Publications by authors named "Masashi Muramatsu"

23 Publications

  • Page 1 of 1

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

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

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

SSeCKS/Akap12 suppresses metastatic melanoma lung colonization by attenuating Src-mediated pre-metastatic niche crosstalk.

Oncotarget 2018 Sep 11;9(71):33515-33527. Epub 2018 Sep 11.

Department of Cancer Genetics and Genomics, Roswell Park Comprehensive Cancer Center, Buffalo 14263, NY, USA.

SSeCKS/Gravin/AKAP12 (SSeCKS) controls metastasis-associated PKC and Src signaling through direct scaffolding activity. SSeCKS is downregulated in the metastases of many human cancer types, and its forced re-expression suppresses the metastatic behavior of prostate cancer cells. SSeCKS is also downregulated in breast and prostate cancer stroma, and SSeCKS-null mice (KO) are metastasis-prone, suggesting a role in suppressing formation of the pre-metastatic niche. Here, we show that lung colonization and metastasis formation by B16F10 and SM1WT1[ ] mouse melanoma cells is 9-fold higher in syngeneic KO compared to WT hosts, although there is no difference in orthotopic tumor volumes. Although melanoma cells adhered equally to KO or WT lung fibroblasts (LF), co-injection of melanoma cells with KO (vs. WT) LF increased lung macrometastasis formation in WT hosts, marked by increased melanoma colonization at foci of leaky vasculature. Increased melanoma adhesion on KO lung endothelial cells (LEC) was facilitated by increased E-Selectin levels and by increased STAT3-regulated secretion of senescence-associated factors from KO-LF, such as Vegf. Finally, the ability of SSeCKS to attenuate IFNα-induced Stat3 activation in KO-LF required its Src-scaffolding domain. Taken together, these data suggest that SSeCKS normally suppresses metastatic colonization in the lung by attenuating the expression of Selectin adhesion proteins, which can be controlled autonomously by local endothelial cells or enhanced by senescence factors secreted by neighboring fibroblasts in a SSeCKS-regulated, Src/Stat3-dependent manner.
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http://dx.doi.org/10.18632/oncotarget.26067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173366PMC
September 2018

Defensive effect of microRNA-200b/c against amyloid-beta peptide-induced toxicity in Alzheimer's disease models.

PLoS One 2018 8;13(5):e0196929. Epub 2018 May 8.

Medical Genome Center, National Center for Geriatrics and Gerontology, Obu, Aichi, Japan.

MiRNA molecules are important post-transcriptional regulators of gene expression in the brain function. Altered miRNA profiles could represent a defensive response against the pathogenesis of neurodegenerative disorders, such as Alzheimer's disease (AD). Endogenous miRNAs have lower toxic effects than other gene silencing methods, thus enhancing the expression of defensive miRNA could be an effective therapy. However, little is known about the potential of targeting miRNAs for the treatment of AD. Here, we examined the function of the miR-200 family (miR-200a, -141, -429, -200b, -200c), identified using miRNA microarray analysis of cortical tissue from Tg2576 transgenic mice. In murine primary neurons, we found that upregulation of miR-200b or -200c was induced by the addition of amyloid beta (Aβ). Neurons transfected with miR-200b or -200c reduced secretion of Aβ in conditioned medium. Moreover, mice infused with miR-200b/c into the brain were relieved of memory impairments induced by intracerebroventricular injection of oligomeric Aβ, and demonstrated proper spatial learning in the Barnes maze. To gain further understanding of the relationship between miR-200b/c and Aβ, we identified target mRNAs via an RNA-binding protein immunoprecipitation-microarray assay. Western blot analysis showed that expression of ribosomal protein S6 kinase B1 (S6K1), a candidate target, was inhibited by miR-200c. S6K1, a downstream effector of mammalian target of rapamycin (mTOR), serves as a negative feedback mediator that phosphorylates insulin receptor substrate 1 at serine residues (IRS-1pSer). S6K1-dependent IRS-1pSer suppresses insulin signaling leading to insulin resistance, which is frequently observed in AD brains. Notably, miR-200b/c transfection of SH-SY5Y cells reduced the levels of IRS-1pSer. This finding indicates that miR-200b/c has the potential to alleviate insulin resistance via modulation of S6K1. Taken together, miR-200b/c may contribute to reduce Aβ secretion and Aβ-induced cognitive impairment by promoting insulin signaling.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0196929PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940223PMC
August 2018

SSeCKS/AKAP12 scaffolding functions suppress B16F10-induced peritoneal metastasis by attenuating CXCL9/10 secretion by resident fibroblasts.

