Publications by authors named "Takashi Minami"

91 Publications

Calcineurin-nuclear factor for activated T cells (NFAT) signaling in pathophysiology of wound healing.

Inflamm Regen 2021 Aug 18;41(1):26. Epub 2021 Aug 18.

Division of Molecular and Vascular Biology, IRDA, Kumamoto University, 2-2-1 Honjyo Chuo-ku, Kumamoto, 860-0811, Japan.

Wound healing occurred with serial coordinated processes via coagulation-fibrinolysis, inflammation following to immune-activation, angiogenesis, granulation, and the final re-epithelization. Since the dermis forms critical physical and biological barriers, the repair system should be rapidly and accurately functioned to keep homeostasis in our body. The wound healing is impaired or dysregulated via an inappropriate microenvironment, which is easy to lead to several diseases, including fibrosis in multiple organs and psoriasis. Such a disease led to the dysregulation of several types of cells: immune cells, fibroblasts, mural cells, and endothelial cells. Moreover, recent progress in medical studies uncovers the significant concept. The calcium signaling, typically the following calcineurin-NFAT signaling, essentially regulates not only immune cell activations, but also various healing steps via coagulation, inflammation, and angiogenesis. In this review, we summarize the role of the NFAT activation pathway in wound healing and discuss its overall impact on future therapeutic ways.
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http://dx.doi.org/10.1186/s41232-021-00176-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8371293PMC
August 2021

NFAT indicates nucleocytoplasmic damped oscillation via its feedback modulator.

Biochem Biophys Res Commun 2021 Sep 28;571:201-209. Epub 2021 Jul 28.

Divison of Molecular and Vascular Biology, Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan. Electronic address:

Cell signaling and the following gene regulation are tightly regulated to keep homeostasis. NF-κB is a famous key transcription factor for inflammatory cell regulations that obtain a closed feedback loop with IκB. Similarly, we show here, NFAT is also tightly regulated via its downstream target, down syndrome critical region (DSCR)-1. In primary cultured endothelium, either shear stress or VEGF treatment revealed quick NFAT1 nuclear localization following the DSCR-1 transactivation, which in turn induced NFAT1 cytoplasm sequestration. Interestingly, both NFAT and DSCR-1 can be competitive substrates for calcineurin phosphatase and DSCR-1 is known to unstable protein, which caused NFAT1-nucleocytoplasmic damped oscillation via sustained shear stress or VEGF stimulation in endothelial cell (EC)s. To understand the molecular mechanism underlying the NFAT1 oscillation, we built a mathematical model of spatiotemporal regulation of NFAT1 combined with calcineurin and DSCR-1. Theoretically, manipulation of DSCR-1 expression in simulation predicted that DSCR-1 reduction would cause nuclear retention of dephosphorylated NFAT1 and disappearance of NFAT1 oscillation. To confirm this in ECs, DSCR-1 knockdown analysis was performed. DSCR-1 reduction indeed increased dephosphorylated NFAT1 in both the nucleus and cytoplasm, which eventually led to nuclear retention of NFAT1. Taken together, these studies suggest that DSCR-1 is a responsible critical factor for NFAT1 nucleocytoplasmic oscillation in shear stress or VEGF treated ECs. Our mathematical model successfully reproduced the experimental observations of NFAT1 dynamics. Combined mathematical and experimental approaches would provide a quantitative understanding way for the spatiotemporal NFAT1 feedback system.
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http://dx.doi.org/10.1016/j.bbrc.2021.07.072DOI Listing
September 2021

Lysine Demethylase 5A is Required for MYC Driven Transcription in Multiple Myeloma.

Blood Cancer Discov 2021 07 10;2(4):370-387. Epub 2021 Apr 10.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA 02215.

Lysine demethylase 5A (KDM5A) is a negative regulator of histone H3K4 trimethylation, a histone mark associated with activate gene transcription. We identify that KDM5A interacts with the P-TEFb complex and cooperates with MYC to control MYC targeted genes in multiple myeloma (MM) cells. We develop a cell-permeable and selective KDM5 inhibitor, JQKD82, that increases histone H3K4me3 but paradoxically inhibits downstream MYC-driven transcriptional output and . Using genetic ablation together with our inhibitor, we establish that KDM5A supports MYC target gene transcription independent of MYC itself, by supporting TFIIH (CDK7)- and P-TEFb (CDK9)-mediated phosphorylation of RNAPII. These data identify KDM5A as a unique vulnerability in MM functioning through regulation of MYC-target gene transcription, and establish JQKD82 as a tool compound to block KDM5A function as a potential therapeutic strategy for MM.
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http://dx.doi.org/10.1158/2643-3230.BCD-20-0108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8265280PMC
July 2021

miRNA-1246 in extracellular vesicles secreted from metastatic tumor induces drug resistance in tumor endothelial cells.

Sci Rep 2021 Jul 5;11(1):13502. Epub 2021 Jul 5.

Department of Vascular Biology, Hokkaido University Graduate School of Dental Medicine, Sapporo, 060-8586, Japan.

