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【摘要】
Objective- Tumor necrosis factor (TNF)- initiates numerous changes in endothelial cell (EC) gene expression that contributes to the pathology of various diseases including inflammation. We hypothesized that TNF- -mediated gene induction involves multiple signaling pathways, and that inhibition of one or more of these pathways may selectively target subsets of TNF- -responsive genes and functions.
Methods and Results- Human umbilical vein endothelial cells (ECs) were preincubated with inhibitors of PI3 kinase (LY294002 ), histone deacetylases (HDAC) (trichostatin A ), de novo protein synthesis (CHX), proteasome (MG-132), and GATA factors (K-11430) before exposure to TNF- at 4 hours and analyzed by microarray. TNF- -mediated induction of vascular cell adhesion molecule-1 (VCAM-1) was attenuated by all of these inhibitors, whereas in contrast, stimulation of intercellular adhesion molecule-1 (ICAM-1) was blocked by MG-132 alone. Moreover TSA blocked TNF- -mediated induction of monocyte adhesion both in vitro and in vivo through the suppression of VCAM-1. Further analysis demonstrated that HDAC3 plays a significant role in the regulation of TNF- -mediated VCAM-1 expression.
Conclusions- TNF- activates ECs via multiple signaling pathways, and these pathways may be selectively targeted to modulate EC function. Moreover, TSA treatment reduced monocyte adhesion via VCAM-1 suppression in vitro and in vivo, suggesting that TSA might be useful for the attenuation of the inflammatory response in EC.
A global-survey of TNF- -signaling in ECs revealed that VCAM-1 expression is highly induced in a process dependent on PI3-K, GATA, HDAC, and new protein synthesis. HDAC inhibition abrogated monocyte adhesion to inflamed endothelium suggesting that HDAC, specifically HDAC3, might be useful for the attenuation of the inflammatory response in ECs.
【关键词】 cDNA microarray HDAC HUVEC trichostatin A VCAM
Introduction
The proinflammatory cytokine, tumor necrosis factor (TNF)-, is a prototype "activation agonist" that modulates leukocyte adhesion and transmigration in vascular inflammatory diseases including atherosclerosis. The biological activities of TNF- are mediated in part by the induction in endothelial cells of a suite of genes that regulate leukocyte-endothelial cell interactions, such as adhesions (intercellular adhesion molecule -1, vacular cell adhesion molecule -1, and E-selectin), procoagulants (tissue factor), and additional inflammatory cytokines and chemokines. 1 Previous studies have shown that TNF- -mediated induction of gene transcription in endothelial cells (ECs) involves several transcription factors including NF- B, GATA-2, IRF-1, and AP-1. 2-7
In addition to the specific transcription factor-mediated signaling axis, general protein acetylation has been shown to influence a broad set of cellular responses including diverse aspects of transcriptional regulation through the recruitment of 2 classes of enzymes, histone deacetylases (HDAC) and histone acetyltransferases (HAT). 8 The packaging of eukaryotic DNA into chromatin plays an active role in transcriptional repression by interfering with the accessibility of transcription factors to that region. Mammalian deacetylases can be grouped into 3 major classes. These classes are distinguished by size, catalytic domain, and subcellular localization. Class I HDACs, such as HDAC1, 2, 3, 8, and 11, are primary found in the cell nucleus. The class II deacetylases HDAC4, 5, 6, 7, 9, and 10 are found both in the cytoplasm and nucleus. 9 Acetylation of specific lysine residues within the amino-terminal tails of nucleosomal histones is generally linked to chromatin disruption and transcriptional activation of genes. In fact, Trichostatin A (TSA), a class I and II HDAC inhibitor, treatment increases NF- B-dependent gene expression. 10,11
In this study we wished to test the hypothesis that different signal intermediates couple TNF- to distinct subsets of target genes. We show that TNF- -mediated gene induction was indeed governed by transcript-specific signaling pathways. In addition, we demonstrated that TSA and other HDAC inhibitors block TNF- induction of VCAM-1, but not ICAM-1 and E-selectin. Furthermore, TSA effectively inhibited VCAM-1-dependent leukocyte adhesion under in vitro and in vivo conditions. Among HDACs, we found that HDAC3 was most important in the regulation of TNF- -mediated VCAM-1 expression in endothelium. Collectively, these findings suggest that TSA might be beneficial for the prevention of monocyte adhesion to EC during the inflammatory response.