Oncotarget 2017 Sep 9;8(41):70281-70298. Epub 2017 Aug 9.

Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo 14263, NY, USA.

SSeCKS/Gravin/AKAP12 (SSeCKS) is a kinase scaffolding protein known to suppress metastasis by attenuating tumor-intrinsic PKC- and Src-mediated signaling pathways [1]. In addition to downregulation in metastatic cells, analyses identified SSeCKS downregulation in prostate or breast cancer-derived stroma, suggesting a microenvironmental cell role in controlling malignancy. Although orthotopic B16F10 and SM1WT1[] mouse melanoma tumors grew similarly in syngeneic WT or SSeCKS-null (KO) mice, KO hosts exhibited 5- to 10-fold higher levels of peritoneal metastasis, and this enhancement could be adoptively transferred by pre-injecting naïve WT mice with peritoneal fluid (PF), but not non-adherent peritoneal cells (PC), from naïve KO mice. B16F10 and SM1WT1 cells showed increased chemotaxis to KO-PF compared to WT-PF, corresponding to increased PF levels of multiple inflammatory mediators, including the Cxcr3 ligands, Cxcl9 and 10. knockdown abrogated enhanced chemotaxis to KO-PF and peritoneal metastasis in KO hosts. Conditioned media from KO peritoneal membrane fibroblasts (PMF), but not from KO-PC, induced increased B16F10 chemotaxis over controls, which could be blocked with Cxcl10 neutralizing antibody. KO-PMF exhibited increased levels of the senescence markers, SA-β-galactosidase, p21 and p16, and enhanced Cxcl10 secretion induced by inflammatory mediators, lipopolysaccharide, TNFα, IFNα and IFNγ. SSeCKS scaffolding-site mutants and small molecule kinase inhibitors were used to show that the loss of SSeCKS-regulated PKC, PKA and PI3K/Akt pathways are responsible for the enhanced Cxcl10 secretion. These data mark the first description of a role for stromal SSeCKS/AKAP12 in suppressing metastasis, specifically by attenuating signaling pathways that promote secretion of tumor chemoattractants in the peritoneum.
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http://dx.doi.org/10.18632/oncotarget.20092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5642554PMC
September 2017

A subset of cerebrovascular pericytes originates from mature macrophages in the very early phase of vascular development in CNS.

Sci Rep 2017 06 20;7(1):3855. Epub 2017 Jun 20.

Department of Pathology, University of Toyama, Toyama, Japan.

Pericytes are believed to originate from either mesenchymal or neural crest cells. It has recently been reported that pericytes play important roles in the central nervous system (CNS) by regulating blood-brain barrier homeostasis and blood flow at the capillary level. However, the origin of CNS microvascular pericytes and the mechanism of their recruitment remain unknown. Here, we show a new source of cerebrovascular pericytes during neurogenesis. In the CNS of embryonic day 10.5 mouse embryos, CD31F4/80 hematopoietic lineage cells were observed in the avascular region around the dorsal midline of the developing midbrain. These cells expressed additional macrophage markers such as CD206 and CD11b. Moreover, the CD31F4/80 cells phagocytosed apoptotic cells as functionally matured macrophages, adhered to the newly formed subventricular vascular plexus, and then divided into daughter cells. Eventually, these CD31F4/80 cells transdifferentiated into NG2/PDGFRβ/desmin-expressing cerebrovascular pericytes, enwrapping and associating with vascular endothelial cells. These data indicate that a subset of cerebrovascular pericytes derive from mature macrophages in the very early phase of CNS vascular development, which in turn are recruited from sites of embryonic hematopoiesis such as the yolk sac by way of blood flow.
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http://dx.doi.org/10.1038/s41598-017-03994-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478595PMC
June 2017

Significance of Extracellular Vesicles: Pathobiological Roles in Disease.

