Takahide Kohro

Vascular endothelial growth factor- and thrombin-induced termination factor, Down syndrome critical region-1, attenuates endothelial cell proliferation and angiogenesis.

Activation and dysfunction of the endothelium underlie many vascular disorders including atherosclerosis, tumor growth, and inflammation. Endothelial cell activation is mediated by many different extra-cellular signals, which result in overlapping yet distinct patterns of gene expression. Here we show, in DNA microarray analyses, that vascular endothelial growth factor (VEGF) and thrombin result in dramatic and rapid upregulation of Down syndrome critical region (DSCR)-1 gene encoding exons 4–7, a negative feedback regulator of calcium-calcineurin-NF-AT signaling. VEGF- and thrombin-mediated induction of DSCR-1 involves the cooperative binding of NF-ATc and GATA-2/3 to neighboring consensus motifs in the upstream promoter. Constitutive expression of DSCR-1 in endothelial cells markedly impaired NF-ATc nuclear localization, proliferation, and tube formation. Under in vivo conditions, overexpression of DSCR-1 reduced vascular density in matrigel plugs and melanoma tumor growth in mice. Taken together, these findings support a model in which VEGF- and thrombin-mediated induction of endothelial cell proliferation triggers a negative feedback loop consisting of DSCR-1 gene induction and secondary inhibition of NF-AT signaling. As a natural brake in the angiogenic process, this negative pathway may lend itself to therapeutic manipulation in pathological states.

Dynamic Change of Chromatin Conformation in Response to Hypoxia Enhances the Expression of GLUT3 (SLC2A3) by Cooperative Interaction of Hypoxia-Inducible Factor 1 and KDM3A

ABSTRACT Hypoxia-inducible factor 1 (HIF1) is a master regulator of adaptive gene expression under hypoxia. However, a role for HIF1 in the epigenetic regulation remains unknown. Genome-wide analysis of HIF1 binding sites (chromatin immunoprecipitation [ChIP] with deep sequencing) of endothelial cells clarified that HIF1 mainly binds to the intergenic regions distal from transcriptional starting sites under both normoxia and hypoxia. Next, we examined the temporal profile of gene expression under hypoxic conditions by using DNA microarrays. We clarified that early hypoxia-responsive genes are functionally associated with glycolysis, including GLUT3 (SLC2A3). Acetylated lysine 27 of histone 3 covered the HIF1 binding sites, and HIF1 functioned as an enhancer of SLC2A3 by interaction with lysine (K)-specific demethylase 3A (KDM3A). Knockdown of HIF1α and KDM3A showed that glycolytic genes are regulated by both HIF1 and KDM3A and respond to hypoxia in a manner independent of cell type specificity. We elucidated that both the chromatin conformational structure and histone modification change under hypoxic conditions and enhance the expression of SLC2A3 based on the combined results of chromatin conformation capture (3C) and ChIP assays. KDM3A is recruited to the SLC2A3 locus in an HIF1-dependent manner and demethylates H3K9me2 so as to upregulate its expression. These findings provide novel insights into the interaction between HIF1 and KDM3A and also the epigenetic regulation of HIF1.

A comparison of gene expression in murine cardiac allografts and isografts by means DNA microarray analysis.

BACKGROUND Acute rejection of allografts remains a significant problem in clinical transplantation, and the fundamental mechanism underlying this rejection are as yet only poorly elucidated. Recently, DNA microarrays have come into use for the study of gene expression profiles, and we have taken advantage of this new technology to investigate acute rejection. We compared mRNA profiles in murine cardiac allografts with isografts using DNA microarrays with probe sets corresponding to more than 11,000 mice genes. METHODS We screened for gene expression changes in murine cardiac allografts between fully incompatible mice strains (BALB/c H2d to C3H/He H2k) using a DNA microarray. The heart was heterotopically transplanted. Allografts (BALB/c to C3H/He) were removed on days 1, 3, and 5. As a control, isografts (C3H/He to C3H/He) harvested on days 1, 3, and 5 and native hearts of both strain mice (C3H/He and BALB/c) were obtained. RESULTS On day 5, interferon-gamma (IFN-gamma) and many IFN-gamma-inducible genes were profoundly induced in the allograft relative to isograft. Monokine induced by IFN-gamma was most profoundly induced followed by inducibly expressed GTPase and Lmp-2. IFN-gamma was also profoundly induced. The induction was detectable from day 3. In contrast, genes regulated by other cytokines exhibited only modest changes. CONCLUSION IFN-gamma-inducible genes are specifically up-regulated in murine cardiac allografts, suggesting that signaling mediated by IFN-gamma may play an important role in the late phase of acute rejection in vivo.

TNFα signals through specialized factories where responsive coding and miRNA genes are transcribed.