Materials and Methods
Purchased reagents and detailed experimental conditions were as described online (please see http://atvb.ahajournals.org).
Affymetrix Oligonucleotide Microarray Analysis
Human umbilical vein endothelial cells (HUVECs) were serum-starved in EBM-2 basal medium (Clonetics) containing 0.5% fetal bovine serum (FBS); 18 hours later, HUVECs were pretreated for 30 minutes with 10 µmol/L MG-132, 50 µmol/L LY294002, 20 µmol/L TSA, 70 µmol/L CHX, 50 µmol/L PD98059, or 1 µmol/L K-11430, and then incubated in the absence or presence of 10 ng/mL human TNF- for 4 hours. Preparation of cRNA and hybridization of probe arrays (U133.2.plus) were performed according to the manufacturer?s instructions (Affymetrix, Santa Clara, Calif).
Animals
Mice were housed in a room with a 12-hour light cycle and fed a normal chow diet. Fms-EGFP (MacGreen) mice were generated as previously described. 12 These mice carry a transgene containing the c-fms promoter (3.5kb of 5'-flanking sequence and the downstream second intron) coupled to EGFP.
Intravital Microscopy Studies
Mice were pretreated with an intraperitoneal injection of TSA (5 mg/kg) or DMSO twice, 25 hours and 1 hour before intraperitoneal lipopolysaccharide (LPS) (0.35 mg/kg) or mouse recombinant TNF- (20 µg/kg) injection. The selected doses of LPS and TNF- were based on a previous report demonstrating a significant increase in monocyte adhesion to venules in mice in response to LPS or TNF- stimulation. 13 The mesentery was surgically exposed and positioned over a stage for viewing. GFP-expressing monocytes in mesenteric venules were observed. Examination was performed within 15 minutes after exteriorization of the intestine to avoid an procedure-related adhesion of monocytes. 14 The number 30 seconds was counted in vascular segments of defined length. To examine the effect of VCAM-1 blockage on monocyte adhesion, 30 µg of mouse anti-mouse VCAM-1 15 or control mouse IgG were injected via a tail vein 20 minutes before intraperitoneal injection of LPS.
Results
TNF- -Mediated Gene Expression in Human Primary ECs
HUVECs were treated with TNF- for 4 hours. Total RNA was obtained from 3 independent experiments and processed for cDNA microarray hybridization. A total of 64 genes were induced by TNF- by at least 2.5-fold in all 3 experiments (as shown in supplemental Table I). Most of the genes could be assigned to functional groups including chemokines/cytokines, cell surface receptors, signaling and transcription factors, and metabolic factors (supplemental Table I).