Tumor endothelial cells (TECs) reportedly exhibit altered phenotypes. We have demonstrated that TECs acquire drug resistance with the upregulation of P-glycoprotein (P-gp, ABCB1), contrary to traditional assumptions. Furthermore, P-gp expression was higher in TECs of highly metastatic tumors than in those of low metastatic tumors. However, the detailed mechanism of differential P-gp expression in TECs remains unclear. miRNA was identified in highly metastatic tumor extracellular vesicles (EVs) and the roles of miRNA in endothelial cell resistance were analyzed in vitro and in vivo. In the present study, we found that treatment of highly metastatic tumor-conditioned medium induced resistance to 5-fluorouracil (5-FU) with interleukin-6 (IL-6) upregulation in endothelial cells (ECs). Among the soluble factors secreted from highly metastatic tumors, we focused on EVs and determined that miR-1246 was contained at a higher level in highly metastatic tumor EVs than in low metastatic tumor EVs. Furthermore, miR-1246 was transported via the EVs into ECs and induced IL-6 expression. Upregulated IL-6 induced resistance to 5-FU with STAT3 and Akt activation in ECs in an autocrine manner. These results suggested that highly metastatic tumors induce drug resistance in ECs by transporting miR-1246 through EVs.
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http://dx.doi.org/10.1038/s41598-021-92879-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8257582PMC
July 2021

Loss of Down syndrome critical region-1 leads to cholesterol metabolic dysfunction that exaggerates hypercholesterolemia in ApoE-null background.

J Biol Chem 2021 Jan-Jun;296:100697. Epub 2021 Apr 23.

Division of Molecular and Vascular Biology, IRDA, Kumamoto University, Kumamoto, Japan. Electronic address:

Down syndrome critical region (DSCR)-1 functions as a feedback modulator for calcineurin-nuclear factor for activated T cell (NFAT) signals, which are crucial for cell proliferation and inflammation. Stable expression of DSCR-1 inhibits pathological angiogenesis and septic inflammation. DSCR-1 also plays a critical role in vascular wall remodeling associated with aneurysm development that occurs primarily in smooth muscle cells. Besides, Dscr-1 deficiency promotes the M1-to M2-like phenotypic switch in macrophages, which correlates to the reduction of denatured cholesterol uptakes. However, the distinct roles of DSCR-1 in cholesterol and lipid metabolism are not well understood. Here, we show that loss of apolipoprotein (Apo) E in mice with chronic hypercholesterolemia induced Dscr-1 expression in the liver and aortic atheroma. In Dscr-1-null mice fed a high-fat diet, oxidative- and endoplasmic reticulum (ER) stress was induced, and sterol regulatory element-binding protein (SREBP) 2 production in hepatocytes was stimulated. This exaggerated ApoE-mediated nonalcoholic fatty liver disease (NAFLD) and subsequent hypercholesterolemia. Genome-wide screening revealed that loss of both ApoE and Dscr-1 resulted in the induction of immune- and leukocyte activation-related genes in the liver compared with ApoE deficiency alone. However, expressions of inflammation-activated markers and levels of monocyte adhesion were suspended upon induction of the Dscr-1 null background in the aortic endothelium. Collectively, our study shows that the combined loss of Dscr-1 and ApoE causes metabolic dysfunction in the liver but reduces atherosclerotic plaques, thereby leading to a dramatic increase in serum cholesterol and the formation of sporadic vasculopathy.
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http://dx.doi.org/10.1016/j.jbc.2021.100697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8142255PMC
August 2021

Loss of Down Syndrome Critical Region-1 Mediated-Hypercholesterolemia Accelerates Corneal Opacity Via Pathological Neovessel Formation.

Arterioscler Thromb Vasc Biol 2020 10 13;40(10):2425-2439. Epub 2020 Aug 13.

Division of Molecular and Vascular Biology, IRDA, Kumamoto University, Japan (M.M., T.M.).

Objective: The calcineurin-NFAT (nuclear factor for activated T cells)-DSCR (Down syndrome critical region)-1 pathway plays a crucial role as the downstream effector of VEGF (vascular endothelial growth factor)-mediated tumor angiogenesis in endothelial cells. A role for DSCR-1 in different organ microenvironment such as the cornea and its role in ocular diseases is not well understood. Corneal changes can be indicators of various disease states and are easily detected through ocular examinations. Approach and Results: The presentation of a corneal arcus or a corneal opacity due to lipid deposition in the cornea often indicates hyperlipidemia and in most cases, hypercholesterolemia. Although the loss of Apo (apolipoprotein) E has been well characterized and is known to lead to elevated serum cholesterol levels, there are few corneal changes observed in mice. In this study, we show that the combined loss of ApoE and DSCR-1 leads to a dramatic increase in serum cholesterol levels and severe corneal opacity with complete penetrance. The cornea is normally maintained in an avascular state; however, loss of is sufficient to induce hyper-inflammatory and -oxidative condition, increased corneal neovascularization, and lymphangiogenesis. Furthermore, immunohistological analysis and genome-wide screening revealed that loss of in mice triggers increased immune cell infiltration and upregulation of SDF (stromal derived factor)-1 and its receptor, CXCR4 (C-X-C motif chemokine ligand receptor-4), potentiating this signaling axis in the cornea, thereby contributing to pathological corneal angiogenesis and opacity.

Conclusions: This study is the first demonstration of the critical role for the endogenous inhibitor of calcineurin, DSCR-1, and pathological corneal angiogenesis in hypercholesterolemia induced corneal opacity.
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http://dx.doi.org/10.1161/ATVBAHA.120.315003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518298PMC
October 2020

Organ/Tissue-Specific Vascular Endothelial Cell Heterogeneity in Health and Disease.

Biol Pharm Bull 2019 ;42(10):1609-1619

Div. of Molecular and Vascular Biology, IRDA, Kumamoto University.

