Microarray analysis of human vascular smooth muscle cell responses to bacterial lipopolysaccharide.
Article Type: Report
Subject: Vascular smooth muscle (Health aspects)
Microbial polysaccharides (Health aspects)
DNA microarrays (Research)
Polymerase chain reaction (Methods)
Atherosclerosis (Research)
Muscle cells (Health aspects)
RNA (Health aspects)
Authors: Minta, Joe
Yun, J.
Der, Sandy
Rana, Shikha
Pub Date: 04/01/2007
Publication: Name: American Journal of Immunology Publisher: Science Publications Audience: Professional Format: Magazine/Journal Subject: Biological sciences Copyright: COPYRIGHT 2007 Science Publications ISSN: 1553-619X
Issue: Date: April, 2007 Source Volume: 3 Source Issue: 2
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: Canada Geographic Code: 1CANA Canada
Accession Number: 182406510
Full Text: Abstract: Accumulating evidence suggest a causal role of bacterial and viral infections in atherogenesis. Bacterial lipopolysaccharide (LPS) has been shown to stimulate resting vascular smooth muscle cells (SMC) with the production of inflammatory cytokines and modulation of quiescent cells to the proliferative and synthetic phenotype. To comprehensively identify biologically important genes associated with LPS-induced SMC phenotype modulation, we compared the transcriptomes of quiescent human coronary artery SMC and cells treated with LPS for 4 and 22 h. The SMCs responded robustly to LPS treatment by the differential regulation of several genes involved in chromatin remodeling, transcription regulation, translation, signal transduction, metabolism, host defense, cell proliferation, apoptosis, matrix formation, adhesion and motility and suggest that the induction of clusters of genes involved in cell proliferation, migration and ECM production may be the main force that drives the LPS-induced phenotypic modulation of SMC rather than the differential expression of a single gene or a few genes. An interesting observation was the early and dramatic induction of four tightly clustered interferon-induced genes with tetratricopeptide repeats (IFIT1, 2, 4, 5). siRNA knock-down of IFIT1 in SMC was found to be associated with a remarkable up-regulation of TP53, CDKN1A and FOS, suggesting that IFIT1 may play a role in cell proliferation. Our data provide a comprehensive list of genes involved in LPS biology and underscore the important role of LPS in SMC activation and phenotype modulation which is a pivotal event in the onset of atherogenesis.

Key words: Vascular smooth muscle cells, lipopolysaccharide, transcriptome, microarray, quantitative PCR, siRNA, IFIT1


Atherosclerosis is a complex, chronic inflammatory disease of the arterial vessel wall manifested at predilected locations of the vasculature [1]. It is initiated by multiple local and systemic risk factors (including mechanical shear stress due to hemodynamic changes, hypercholesterolemia, hypertension, high plasma levels of inflammatory markers, etc.) which induce endothelial dysfunction and vascular injury [1]. The disease is characterized by complex interactions between a variety of lipids, mononuclear phagocytes and their soluble mediators in the intima and by intimal hyperplasia. The accumulation of excess cholesterol and cholesteryl esters by macrophages at these sites results in the formation of foam cells that are the hallmarks of early fatty streak lesions and atheroma development [1,2]. Accumulating evidence suggests that chronic viral or bacterial infections, particularly infections with the ubiquitous gram-negative respiratory pathogen, Chlamydia pneumoniae, may be an additional risk factor that may initiate and/or promote atherosclerosis. Patients with acute myocardial infarction and coronary artery disease have been reported to have significantly higher titers of antibodies against C. pneumoniae compared to healthy controls and C. pneumoniae has been localized to the intima of vascular smooth muscle cells (SMCs) in many atherosclerotic lesions [3,4]. C. pneumoniae has been shown to infect and multiply within macrophages, endothelial cells and SMCs [3] and C. pneumoniae and Chlamydial lipopolysaccharide (LPS) have been reported to induce the release of inflammatory cytokines from mononuclear cells, endothelial cells and SMCs [5,6] and to stimulate oxidation of low-density lipoprotein and formation of macrophage foam cells [7,8]. Failure of coronary artery bridge grafts or restenosis has been associated with chronic inflammation induced by LPS [9,10]. Systemic inflammatory responses induced by LPS have been shown to increase neointimal formation after ballon injury and stent implantation and the induced proliferation of SMCs may play a key role in atherogenesis [11].

LPS is a complex glycolipid component of the gram-negative bacterial cell wall composed of a hydrophilic polysaccharide and a hydrophobic Lipid A moiety [12]. The interaction of serum LPS-binding protein (LBP) with LPS facilitates the binding of the lipid A moiety to CD14, a 55 kDa glycoprotein present as a soluble factor (sCD14) or as a glycosylphosphatidylinositol (GPI)-anchored membrane protein [13,14,15]. This complex, which also contains MD-2, interacts with Toll-like receptor 4 (TLR4) which mediates the cellular activation by LPS. TLR4 signaling induces the activation of NFkB and MAPK pathways and proliferation of SMCs [16,17,18,19,20,21]. Recently, Engelmann et al. [22,23] have reported that repeated perivascular administration of LPS or inoculation of Chlamydophila pneumoniae into rabbits induce formation of atheromatous lesions in vivo.

The ability of VSMCs in the media of arteries to undergo phenotype modulation from the quiescent and contractile state to the proliferative, migratory and synthetic phenotype that elaborates extracellular matrix underlies their crucial role in the development and progression of vascular pathology such as atherosclerosis and restenosis. Phenotype modulation involves a cascade of events in which different genes are turned on or off in a regulated manner. Thus to gain further insight into the molecular events associated with LPS-mediated activation of SMCs, we have analyzed the transcriptomes of quiescent and LPS-treated human coronary artery SMCs using oligonucleotide microarray analysis. Our results show that the transcriptional effects of LPS on SMCs were particularly far-reaching and many genes that are involved in various biological mechanisms such as chromatin remodeling, transcriptional regulation, translation, signal transduction, metabolism, innate and adaptive immune responses, cell proliferation, apoptosis, matrix formation, cell adhesion and motility, were regulated in a coordinated fashion. The LPS effect appeared to be mediated via the transcriptional induction of proinflammatory cytokines and growth factors and these in turn initiated multiple signal transduction pathways that induced transcriptional activation of genes that are effectors of cell proliferation, migration and extracellular matrix formation. The data presented is thus in agreement with accumulating evidence that LPS may be a risk factor that initiates and/or promotes atherosclerosis.


Smooth muscle cell culture and reagents: Human coronary artery SMCs (Clonetics, Walkersville, MD) were cultured in SMC basal medium (SmBM) containing SmBM-3 growth supplements [FBS (5%), bovine insulin (50 ng [mL.sup.-1]), human recombinant (hr)-EGF (5.0 ng [mL.sup.-1]), hr-FGF-B (20 ng [m.sup.L-1]) and GA-1000 (Gentamicin, Amphotericin B)] supplied by BioWhittaker Inc. Walkersville, MD. SMCs were characterized by their typical hill and valley growth pattern, by staining with anti-SM-[alpha]-actin antibody (Dako Diagnostics) and by the lack of staining of Factor VIII related antigen, an endothelial cell marker, using anti-factor VIII antibody (Dako Diagnostics). SMCs at passage 4 were seeded in growth supplemented SmBM-3 medium for 24 h and the medium was replaced with basal mediun containing 0.5% FBS for 48 h to induce quiescence before treated with LPS (E. coli 055.B5, Sigma/Aldrich, St. Louis, MO). Human AB serum was purchased from Sigma/Aldrich, St. Louis, MO. For siRNA experiments, human aorta SMCs (ATCC, Manassas, VA) were cultured in growth supplemented F12K medium according to the instructions of the supplier.