To determine which signaling pathways couple TNF- to downstream genes, HUVECs were preincubated in the presence or absence with inhibitors of proteasomes (10 µmol/L MG-132), MEK1/2 (50 µmol/L PD98059), PI3K (50 µmol/L LY294002 ), histone deacetylases (20 µmol/L TSA), de novo synthesis protein (70 µmol/L CHX), and GATA (1 µmol/L K-11430 16,17 ), and then incubated with or without 10 ng/mL TNF-. None of these 0.5-fold induction of genes in untreated control HUVECs (data not shown). MG-132, which blocks the proteasome-mediated degradation of I B and subsequent NF- B activation, 18 inhibited many TNF- -stimulated genes (54 out of 64 genes) by at least 40% ( P <0.05) (supplemental Table I). Pretreatment with LY294002 40% ( P <0.05) inhibition of 24, 33, 16, and 26 TNF- -responsive genes, respectively. PD98059 inhibited the effect of TNF- on only one gene, namely TRAF1 (supplemental Table I, full gene lists). All TNF- -responsive genes were clustered and grouped according to inhibitor effects. As shown in Figure 1 A, TNF- -mediated induction of 16 genes (upper column, lane 1) were significantly attenuated by LY294002, K-11430, and TSA (upper column, lanes 2 to 4, respectively); 11 of these 16 genes were also downregulated by CHX (upper column, lane 5). In contrast, a total of 21 TNF- -inducible genes were unaffected by these three inhibitors (lower column, lanes 2 to 5).
Figure 1. Transcriptional profiles of TNF- -treated HUVECs. A, The triplicate array data were summarized and clustered. HUVECs were preincubated with vehicle (lane 1); 50 µmol/L LY294002 (lane 2), 1 µmol/L K-11430 (lane 3), 20 µmol/L TSA (lane 4), and 70 µmol/L CHX (lane 5), and then treated with 10 ng/mL TNF- for 4 hours. The cluster data with CHX was derived from independent experiments. Colors represent relative log-fold gene expression levels based on normalized hybridization signals. Red or blue and green or yellow represent higher and lower expression than the median for that particular gene, respectively. B, HUVECs were treated as noted, and then VCAM-1, ICAM-1, and E-selectin mRNAs were quantified with real-time PCR. Values were normalized to cyclophilin A mRNA.
However, there are 85 genes that were suppressed by TNF- significantly (supplemental Table I). Bone morphogenic protein 4 was inhibited most (90%). In this list, we could demonstrate many genes, including thrombomodulin, GATA-3, MEF2C, KLF2, endothelial nitric oxide synthase (eNOS), and HoxA9, which have already been reported to be suppressed by TNF-. 1
TNF- Stimulation of VCAM-1, ICAM-1, and E-selectin mRNA Is Mediated by Distinct Signaling Pathways in Human Primary ECs
The DNA microarray data suggested that the TNF- -mediated induction of cell adhesion molecules involves distinct signaling pathways. Specifically, TNF- stimulation of VCAM-1 was inhibited by MG-132, LY294002, TSA, K-11430, and CHX, whereas the effect of TNF- on ICAM-1 mRNA was attenuated by MG-132 but not others. E-selectin followed a similar pattern to ICAM-1, with the exception that it was partially inhibited by K-11430. To confirm these results, we performed quantitative real-time reverse-transcription polymerase chain reaction (PCR). In the absence of inhibitors, TNF- resulted in 150-fold induction of VCAM-1, 76-fold induction of ICAM-1, and 560-fold induction of E-selectin in HUVECs, compared with each gene?s expression in the absence of TNF-, normalized to constitutively expressed cyclophilin A mRNA ( Figure 1 B). Consistent with the DNA microarray results, TNF- induction of VCAM-1 was markedly inhibited by MG-132 (98%), LY294002 (78%), TSA (86%), K-11430 (91%), and CHX (60%); ICAM-1 by MG-132 alone (96%); and E-selectin by MG-132 (99%) and K-11430 (18%). PD98059 had no reduction effect on any of these 3 genes ( Figure 1 B). Moreover, these inhibitor studies were performed in human coronary artery ECs. Similarly in HUVECs, VCAM-1 was significantly inhibited by MG-132, LY294002, TSA, and K-11430; ICAM-1 and E-selectin were inhibited by MG-132 (supplemental Figure I).