Tumour necrosis factor alpha (TNFα) is a potent cytokine that signals through nuclear factor kappa B (NFκB) to activate a subset of human genes. It is usually assumed that this involves RNA polymerases transcribing responsive genes wherever they might be in the nucleus. Using primary human endothelial cells, variants of chromosome conformation capture (including 4C and chromatin interaction analysis with paired‐end tag sequencing), and fluorescence in situ hybridization to detect single nascent transcripts, we show that TNFα induces responsive genes to congregate in discrete ‘NFκB factories’. Some factories further specialize in transcribing responsive genes encoding micro‐RNAs that target downregulated mRNAs. We expect all signalling pathways to contain this extra leg, where responding genes are transcribed in analogous specialized factories.

Detection of an up-regulation of a group of chemokine genes in murine cardiac allograft in the absence of interferon-gamma by means of DNA microarray.

Backgound. Interferon (IFN)-&ggr; and the IFN-&ggr;-dependent pathway are prominent in vascularized allograft during acute rejection. However, IFN-&ggr; deficient (IFN-&ggr;−/−) mice can rapidly reject cardiac allografts. To bring the alternative pathway during allograft rejection into more precise focus, we investigated the gene expression profile in murine cardiac allografts in IFN-&ggr;−/− mice by means of DNA microarray. Material and Method. We screened for gene expression changes in murine cardiac allografts of BALB/c H-2d into both wild-type C57BL/6 H-2b (n=3) and IFN-&ggr;−/− C57BL/6 H-2b(IFN-&ggr;−/−, n=4) using Affymetrix oligonucleotide arrays to monitor more than 11,000 genes and expressed sequence tag (ESTs). The heart was heterotopically transplanted. Transplanted hearts were harvested on day 5. As a control, isografts (C57BL/6 to C57BL/6) were also harvested on day 5. Results. On day 5, 64 of the 84 genes induced in the allografts in wild-type mice were not up-regulated in IFN-&ggr;−/− mice. We identified a group of 54 genes that were up-regulated in allografts in IFN-&ggr;−/− mice. Several chemokine genes, including monocyte chemoattractant protein=1 and macrophage inflammatory protein, were induced in the allografts in both wild-type and IFN-&ggr;−/− mice. Interestingly, a group of genes, including C10-like chemokine and platelet factor 4, were specifically induced in the IFN-&ggr;−/− mice. Conclusion. DNA microarray analysis reveals a unique pattern of mRNA expression in allografts in IFN-&ggr;−/− mice as well as a group of genes induced in cardiac allografts in both wild-type and IFN-&ggr;−/− mice, including monocyte chemoattractant protein-1 and monocyte chemoattractant protein-1.

Epigenetically coordinated GATA2 binding is necessary for endothelium-specific endomucin expression.

GATA2 is well recognized as a key transcription factor and regulator of cell‐type specificity and differentiation. Here, we carried out comparative chromatin immunoprecipitation with comprehensive sequencing (ChIP‐seq) to determine genome‐wide occupancy of GATA2 in endothelial cells and erythroids, and compared the occupancy to the respective gene expression profile in each cell type. Although GATA2 was commonly expressed in both cell types, different GATA2 bindings and distinct cell‐specific gene expressions were observed. By using the ChIP‐seq with epigenetic histone modifications and chromatin conformation capture assays; we elucidated the mechanistic regulation of endothelial‐specific GATA2‐mediated endomucin gene expression, that was regulated by the endothelial‐specific chromatin loop with a GATA2‐associated distal enhancer and core promoter. Knockdown of endomucin markedly attenuated endothelial cell growth, migration and tube formation. Moreover, abrogation of GATA2 in endothelium demonstrated not only a reduction of endothelial‐specific markers, but also induction of mesenchymal transition promoting gene expression. Our findings provide new insights into the correlation of endothelial‐expressed GATA2 binding, epigenetic modification, and the determination of endothelial cell specificity.

Lipid Accumulation in Smooth Muscle Cells Under LDL Loading Is Independent of LDL Receptor Pathway and Enhanced by Hypoxic Conditions

Objective—The effect of a variety of hypoxic conditions on lipid accumulation in smooth muscle cells (SMCs) was studied in an arterial wall coculture and monocultivation model. Methods and Results—Low density lipoprotein (LDL) was loaded under various levels of oxygen tension. Oil red O staining of rabbit and human SMCs revealed that lipid accumulation was greater under lower oxygen tension. Cholesterol esters were shown to accumulate in an oxygen tension–dependent manner by high-performance liquid chromatographic analysis. Autoradiograms using radiolabeled LDL indicated that LDL uptake was more pronounced under hypoxia. This result holds in the case of LDL receptor–deficient rabbit SMCs. However, cholesterol biosynthesis and cellular cholesterol release were unaffected by oxygen tension. Conclusions—Hypoxia significantly increases LDL uptake and enhances lipid accumulation in arterial SMCs, exclusive of LDL receptor activity. Although the molecular mechanism is not clear, the model is useful for studying lipid accumulation in arterial wall cells and the difficult-to-elucidate events in the initial stage of atherogenesis.