RNA isolation and gene expression profiling: Total RNA was extracted from cells using TRIzol reagent (Invitrogen Life Technologies, Inc. Burlington, ON) and contaminating chromosomal DNA was removed using Rnasefree DNase Message Clean Kit (GenHunter Corp., Nashville, TN). The integrity of RNA preparations was assured by comparing the intensities of 28 S and 18 S rRNA bands on 1% formamidealdehyde agarose gel. mRNA was isolated from the total RNA using Oligotex according to the manufacturer's instructions (Qiagen Inc., Mississauga, ON). Biotinylated complementary RNA (cRNA) samples for chip hybridization were prepared according to protocols supplied by Affymetrix (Affymetrix, Santa Clara, CA) and then hybridized to HG-U133A oligonucleotide array Gene Chip (Affymetrix, Santa Clara, CA) following the manufacturer's protocol. The arrays were washed, stained with streptavidin-phycoerythrin and scanned. Data files were analyzed using Affymetrix GeneChip[R] Operating Software (GCOS) version 1.0 (Affymetrix, Santa Clara, CA).

Gene cluster analysis: Gene expression pattern analysis was performed using GENESIS ver. 1.3.0. Raw expression values of differentially regulated genes (above signal log ratio of 1.0) from microarray analysis were adjusted with log 2 transform data and 'median centre genes, and clustered I nto 40 groups using k-mean clustering according to the method of Eisen et al. [24] Groups with similar gene expression pattern were combined together to generate three gene expression patterns: up-regulated at both 4 and 22 h, upregulated only at 4 h and down-regulated only at 4 h.

Quantitative real-time PCR: Quantitative real-time PCR was performed with an ABI Prism 7900HT Sequence Analyzer using the manufacturer's recommended protocol (Perkin-Elmer Applied Biosystems, Foster City, CA) to validate differential expression of selected genes. Two different primer sets were designed for each investigated gene using Primer Express version 2.0 (Perkin-Elmer Applied Biosystems, Foster City, CA). Each reactions was run in triplicates in 10 [micro]L volumes containing 4 [micro]L of diluted first strand cDNA template, 5 [micro]L of SYBR Green PCR Master Mix, 0.1 [micro]L (50 [micro]M) of each forward and reverse primers and 0.8 [micrp]L [H.sub.2]O. Samples were incubated at 95 [degrees]C for 3 min to activate Taq polymerase and 40 cycles were performed at 95 [degrees]C for 10 sec, at 65 [degrees]C for 15 sec and at 70 oC for 20 sec. A standard curve of each primer set was generated using human tonsil genomic DNA [25]. Sequences for the primers used in this study are available on request.

siRNA preparation and transfection: Cell cultures at 60-80% confluence were transfected with custom designed and synthesized IFIT1-specific siRNA primers (Sense: GGC UGU CCG UUU AAA UCC A; Antisense: UGG AUU UAA GCG GAC AGC C) using RNAi Human/Mouse Control Kit and instructions supplied by Qiagen. siRNA (30 [micro]L of 20 [micro]M solution), RNAiFect (22.5 [micro]L) and buffer EC-R buffer (70 [micro]L) were mixed together and incubated for 15 min at room temperature to allow formation of tranfection complexes. The complexes were then added drop-wise onto the cells in the fresh medium. A scrambled nonsilencing siRNA that has no known homology with mammalian genes was used to control for non-specific silencing effects.

RT-PCR: RT-PCR was performed using the Superscript first strand synthesis system (Invitrogen), Hotstart Taq Master Mix Kit (Qiagen) and gene specific primers designed from published cDNA sequences. IFIT1 forward primer (5'TCAGAATTCGGATCCATGAGTGAAATTCGTA AG3') and reverse primer (5'CAAGCTTGCCTGCAGTTAAATGGAAAGTCGC AG 3') amplifies a 1.5 kb cDNA fragment. GAPDH forward primer (5'CCACCCAGAAGACTGTGGAT3') and reverse primer (5'CCCCTCTTCAAGGGGTCTAC3') amplifies a 600 bp cDNA fragment. PCR amplification was initiated with a hot start at 95[degrees]C for 15 min followed by 35 cycles of denaturation at 94[degrees]C for 3 min, annealing at 56 [degrees]C for 1 min and extension at 72[degrees]C for 3 min and ended with an extension phase at 72 [degrees]C for 10 min. PCR products were collected and analyzed by electrophoresis on 1% agarose gel to determine the levels of IFIT1 and GAPDH.


Effect of LPS on the proliferation of SMC: In order to access the effect of LPS on SMC proliferation, quiescent SMCs in microtiter plates were stimulated with LPS (100 ng [mL.sup.-1]) in the presence of 0.5% FBS or 5% AB serum as source of sCD14 [26]. The culture media were replaced at day 2 and the cells were detached at day 5 and counted. The mean and standard error of the mean were determined for three experiments, each performed in quadruplicates. LPS stimulated SMC proliferation as determined by the increase in cell number over a 5 day culture period Fig. 1. The increase in cell number in cultures containing 0.5% FBS or 5% AB serum, were comparable. In the presence of 0.5% FBS and 100 ng [mL.sup.-1] LPS, SMC proliferation was increased 2.4 fold relative to cells in basal medium containing 0.5% FBS alone and SMC proliferation in medium containing 5% AB serum and 100 ng [mL.sup.-1] LPS was 5.1 fold greater than in the presence of AB serum alone. Our results on the mitogenic effect of LPS on SMCs is in agreement with earlier reports [27, 28].

Microarray analysis of differential gene expression in LPS-treated SMCs: Confluent SMCs cultures were synchronized to quiescence by incubation for 48 h in SmBM+0.5% FBS. To obtain a global profile of the transcriptional changes induced by LPS, we examined the gene expression responses of human coronary artery SMCs stimulated with 5% AB serum alone or AB serum containing LPS (100 ng [mL.sup.-1]) for 4 and 22 h using Affymetrix oligonucleotide arrays (HGU133A). Of the 18,400 transcripts (14,500 well-characterized genes) present on the chips, 1015 and 3749 genes (including ests and redundant gene probes) were found to be differentially regulated by LPS at 4 and 22 h, respectively. 469 genes were induced and 546 were suppressed at 4 h and 2526 were induced and 1223 were suppressed at 22 h.


Quantitative real-time pcr validation of results of gene array analysis: To validate the gene array results, the expression of 13 regulated genes was analyzed using reverse transcription followed by quantitative real-time PCR. The expression levels of human GAPDH and beta-2-microglobulin were used as internal housekeeping gene controls to normalize technical variability between samples. The expression of 9 genes was shown to correlate well in microarray and real-time PCR whereas the magnitudes of differential expression were somewhat different for 4 genes (data now shown). Overall, the real-time PCR data was more sensitive than the microarray data.

Scatter plot analysis: Scatter plots of log-transformed expression values from microarray analysis for pairwise comparisons of AB serum+LPS treated SMCs (ordinate) versus the control cells treated with AB serum alone (abscissa) for 4 and 22 h were used to display the regulated genes (Fig. 2). The data points along the diagonal represent genes expressed at comparable levels in both sets whereas those that lie off the diagonal represent genes differentially expressed between the samples identified by the two axes. The data indicate that the number of regulated genes increased with time. Also, whereas the number of induced and repressed genes at 4 h was comparable, the number of induced genes at 22 h was much greater than that of the suppressed genes. The plots confirmed that LPS induced and suppressed a greater numbers of genes at 22 h compared to 4 h (Fig. 2A and B). Scatter plot analysis of SMC treated with AB serum for 4 and 22 h shows that AB serum induces regulation of genes (Fig. 2C). This effect was more pronounced at 4 h and appeared to subside by 22 h. Comparison of the scatter plots of SMC treated with AB+LPS for 4 and 22 h showed that the magnitude of expression appeared to be similar for most genes. This suggests that the robust induction of genes in LPS 22 h compared to AB 22 h may be due to the prolonged and sustained activation of genes by LPS which compensates for the diminished AB serum effect at 22 h (Fig. 2D).