The vascular system forms the largest surface in our body, serving as a critical interface between blood circulation and our diverse organ/tissue environments. Thus, the vascular system performs a gatekeeper function for organ/tissue homeostasis and the body's adjustment to pathological challenges. The endothelium, as the most inner layer of the vasculature, regulates the tissue microenvironment, which is critical for development, hemostatic balance, inflammation, and angiogenesis, with a role as well in tumor malignancy and metastasis. These multitudinous functions are primarily mediated by organ/tissue-specifically differentiated endothelial cells, in which heterogeneity has long been recognized at the molecular and histological level. Based on these general principles of vascular-bed heterogeneity and characterization, this review largely covers landmark discoveries regarding organ/tissue microenvironment-governed endothelial cell phenotypic changes. These involve the physical features of continuous, discontinuous, fenestrated, and sinusoidal endothelial cells, in addition to the more specialized endothelial cell layers of the lymphatic system, glomerulus, tumors, and the blood brain barrier (BBB). Major signal pathways of endothelial specification are outlined, including Notch as a key factor of tip/stalk- and arterial-endothelial cell differentiation. We also denote the shear stress sensing machinery used to convey blood flow-mediated biophysical forces that are indispensable to maintaining inert and mature endothelial phenotypes. Since our circulatory system is among the most fundamental and emergent targets of study in pharmacology from the viewpoint of drug metabolism and delivery, a better molecular understanding of organ vasculature-bed heterogeneity may lead to better strategies for novel vascular-targeted treatments to fight against hitherto intractable diseases.
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http://dx.doi.org/10.1248/bpb.b19-00531DOI Listing
February 2020

Phosphoethanolamine Accumulation Protects Cancer Cells under Glutamine Starvation through Downregulation of PCYT2.

Cell Rep 2019 10;29(1):89-103.e7

Division of Metabolic Medicine, RCAST, The University of Tokyo, Tokyo 153-8904, Japan; Division of Molecular Physiology and Metabolism, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan.

Tolerance to severe tumor microenvironments, including hypoxia and nutrient starvation, is a common feature of aggressive cancer cells and can be targeted. However, metabolic alterations that support cancer cells upon nutrient starvation are not well understood. Here, by comprehensive metabolome analyses, we show that glutamine deprivation leads to phosphoethanolamine (PEtn) accumulation in cancer cells via the downregulation of PEtn cytidylyltransferase (PCYT2), a rate-limiting enzyme of phosphatidylethanolamine biosynthesis. PEtn accumulation correlated with tumor growth under nutrient starvation. PCYT2 suppression was partially mediated by downregulation of the transcription factor ELF3. Furthermore, PCYT2 overexpression reduced PEtn levels and tumor growth. In addition, PEtn accumulation and PCYT2 downregulation in human breast tumors correlated with poor prognosis. Thus, we show that glutamine deprivation leads to tumor progression by regulating PE biosynthesis via the ELF3-PCYT2 axis. Furthermore, manipulating glutamine-responsive genes could be a therapeutic approach to limit cancer progression.
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http://dx.doi.org/10.1016/j.celrep.2019.08.087DOI Listing
October 2019

Downregulation of ERG and FLI1 expression in endothelial cells triggers endothelial-to-mesenchymal transition.

PLoS Genet 2018 11 30;14(11):e1007826. Epub 2018 Nov 30.

Division of Molecular and Vascular Biology, IRDA, Kumamoto University, Kumamoto, Japan.

Endothelial cell (EC) plasticity in pathological settings has recently been recognized as a driver of disease progression. Endothelial-to-mesenchymal transition (EndMT), in which ECs acquire mesenchymal properties, has been described for a wide range of pathologies, including cancer. However, the mechanism regulating EndMT in the tumor microenvironment and the contribution of EndMT in tumor progression are not fully understood. Here, we found that combined knockdown of two ETS family transcription factors, ERG and FLI1, induces EndMT coupled with dynamic epigenetic changes in ECs. Genome-wide analyses revealed that ERG and FLI1 are critical transcriptional activators for EC-specific genes, among which microRNA-126 partially contributes to blocking the induction of EndMT. Moreover, we demonstrated that ERG and FLI1 expression is downregulated in ECs within tumors by soluble factors enriched in the tumor microenvironment. These data provide new insight into the mechanism of EndMT, functions of ERG and FLI1 in ECs, and EC behavior in pathological conditions.
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http://dx.doi.org/10.1371/journal.pgen.1007826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6291168PMC
November 2018

Loss of Endogenous HMGB2 Promotes Cardiac Dysfunction and Pressure Overload-Induced Heart Failure in Mice.

Circ J 2019 01 27;83(2):368-378. Epub 2018 Nov 27.

Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University.

Background: The rapid increase in the number of heart failure (HF) patients in parallel with the increase in the number of older people is receiving attention worldwide. HF not only increases mortality but decreases quality of life, creating medical and social problems. Thus, it is necessary to define molecular mechanisms underlying HF development and progression. HMGB2 is a member of the high-mobility group superfamily characterized as nuclear proteins that bind DNA to stabilize nucleosomes and promote transcription. A recent in vitro study revealed that HMGB2 loss in cardiomyocytes causes hypertrophy and increases HF-associated gene expression. However, it's in vivo function in the heart has not been assessed. Methods and Results: Western blotting analysis revealed increased HMGB2 expression in heart tissues undergoing pressure overload by transverse aorta constriction (TAC) in mice. Hmgb2 homozygous knockout (Hmgb2) mice showed cardiac dysfunction due to AKT inactivation and decreased sarco(endo)plasmic reticulum Ca-ATPase (SERCA)2a activity. Compared to wild-type mice, Hmgb2 mice had worsened cardiac dysfunction after TAC surgery, predisposing mice to HF development and progression.

