a member of the tumor necrosis factor family, is strongly related with the onset of acute myocardial infarction; however, the roles of LTA in the development of atherosclerosis remain unclear. Methods and Results: Changes in gene expression profile in cultured human umbilical vein (HUVEC) and coronary artery endothelial cells (HCAEC) treated with LTA were analyzed with high density oligonucleotide arrays comprised of 8,500 genes. LTA stimulation at 10 ng/mL for 2 hours profoundly induced gene expression associated with signal transduction, cell adhesion and chemoattraction, such as the nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFkB), endothelial adhesion molecule 1 (E-Selectin), vascular cell adhesion molecule 1 (VCAM1), and monocyte chemotactic protein 1 (MCP1) (2.6, 55.7, 45.3 and 2.8 fold in HUVEC, and 2.6, 137.2, 64.0 and 13.0 fold in HCAEC, respectively). Quantitative real-time reverse transcriptase-polymerase chain reaction analysis confirmed that LTA increased the expressions of E-Selectin and VCAM1 in a dose- dependent manner both in HUVEC and HCAEC. Conclusion: LTA increased the expression of various genes involved in the process of atherosclerosis or inflammation in human endothelial cells, suggesting the roles of LTA in the development of atherosclerosis.

J Atheroscler Thromb, 2008; 15:160-165. Key words; Lymphotoxin alpha, DNA microarray analysis, Atherosclerosis, Cell adhesion molecule

LTα2β1 binds to the LTβ receptor (LTβR) and car- Introduction ries out unique activities5). LTA is also involved in a Lymphotoxin (LT) is a member of the LT/tumor large variety of inflammatory, immunostimulatory, and necrosis factor (TNF) family and can be produced as a antiviral responses as well as in secondary lymphoid secreted LTα3 homotrimer or a cell-associated LTα2β1 organ development or apoptosis6, 7). or LTα1β2 heterotrimer1-4). These homotrimer LTα3 We and others recently reported that polymor- and heterotrimer LTα2β1 interact with the tumor phisms in the LTA and its secretion regulator protein necrosis factor receptor (TNFR) Ⅰ (p55) and TNFRⅡ LGALS2 genes are significantly related with the onset (p75), accounting for activities similar to those of of and/or prognosis after acute myocardial infarction TNFα. On the other hand, heterotrimer LTα1β2 or (AMI)8-14). However, although it is reported that LTA induces cell adhesion molecules from several kinds of Address for correspondence: Hiroshi Sato, Department of Cardiovascular Medicine, Osaka University Graduate School cells, it has not been systematically investigated how of Medicine, 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan. LTA contributes to cardiovascular events or atheroscle- E-mail: [email protected] rosis via the stimulation of human endothelial cells. Received: October 25, 2007 In the present study, we examined whether LTA Accepted for publication: February 2, 2008 up-regulates factors involved in cell adhesion or inflam- 160 Journal of Atherosclerosis and Thrombosis Vol.15, No.3 Gene Regulation by LTA in Endothelial Cells 161 Original Article