Profile of down-regulated genes: Since it is possible that the SMC genes that were down-regulated by LPS may be implicated in the maintenance of the quiescent phenotype, we have displayed in Tables 1A and 1B the induction and functional profiles of the top 10 SMC genes down-regulated by LPS treatment at 4 and 22 h. Two transcripts with the greatest fold decrease in expression in 4 h were albumin and cannabinoid receptor 1 (CNR1). Albumin is the serum carrier protein that plays an important role in stabilizing extracellular fluid volume and is known to be down-regulated during acute inflammation. CNR1 is a G-coupled receptor in brain and some peripheral tissues that recognize plant derived cannabinoids (marijuana) as well as endogenous lipid-like mediators (such as arachidonyl ethanolamide [anandamide] and 2-arachidonyl glycerol) that are potent modulators of neurobehavioral functions [29]. CNR1 and its endogenous ligands have role in regulating cerebral arterial tone and reactivity by modulating influx of calcium through Ltype calcium channels. Todate, the role of CNRs in SMC phenotype modulation is unknown. Among the down-regulated genes were two prostanoid receptors, PTGDR and PTGER4, a transcription-related gene (HCAP-G), two structural-related genes (D2LIC, PPFIA3), a chloride transporter (CLCN3) and negative regulators of cell proliferation and apoptosis (GW112 [30], INHBE [31]).


The expression of the top 10 down-regulated genes at 4 h had returned to near normal levels at 22 h and thus the profile of the genes repressed at 22 h was different from the genes repressed at 4 h (Table 1). Three of the top 10 genes that were most highly down-regulated in 22 h were signal transduction-related genes (KRAS2, PAK1IP1, SH3BP2). This group also contained genes involved in various functions such as transcription repression (HDAC4), ribosome macromolecular assembly (RPS10), cytochrome c oxidase respiratory chain complex (COX5), glycine metabolism (GCSH), actin polymerization and cell adhesion to extracellular matrix (ITGB5) and two hypothetical genes (C6orf79, KIAA0507).

Profile of up-regulated genes: SMC genes upregulated by LPS treatment may be associated with antibacterial or anti-viral activity and inflammation and/or the induction of the proliferative, migratory and synthetic phenotype. The top 10 up-regulated genes in SMC treated with LPS for 4 h were mostly LPS-, interferon-, TNF- or IL1-inducible proinflammatory and antiviral genes that participate in host defense (Table 2). The level of expression of many of the genes up-regulated at 4 h was found to have subsided by 22 h. The gene that was most highly up-regulated in 4 h LPS-stimulated SMCs was IFIT1. It has been suggested that IFIT1 is an anti-viral protein; however, its exact mechanism of action is at present unclear [32]. IFIT1 has recently been reported to bind to Rho guanine nucleotide exchange factor 10 [33] and also to block the interaction between eukaryotic translation initiation factors, eIF2 and eIF3 and thereby regulate translation initiation and protein synthesis [34]. IFIT4 which is a member of IFIT1 family of closely linked genes was also highly up-regulated [35]. Other genes encoding antiviral proteins that were highly induced in 4 h include MX1, cig5 and MDA5. Pro-inflammatory cytokines and chemokines known to be induced in response to bacterial and viral infections (CCL3, CCL20) were upregulated in 4 h LPS-stimulated SMCs. Some of the cytokine up-regulated genes may not be directly involved in defense response and these include USP18 (deubiquitinating protease)[36] and SAMHD1 (implicated in shifting dendritic cells from antigen uptake and processing phenotype to the antigen presentation phenotype)[37]. Two cytokine inducible genes of unknown functions (IFIT4, IFI44) were also upregulated at 4 h. The overall profile of the top 10 genes regulated in 4 h LPS-stimulated SMC is reminiscent of the induction of acute inflammation and a proliferative response.

In contrast with the profile of the genes induced by LPS in 4 h, most of the genes up-regulated in 22 h were probably not the direct result of cytokine activity but rather the effects of induction or activation of transcription factors. The top 10 over-expressed genes were predominantly involved in transcriptional activation, extracellular matrix formation and cell proliferation. LOXL2, a copper-dependent enzyme that initiates cross-linking of collagens and elastin [38], had the greatest expression among the top 10 highly up-regulated genes in 22 h LPS-treated SMCs (Table 2). Recently a direct link has been established between LOXL2 and SNAIL, a transcription factor that represses E-cadherin expression, in inducing epithelial-mesenchymal cell transitions and carcinoma progression [39]. Thus there is a possibility that the up-regulated expression of LOXL2 in LPS treated SMC may be associated with the induction of the proliferative response. Several genes involved in extracellular matrix formation (COL6A2, EMILIN1, PLEC1) were also highly up-regulated. The other genes in the 22 h top 10 up-regulated category include genes involved in transcription regulation or chromatin modification (POLR2E, MAZ, BTBD2), IFN-inducible genes (IFI27), tetracyclin transporter-like protein (TETRAN) and the hypothetical C1orf29 gene.

Functional characterization of the LPS regulated genes: In order to discover the classes of genes that were involved in LPS-mediated SMC activation, we applied a signal log ratio cut-off of > [+ or -] 1.0, eliminated all redundant probes that had less significant statistical values for each gene and categorized the remaining 1541 differentially regulated genes into functional groups/subgroups based on gene annotation information from Affymetrix database. Genes of unknown or miscellaneous functions were grouped together as Others. As shown in Figure 3, differential expression of genes belonging to functional categories such as transcription and chromatin remodeling, translation, signal transduction, host defense, proliferation, apoptosis, metabolism, ubiquitination, transporters and structural proteins indicates a robust SMC response to LPS treatment. The complete tables of the functional categories of LPS regulated SMC genes are available on request Table 3. A detailed discussion of all regulated genes is beyond the scope of this report and instead we focus on the specific sets of genes that are involved in host defense, transcription and chromatin remodeling, signal transduction, proliferation, apoptosis and cytoskeleton and matrix formation which we believe drive SMC phenotype modulation.

Host defense: Treatment of SMC with LPS induced the expression of several ligands and receptors of C-C and C-X-C chemokines and interleukins. Although the changes in the expression of various interferon subtypes were not deemed significant when analyzed using GCOS version 1.0, several interferon-inducible genes (IFIT1, IFIT2, IFIT4, IFI27, STIP1, G1P2, G1P3, IFI44, IFI35, OAS1-3, MX1), granulocyte colony stimulating factor 3 (CSF3), neutrophil chemokines, IL8, CCL20, CCL3, CXCL3, CXCL10 and MIF were highly expressed. Other inflammatory and immune-related genes such as the toll-interacting protein (TOLLIP) [40] and antiviral proteins (OAS1, 2, 3) were more up-regulated at 22 h compared to 4 h. Serine proteinase inhibitor (SERPINB2) was induced possibly to protect the SMCs from excessive inflammation. Taken together, the results suggest that LPS induces SMCs to express inflammatory cytokines, chemokines and cell surface receptors that could induce chemotactic attraction of hematopoietic cells, neutrophils and mononuclear cells which contribute to inflammatory and immune responses. This is consistent with the well known function of LPS as a potent activator of inflammation and immune response.

Transcription and chromatin remodeling: LPS regulated the expression of many transcription factors and molecules involved in chromatin remodeling. The most highly up-regulated transcription-related gene was RNA polymerase II polypeptide E (POLR2E), a component of the multisubunit eukaryotic DNA-dependent RNA polymerase II complex [41]. Several other components of the POLR2 holoenzyme were up-regulated. These include DNA polymerases (PDIP46, POLD4), core promoter binding proteins (COPEB, TAF13), initiator binding protein (GTF2I) and transcription complex subunit 3 (CNOT3).