To confirm these results at the protein level, HUVECs were preincubated with chemical inhibitors, treated with TNF- for 4 hours, and then processed for Western blot analysis of VCAM-1 and ICAM-1. As shown in Figure 2, and consistent with the mRNA results, TNF- -mediated induction of VCAM-1 was significantly inhibited by MG-132, LY294002, TSA, K-11430, but not PD98059; whereas TNF- stimulation of ICAM-1 was inhibited by MG-132 alone. Together with the real-time reverse-transcription PCR data, these results suggest that while NF- B is necessary for TNF- induction of VCAM-1 and ICAM-1, the TNF- -VCAM-1 signaling axis is additionally dependent on PI3K, GATA factor(s), HDAC, and de novo protein synthesis.
Figure 2. TNF- -mediated induction of VCAM-1, ICAM-1, in HUVECs is mediated by distinct signaling pathways. Western blot analysis was shown with anti-VCAM-1 or anti-ICAM-1 antibody. Anti-ß-actin antibody was used as an internal control. The results are representative of three independent experiments.
TNF- Stimulated Monocyte Adhesion Is Inhibited by LY294002, TSA, and K-11430
To test the functional consequences of selective signaling inhibition, we performed cell adhesion assays using U937 monocytes and HUVECs. Very few fluorescent-labeled U937 cells adhered to HUVECs over 4 hours in the absence of TNF-, whereas in the presence of TNF-, the number of adherent U937 cells dramatically increased. As shown in Figure 3, pretreatment with MEK1/2 inhibitor (PD98059) failed to reduce TNF- -mediated monocyte adhesion. In contrast, U937 adhesion was inhibited by pretreatment of ECs with LY294002 (1.6-fold), TSA (2.5-fold), or K-11430 (3-fold), compared with mock treatment.
Figure 3. Inhibitors of HDAC, PI3K, and GATA, but not MEK1/2, inhibit TNF- -mediated induction of monocyte adhesion to HUVECs. Adherent U937 cells were lysed and assayed for fluorescent intensities relative to control (treatment with TNF- alone). Values represent mean±SD (n=3). * P <0.01.
To determine whether TSA more profoundly attenuates the adherent function via its effect on monocytes or ECs, we treated U937 monocytes with TSA for 30 minutes (same conditions to HUVECs) in advance of incubation with TNF- -stimulated HUVECs. However, the pretreatment of monocytes with TSA did not lead to a significant reduction in adhesion to inflamed endothelial cells (supplemental Figure II). Therefore, the downregulation of VCAM-1 expression by TSA in endothelial cells appears to be more important for the reduction in monocyte adherent levels. These results suggest that selective inhibition of TNF- -VCAM-1 signaling is associated with a significant attenuation in monocyte adhesion to cultured ECs.
TSA Blocks Monocyte Adhesion in Inflamed Venules in Mice
Recent studies have demonstrated a potential role for TSA in inhibiting VEGF expression, hypoxia-stimulated angiogenesis, and tumor progression in mice. 19-22 Our in vitro data suggest that TSA may also target inflammation, by inhibiting TNF- induction of VCAM-1. To determine whether TSA reduces monocyte adhesion in vivo, we employed fluorescence microscopy to directly visualize circulating monocytes in living mice. In these experiments, we used fms-EGFP transgenic mice (provided by Dr Hume DA, University of Queensland, Australia), in which the monocyte/macrophage specific c-fms-promoter (3.5kb 5'-flanking region plus second intron) is coupled to EGFP. 12 Mice were administered TSA (5 mg/kg) subcutaneously 25 hours and 1 hour before intraperitoneal injection of 20 µg/kg of TNF-; 3 hours later, mesenteric venules were observed under fluorescence microscopy. TNF- treatment caused intense monocyte adhesion in venules (supplemental Video I). In contrast, TSA administration resulted in a marked attenuation (85%) of circulating monocyte adherence to mesenteric venule ECs (supplemental Video I).