Conclusions: This study demonstrates that upregulation of cardiac HMGB2 is an adaptive response to cardiac stress, and that loss of this response could accelerate cardiac dysfunction, suggesting that HMGB2 plays a cardioprotective role.
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http://dx.doi.org/10.1253/circj.CJ-18-0925DOI Listing
January 2019

Development of a high-speed full digital processing phase detector for interferometry.

Rev Sci Instrum 2018 Oct;89(10):10K108

National Institute for Fusion Science, Oroshi, Toki-shi, Gifu 509-5292, Japan.

This study describes the development of a fully digital-type phase detector for plasma interferometry. This detector functions even in situations in which the phase changes rapidly or the input signal is too small to derive the correct phase shift from the intermediate frequency (IF) signal. The detector directly converts the IF signal waveform of the interferometer to the phase shift signal by means of data processing in a logic circuit. Thus, the phase is derived from the whole waveform of the IF signal. The IF signal of the interferometer is converted to in-phase and quadrature-phase signals by Hilbert transformation, processed by a digital low-pass filter, and converted to polar coordinates by a coordinate rotation digital computer algorithm to obtain the phase shift. A simulation of the high-speed full digital processing phase detector shows that a fringe jump does not occur unless the phase change rate exceeds 0.8 × 10 rad/s. This value is sufficiently large compared to the phase change velocity in rapid density increase resulting from a pellet injection. The phase conversion is simulated using a real IF signal from an interferometer measured with a Heliotron J device. The results show that the phase signal is correctly calculated by the full digital processing method from the IF signal, the phase derivation of which is typically difficult to obtain when using a conventional analog phase detector.
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http://dx.doi.org/10.1063/1.5038838DOI Listing
October 2018

Folliculin Regulates Osteoclastogenesis Through Metabolic Regulation.

J Bone Miner Res 2018 10 26;33(10):1785-1798. Epub 2018 Jun 26.

International Research Center for Medical Sciences (IRCMS), Kumamoto University, Kumamoto, Japan.

Osteoclast differentiation is a dynamic differentiation process, which is accompanied by dramatic changes in metabolic status as well as in gene expression. Recent findings have revealed an essential connection between metabolic reprogramming and dynamic gene expression changes during osteoclast differentiation. However, the upstream regulatory mechanisms that drive these metabolic changes in osteoclastogenesis remain to be elucidated. Here, we demonstrate that induced deletion of a tumor suppressor gene, Folliculin (Flcn), in mouse osteoclast precursors causes severe osteoporosis in 3 weeks through excess osteoclastogenesis. Flcn-deficient osteoclast precursors reveal cell autonomous accelerated osteoclastogenesis with increased sensitivity to receptor activator of NF-κB ligand (RANKL). We demonstrate that Flcn regulates oxidative phosphorylation and purine metabolism through suppression of nuclear localization of the transcription factor Tfe3, thereby inhibiting expression of its target gene Pgc1. Metabolome studies revealed that Flcn-deficient osteoclast precursors exhibit significant augmentation of oxidative phosphorylation and nucleotide production, resulting in an enhanced purinergic signaling loop that is composed of controlled ATP release and autocrine/paracrine purinergic receptor stimulation. Inhibition of this purinergic signaling loop efficiently blocks accelerated osteoclastogenesis in Flcn-deficient osteoclast precursors. Here, we demonstrate an essential and novel role of the Flcn-Tfe3-Pgc1 axis in osteoclastogenesis through the metabolic reprogramming of oxidative phosphorylation and purine metabolism. © 2018 The Authors Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).
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http://dx.doi.org/10.1002/jbmr.3477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220829PMC
October 2018

Pathophysiology Derived from Down Syndrome Related Genes on the 21st Chromosome.

Authors:
Takashi Minami

Yakugaku Zasshi 2017;137(7):791-793

Division of Vascular Biology, Research Center for Advanced Science and Technology (RCAST), The University of Tokyo.

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http://dx.doi.org/10.1248/yakushi.16-00236-FDOI Listing
January 2017

Negative feedback loop of bone resorption by NFATc1-dependent induction of Cadm1.

PLoS One 2017 17;12(4):e0175632. Epub 2017 Apr 17.

Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

Trimethylation of histone H3 lysine 4 and lysine 27 (H3K4me3 and H3K27me3) at gene promoter regions critically regulates gene expression. Key developmental genes tend to exhibit changes in histone modification patterns from the H3K4me3/H3K27me3 bivalent pattern to the H3K4me3 monovalent pattern. Using comprehensive chromatin immunoprecipitation followed by sequencing in bone marrow-derived macrophages (BMMs) and mature osteoclasts, we found that cell surface adhesion molecule 1 (Cadm1) is a direct target of nuclear factor of activated T cells 1 (NFATc1) and exhibits a bivalent histone pattern in BMMs and a monovalent pattern in osteoclasts. Cadm1 expression was upregulated in BMMs by receptor activator of nuclear factor kappa B ligand (RANKL), and blocked by a calcineurin/NFATc1 inhibitor, FK506. Cadm1-deficient mice exhibited significantly reduced bone mass compared with wild-type mice, which was due to the increased osteoclast differentiation, survival and bone-resorbing activity in Cadm1-deficient osteoclasts. These results suggest that Cadm1 is a direct target of NFATc1, which is induced by RANKL through epigenetic modification, and regulates osteoclastic bone resorption in a negative feedback manner.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0175632PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393607PMC
May 2017

Dynamically and epigenetically coordinated GATA/ETS/SOX transcription factor expression is indispensable for endothelial cell differentiation.