Parathymosin (PTMS) and chromodomain helicase DNA binding proteins (CHD1L) and several nucleosomal histones (H1FX, HIST1H4H, HISTH2BE, HIST2H2AA) were highly up-regulated in LPS-treated cells. Particularly PTMS affects the interaction of linker histone H1 which is the major factor that stabilizes higher order chromatin structure with nucleosomes and thereby modulates the action of chromatin-remodeling enzymes [42]. Zinc finger protein transcriptional regulators (NAKAP95 and MAZ) and members of NFkB family (NFKB2, RELA) were induced at 22 h. IRF7 (a regulatory factor of viral/interferon inducible genes) and a member of the STAT family of transcription activators (STAT 6) were induced at 22 h. These results are in agreement with previous reports of activation of the JAK-STAT signaling pathway in LPS treated cells. The above compendium of LPS-mediated differential expression of several chromatin remodeling genes and transcription regulators have profound effects on gene expression in LPS treated SMCs and subsequently on inflammation, proliferation, apoptosis, ECM production, cell adhesion and cell migration responses and the modulation of the quiescent cells to the activated SMC phenotype.

Signal transduction: Several genes involved in a variety of signal transduction pathways were up-regulated in SMCs following treatment with LPS for 22 h. The Ras/Rho/CDC42 signaling pathway was activated as judged by the increase in the expression of genes encoding Rho guanine nucleotide exchange factors and dissociation inhibitors (ARHGEF16, ARHGDIA), G-protein coupled receptors (GPR, GPR51, GPR161, MUC20) and GTP-binding proteins of the Ras superfamily (GNB2, GPS1, DRG2, RABAC1, RAB3GAP, RAC2). Plexin B1 (PLXNB1) was the second highest induced gene in this category. PLXNB1 has been reported to mediate Sema4D-induced repulsive axon guidance signaling by acting as a GTPase activating protein for R-Ras, a member of the Ras superfamily of small GTP-binding proteins, which plays a key role in cell adhesion by activating integrins and promoting cell migration and neurite outgrowth [43, 44].

The transforming growth factor signaling pathway was recruited as judged by the induction of TGFB1, TGFBR2, LTBP3 and BMPR2. Binding of TGFB or BMPs to cognate type II receptors leads to the phosphorylation of the type I subunit by the constitutively active serine-threonine kinase type II receptor. Phosphorylated receptor I is an active kinase that subsequently phosphorylates and propagates intracellular downstream signals, including Smad proteins and mitogen-activated protein kinases [45, 46]. By the regulation of TGF inducible genes, the TGFB signaling pathway may mediate regulatory effects on a multitude of cellular processes in the LPS-treated SMCs including proliferation, differentiation, migration and extracellular matrix accumulation [47].

LPS also stimulated the expression of several transcriptional activators. The NF-kB subunits NFKB2 (p49/p100) and RELA (p65) as well as a putative NFkB activating protein (FLJ20241) and an inhibitor of inducible I-kappa B-kinase epsilon (IKBKE) were up-regulated and their activation suggests NFkB pathway signaling [48]. The MAPK pathway was activated in LPS-treated SMCs as judged by the increased expression of MAPK enzymes [48] (MAP2K2, MAPKAPK2). The phosphatidylinositol-3-kinase (PI3K) signaling pathway was also activated as judged by the increased expression of PIK4CB and protein kinase isotypes (PRKCD, PRKCL2).

Proliferation: Several growth factor genes (including VEGF, NRG1, NGFB) and growth factor receptor genes (e.g. FGFR1, MET, PDAP1) were induced by LPS at 22 h. In mammals, the progression through the cell cycle is regulated by the association of cyclins and their cyclin-dependent kinase (CDK) partners. Activity of these cyclin/cyclin-dependent kinase complexes is inhibited by the action of cyclin-dependent kinase inhibitors (CDKIs) that bind to these complexes and thereby inhibit progression through the restriction points of the cell cycle and cellular proliferation. Treatment of SMCs with LPS for 22 h resulted in up-regulation of several cell cycle-dependent genes (CCNL2, CDC2L2, CDC37, CDK5, CDK10), cell cycle regulatory genes (BUB1, human DOC-1R, NF2, FZR1, WISP1) and regulators of proliferation (MAD1L1, TMPO, CAPNS1, ANAPC5, NOTCH2). In contrast, negative regulators of growth such as cell cycle arrest genes including HCAP-G (mitosis-related chromosome condensation protein G), MAD1L1 (a G1 to S cell cycle checkpoint protein) and several cyclin-dependent kinase inhibitors (CKS2, p27, p21, p18, p16) were repressed at 4 h. These results are consistent with the pro-proliferative effect of LPS on SMCs.

Apoptosis: Six anti-apoptotic genes (BIRC3, RIG-I, BCL2A1, TNFAIP2, TNFSF7, TNFSF10) and 3 proapoptotic genes (HSXIAPAF1, MDA5, TARSH) were up-regulated within 4 h of LPS treatment whereas GW112 were repressed at 4 h. Pro-apoptotic genes such as BAX, RIPK1, TP53I11, BAD, DAPK3, PRF1, MCL1, SH3GLB2, SNCA and TNFRSF10D were induced at 22 h and HSXIAPAF1 was induced at both 4 and 22 h.

Structural proteins: The changes in expression levels of structural genes occurred predominantly at 22 h. Among the up-regulated genes, extracellular matrix collagen 6A2, 6A1, 1A1 and 4A2 were greatly increased. The different types of collagens i) may be associated with laminin, entactin and heparan sulfate proteoglycans to form the sheet-like basement membranes that separate epithelium from connective tissue or ii) may be components of microfibrillar structures in many tissues that localize close to cells where they may provide an anchoring function and may be involved in cell migration, differentiation and embryonic development. Several non-collagenous ECM components were also up-regulated. LOXL2, a copper-dependent enzyme that initiates cross-linking of collagens and elastin, was among the highly up-regulated structural genes in 22 h LPS-treated SMCs (Table 1B). Genes encoding microfibril associated proteins (EMILIN1, THBS3) and heparin sulfate proteoglycans of basement membranes (HSPG2, BGN, SDC1, SDC4) were also up-regulated at 22 h. These proteins may play a role in cell adhesion, cell morphology, intracellular communication and anchorage of SMC to elastic lamellae. The expression of several genes encoding cytoplasmic cytoskeletal proteins involved in actin reorganization (PLEC1, TUBB, GSN, NF2, VIL2, FLNA, ARPC4, TAGLN2, MYO1B, NRP2, TMOD3) were up-regulated after 22 h of LPS stimulation. These cytoskeletal proteins are central to the modulation of cell shape, adhesion, migration and the organization of cell surface structures.

Cell adhesion is an essential requirement for cell migration and morphogenesis during development. Genes encoding integral membrane proteins localized at focal adhesions and cell-cell junctions, such as VIL2, ASTN2, CLDN1, CLDN11, ITGB3, SCAM-1, ZYX, PCDHGC3, THY1, CD151, ICAM1 were up-regulated at 22 h in LPS stimulated SMCs. These proteins regulate a variety of cellular events, including cell adhesion, spreading and migration, cell proliferation and differentiation and anchorage dependence of signal transduction that may lead to cell proliferation, migration or transformation and metastasis [49,50,51]. Secretory metalloproteinases (such as MMP14, ADAM17) which are Zn(2+)-binding endopeptidases that degrade various components of the extracellular matrix such as collagens, pro-collagens and hyaluronan binding proteoglycans and probably facilitate SMC migration were over-expressed in LPS stimulated SMC.