Next, we studied the effect of TSA in a second model of inflammation, namely mouse endotoxemia. 23 Compared with controls, LPS administration (0.35 mg/kg, intraperitoneal) resulted in a significant increase 80% by TSA pretreatment (supplemental Video II). A similar effect was observed when mice were pretreated with anti-VCAM-1 monoclonal antibodies (30 µg intravenous) (supplemental Video II). Combined pretreatment with both TSA and anti-VCAM-1 antibody did not result in further inhibition.
To verify that TSA inhibits VCAM-1 expression in inflamed blood vessels, tissue sections were collected from the aorta and lung, and immunostained with anti-VCAM-1 antibody. As shown in Figure 4A and 4 B, basal levels of VCAM-1 was detected, but not induced by mock treatment (upper right columns, parallel shown VCAM-1 in green and von Willebrand factor in red). LPS treatment markedly induced VCAM-1 expression in the endothelium of larger vessels in lung and aorta (semiquantitatively, 2.1- and 5-fold induction, respectively; middle right columns and supplemental Figure III). However, TSA pretreatment almost completely inhibited LPS-induced VCAM-1 expression (lower right columns) Semiquantitatively, 88% and 97% inhibition in lung and aorta, respectively (supplemental Figure III). In contrast, although LPS treatment also resulted in increased ICAM-1 expression, this effect was unaltered by TSA in lung and aorta (left columns). Taken together, these findings suggest that acute monocyte accumulations in inflamed mice are significantly inhibited in the presence of TSA via the reduction in EC surface VCAM-1 expression.
Figure 4. Double immunofluorescent stains for ICAM-1 and von Willebrand factor (vWF) and for VCAM-1 revealed a pronounced inhibitory effect of TSA on LPS-mediated VCAM-1 protein expression, but not ICAM-1 expression in aorta (A) and lung (B) endothelial cells.
TSA Inhibition of HDAC, Specifically HDAC3, Is Responsible for the Suppression of VCAM-1 Expression in Human Primary ECs
To verify that the inhibition of HDAC by TSA is responsible for the downregulation in VCAM-1, but not ICAM-1, expression in activated endothelium, we treated HUVECs with TNF- in the presence of other HDAC inhibitors, CHAP31; HDAC class I and II inhibitor, SCOP307 24; HDAC class I and HDAC4 selective inhibitor, and SAHA; HDAC1 and 3 selective inhibitor. 25,26 As shown in Figure 5 A, 10 nomol/L CHAP31, 0.6 µmol/L SCOP307, or 0.2 mmol/L SAHA markedly augmented the acetylation of cellular histone in HUVECs (inhibiting the HDAC activities). These inhibiting activities were similar to treatment with 10 µmol/L TSA. Importantly, CHAP31, SCOP307, and SAHA, as well as TSA, provided the nearly complete inhibition of TNF- -mediated VCAM-1 gene expression. In contrast, these treatments did not lead to the reduction of TNF- -stimulated ICAM-1 and E-selectin expression ( Figure 5 B). From these findings, TNF- -mediated VCAM-1 expression was mediated by HDAC1, 3, and 4.
Figure 5. HDAC inhibitors and knock down to HDAC3 suppress VCAM-1 expression in HUVECs A, HUVECs were preincubated for 30 minutes with TSA, CHAP31, SCOP307, or SAHA at the indicated concentrations, and then treated with TNF- for 4 hours; 4 µg of each cellular protein was loaded and detected by acetyl histone 3 antibody. B, HUVECs total RNA was harvested and assayed for VCAM-1, ICAM-1, and E-selectin mRNA by quantitative real-time PCR. Values were normalized to cyclophilin A, and represent mean±SE (n=3). * P <0.01 compared with the treatment with TNF- alone. C, Real-time PCR data showing the VCAM-1 mRNA in the HUVECs treated with either control or siRNAs against HDAC1, 3, and 4. Values were normalized to cyclophilin A. D, Western blot analysis of VCAM-1, HDAC1, and 3. HUVECs were transfected with 60 nmol/L si-control, HDAC1, 3, and 4, then incubated in the presence or absence of TNF-. Anti-ß-actin antibody was used as an internal control. The results are representative of 4 independent experiments.