matory process, which might trigger atherosclerosis instructions. RT-PCR conditions were 2 min at 50℃, in human vessels. DNA microarray and quantitative 10 min at 95℃, and 40 cycles of 15 sec at 95℃ and 1 RT-PCR analyses revealed the changes in gene expres- min at 60℃. Data were normalized to the glyceralde- sion induced by LTA in cultured human umbilical hydes phosphate dehydrogenase (GAPDH) level. Each vein endothelial cells (HUVEC) and human coronary sample was analyzed in duplicate and the experiments artery endothelial cells (HCAEC) and showed that were replicated more than twice. For GAPDH, endo- LTA induced various genes involved in signal trans- thelial adhesion molecule 1 (E-Selectin) and vascular duction, leukocyte adhesion, and chemoattraction. cell adhesion molecule 1 (VCAM1), primers and probes were purchased from Applied Biosystems via Taqman Assays-on-Demand gene expression products. The PCR Material and Methods probe sequences were as follows: hGAPDH; Assay ID: Cell Culture 4326317E, E-Selectin; Assay ID: Hs00174057_m1, HUVEC and HCAEC were purchased from Clo- probe 5’-GTGTGAGCAAATTGTGAACTGTACA, netics (San Diego, CA, USA), and cultured in endo- hVCAM1; Assay ID: Hs00174239_m1, probe 5’- thelial cell medium EGM2, including 2% fetal bovine CAGGTGGAGCTCTACTCATTCCCTA. serum, 0.04% hydrocortisone, 0.4% hFGF-B, 0.1% VEGF, 0.1% R3-IGF-1, 0.1% Ascorbic acid, 0.1% Western Blotting hEGF, 0.1% GA-1000 and 0.1% heparin (Clonetics, For VCAM1 assay, membranous and cytosolic San Diego, CA, USA). Cells passaged four to ten times protein was separated with Mem-PER® Eukaryotic were used for all experiments. LTA was purchased from Membrane Protein Extraction Reagent Kit (PIERCE, R&D Systems (Minneapolis, MN, USA), and was used Rockford, IL, USA) after stimulation for 6 hours by at 10 ng/mL in EGM2 medium. HUVEC and HCAEC LTA at a concentration of 10 ng/mL. were stimulated by LTA for 2 hours for DNA micro- For NFkB p65 assay, nucleus protein was sepa- array analysis. rated with NE-PER® Nuclear and Cytoplasmic Extrac- tion Reagent Kit (PIERCE) after stimulation by LTA RNA Extraction for 0, 5, 15, 30, 45, 60 minutes at 10 ng/mL. Total RNA was extracted using RNA-Bee RNA For Western blot analysis, 15 μg of protein extract isolation Reagent (Tel-Test Inc., Friendswood, TX, USA) was separated on 10% SDS PAGE and transferred to according to the manufacturer’s protocol. polyvinylidene difuoride membranes, and membranes were blocked with 3% non-fat milk in PBS containing Microarray Analysis 0.2% Tween 20. Thereafter, membranes were labeled DNA microarray analysis was performed with with the indicated primary antibodies, washed with GeneChip® Human Genome Focus Array (GeneChip®, 0.2% Tween 20 in PBS, and stained with horseradish Affymetrix, Santa Clara, CA, USA) according to the peroxidase-conjugated secondary antibody, which was instruction manual15). GeneChip Human Genome visualized by enhanced chemiluminescence (Amersham Focus Array is comprised of 8,500 genes. Derived data Pharmacia Biotech). VCAM1 was detected by anti- were analyzed using Affymetrix GCOS ver1.2. In order VCAM1 antibodies (Santa Cruz Biothecnology, Santa to avoid including ambiguously induced genes, we Cruz, CA, USA), actin by anti-Actin antibodies (Santa set a threshold of 10 units for any gene because dis- Cruz Biotechnology), NFkB p65 by anti-NFkB p65 crimination of expression below this level cannot be antibodies (Santa Cruz Biotechnology), and HP1α by performed with confidence, and also considered a anti-HP1α antibodies (Sigma Aldrich, Saint Louis, more than 2.6-fold increase or decrease on average of Missouri, USA). HUVEC and HCAEC in gene expression as significant. Statistical Analysis Analyses were performed using SPSS 11.0 (SPSS Quantitative Real-Time Reverse Transcriptase – Inc., Tokyo, Japan). Statistical analysis was performed Polymerase Chain Reaction (RT-PCR) by a two-tailed Student’s t-test. A p-value smaller than A 1.5 μg sample of total RNA was reverse-tran- 0.05 was considered statistically significant. scribed with Omniscript RTkit (Qiagen, Tokyo, Japan) according to the manufacturer’s protocol. Quantitative Results RT-PCR was performed with Taqman technology with the ABI Prism 7,000 detection system (Applied Biosys- DNA Microarray Anlysis of LTA Stimulus tems, Tokyo, Japan) according to the manufacturer’s In HUVEC, E-Selectin mRNA was most pro- 162 Suna et al. Gene Regulation by LTA in Endothelial Cells 163