Studies using the candidate gene approach has shown that bacterial lipopolysaccharide induces differential induction of the expression of several VSMC genes that encode cytokines (IL-1 [52], IL-6 [53], TNF-alpha [54], IFN [55]), chemokines (MCP-1 [56], IP-10 [57], IL-8, MIP-2 [58]), enzymes involved in prostanoid synthesis (PLA2 [59], COX-2 [60]), adhesion molecules (VCAM-1 [61], E-selectin [62], ICAM-1 [63]), LPS-binding proteins (LBP [64], CD14 [65], TLR4 [17]), human antigen [R.sup.16] (an RNA-binding protein involved in LPS cell activation effects), adrenomedullin [66], heme oxygenase-1 [67], low-density lipoprotein receptor-1 (LOX-1) [68], complement components and regulators (C3, C4, DAF, MCP, CD59 [69]), iNOS [70] and NO production [71], endothelin-receptor A and B [72], antiviral cytomegalovirus inducible gene 5 (viperin) [73], tissue factor and urokinase-type plasminogen activator (u-PA) [74]. LPS has also been shown to mediate some of its effects by the regulation of cytokines [55], activation of NADPH oxidase [17] and MAPK [17,75] and NFKB [70] signaling pathways. The data reported in this study is in agreement with most of these findings and also provides a comprehensive list of genes that have thus far not been implicated in LPS biology.

Clustering analysis of genes displaying similar expression: Differentially regulated genes were grouped according to their expression patterns following treatment with AB (4 h), AB+LPS (4 h), AB (22 h) and AB+LPS (22 h) to determine if a correlation exists between gene function and expression pattern in response to LPS stimulation. Gene expression pattern analysis was performed using GENESIS ver. 1.3.0. Raw expression values of all differentially regulated genes (above signal log ratio of 1.0) from microarray analysis were adjusted with 'log 2 transform data' and 'median centre genes' and clustered into 40 groups using k-mean clustering. We focused on regulation at 4 h because we reasoned that this early time period may better represent direct signaling effect of LPS on gene transcription. Clusters with similar gene expression pattern were combined together to generate 3 groups: up-regulated at both 4 and 22 h, up-regulated at 4 h and down-regulated at 4 h. Detailed results of clustering analysis are available on request.

The majority of 83 genes up-regulated at both 4 and 22 h are IFN- or TNF-inducible genes (IFI-15K, IFIT1, IFN p78, IFNAI27, IFNR2, ISTF3, IFNIP44, IFIT4, OAS1-3, IRF7, TNFSF7, 10, TNFAIP2, etc.) or cytokine/chemokine genes (CCL3, CXCL3, CSF3, CXCL10, CCL8, IL8, etc.) associated with the inflammatory response. This observation re-emphasizes the important role of LPS in the regulation of SMC activation and phenotype modulation and the initiation and/or augmentation of inflammation in cardiovascular diseases.

Fourteen out of 41 genes in the 4 h up-regulated group belong to a subgroup containing growth factors (FGF2, BMP2, LIM), cytokines (IL-1b, IL-15) and cytokine-induced genes (IFIT5, VCAM1, TNFAIP6, IKKa, CC2, CC7, CXC11, OASL, SOD2). The 4 h down-regulated gene group (116 genes) contained genes that mediate diverse biological functions such as chromosomal modification, signal transduction, growth and differentiation, metabolism, translation, and nuclear transport.

siRNA knockdown of IFIT1: Since IFIT1 was an early response gene that was most dramatically induced by LPS, we hypothesized that it may have an important role in the early events of the activation and proliferation of SMCs. Thus to gain insight into the biological significance of IFIT1 in LPS-induced SMCs, we knocked down IFIT1 transcripts using small interference RNA and then compared the expression of candidate genes in knock-down cells with wild type cells using real-time PCR. Human aortic SMCs (T/G HA-VSMC) were transfected with scrambled siRNA or IFIT1 siRNA as described and IFIT1 expression was assessed using semi-quantitative RT-PCR. Aliquots of the amplification mixtures were collected after 25, 30 and 35 cycles and analyzed by 1% agarose gel electrophoresis. IFIT1 expression was almost completely suppressed by siRNA relative to scrambled siRNA control (data not shown). To gain further insight into the biological functions of IFIT1, the effect of IFIT1 knock-down on the expression of 14 different candidate genes such as IFIT1, IFIT4, FOS, B2M, cytokines (TNF, TGF[beta]), adhesion molecules (ICAM1, VCAM), cell cycle progression/control (CCND1, p53, p15, p16, p21, p27) was analyzed. The expression of each gene in IFIT1 knock-down (KD) SMCs was calculated relative to the expression level in control cells treated with the scrambled siRNA, which was taken as 100%. The expression of IFIT1 in SMC IFIT1 knock-down SMCs was reduced to 8% and interestingly the expression of the closely linked paralogous gene, IFIT4, was also coordinately down-regulated to 35% (Fig. 4A). The expression of TGF[beta], ICAM1, VCAM, p15, p16, p27 and B2M was not significantly affected by IFIT1 KD. While TNF was moderately down-regulated to 65% (Fig. 4A), IFIT1 knock-down resulted in a remarkable up-regulation of FOS (8 fold), TP53 (11 fold), CDKN1A/p21 (866 fold) (Fig. 4B). The human c-fos oncogene heterodimerizes with c-jun to form the immediate-early transcription factor AP-1 that stimulates the transcription of genes containing AP-1 regulatory elements [76]. The tumor suppressor protein, TP53, which is undetectable or present in low levels in resting cells but is generally found in increasing amounts in proliferating non-transformed and transformed cells, is up-regulated in response to stress signals, DNA damage and is transcriptionally induced by IFNA/B through ISGF3 [77]. It has been proposed that TP53 binds as a tetramer to a p53-binding site and activates the transcription of downstream target genes that induce apoptosis or cell cycle arrest and/or invasion [78,79]. The cyclin-dependent kinase inhibitor, CDKN1A/p21, participates in the regulation of the G2 checkpoint of the cell cycle and probably serves as the effector of TP53 cell cycle control [76]. The up-regulation of these genes in IFIT1 knock-down cells suggests that IFIT1 may be implicated in the regulation of the expression of the above genes during SMC proliferation.



In this study, LPS was found to have a profound effect on SMC gene expression profile and the induction of many genes involved in cellular transcription, translation, signal transduction and metabolism indicates the activated state of SMC. This global change in genetic profile may be the main force that drives the LPS-induced phenotypic transition of SMC rather than the differential expression of a single gene or a few genes. The induction of genes involved in cell proliferation, adhesion, motility and matrix formation suggests that LPS mediated SMC transition from the quiescent to proliferative, synthetic and migratory phenotype. Many genes associated with apoptosis, both pro- and anti-, were also differentially regulated by LPS suggesting that the balance in cell survival and death was somehow tilted although the direction is not clear. Since LPS is the ligand for TLR4 that is a part of host innate immune system, genes involved in host defense are activated expectedly. The LPS response is not due solely to primary TLR4 signaling pathway but also the combinatory effect of various signaling pathways activated by the interaction of several ligands with their cognate receptors that are induced in this system. This will consist of LPS interacting with TLR4 in the presence of CD14 and LBP as well as the interaction of growth factors, interleukins, interferons and TGFb with their receptors.


This work was supported by grants from the University of Toronto, Faculty of Medicine, Dean's Research Fund (to J.M) and Genome Canada (to S.D).