Therefore, to determine which HDAC (HDAC1, 3, or 4) is important for the regulation of VCAM-1 expression, we examined knockdown experiments using siRNA against HDAC1, 3, and 4. SiRNA treatment 75%) inhibition in the expression of the targeted protein (supplemental Figure IV). Under these conditions, TNF- -stimulated VCAM-1 mRNA and protein were downregulated (59% and 70%, respectively) in the presence of siRNA against HDAC3, but neither HDAC1 nor 4, compared with si-Control ( Figure 5C and 5 D). Finally, to determine whether siRNA-mediated reduction of HDAC3 attenuates inflammation on TNF- -stimulated ECs via a VCAM-1-dependent manner, U937 monocyte adhesion assays were performed. As shown in Figure 6 A, TNF- augmented cell adhesion, which was 79% reduced in the presence of anti-VCAM-1 neutral antibodies. Similarly, a large reduction effect was obtained with the pretreatment of TSA (84% reduction), whereas only a weak reduction occurred in the presence of anti-ICAM-1 or E-selectin neutral antibodies ( Figure 6 A and data not shown). Moreover, as shown in Figure 6 B, siRNA mediated knockdown of HDAC3 in HUVECs resulted in a 64% reduction in TNF- -mediated monocyte adhesion compared with control siRNA. Consistent with our VCAM-1 mRNA and protein data, siRNA against HDAC1 did not show an inhibitory effect of TNF- -stimulated monocyte adhesion. Treatment of HUVEC with siRNA, neutralizing antibody, or TSA did not demonstrate any morphological abnormalities (data not shown). Taken together, these findings suggest that the effect of TNF- on U937 monocyte adhesion is mediated primarily through the inducible expression of VCAM-1 expression on endothelial cells. Among the HDAC families, HDAC3 plays an important role in TNF- -mediated VCAM-1 expression in endothelium.
Figure 6. Knockdown to HDAC3 attenuates monocyte adhesion to the activated HUVECs treated with TNF-. Quantification of the adherent monocytes. The mean and standard deviation values were calculated using the MetaMorph and cell image analyzer derived from six arbitral optical images with 2 independent experiments.
Discussion
The functional state of ECs is the major determinant of leukocyte extravasation in health and disease. Various agonists induce EC activation, including cytokines such as TNF-, chemokines, and thrombin. 5,27 Activated ECs typically display pro-adhesive and pro-coagulant properties, although the precise nature of the activation phenotype depends on the type of stimulus and the location within the vascular tree. 28
In this study, we have shown that endothelial cell activation is mediated by overlapping yet distinct signaling pathways. Although the majority of TNF- -responsive genes at 4 hour are dependent on NF- B activity, there is variable requirement for proteasomes, PI3K, histone deacetylases, de novo synthesis protein, and GATA. Only one gene (TRAF1) was inhibited by PD98059, indicating a minor role for MEK1/2 in TNF- signaling in ECs. Recently, Lee et al reported that TNF- -stimulated VCAM-1 expression in tracheal smooth muscle cells was dependent on MEK1/2 signaling as well as NF- B. 29 Here, VCAM-1 induction patterns indicated prolonged and sustained expression. While in contrast, TNF- -stimulated VCAM-1, ICAM-1, and E-selectin were dramatically and transiently (with peak at 2 to 4 hours after the treatment) induced in endothelial cells (supplemental Figure V). In addition, E-selectin was upregulated specifically in ECs, but not in smooth muscle cells. 30 These data suggest that TNF- signaling in ECs is modular and cell type-dependent, in the sense that each signal intermediate couples TNF- to a different subset of target genes.