Nucleic Acids Res 2017 05;45(8):4344-4358

Division of Vascular Biology, RCAST, The University of Tokyo, Tokyo 153-8904, Japan.

Although studies of the differentiation from mouse embryonic stem (ES) cells to vascular endothelial cells (ECs) provide an excellent model for investigating the molecular mechanisms underlying vascular development, temporal dynamics of gene expression and chromatin modifications have not been well studied. Herein, using transcriptomic and epigenomic analyses based on H3K4me3 and H3K27me3 modifications at a genome-wide scale, we analysed the EC differentiation steps from ES cells and crucial epigenetic modifications unique to ECs. We determined that Gata2, Fli1, Sox7 and Sox18 are master regulators of EC that are induced following expression of the haemangioblast commitment pioneer factor, Etv2. These master regulator gene loci were repressed by H3K27me3 throughout the mesoderm period but rapidly transitioned to histone modification switching from H3K27me3 to H3K4me3 after treatment with vascular endothelial growth factor. SiRNA knockdown experiments indicated that these regulators are indispensable not only for proper EC differentiation but also for blocking the commitment to other closely aligned lineages. Collectively, our detailed epigenetic analysis may provide an advanced model for understanding temporal regulation of chromatin signatures and resulting gene expression profiles during EC commitment. These studies may inform the future development of methods to stimulate the vascular endothelium for regenerative medicine.
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http://dx.doi.org/10.1093/nar/gkx159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5416769PMC
May 2017

Sustained inflammation after pericyte depletion induces irreversible blood-retina barrier breakdown.

JCI Insight 2017 02 9;2(3):e90905. Epub 2017 Feb 9.

Department of Retinal Vascular Biology.

In the central nervous system, endothelial cells (ECs) and pericytes (PCs) of blood vessel walls cooperatively form a physical and chemical barrier to maintain neural homeostasis. However, in diabetic retinopathy (DR), the loss of PCs from vessel walls is assumed to cause breakdown of the blood-retina barrier (BRB) and subsequent vision-threatening vascular dysfunctions. Nonetheless, the lack of adequate DR animal models has precluded disease understanding and drug discovery. Here, by using an anti-PDGFRβ antibody, we show that transient inhibition of the PC recruitment to developing retinal vessels sustained EC-PC dissociations and BRB breakdown in adult mouse retinas, reproducing characteristic features of DR such as hyperpermeability, hypoperfusion, and neoangiogenesis. Notably, PC depletion directly induced inflammatory responses in ECs and perivascular infiltration of macrophages, whereby macrophage-derived VEGF and placental growth factor (PlGF) activated VEGFR1 in macrophages and VEGFR2 in ECs. Moreover, angiopoietin-2 (Angpt2) upregulation and Tie1 downregulation activated FOXO1 in PC-free ECs locally at the leaky aneurysms. This cycle of vessel damage was shut down by simultaneously blocking VEGF, PlGF, and Angpt2, thus restoring the BRB integrity. Together, our model provides new opportunities for identifying the sequential events triggered by PC deficiency, not only in DR, but also in various neurological disorders.
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http://dx.doi.org/10.1172/jci.insight.90905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291729PMC
February 2017

High time resolved electron temperature measurements by using the multi-pass Thomson scattering system in GAMMA 10/PDX.

Rev Sci Instrum 2016 Nov;87(11):11D617

Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.

High time resolved electron temperature measurements are useful for fluctuation study. A multi-pass Thomson scattering (MPTS) system is proposed for the improvement of both increasing the TS signal intensity and time resolution. The MPTS system in GAMMA 10/PDX has been constructed for enhancing the Thomson scattered signals for the improvement of measurement accuracy. The MPTS system has a polarization-based configuration with an image relaying system. We optimized the image relaying optics for improving the multi-pass laser confinement and obtaining the stable MPTS signals over ten passing TS signals. The integrated MPTS signals increased about five times larger than that in the single pass system. Finally, time dependent electron temperatures were obtained in MHz sampling.
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http://dx.doi.org/10.1063/1.4955287DOI Listing
November 2016

Emerging Role of VEGFC in Pathological Angiogenesis.

EBioMedicine 2015 Nov 10;2(11):1588-9. Epub 2015 Nov 10.

Div. phenotype diseases analysis, IRDA, Kumamoto University, Kumamoto, Japan.

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http://dx.doi.org/10.1016/j.ebiom.2015.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4740333PMC
November 2015

Suppression of Slit2/Robo1 mediated HUVEC migration by Robo4.

Biochem Biophys Res Commun 2016 Jan 20;469(4):797-802. Epub 2015 Dec 20.

Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan. Electronic address:

Slit proteins and their receptors, the Roundabout (Robo) family, are known to have a pivotal role in the vascular system. Slit2/Robo1 regulates the migration of human umbilical vein endothelial cells (HUVECs) and tumor-associated endothelial cells. Robo4, the endothelial-specific Robo, is also considered to be involved in vascular cell migration. However, the Slit/Robo signaling pathway is still unclear. Using a Boyden chamber assay, we found that Slit2 induces the migration of HUVECs under a Robo4 knockdown condition. This effect disappeared in Robo1 knockdown cells. The co-existence of the N-terminal extracellular portion of Robo1 blocked the Slit2-evoked migration of HUVECs, while that of Robo4 caused no effect. These results show that the Slit2 signal is transduced through Robo1, while the negative regulation of Robo4 is an intracellular event. Targeted proteomics using an anti-Robo1 monoclonal antibody identified CdGAP, an adhesion-localized Rac1-and Cdc42-specific GTPase activating protein, as a candidate for Slit2/Robo1 signaling. Robo1 and CdGAP were co-immunoprecipitated from CHO cells co-transfected with Robo1 and CdGAP genes. These results suggest that Slit2/Robo1 binding exerts an effect on cell migration, which is negatively regulated by Robo4, and Robo1 may function by interacting with CdGAP in HUVECs.
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http://dx.doi.org/10.1016/j.bbrc.2015.12.075DOI Listing
January 2016

GATA2 facilitates steroid receptor coactivator recruitment to the androgen receptor complex.