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(1) Joe Minta, James (1,2) J. Yun, (1) Sandy Der and (1) Shikha Rana

(1) Department of Laboratory Medicine and Pathobiology, Faculty of Medicine

(2) Applied Molecular Oncology, Ontario Cancer Institute/University Health Network, Toronto, Ontario, Canada

Corresponding Author: Joe Minta, Department of Laboratory Medicine and Pathobiology, 1 King's College Circle, Medical Sciences Building Room 6211, Faculty of Medicine, University of Toronto, Toronto, ON Canada M5S1A8, Tel: 416-946-0316
Table 1: Ten SMC genes highly down-regulated by LPS treatment at
4 h (A) and 22 h (B). (All numbers are expressed as signal log ratio.)

Symbol     LPS 4h   LPS 22h    Gene

CNR1       -3.5     1.3        cannabinoid receptor 1 (brain)
ALB        -3.2     0          albumin
HCAP-G     -3.1    -1.1        chromosome condensation protein G
GW112      -3.1     0.1        differentially expressed in
                                 hematopoietic lineages
PTGDR      -2.9     0.1        prostaglandin D2 receptor (DP)
CLCN3      -1.8     0.5        chloride channel 3
INHBE      -1.7    -0.4        activin beta E
D2LIC      -1.6    -0.9        dynein 2 light intermediate chain
PTGER4     -1.6    -0.6        prostaglandin E receptor 4
                                 (subtype EP4)
PPFIA3     -1.4    -1.9        protein tyrosine phosphatase,
                                 receptor type, f polypeptide
                                 (PTPRF), interacting protein
                                 (liprin), alpha 3
KRAS2      -0.2    -3.3        v-Ki-ras2 Kirsten rat sarcoma 2
                               viral oncogene homolog
KIAA0507    0.3    -2.4        Homo sapiens mRNA, chromosome 1
                                 specific transcript KIAA0507.
HDAC4       0.3    -2.2        histone deacetylase 4
COMA       -0.2    -2.1        cytochrome c oxidase subunit Va
PAKIIPI    -0.1    -2.1        PAKI interacting protein 1
ITGB5       0.3    -2          integrin, beta 5
SH3BP2      2.1    -2          SH3-domain binding protein 2
GCSH       -0.2    -1.9        Homo sapiens transcribed sequence
                                 with strong similarity to protein
                                 pir:GCHUH (H.sapiens) GCHUH
                                 glycine cleavage system protein
                               H precursor--human
C6orf79     0.1    -1.9        chromosome 6 open reading frame 79
RPS10       0.3    -1.9        ribosomal protein S10

Table 2. Ten SMC genes highly up-regulated by LPS treatment at 4 h (A)
and 22 h (B). (All numbers are expressed as signal log ratio.)

Symbol        LPS 4h    LPS 22h   Gene

IFITI         7.3       5         interferon-induced protein with
                                    tetratricopeptide repeats 1
USP18         4.3       5         ubiquitin specific protease 18
SAMHDI        3.9       3.1       SAM domain and HD domain 1
cig5          3.8       0.9       viperin
MX1           3.7       3.8       myxovirus (influenza virus)
                                    resistance 1, interferon-
                                    inducible protein
p78 (mouse)
MDA5          3.7       1.9       melanoma differentiation associated
IFIT4         3.6       1.6       interferon-induced protein with
                                    tetratricopeptide repeats 4
CCL3          3.6       1.3       chemokine (C-C motif) ligand 3
CCL20         3.4       4.3       chemokine (C-C motif) ligand 20
IFI44         3.4       2.6       interferon-induced protein 44
LOXL2         0.1       7         lysyl oxidase-like 2
POLR2E        0         6.8       polymerase (RNA)11(DNA directed)
                                    polypeptide E, 25kDa
CIorf29       3.1       6.2       chromosome 1 open reading frame 29
COL6A2        -0.1      6.1       collagen, type VI, alpha 2
IFI27         2.4       5.7       interferon, alpha-inducible
                                    protein 27
TETRAN        -0.5      5.7       tetracycline transporter-like
EMILINI       -0.7      5.7       elastin microfibril interfacer 1
MAZ           0.2       5.6       MYC-associated zinc finger protein
                                    (purine-binding transcription
PLECI         -0.4      5.4       plectin 1, intermediate filament
                                    binding protein 500kDa
BTBD2         0         5.3       BTB (POZ) domain containing 2

Table 3. VSMC genes regulated by LPS treatment at 4 and 22 h. (Gene
expressions are shown in signal log ratio.)

A. Genes involved in host defense

Probe Set ID      Gene

IFN Inducible
  202411_at       interferon, alpha-inducible protein 27
  203153_at       interferon-induced protein with tetratricopeptide
                    repeats 1
  212009_s_at     stress-induced-phosphoprotein 1
                    (Hsp70/Hsp90-organizing protein)
  205483_s_at     interferon, alpha-inducible protein (clone IFI-15K)
  202086_at       myxovirus (influenza virus) resistance 1,
                    interferon-inducible protein p78 (mouse)
  204972_at       2'-5'-oligoadenylate synthetase 2, 69/71kDa
  205552_s_at     2',5'-oligoadenylate synthetase 1, 40/46kDa
  217502_at       interferon-induced protein with tetratricopeptide
                    repeats 2
  218400_at       2'-5'-oligoadenylate synthetase 3, 100kDa
  204415_at       interferon, alpha-inducible protein (clone
  214453_s_at     interferon-induced protein 44
  209417_s_at     interferon-induced protein 35
  204747_at       interferon-induced protein with tetratricopeptide
                    repeats 4

Innate Immunity and Inflammation
  205476_at       chemokine (C-C motif) ligand 20
  204614_at       serine (or cysteine) proteinase inhibitor, clade B
                    (ovalbumin), member 2
  217871_s_at     macrophage migration inhibitory factor
                    (glycosylation-inhibiting factor)
  217930_s_at     toll interacting protein
  211506 s at     interleukin 8
  207442_at       colony stimulating factor 3 (granulocyte)
  205114_s_at     chemokine (C-C motif) ligand 3
  204533_at       chemokine (C-X-C motif) ligand 10
  207850_at       chemokine (C-X-C motif) ligand 3

B. Genes involved transcription and chromatin remodeling
Chromatin Remodeling
  207824_s_at     MYC-associated zinc finger protein (purine-binding
                    transcription factor)
  218045_x_at     parathymosin
  218064_s_at     neighbor of A-kinase anchoring protein 95
  204805_s_at     Hl histone family, member X
  208180_s_at     histone 1, H4h
  207645_s_at     chromodomain helicase DNA binding protein 1-like
  208527_x_at     histone 1, H2be
  214290_s_at     histone 2, H2aa
Transcription Regulation
  209636_at       nuclear factor of kappa light polypeptide gene
                  enhancer in B-cells 2 (p49/p100)
  209878_s_at     v-rel reticuloendotheliosis viral oncogene homolog
                    A, nuclear factor of kappa light polypeptide
                    gene enhancer in B-cells 3, p65 (avian)
  201332_s_at     signal transducer and activator of transcription 6,
                    interleukin-4 induced
  208436_s_at     interferon regulatory factor 7
Basal TF
  213887_s_at     polymerase (RNA)11(DNA directed) polypeptide E, 25kDa
  203239_s_at     CCR4-NOT transcription complex, subunit 3
  215357_s_at     polymerase delta interacting protein 46
  202996_at       polymerase (DNA-directed), delta 4
  208960_s_at     core promoter element binding protein
  205966_at       TAF13 RNA polymerase 11, TATA box binding protein
                    (TBP)-associated factor, 18kDa TAF13
  210892_s_at     general transcription factor II, i