Cell Struct Funct 2016 Nov 29;41(2):137-143. Epub 2016 Sep 29.

Department of Pathology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama.

Over the past decade, many studies have been conducted on extracellular vesicles (EVs) in the fields of basic and clinical research. EVs are small sized membranous vesicles generated from many type of cells upon activation by environmental stresses such as heat, hypoxia, and irradiation. EVs theoretically consist of microparticles/microvesicles, exosomes, and apoptotic bodies by different productive mechanisms. Clinically, EVs are observed in the blood stream of patients suffering from acute and chronic inflammation evoked by various diseases, and number of EVs in blood flow is often dependent on the inflammatory status and severity of the diseases. To date, it has been reported that small molecules such as RNAs and proteins are encapsulated in EVs; however, the functions of EVs are still unclear in the biological, pathological, and clinical aspects. In this review, we summarize and discuss the biogenesis-based classification, expected function, and pathobiological activities of EVs.
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http://dx.doi.org/10.1247/csf.16014DOI Listing
November 2016

Inflammation-induced endothelial cell-derived extracellular vesicles modulate the cellular status of pericytes.

Sci Rep 2015 Feb 17;5:8505. Epub 2015 Feb 17.

Department of Pathology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.

Emerging lines of evidence have shown that extracellular vesicles (EVs) mediate cell-to-cell communication by exporting encapsulated materials, such as microRNAs (miRNAs), to target cells. Endothelial cell-derived EVs (E-EVs) are upregulated in circulating blood in different pathological conditions; however, the characteristics and the role of these E-EVs are not yet well understood. In vitro studies were conducted to determine the role of inflammation-induced E-EVs in the cell-to-cell communication between vascular endothelial cells and pericytes/vSMCs. Stimulation with inflammatory cytokines and endotoxin immediately induced release of shedding type E-EVs from the vascular endothelial cells, and flow cytometry showed that the induction was dose dependent. MiRNA array analyses revealed that group of miRNAs were specifically increased in the inflammation-induced E-EVs. E-EVs added to the culture media of cerebrovascular pericytes were incorporated into the cells. The E-EV-supplemented cells showed highly induced mRNA and protein expression of VEGF-B, which was assumed to be a downstream target of the miRNA that was increased within the E-EVs after inflammatory stimulation. The results suggest that E-EVs mediate inflammation-induced endothelial cell-pericyte/vSMC communication, and the miRNAs encapsulated within the E-EVs may play a role in regulating target cell function. E-EVs may be new therapeutic targets for the treatment of inflammatory diseases.
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http://dx.doi.org/10.1038/srep08505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4330530PMC
February 2015

Delphinidin, one of the major anthocyanidins, prevents bone loss through the inhibition of excessive osteoclastogenesis in osteoporosis model mice.

PLoS One 2014 13;9(5):e97177. Epub 2014 May 13.

Laboratory of Genomics and Proteomics, National Center for Geriatrics and Gerontology (NCGG), Aichi, Japan; Biobank Omics Unit, National Center for Geriatrics and Gerontology (NCGG), Aichi, Japan.

Anthocyanins, one of the flavonoid subtypes, are a large family of water-soluble phytopigments and have a wide range of health-promoting benefits. Recently, an anthocyanin-rich compound from blueberries was reported to possess protective property against bone loss in ovariectomized (OVX) animal models. However, the active ingredients in the anthocyanin compound have not been identified. Here we show that delphinidin, one of the major anthocyanidins in berries, is a potent active ingredient in anti-osteoporotic bone resorption through the suppression of osteoclast formation. In vitro examinations revealed that delphinidin treatment markedly inhibited the differentiation of RAW264.7 cells into osteoclasts compared with other anthocyanidins, cyanidin and peonidin. Oral administration of delphinidin significantly prevented bone loss in both RANKL-induced osteoporosis model mice and OVX model mice. We further provide evidence that delphinidin suppressed the activity of NF-κB, c-fos, and Nfatc1, master transcriptional factors for osteoclastogenesis. These results strongly suggest that delphinidin is the most potent inhibitor of osteoclast differentiation and will be an effective agent for preventing bone loss in postmenopausal osteoporosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0097177PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4019566PMC
December 2014

Inhibition of histone demethylase JMJD1A improves anti-angiogenic therapy and reduces tumor-associated macrophages.