Proc Natl Acad Sci U S A 2014 Dec 8;111(51):18261-6. Epub 2014 Dec 8.

Departments of Medicine, Molecular and Cellular Biology,

The androgen receptor (AR) is a key driver of prostate cancer (PC), even in the state of castration-resistant PC (CRPC) and frequently even after treatment with second-line hormonal therapies such as abiraterone and enzalutamide. The persistence of AR activity via both ligand-dependent and ligand-independent mechanisms (including constitutively active AR splice variants) highlights the unmet need for alternative approaches to block AR signaling in CRPC. We investigated the transcription factor GATA-binding protein 2 (GATA2) as a regulator of AR signaling and an actionable therapeutic target in PC. We demonstrate that GATA2 directly promotes expression of both full-length and splice-variant AR, resulting in a strong positive correlation between GATA2 and AR expression in both PC cell lines and patient specimens. Conversely, GATA2 expression is repressed by androgen and AR, suggesting a negative feedback regulatory loop that, upon androgen deprivation, derepresses GATA2 to contribute to AR overexpression in CRPC. Simultaneously, GATA2 is necessary for optimal transcriptional activity of both full-length and splice-variant AR. GATA2 colocalizes with AR and Forkhead box protein A1 on chromatin to enhance recruitment of steroid receptor coactivators and formation of the transcriptional holocomplex. In agreement with these important functions, high GATA2 expression and transcriptional activity predicted worse clinical outcome in PC patients. A GATA2 small molecule inhibitor suppressed the expression and transcriptional function of both full-length and splice-variant AR and exerted potent anticancer activity against PC cell lines. We propose pharmacological inhibition of GATA2 as a first-in-field approach to target AR expression and function and improve outcomes in CRPC.
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http://dx.doi.org/10.1073/pnas.1421415111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4280633PMC
December 2014

Protective effect of the long pentraxin PTX3 against histone-mediated endothelial cell cytotoxicity in sepsis.

Sci Signal 2014 Sep 16;7(343):ra88. Epub 2014 Sep 16.

Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan.

Pentraxin 3 (PTX3), a member of the long pentraxin subfamily within the family of pentraxins, is a soluble pattern recognition molecule that functions in the innate immune system. Innate immunity affords the infected host protection against sepsis, a potentially life-threatening inflammatory response to infection. Extracellular histones are considered to be the main cause of septic death because of their cytotoxic effect on endothelial cells, which makes them a potential therapeutic target. We found that PTX3 interacted with histones to form coaggregates, which depended on polyvalent interactions and disorder in the secondary structure of PTX3. PTX3 exerted a protective effect, both in vitro and in vivo, against histone-mediated cytotoxicity toward endothelial cells. Additionally, the intraperitoneal administration of PTX3 reduced mortality in mouse models of sepsis. The amino-terminal domain of PTX3, which was required for coaggregation with histones, was sufficient to protect against cytotoxicity. Our results suggest that the host-protective effects of PTX3 in sepsis are a result of its coaggregation with histones rather than its ability to mediate pattern recognition. This long pentraxin-specific effect provides a potential basis for the treatment of sepsis directed at protecting cells from the toxic effects of extracellular histones.
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http://dx.doi.org/10.1126/scisignal.2005522DOI Listing
September 2014

Genome-wide approaches reveal functional vascular endothelial growth factor (VEGF)-inducible nuclear factor of activated T cells (NFAT) c1 binding to angiogenesis-related genes in the endothelium.

J Biol Chem 2014 Oct 25;289(42):29044-59. Epub 2014 Aug 25.

From the Division of Vascular Biology,

VEGF is a key regulator of endothelial cell migration, proliferation, and inflammation, which leads to activation of several signaling cascades, including the calcineurin-nuclear factor of activated T cells (NFAT) pathway. NFAT is not only important for immune responses but also for cardiovascular development and the pathogenesis of Down syndrome. By using Down syndrome model mice and clinical patient samples, we showed recently that the VEGF-calcineurin-NFAT signaling axis regulates tumor angiogenesis and tumor metastasis. However, the connection between genome-wide views of NFAT-mediated gene regulation and downstream gene function in the endothelium has not been studied extensively. Here we performed comprehensive mapping of genome-wide NFATc1 binding in VEGF-stimulated primary cultured endothelial cells and elucidated the functional consequences of VEGF-NFATc1-mediated phenotypic changes. A comparison of the NFATc1 ChIP sequence profile and epigenetic histone marks revealed that predominant NFATc1-occupied peaks overlapped with promoter-associated histone marks. Moreover, we identified two novel NFATc1 regulated genes, CXCR7 and RND1. CXCR7 knockdown abrogated SDF-1- and VEGF-mediated cell migration and tube formation. siRNA treatment of RND1 impaired vascular barrier function, caused RhoA hyperactivation, and further stimulated VEGF-mediated vascular outgrowth from aortic rings. Taken together, these findings suggest that dynamic NFATc1 binding to target genes is critical for VEGF-mediated endothelial cell activation. CXCR7 and RND1 are NFATc1 target genes with multiple functions, including regulation of cell migration, tube formation, and barrier formation in endothelial cells.
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http://dx.doi.org/10.1074/jbc.M114.555235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200259PMC
October 2014

Myocardium-derived angiopoietin-1 is essential for coronary vein formation in the developing heart.