C. Genes involved signal transduction
  213490_s_at     mitogen-activated protein kinase kinase 2
  215050_x_at     mitogen-activated protein kinase-activated protein
                    kinase 2
  209991_x_at     G protein-coupled receptor 51
  215807_s_at     plexin B1
  207419_s_at     ras-related C3 botulinum toxin substrate 2 (rho
                    family, small GTP binding protein Rac2)
  217782_s_at     G protein pathway suppressor 1
  203267_s_at     developmentally regulated GTP binding protein 2
  208009_s_at     Rho guanine exchange factor (GEF) 16
  201167_x_at     Rho GDP dissociation inhibitor (GDI) alpha
  206971_at       G protein--coupled receptor 161
  203136_at       Rab acceptor 1 (prenylated)
  215249_at       mucin 20
  212932_at       RAB3 GTPase-ACTIVATING PROTEIN
  206673_at       putative G protein coupled receptor
  200852_x_at     guanine nucleotide binding protein (G protein), beta
                    polypeptide 2
  210969_at       protein kinase C-like 2
  202545_at       protein kinase C, delta
  210417_s_at     phosphatidylinositol 4-kinase, catalytic, beta
  58994_at        putative NFkB activating protein
  214398_s_at     inhibitor of kappa light polypeptide gene enhancer
                    in B-cells, kinase epsilon
TGFB Related
  203085_s_at     transforming growth factor, beta 1
                    (Camurati-Engelmann disease)
  209920_at       bone morphogenetic protein receptor, type
                    11(serine/threonine kinase)

D. Genes involved proliferation
Cell Cycle
  221427_s_at     cyclin L2
  204247_s_at     cyclin-dependent kinase 5
  204857_at       MADI mitotic arrest deficient-like 1 (yeast)
  211017_s_at     neurofibromin 2 (bilateral acoustic neuroma)
  209754_s_at     thymopoietin
  207428_x_at     cell division cycle 2-like 2
  203252_at       tumor suppressor deleted in oral cancer-related 1
  209414_at       Fzrl protein
  210622_x_at     cyclin-dependent kinase (CDC2-like) 10
  209953_s_at     CDC37 cell division cycle 37 homolog (S. cerevisiae)
  208721_s_at     anaphase promoting complex subunit 5
  216277_at       BUB1 budding uninhibited by benzimidazoles 1 homolog
  209112_at       cyclin-dependent kinase inhibitor 1B (p27, Kipl)
  204170_s_at     CDC28 protein kinase regulatory subunit 2
  202284_s_at     cyclin-dependent kinase inhibitor 1A (p21, CipI)
  204159_at       cyclin-dependent kinase inhibitor 2C (p18, inhibits
  207039_at       cyclin-dependent kinase inhibitor 2A (melanoma, p16,
                    inhibits CDK4)
GF and Angiogenesis
  219922_s_at     latent transforming growth factor beta binding
                    protein 3
  207334_s_at     transforming growth factor, beta receptor II
  208231_at       neuregulin 1
  206796_at       WNTI inducible signaling pathway protein 1
  206814_at       nerve growth factor, beta polypeptide
  210513_s_at     vascular endothelial growth factor
  210973_s_at     fibroblast growth factor receptor 1 (fins-related
                    tyrosine kinase 2, Pfeiffer syndrome)
  213807_x_at     met proto-oncogene (hepatocyte growth factor
  202290_at       PDGFA associated protein 1
  210756_s_at     Notch homolog 2 (Drosophila)
  200001_at       calpain, small subunit 1
  218663_at       chromosome condensation protein G

E. Genes involved in apoptosis
  211833_s_at     BCL2-associated X protein
  214667_s_at     tumor protein p53 inducible protein 11
  209941_at       receptor (TNFRSF)-interacting serine-threonine
                    kinase 1
  1861_at         BCL2-antagonist of cell death
  206133_at       XIAP associated factor-1
  203890_s_at     death-associated protein kinase 3
  214617_at       perforin 1 (pore forming protein)
  219209_at       melanoma differentiation associated protein-5
  220518_at       target of Nesh-SH3
  200796_s_at     myeloid cell leukemia sequence 1 (BCL2-related)
  210538_s_at     baculoviral IAP repeat-containing 3
  218813_s_at     SH3-domain GRB2-like endophilin B2
  211546_x_at     synuclein, alpha (non A4 component of amyloid
  218943_s_at     DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide
  202510_s_at     tumor necrosis factor, alpha-induced protein 2
  205681_at       BCL2-related protein Al
  212768_s_at     differentially expressed in hematopoietic lineages
TNF Related
  210654_at       tumor necrosis factor receptor superfamily, member
                    10d, decoy with truncated death domain
  206508_at       tumor necrosis factor (ligand) superfamily,
                    member 7
  202688_at       tumor necrosis factor (ligand) superfamily,
                    member 10
F. Genes involved in structural proteins
  202997_s_at     lysyl oxidase-like 2
  209156_s_at     collagen, type VI, alpha 2
  204163_at       elastin microfibril interfacer 1
  217430_x_at     collagen, type I, alpha 1
  212091_s_at     collagen, type VI, alpha 1
  211966_t        collagen, type IV, alpha 2
  201654_s_at     heparan sulfate proteoglycan 2 (perlecan)
  201262_s_at     biglycan
  202827_s at     matrix metalloproteinase 14 (membrane-inserted)
  201286_at       syndecan 1
  209561_at       thrombospondin 3
  205746_s_at     a disintegrin and metalloproteinase domain 17
                    (tumor necrosis factor, alpha, converting enzyme)
  216971_s_at     plectin 1, intermediate filament binding protein
  209372_x_at     tubulin, beta polypeptide
  214040_s_at     gelsolin (amyloidosis, Finnish type)
  214752_x_at     filamin A, alpha (actin binding protein 280)
  217817_at       actin related protein 2/3 complex, subunit 4, 20kDa
  210978_s_at     transgelin 2
  215074_at       myosin 113
  220800_s_at     tropomodulin 3 (ubiquitous)
Adhesion and Migration
  217234_s_at     villin 2 (ezrin)
  218182_s_at     claudin 1
  209693_at       astrotactin 2
  204627_s_at     integrin, beta 3 (platelet glycoprotein IIIa,
                    antigen CD61)
  207788_s_at     vinexin beta (SH3-containing adaptor molecule-1)
  200808_s_at     zyxin
  202071_at       syndecan 4 (amphiglycan, ryudocan)
  210841_s_at     neuropilin 2
  215836_s_at     protocadherin gamma subfamily C, 3
  206908_s_at     claudin 11 (oligodendrocyte transmembrane protein)
  202638_s_at     intercellular adhesion molecule 1 (CD54), human
                    rhinovirus receptor
  204306_s_at     CD151 antigen
  208850_s_at     Thy-1 cell surface antigen

Probe Set ID      Symbol            4h           22h

IFN Inducible
  202411_at       IFI27            2.4           5.7
  203153_at       IFITI            7.3           5
  212009_s_at     STIP1           -0.1           4.6
  205483_s_at     GIP2             2.3           3.9
  202086_at       MX1              3.7           3.8
  204972_at       OAS2             1.8           2.9
  205552_s_at     OAS1             2.5           2.8
  217502_at       IFIT2            2.3           2.8
  218400_at       OAS3             1.1           2.8
  204415_at       GIP3             1.3           2.7
  214453_s_at     IFI44            3.4           2.6
  209417_s_at     IFI35            0.6           2
  204747_at       IFIT4            3.6           1.6

Innate Immunity and Inflammation
  205476_at       CCL20            3.4           4.3
  204614_at       SERPINB2         1.3           3.2
  217871_s_at     MIF             -0.1           2.9
  217930_s_at     TOLLIP           0.1           2.8
  211506 s at     IL8              1.9           2.6
  207442_at       CSF3             2.6           1.6
  205114_s_at     CCL3             3.6           1.3
  204533_at       CXCL10           2.6           0.9
  207850_at       CXCL3            2.5           0.2