Cancer Res 2013 May 14;73(10):3019-28. Epub 2013 Mar 14.

Laboratory for Vascular Biology, Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Antiangiogenic strategies can be effective for cancer therapy, but like all therapies resistance poses a major clinical challenge. Hypoxia and nutrient starvation select for aggressive qualities that may render tumors resistant to antiangiogenic attack. Here, we show that hypoxia and nutrient starvation cooperate to drive tumor aggressiveness through epigenetic regulation of the histone demethylase JMJD1A (JHDM2A; KDM3A). In cancer cells rendered resistant to long-term hypoxia and nutrient starvation, we documented a stimulation of AKT phosphorylation, cell morphologic changes, cell migration, invasion, and anchorage-independent growth in culture. These qualities associated in vivo with increased angiogenesis and infiltration of macrophages into tumor tissues. Through expression microarray analysis, we identified a cluster of functional drivers such as VEGFA, FGF18, and JMJD1A, the latter which was upregulated in vitro under conditions of hypoxia and nutrient starvation and in vivo before activation of the angiogenic switch or the prerefractory phase of antiangiogenic therapy. JMJD1A inhibition suppressed tumor growth by downregulating angiogenesis and macrophage infiltration, by suppressing expression of FGF2, HGF, and ANG2. Notably, JMJD1A inhibition enhanced the antitumor effects of the anti-VEGF compound bevacizumab and the VEGFR/KDR inhibitor sunitinib. Our results form the foundation of a strategy to attack hypoxia- and nutrient starvation-resistant cancer cells as an approach to leverage antiangiogenic treatments and limit resistance to them.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-3231DOI Listing
May 2013

Increased expression of histone demethylase JHDM1D under nutrient starvation suppresses tumor growth via down-regulating angiogenesis.

Proc Natl Acad Sci U S A 2011 Dec 5;108(51):20725-9. Epub 2011 Dec 5.

Department of Molecular Oncology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, 1-5-45 Bunkyo-ku, Yushima, Tokyo 113-8519, Japan.

Histone demethylase JHDM1D (also known as KDM7A) modifies the level of methylation in histone and participates in epigenetic gene regulation; however, the role of JHDM1D in tumor progression is unknown. Here, we show that JHDM1D plays a tumor-suppressive role by regulating angiogenesis. Expression of JHDM1D was increased in mouse and human cancer cells under long-term nutrient starvation in vitro. Expression of JHDM1D mRNA was increased within avascular tumor tissue at the preangiogenic switch, along with increased expression of angiogenesis-regulating genes such as Vegf-A. Stable expression of JHDM1D cDNA or siRNA silencing of JHDM1D in cancer cells did not affect cell proliferation, anchorage-independent cell growth, or cell cycle progression in vitro. Notably, JHDM1D-expressing mouse melanoma (B16) and human cervical carcinoma (HeLa) cells exhibited significantly slower tumor growth in vivo compared with the original cells. This reduction in tumor growth was associated with decreased formation of CD31(+) blood vessels and reduced infiltration of CD11b(+) macrophage linage cells into tumor tissues. Expression of multiple angiogenic factors such as VEGF-B and angiopoietins was decreased in tumor xenografts of JHDM1D-expressing B16 and HeLa cells. Our results provide evidence that increased JHDM1D expression suppressed tumor growth by down-regulating angiogenesis under nutrient starvation.
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http://dx.doi.org/10.1073/pnas.1108462109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3251107PMC
December 2011

RACK1 regulates VEGF/Flt1-mediated cell migration via activation of a PI3K/Akt pathway.

J Biol Chem 2011 Mar 6;286(11):9097-106. Epub 2011 Jan 6.

Department of Molecular Oncology, Graduate School of Medicine and Dentistry, Tokyo Dental and Medical University, 1-5-45 Yushima Bunkyo-ku Tokyo 113-8519, Japan.