Nat Commun 2014 Jul 29;5:4552. Epub 2014 Jul 29.

1] Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan [2] Core Research for Evolutional Science and Technology (CREST), Japan Science Technology Agency, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan.

The origin and developmental mechanisms underlying coronary vessels are not fully elucidated. Here we show that myocardium-derived angiopoietin-1 (Ang1) is essential for coronary vein formation in the developing heart. Cardiomyocyte-specific Ang1 deletion results in defective formation of the subepicardial coronary veins, but had no significant effect on the formation of intramyocardial coronary arteries. The endothelial cells (ECs) of the sinus venosus (SV) are heterogeneous population, composed of APJ-positive and APJ-negative ECs. Among these, the APJ-negative ECs migrate from the SV into the atrial and ventricular myocardium in Ang1-dependent manner. In addition, Ang1 may positively regulate venous differentiation of the subepicardial APJ-negative ECs in the heart. Consistently, in vitro experiments show that Ang1 indeed promotes venous differentiation of the immature ECs. Collectively, our results indicate that myocardial Ang1 positively regulates coronary vein formation presumably by promoting the proliferation, migration and differentiation of immature ECs derived from the SV.
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http://dx.doi.org/10.1038/ncomms5552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4124867PMC
July 2014

PPARβ/δ activation of CD300a controls intestinal immunity.

Sci Rep 2014 Jun 24;4:5412. Epub 2014 Jun 24.

Division of Metabolic Medicine, Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, Tokyo 153-8904, Japan.

Macrophages are important for maintaining intestinal immune homeostasis. Here, we show that PPARβ/δ (peroxisome proliferator-activated receptor β/δ) directly regulates CD300a in macrophages that express the immunoreceptor tyrosine based-inhibitory motif (ITIM)-containing receptor. In mice lacking CD300a, high-fat diet (HFD) causes chronic intestinal inflammation with low numbers of intestinal lymph capillaries and dramatically expanded mesenteric lymph nodes. As a result, these mice exhibit triglyceride malabsorption and reduced body weight gain on HFD. Peritoneal macrophages from Cd300a-/- mice on HFD are classically M1 activated. Activation of toll-like receptor 4 (TLR4)/MyD88 signaling by lipopolysaccharide (LPS) results in prolonged IL-6 secretion in Cd300a-/- macrophages. Bone marrow transplantation confirmed that the phenotype originates from CD300a deficiency in leucocytes. These results identify CD300a-mediated inhibitory signaling in macrophages as a critical regulator of intestinal immune homeostasis.
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http://dx.doi.org/10.1038/srep05412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4067692PMC
June 2014

Direct evidence for pitavastatin induced chromatin structure change in the KLF4 gene in endothelial cells.

PLoS One 2014 5;9(5):e96005. Epub 2014 May 5.

Research Center for Advanced Science and Technology, The University of Tokyo, Meguro-ku, Tokyo, Japan.

Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor (KLF) family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs) treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2) family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq) followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET), and real time chromosome conformation capture (3C) assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS) of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH) imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0096005PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4010393PMC
June 2015

Role of endothelial cell-derived angptl2 in vascular inflammation leading to endothelial dysfunction and atherosclerosis progression.

Arterioscler Thromb Vasc Biol 2014 Apr 13;34(4):790-800. Epub 2014 Feb 13.

From the Department of Molecular Genetics (E.H., T.K., K.M., H.H., M.E., M. Tabata, H.T., Z.T., O.T., K.T., Y.O.), Department of Cardiovascular Medicine (E.H., H.O., K.K.), and Department of Cell Pathology (M. Takeya), Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; Division of Geriatric Medicine, Department of Internal Medicine (Y.A., N.H.) and Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine (K.H., T.S.), Keio University School of Medicine, Tokyo, Japan; Department of Environmental Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan (Y.D., T.N., Y.K.); Department of Surgical Pathology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan (H.H.); Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan (T.M.); and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo, Japan (Y.O.).

Objective: Cardiovascular disease (CVD), the most common morbidity resulting from atherosclerosis, remains a frequent cause of death. Efforts to develop effective therapeutic strategies have focused on vascular inflammation as a critical pathology driving atherosclerosis progression. Nonetheless, molecular mechanisms underlying this activity remain unclear. Here, we ask whether angiopoietin-like protein 2 (Angptl2), a proinflammatory protein, contributes to vascular inflammation that promotes atherosclerosis progression.

Approach And Results: Histological analysis revealed abundant Angptl2 expression in endothelial cells and macrophages infiltrating atheromatous plaques in patients with cardiovascular disease. Angptl2 knockout in apolipoprotein E-deficient mice (ApoE(-/-)/Angptl2(-/-)) attenuated atherosclerosis progression by decreasing the number of macrophages infiltrating atheromatous plaques, reducing vascular inflammation. Bone marrow transplantation experiments showed that Angptl2 deficiency in endothelial cells attenuated atherosclerosis development. Conversely, ApoE(-/-) mice crossed with transgenic mice expressing Angptl2 driven by the Tie2 promoter (ApoE(-/-)/Tie2-Angptl2 Tg), which drives Angptl2 expression in endothelial cells but not monocytes/macrophages, showed accelerated plaque formation and vascular inflammation because of increased numbers of infiltrated macrophages in atheromatous plaques. Tie2-Angptl2 Tg mice alone did not develop plaques but exhibited endothelium-dependent vasodilatory dysfunction, likely because of decreased production of endothelial cell-derived nitric oxide. Conversely, Angptl2(-/-) mice exhibited less severe endothelial dysfunction than did wild-type mice when fed a high-fat diet. In vitro, Angptl2 activated proinflammatory nuclear factor-κB signaling in endothelial cells and increased monocyte/macrophage chemotaxis.