Identification of CCR2, flotillin, and gp49B genes as new G-CSF targets during neutrophilic differentiation

Granulocyte colony‐stimulating factor (G‐CSF) is a cytokine that stimulates myeloid progenitor cells to proliferate and differentiate into neutrophilic granulocytes. To identify genes induced by G‐CSF during neutrophil differentiation, interleukin‐3‐dependent murine myeloid precursor FDC‐P1 cells expressing the G‐CSF receptor were stimulated with G‐CSF, and the gene expression profile was characterized by DNA microarray analysis. In addition to known signal transducer and activator of transcription‐3 target genes, such as suppressor of cytokine signaling‐3 (SOCS3), JunB, and p19INK4D, we newly identified several G‐CSF targets, including genes for the CC chemokine receptor‐2 (CCR2), raft proteins flotillin‐1 and flotillin‐2, and immunoglobulin‐like receptor gp49B. Real‐time, quantitative polymerase chain reaction analyses revealed that the expression of these genes was induced in various myeloid cell lines by G‐CSF. Furthermore, when HoxA9‐immortalized bone marrow progenitors were induced by G‐CSF to differentiate into mature neutrophils, all of these genes were strongly activated. These genes could be categorized into three groups based on their time‐course of expression: immediate‐early (∼20 min, SOCS3), mid‐early (2–4 h, flotillin‐1/2 and gp49B), and late (>12 h, CCR2). This suggests that different transcriptional mechanisms are involved in the regulation of these genes. We show that bone marrow neutrophils express functional CCR2, which suggest that CC chemokines may play previously unknown roles in neutrophil activation and chemotaxis.

Genome-Wide Approaches Reveal Functional Interleukin-4-Inducible STAT6 Binding to the Vascular Cell Adhesion Molecule 1 Promoter

ABSTRACT Endothelial cell activation and dysfunction underlie many vascular disorders, including atherosclerosis and inflammation. Here, we show that interleukin-4 (IL-4) markedly induced vascular cell adhesion molecule 1 (VCAM-1), both in cultured endothelial cells and in the intact endothelium in mice. Combined treatment with IL-4 and tumor necrosis factor alpha (TNF-α) resulted in further, sustained induction of VCAM-1 expression. IL-4-mediated induction of VCAM-1 and secondary monocyte adhesion was predominantly regulated by the transcription factor STAT6. Genome-wide survey of IL-4-mediated STAT6 binding from sequential chromatin-immunoprecipitation with deep sequencing (chromatin immunoprecipitation sequencing [ChIP-seq]) in endothelial cells revealed regions of transient and sustained transcription factor binding. Through the combination of DNA microarrays and ChIP-seq at the same time points, the majority of IL-4-responsive genes were shown to be STAT6 dependent and associated with direct STAT6 binding to their promoter. IL-4-mediated stable binding of STAT6 led to sustained target gene expression. Moreover, our strategy led to the identification of a novel functionally important STAT6 binding site within 16 kb upstream of the VCAM-1 gene. Taken together, these findings support a critical role for STAT6 in mediating IL-4 signal transduction in endothelial cells. Identification of a novel IL-4-mediated VCAM-1 enhancer may provide a foundation for targeted therapy in vascular disease.

Serum levels of IgG4 and soluble interleukin-2 receptor in patients with coronary artery disease.

BACKGROUND Immunoglobulin G4 (IgG4)-related immuno-inflammation has been suggested to play a role in the development of remodeling of arterial wall. We investigated the association between serum concentrations of IgG4 or soluble interleukin-2 receptor (sIL-2R) and coronary artery disease (CAD). METHODS Serum concentrations of IgG4 and sIL-2R were measured in 286 patients who underwent coronary angiography. RESULTS In patients with CAD, the medians of serum concentrations of IgG4 (39.3 mg/dl) and sIL-2R (388 U/ml) were significantly higher than corresponding values in patients without CAD (IgG4 27.0 mg/dl, sIL-2R 312 U/ml). In receiver-operating characteristic curve analysis, the area under the curve of sIL-2R and IgG4 for the presence of CAD was 0.634 and 0.632, respectively. Age- and gender-adjusted logistic regression analysis showed that both of the fourth quartile of sIL-2R concentrations (≥509 U/ml) and that of IgG4 concentrations (≥57.7 mg/dl) were found to be associated with CAD with an odds ratio of 2.82 and 4.08, respectively, compared with the corresponding lowest quartile. CONCLUSIONS Serum concentrations of IgG4 and sIL-2R were increased in patients with angiographically-proven CAD, suggesting that IgG4-related immuno-inflammation may also have a role in the development and/or progression of coronary artery atherosclerosis.