Vascular endothelial growth factor (VEGF) is vital to physiological as well as pathological angiogenesis, and regulates a variety of cellular functions, largely by activating its 2 receptors, fms-like tyrosine kinase (Flt1) and kinase domain receptor (KDR). KDR plays a critical role in the proliferation of endothelial cells by controlling VEGF-induced phospholipase Cγ-protein kinase C (PLCγ-PKC) signaling. The function of Flt1, however, remains to be clarified. Recent evidence has indicated that Flt1 regulates the VEGF-triggered migration of endothelial cells and macrophages. Here, we show that RACK1, a ubiquitously expressed scaffolding protein, functions as an important regulator of this process. We found that RACK1 (receptor for activated protein kinase C 1) binds to Flt1 in vitro. When the endogenous expression of RACK1 was attenuated by RNA interference, the VEGF-driven migration was remarkably suppressed whereas the proliferation was unaffected in a stable Flt1-expressing cell line, AG1-G1-Flt1. Further, we demonstrated that the VEGF/Flt-mediated migration of AG1-G1-Flt1 cells occurred mainly via the activation of the PI3 kinase (PI3K)/Akt and Rac1 pathways, and that RACK1 plays a crucial regulatory role in promoting PI3K/Akt-Rac1 activation.
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http://dx.doi.org/10.1074/jbc.M110.165605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3058986PMC
March 2011

Vascular endothelial growth factor receptor-1 signaling promotes mobilization of macrophage lineage cells from bone marrow and stimulates solid tumor growth.

Cancer Res 2010 Oct 5;70(20):8211-21. Epub 2010 Oct 5.

Department of Molecular Oncology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, Tokyo, Japan.

Vascular endothelial growth factor and its receptors, including Flt-1 and Flk-1, are involved in angiogenesis under physiologic and pathologic conditions. Recently, Flt-1-expressing cells were reported to contribute to the intracranial growth of glioma cells. However, the role of Flt-1 signaling in solid tumor growth in s.c. tissue has not been elucidated. To investigate how Flt-1 signaling is involved in the proliferation of solid tumors, we implanted tumor cells into wild-type (Wt) and Flt-1 tyrosine kinase (TK)-deficient (Flt-1 TK(-/-)) mice. Growth of HSML and B16 but not Lewis lung carcinoma cell in s.c. tissue was significantly decreased in Flt-1 TK(-/-) mice. Angiogenesis in HSML and B16 tumors was remarkably reduced in Flt-1 TK(-/-) mice. Moreover, the infiltration of macrophage lineage cells into HSML and B16 tumors was clearly suppressed in Flt-1 TK(-/-) mice. Pericyte marker(+) cells were also reduced in Flt-1 TK(-/-) mice. However, in the border area of tumor, angiogenesis and the infiltration of macrophage lineage cell were basically similar between Wt and Flt-1 TK(-/-) mice. In bone marrow (BM) transplantation experiments, tumor angiogenesis, infiltration of macrophage lineage cells, and tumor growth were significantly suppressed in Wt/Flt-1 TK(-/-) mice implanted with Flt-1 TK(-/-) BM cells compared with those implanted with Wt BM cells. We conclude that Flt-1 signaling is involved in the function of BM-derived cell, such as the migration of macrophages into cancerous tissues, and significantly contributes to angiogenesis and tumor progression.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-0202DOI Listing
October 2010

Hypoxia and low-nutrition double stress induces aggressiveness in a murine model of melanoma.

Cancer Sci 2009 May 10;100(5):844-51. Epub 2009 Feb 10.

Department of Molecular Oncology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, Tokyo, Japan.

Antiangiogenic therapy is a potent cancer treatment, however, the possibility of recurrence and resistance to this approach remains. Here we show that hypoxia and low-nutrition double-deprivation stress induces reversible tumor aggressiveness. In a stress-cycle-dependent manner, murine melanoma cells showed morphological changes, up-regulated phospho-Akt, and abnormal regulation of multiple genes including fibroblast growth factor-21, a metabolic regulator, resulting in increased cell proliferation in vitro, and increased tumorigenesis and invasive potential in vivo. In this system, altered cellular metabolism participates in the adaptation of tumor to the double-deprivation stress. Our results suggest the targeting of a minor population of cancer cells resistant to both hypoxia and low nutrition to be an effective new antitumor strategy in combination with antiangiogenic therapy.
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http://dx.doi.org/10.1111/j.1349-7006.2009.01105.xDOI Listing
May 2009

Downregulation of angiopoietin-1 and Tie2 in chronic hypoxic pulmonary hypertension.