Conclusions: Endothelial cell-derived Angptl2 accelerates vascular inflammation by activating proinflammatory signaling in endothelial cells and increasing macrophage infiltration, leading to endothelial dysfunction and atherosclerosis progression.
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http://dx.doi.org/10.1161/ATVBAHA.113.303116DOI Listing
April 2014

Calcineurin-NFAT activation and DSCR-1 auto-inhibitory loop: how is homoeostasis regulated?

Authors:
Takashi Minami

J Biochem 2014 Apr 5;155(4):217-26. Epub 2014 Feb 5.

Div. of Vascular Biology, RCAST, The University of Tokyo, Tokyo 153-8904, Japan.

Calcineurin-nuclear factor of activated T cells (NFAT) signalling plays a critical role not only in the immune and nervous systems, but also in cardiovascular development and pathological endothelial cell activation during angiogenesis or inflammation. Studies in NFAT-null mice demonstrated that there is high redundancy between functions of the different NFAT family members. Deletion of only one NFAT causes mild phenotypes, but compound deletions of multiple NFAT family members leads to severe abnormalities in multiple organ systems. Genome-wide transcription analysis revealed that many NFAT target genes are related to cell growth and inflammation, whereas the gene most strongly induced by NFAT in endothelial cells is an auto-inhibitory molecule, Down syndrome critical region (DSCR)-1. The NFAT-DSCR-1 signalling axis may vary depending on the cell-type or signal dosage level under the microenvironment. In the endothelium, stable expression of the DSCR-1 short isoform attenuates septic inflammatory shock, tumour growth and tumour metastasis to lung. Moreover, dysfunction of DSCR-1 and the NFAT priming kinase, DYRK1A, prevents NFAT nuclear occupancy. This change in NFAT nuclear localization is responsible for many of the features of Down syndrome. Thus, fine-tuning of the NFAT-DSCR-1 negative feedback loop may enable therapeutic manipulation in vasculopathic diseases.
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http://dx.doi.org/10.1093/jb/mvu006DOI Listing
April 2014

A major role for RCAN1 in atherosclerosis progression.

EMBO Mol Med 2013 Dec 15;5(12):1901-17. Epub 2013 Oct 15.

Department of Vascular Biology and Inflammation, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain.

Atherosclerosis is a complex inflammatory disease involving extensive vascular vessel remodelling and migration of vascular cells. As RCAN1 is implicated in cell migration, we investigated its contribution to atherosclerosis. We show RCAN1 induction in atherosclerotic human and mouse tissues. Rcan1 was expressed in lesional macrophages, endothelial cells and vascular smooth muscle cells and was induced by treatment of these cells with oxidized LDLs (oxLDLs). Rcan1 regulates CD36 expression and its genetic inactivation reduced atherosclerosis extension and severity in Apoe(-/-) mice. This effect was mechanistically linked to diminished oxLDL uptake, resistance to oxLDL-mediated inhibition of macrophage migration and increased lesional IL-10 and mannose receptor expression. Moreover, Apoe(-/-) Rcan1(-/-) macrophages expressed higher-than-Apoe(-/-) levels of anti-inflammatory markers. We previously showed that Rcan1 mediates aneurysm development and that its expression is not required in haematopoietic cells for this process. However, transplantation of Apoe(-/-) Rcan1(-/-) bone-marrow (BM) cells into Apoe(-/-) recipients confers atherosclerosis resistance. Our data define a major role for haematopoietic Rcan1 in atherosclerosis and suggest that therapies aimed at inhibiting RCAN1 expression or function might significantly reduce atherosclerosis burden.
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http://dx.doi.org/10.1002/emmm.201302842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3914525PMC
December 2013

The calcineurin-NFAT-angiopoietin-2 signaling axis in lung endothelium is critical for the establishment of lung metastases.

Cell Rep 2013 Aug 15;4(4):709-23. Epub 2013 Aug 15.

Division of Vascular Biology, RCAST, the University of Tokyo, Tokyo 153-8904, Japan.

The premetastatic niche is a predetermined site of metastases, awaiting the influx of tumor cells. However, the regulation of the angiogenic switch at these sites has not been examined. Here, we demonstrate that the calcineurin and nuclear factor of activated T cells (NFAT) pathway is activated specifically in lung endothelium prior to the detection of tumor cells that preferentially metastasize to the lung. Upregulation of the calcineurin pathway via deletion of its endogenous inhibitor Dscr1 leads to a significant increase in lung metastases due to increased expression of a newly identified NFAT target, Angiopoietin-2 (ANG2). Increased VEGF levels specifically in the lung, and not other organ microenvironments, trigger a threshold of calcineurin-NFAT signaling that transactivates Ang2 in lung endothelium. Further, we demonstrate that overexpression of DSCR1 or the ANG2 receptor, soluble TIE2, prevents the activation of lung endothelium, inhibiting lung metastases in our mouse models. Our studies provide insights into mechanisms underlying angiogenesis in the premetastatic niche and offer targets for lung metastases.
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http://dx.doi.org/10.1016/j.celrep.2013.07.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3763962PMC
August 2013
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