This study is the first to our knowledge to show that TSA blocks monocyte adhesion. Based on the data in HUVECs, and the in vivo studies involving neutralization antibodies and immunofluorescence, TSA reduces monocyte adhesion in part by inhibiting TNF- (or LPS) stimulation of VCAM-1, but not ICAM-1, expression through the inhibition of HDAC3 in EC. Our current findings suggest that TSA affects only ECs and not monocyte activation or function, and we cannot rule out the possibility that different doses and times of TSA administration may modulate monocyte function.
TSA was originally identified as an antifungal antibiotic, which arrests cell cycle in the G1 and G2 phase, and induces differentiation. 31 Moreover, it was established that TSA inhibits histone deacetylase inhibitor, and has potent anti-tumor activity in breast 32 and prostate cancer. 33 Recently, it has been reported that TSA augmented NF- B-dependent gene expression. 10 TSA increases histone H3 acetylation of VCAM-1 and ICAM-1 promoters. 34 However, our data strongly indicate that TSA as well as other HDAC inhibitors (CHAP31, SCOP307, and SAHA) inhibit the induction of TNF- -stimulated VCAM-1 but not ICAM-1 or E-selectin. Additionally, HDAC3 siRNA decreased the expression level of VCAM-1 but not ICAM-1. Therefore, understanding the molecular mechanism by which TSA selectively inhibits VCAM-1 expression should be of considerable interest.
It is possible that after chromatin remodeling induced by HDAC inhibitor (TSA) treatment, de novo TNF- -activated transcription factor(s) or co-factor(s) interact with the VCAM-1 promoter to modulate expression. Such a factor could be envisioned to function in a manner independent of ICAM-1 expression. In support of such a mechanism, we have recently shown that GATA binding to the VCAM-1 promoter is important for the thrombin- and TNF- -mediated VCAM-1 expression. 5 Treatment with K-7174 (K-11430 derivative) abrogated VCAM-1 expression, but not ICAM-1 expression, by attenuating GATA binding to the VCAM-1 promoter. 3 In the current study, our microarray analysis, real-time PCR, and Western blot analyses confirmed that K-11430 significantly reduced VCAM-1 but not ICAM-1. Further investigations will be needed to elucidate the exact connection between TSA- and CHX-mediated VCAM-1 suppression with GATA transcription factors. Taken together, our current findings suggest that non-NF- B transcriptional regulation factors function independent and likely downstream of the simple NF- B-driven transcriptional upregulations, and play a further important role in regulating VCAM-1 expression in ECs.
In summary, we have systematically characterized and statistically validated gene expression profiles of TNF- -induced gene expression in primary cultured human ECs. Cluster analysis revealed 2 separate and distinct gene groups. VCAM-1 and ICAM-1 were located in different clustered groups. Moreover, TSA administration reduced monocyte adhesion not only to cultured ECs but also to venules in inflamed mice by mediating the suppression of VCAM-1, but not ICAM-1. On this basis, TSA or related compounds functioning to inhibit HDAC3 may be beneficial for the selective inhibition of adhesion molecules leading to inflammatory diseases, as well as malignancy.
Acknowledgments
We are grateful to Dr Keith B. Glaser (Global Pharmaceutical Research and Development, Abbott Laboratory, Ill) for kindly providing information on the siRNA against HDACs.
Sources of Funding
This study was supported by the Program of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), by NFAT project of New Energy and Industrial Technology Development Organization (NEDO) and by the Science and Technology from Ministry of Education, Culture, Sports, Science and Technology (to T.M. and T.K.) in Japan. This work was also supported in part by the Takeda Science Foundation in Japan (to T.M.) and by NIH/NHLBI HL36028 (to W.C.A.).
Disclosures
None.
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作者单位：From Laboratory for Systems Biology and Medicine (K.I., M.K., M.M., A.I., C.M., T.H., P.C.R., T.K., T.M.), Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan; Tokyo Research Laboratories (T.D.), Kowa Company Ltd, Tokyo, Japan; Institute for Molecular Bioscienc