Table 1. Genes profoundly enhanced by LTA (10 ng/mL) stimulus HUVEC (fold change) HCAEC (fold change) E-Selectin 55.7 137.2 VCAM1 45.3 64.0 IL8CTV (IL8 C-terminal variant) 21.1 27.9 CX3C (chemokine precursor) 19.7 42.2 TNFAIP2 (TNFα-induced protein 2) 17.1 29.9 ICAM1 13.9 36.8 TFNAIP3 (TNFα-induced protein 3) 11.3 36.8 GRO (Growth-related oncogene) beta 7.5 8.6 IRF (interferon regulatory factor 1) 7.0 8.6 GRO1 (Growth-related oncogene) 5.3 4.9 CEBP (CCAATenhancer binding protein) 4.6 3.2 NFKBIA (NFkB inhibitor, alpha) 4.3 5.3 EFNA1 (ephrin-A1) 4.0 4.0 HIVEP2 (HIV type 1 Enhancer-binding Protein 2) 4.0 3.5 ATF3 (activating transcription factor 3) 3.5 5.7 BCL3 (B-cell CLL lymphoma 3) 3.5 4.3 DSCR1 (Down syndrome critical region gene 1) 3.5 4.3 MSX1 (msh (Drosophila) homeo box homolog 1) 3.5 3.7 CSF1 (macrophage-specific colony-stimulating factor) 3.5 2.1 Syndecan4 3.2 8.6 NKX3.1 (homeobox protein) 3.0 3.7 TNFIP (TNFα-induced protein) 3.0 2.6 MCP1 (monocyte chemotactic protein) 2.8 13.0 SerpinB2 (serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 2) 2.8 3.0 NFKBIE (NFkB inhibitor, epsilon) 2.6 2.6 RNA from HUVEC and HCAEC with and without LTA (10 ng/mL) stimulus was subjected to Affimetrix DNA microarray analysis. Genes mark- edly enhanced, with statistical significance, are shown. foundly induced, followed by elevated mRNA levels LTA Induced VCAM1 on the Cell Membrane and for VCAM1, interleukin 8 (IL8) C-terminal variant, NFkB Nuclear Translocation chemokine precursor CX3C, and tumor necrosis fac- Western Blot analysis revealed that VCAM1 was tor (TNF) alpha induced protein 2 (TNFIP2). Simi- induced on the cell membrane and cytosol of HUVEC larly, in HCAEC, E-Selectin mRNA was the most after 6 hours by 10 ng/mL LTA stimulus (Fig.2) and profoundly induced, followed by elevated mRNA lev- that p65 subunit of NFkB protein was increased in els for VCAM1, CX3C, intercellular adhesion mole- the nuclear extract of HUVEC after LTA stimulus at cule 1 (ICAM1), and TNF alpha induced protein 3 10 ng/mL for 15 to 30 minutes (Fig.3). (TNFIP3) (Table 1). Discussion E-Selectin and VCAM1 Expression by LTA by Quantitative RT-PCR In the present study, we investigated the genes In quantitative RT-PCR analysis, the expressions up-regulated by LTA in human endothelial cells with of E-Selectin and VCAM1 were increased by LTA in DNA microarray and real-time RT-PCR analyses. a dose-dependent manner (1 pg/mL to 10 ng/mL) While many reports have elucidated that TNFα, a in HUVEC (p＜0.001; maximum 289-fold and 270- family member of LTA, is a proinflamatory cytokine fold for VCAM1 and E-Selectin, respectively) and and has an important role in atherosclerosis so far, the HCAEC (p＜0.01; maximum 1141-fold and 168-fold roles of LTA as a proinflammatory cytokine have not for VCAM1 and E-Selectin, respectively) (Fig.1). been well understood, which might play an important role in the process of atherosclerosis3, 4, 16). The present study revealed that various factors, including signal 162 Suna et al. Gene Regulation by LTA in Endothelial Cells 163

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�� Fig.1. Quantitative RT-PCR (A) E-Selectin mRNA expression in HUVEC stimulated by LTA stimulus. RNA from HUVEC with LTA (0, 0.001, 0.01, 0.1, 1 and 10 ng/ mL) stimulus was subjected to quantitative RT-PCR. (n=4), (B) VCAM1 mRNA expression in HUVEC stimulated by LTA stimulus. RNA from HUVEC with LTA (0, 0.001, 0.01, 0.1, 1 and 10 ng/mL) stimulus was subjected to quantitative RT-PCR. (n=4), (C) E-Selectin mRNA expression in HCAEC stimulated by LTA stimulus. RNA from HCAEC with LTA (0, 0.001, 0.01, 0.1, 1 and 10 ng/mL) stimulus was subjected to quantitative RT-PCR. (n=2), (D) VCAM1 mRNA expression in HCAEC stimulated by LTA stimulus. RNA from HCAEC with LTA (0, 0.001, 0.01, 0.1, 1 and 10 ng/mL) stimulus was subjected to quantitative RT-PCR. (n=2). ＊p＜0.05, ＊＊p＜0.01, ＊＊＊p＜0.001.

NFkB VCAM1（membrane） p65

HP1α VCAM1（cytosol） LTA 10 ng/mL Control 5 min 15 min 30 min 45 min 60 min Actin（cytosol） Fig.3. LTA stimulus induced NFkB nuclear translocation. HUVEC was stimulated with LTA at 10 ng/mL for 0, 5, 15, 30, Control LTA 10 ng/mL 45, 60 minutes. Nuclear fraction was separated and evaluated by Western Blot analysis. In the nuclear fraction, p65 subunit of NFkB Fig.2. LTA stimulus induced VCAM1 expression on the mem- was increased by LTA stimulus at 10 ng/mL for 30 minutes. brane of endothelial cells. Membrane and cytosol proteins were separated and evaluated by Western Blot analysis. VCAM1 protein increased on the membrane of HUVEC after LTA stimulus at 10 ng/mL for 6 hours. 164 Suna et al. Gene Regulation by LTA in Endothelial Cells 165