B. Genes involved in
Chromatin Remodeling
  207824_s_at     MAZ              0.2           5.6
  218045_x_at     PTMS            -0.1           4.9
  218064_s_at     NAKAP95         -0.3           4.9
  204805_s_at     HIFX            -1.1           4
  208180_s_at     HISTIH4H         1.5           3
  207645_s_at     CHDIL            0.3           3
  208527_x_at     HISTIH2BE       -0.1           2.4
  214290_s_at     HIST2H2AA        0.0           2.3
Transcription Regulation
  209636_at       NFKB2            0.7           4
  209878_s_at     RELA             0.4           3.9

  201332_s_at     STAT6            0.4           3
  208436_s_at     IRF7             1.5           2.5
Basal TF
  213887_s_at     POLR2E           0.0           6.8
  203239_s_at     CNOT3           -0.2           4.1
  215357_s_at     PDIP46           0.3           3.6
  202996_at       POLD4            0.0           3.5
  208960_s_at     COPEB            0.6           2.6
  205966_at                        0.0           2.6
  210892_s_at     GTF2I           -0.2           2.2

C. Genes involved in
  213490_s_at     MAP2K2           0.2           4
  215050_x_at     MAPKAPK2         0.0           3.2
  209991_x_at     GPR51           -0.6           5.2
  215807_s_at     PLXNB1           0.0           4.9
  207419_s_at                      RAC2          0.5
  217782_s_at     GPS1            -0.3           3.9
  203267_s_at     DRG2            -0.1           3.7
  208009_s_at     ARHGEF16         0.1           3.4
  201167_x_at     ARHGDIA          0.8           3.2
  206971_at       GPR161           0.5           3.1
  203136_at       RABAC1           0.1           2.7
  215249_at       MUC20            0.1           2.2
  212932_at       RAB3GAP         -0.3           2.1
  206673_at       GPR             -0.1           2
  200852_x_at     GNB2            -0.4           2
  210969_at       PRKCL2           0.3           3.9
  202545_at       PRKCD           -1.8           3.8
  210417_s_at     PIK4CB          -0.2           2.2
  58994_at        FU20241         -0.2           2.7
  214398_s_at     IKBKE            0.2           2.3
TGFB Related
  203085_s_at     TGFB1           -0.3           3.3
  209920_at       BMPR2           -0.4           2

D. Genes involved in
Cell Cycle
  221427_s_at     CCNL2            0.0           5.1
  204247_s_at     CDK5            -0.6           4.3
  204857_at       MADIL1          -0.8           3.7
  211017_s_at     NF2             -0.4           3.5
  209754_s_at     TMPO             0.1           3.1
  207428_x_at     CDC2L2          -0.4           2.8
  203252_at       DOC-1R           0.0           2.7
  209414_at       FZR1             0.5           2.4
  210622_x_at     CDK10            0.2           2.4
  209953_s_at     CDC37           -0.4           2.4
  208721_s_at     ANAPC5          -0.2           2.3
  216277_at       BUB1             1.4           2
  209112_at       CDKNIB          -1.2          -0.7
  204170_s_at     CKS2            -0.2          -1
  202284_s_at     CDKNIA          -0.3          -1
  204159_at       CDKN2C          -0.7          -1
  207039_at       CDKN2A          -0.1          -1.6
GF and Angiogenesis
  219922_s_at     LTBP3           -0.5           4.5
  207334_s_at     TGFBR2           0.6           4
  208231_at       NRG1            -0.1           2.9
  206796_at       WISP1           -0.3           2.9
  206814_at       NGFB             0.8           2.6
  210513_s_at     VEGF             0.4           2.6
  210973_s_at     FGFR1            0.3           2.2
  213807_x_at     MET              0.9           2
  202290_at       PDAP1            0.2           2
  210756_s_at     NOTCH2           0.6           2
  200001_at       CAPNS1          -0.2           2.7
  218663_at       HCAP-G          -3.1          -1.1

E. Genes involved in apoptosis
  211833_s_at     BAX             -0.2           4.1
  214667_s_at     TP53I11          0.6           3.8
  209941_at       RIPK1           -0.1           3.6
  1861_at         BAD              0.1           3.2
  206133_at       HSXIAPAF1        1.2           2.4
  203890_s_at     DAPK3           -0.1           2
  214617_at       PRF1            -0.6           2
  219209_at       MDA5             3.7           1.9
  220518_at       TARSH            2.0           0.8
  200796_s_at     MCL1             0.7           5
  210538_s_at     BIRC3            2.7           4.9
  218813_s_at     SH3GLB2          0             2.9
  211546_x_at     SNCA             0.8           2
  218943_s_at     RIG-1            3.4           1.6
  202510_s_at     TNFAIP2          2.6           1.2
  205681_at       BCL2A1           2.6           0.6
  212768_s_at     GW112           -3.1           0.1
TNF Related
  210654_at       TNFRSF10D        0.3           4.1

  206508_at       TNFSF7           2.9           1.2
  202688_at       TNFSF10          2.1          -0.3
F. Genes involved
  202997_s_at     LOXL2            0.1           7
  209156_s_at     COL6A2          -0.1           6.1
  204163_at       EMILIN1         -0.7           5.7
  217430_x_at     COLIA1           0.5           5.1
  212091_s_at     COL6A1           0.2           4.7
  211966_t        COL4A2           0.0           4.7
  201654_s_at     HSPG2            0.6           4.5
  201262_s_at     BGN              0.7           4.4
  202827_s at     MMP14            0.7           4.3
  201286_at       SDC1             0.1           3.4
  209561_at       THBS3           -0.1           2.6
  205746_s_at     ADAM17           0.1

  216971_s_at     PLEC1           -0.4           5.4
  209372_x_at     TUBB             0.0           4.3
  214040_s_at     GSN              0.2           4.2
  214752_x_at     FLNA            -0.3           3.3
  217817_at       ARPC4           -0.2           3.2
  210978_s_at     TAGLN2           0.2           3.1
  215074_at       MYOIB           -1.3           3
  220800_s_at     TMOD3            0.0           2.1
Adhesion and Migration
  217234_s_at     VIL2            -0.1           3.9
  218182_s_at     CLDN1            1.1           2.7
  209693_at       ASTN2           -0.7           2.7
  204627_s_at     ITGB3            0.2           2.6
  207788_s_at     SCAM-1           0.4           2.5
  200808_s_at     ZYX             -0.1           2.4
  202071_at       SDC4             1.0           2.3
  210841_s_at     NRP2             0.6           2.2
  215836_s_at     PCDHGC3          0.3           2.1
  206908_s_at     CLDN11           0.3           2.1
  202638_s_at     ICAMI            1.7           2
  204306_s_at     CD151            0             2
  208850_s_at     THYI             0             2

Fig. 3: Functional categories of LPS-regulated VSMC
genes. We applied a signal log ratio cutoff
of >+1.0 to all the genes that were
differentially regulated by LPS treatment by 4 h
and 22 h and all redundant probes that had less
significant statistical values for each gene were
also eliminated. Then the remaining 1541
differentially regulated genes were categorized
into functional groups/subgroups based on gene
annotation information from Affymetrix
database. Genes of unknown or miscellaneous
functions were grouped together as Others.

Distribution of LPS regulated genes into functional groups

Transporters                               163, 11%

Biosynthesis                                75, 5%

remodeling                                 178, 11%

proteins                                   127, 8%

transduction                               125, 8%

Proliferation                              107, 7%

Pro/Antiapoptosis                           57, 4%

Others                                     363, 24%

Metabolism                                 211, 13%

Host defense                               104, 7%

Ubiquitination                              34, 2%

Note: Table made from pie chart.
Gale Copyright: Copyright 2007 Gale, Cengage Learning. All rights reserved.