transduction molecules and adhesion molecules, were ulus induces cell adhesion molecules, including induced by LTA stimulus. Furthermore, E-Selectin and VCAM1, on the membrane of endothelial cells via the VCAM1, both of which mediate leukocyte-endothe- activation of NFkB as in TNF receptor signaling by lial cell adhesion17, 18), were induced by LTA stimulus TNFα26-28). in a dose-dependent manner. Accordingly, the present Differences are reported in the roles in athero- study is one of the first comprehensive studies to elu- genesis between LTA and TNFα in mice. Briefly, loss cidate the roles of LTA in human endothelial cells to of the TNFα gene did not alter atherosclerotic lesion induce various cell adhesion molecules, chemokines development compared with wild-type mice, but LTA and signal transduction molecules. deficiency resulted in a 62% reduction in lesion size 29); Adhesion of monocytes to endothelial cells is one however, genes up-regulated by LTA in the present study of the first and crucial steps in atherosclerosis. Many were almost identical to those induced by TNFα23). reports elucidated that an inflammatory mechanism These observations are possibly explained by the acti- in addition to dyslipidemia contributes to atheroma vation of TNFR Ⅰ and Ⅱ by TNFα and LTA. Besides formation, including the attachment of leukocytes to TNFR Ⅰ and Ⅱ, LTA has another receptor, lympho- endothelial cells19). After attachment to endothelial toxin beta receptor (LTBR) in signal transduction, cells, monocytes migrate beneath endothelial cells into to which LTA binds as a cell-associated LTα2β1 or the tunica intima, becoming foam cells by accumula- LTα1β2 heterotrimer, whereas LTα3 homotrimer or tion of lipid deposits. Thereafter, foam cells secrete TNFα does not. Recently, LTBR signaling has been pro-inflammatory cytokines, including TNFα, a fam- focused on because it has been reported to have a cru- ily member of LTA, which may activate the transcrip- cial role in lipid metabolism5, 30), which may contrib- tion nuclear factor of kappa light polypeptide gene ute to the difference between LTA and TNFα in ath- enhancer in B-cells (NFkB), and up-regulates adhesion erogenesis. Unfortunately, in the present study, how- molecules20-22). ever, we only investigated the roles of soluble LTA It has been reported that TNFα induces cell (LTα3 homotrimer), probably via TNFR Ⅰ or Ⅱ, but adhesion molecules, chemokines and proinflammatory did not investigate the signal transduction via LTBR, cytokines in HUVEC, including E-selectin, ICAM1, which remains to be elucidated. VCAM1, IL8, B cell CLL/lymphoma 3 (BCL3), TNF There are several limitations. Firstly, although we receptor–associated factor 1 (TRAF1) and Lympho- used mRNA stimulated with 10 ng/mL of LTA for 2 toxin β23). E-selectin, ICAM1, VCAM1 are members hours, it is possible that different durations and doses of cell adhesion molecules and IL8 is a member of the of LTA stimulation might have up-regulated different chemokine family. These proteins play an important genes; however, the result may at least suggest that LTA role in the adhesion of leukocytes to endothelial cells. induces cell adhesion-related genes in human endo- BCL3 and TRAF1 are implicated in TNFA signal thelial cells shortly after its stimulation. Secondly, the transduction. BCL3 is a proto-oncogene candidate mechanism of LTA signaling was not fully elucidated and functions as a transcriptional co-activator that is in this study. Further study is needed to elucidate the activated through its association with NFkB homodi- induction mechanism of these molecules by LTA stim- mers24). TRAF1 is a member of the TRAF protein ulus and the induction of monocyte adhesion to endo- family, which associates with and mediates signal trans- thelial cells and transmigration in in vitro studies. duction from various receptors of the TNFR super- In conclusion, the present study showed that family25). These results suggest that TNF signaling LTA has an important role in the expression of various plays an important role in enhancing leukocyte adhe- adhesion molecules and other genes in human endo- sion and chemoattraction by inducing inflammation thelial cells. Further study is needed to elucidate the in vascular endothelial cells. roles of LTA in the step of monocyte adhesion to Besides VCAM1 and E-Selectin, various cell adhe- endothelial cells, which is an initial and crucial step of sion molecules and chemokines, including ICAM1, atherosclerosis. IL8, MCP1, growth-related oncogene (GRO), and chemokine precursor CX3C, were induced by LTA Acknowledgment and stimulus in this study. Interestingly, similar cell adhe- Notice of Grant Support sion molecules and chemokines were induced in both HUVEC and HCAEC. Western Blot analysis revealed This work was supported by a grant-in-aid for that NFkB is activated by LTA stimulus and that scientific research from the Ministry of Education, VCAM1 is up-regulated on the membrane of HUVEC Culture, Sports, Science, and Technology, Japan, to Y. by LTA stimulus. These results suggest that LTA stim- Sakata (19590816) and M. Hori (19390215). 164 Suna et al. Gene Regulation by LTA in Endothelial Cells 165

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