Respiration 2008 10;75(3):328-38. Epub 2007 Dec 10.

Department of Respiratory Medicine, Biomedical Research Center, Juntendo University School of Medicine, Tokyo, Japan.

Background: Angiopoietins, newly discovered vascular-specific growth factors, and vascular endothelial growth factors (VEGF) play distinct and complementary roles in angiogenesis and vascular maturation. However, the exact roles of angiogenic factors in the adult pulmonary vasculature remain unclear.

Objective: To elucidate possible roles of angiopoietins and VEGF in the development of hypoxic pulmonary hypertension (PH), changes in the expression of angiogenic factors were examined.

Methods: The cellular distribution and expression of angiopoietins and their receptor Tie2 and VEGF were investigated by RT-PCR, immunoblot, and immunohistochemical methods in rat lung under normal and hypoxic conditions.

Results: During the development of PH with vascular remodeling characterized by a decrease in vessel density of intrapulmonary arteries, protein expression of angiopoietin-1 (Ang-1), Tie2, and VEGF significantly decreased in the pulmonary arteries, and Tie2 receptor was inactivated in the lung. The expression of angiopoietin-3 (Ang-3), an endogenous antagonist of Ang-1, significantly increased in the intima under hypoxic conditions.

Conclusions: Since both Ang-1/Tie2 and VEGF promote angiogenesis and vascular survival, and play protective roles in the adaptation of microvascular changes during the onset of PH, the downregulation of both Ang-1/Tie2 and VEGF and upregulation of Ang-3 appear to be associated with vascular rarefaction and the development of hypoxic PH.
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http://dx.doi.org/10.1159/000112432DOI Listing
May 2008

Activity of endothelium-derived hyperpolarizing factor is augmented in monocrotaline-induced pulmonary hypertension of rat lungs.

J Vasc Res 2007 16;44(4):325-35. Epub 2007 Apr 16.

Department of Respiratory Medicine, Juntendo University School of Medicine, Tokyo, Japan.

The mechanism of endothelium-dependent vasodilator signaling involves three components such as nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). Although EDHF is distinct from nitric oxide and prostacyclin, it requires activation of Ca(2+)-sensitive K(+) channels (K(Ca)) and cytochrome P(450) metabolites. However, the physiological role of EDHF in the pulmonary circulation is unclear. Thus, we tested if EDHF would regulate vascular tone in rat lungs of control and monocrotaline (MCT)-induced pulmonary hypertension. Inhibition of EDHF with a combination of K(Ca) blockers, charybdotoxin (50 nM) plus apamin (50 nM), increased baseline vascular tone in MCT-induced hypertensive lungs. Thapsigargin (TG; 100 nM), an inhibitor of Ca-ATPase, caused greater EDHF-mediated vasodilation in MCT-induced hypertensive lungs. TG-induced vasodilation was abolished with the charybdotoxin-apamin combination. Sulfaphenazole (10 muM), a cytochrome P(450) inhibitor, reduced the TG-induced vasodilation in MCT-induced hypertensive lungs. RT-PCR analysis exhibited an increase in K(Ca) mRNA in MCT-treated lungs. These results indicate the augmentation of tonic EDHF activity, at least in part, through the alteration in cytochrome P(450) metabolites and the upregulation of K(Ca) expression in MCT-induced pulmonary hypertension.
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http://dx.doi.org/10.1159/000101778DOI Listing
July 2007

Genistein, a phytoestrogen, attenuates monocrotaline-induced pulmonary hypertension.

Respiration 2006 1;73(1):105-12. Epub 2005 Oct 1.

Department of Respiratory Medicine, Juntendo University School of Medicine, Tokyo, Japan.

Background: Pulmonary hypertension is characterized by high pulmonary blood pressure, vascular remodeling, and right ventricular hypertrophy. Although recent studies suggest that an imbalance between endothelial mediators on pulmonary vasculature may contribute to the development of pulmonary hypertension, the pathogenesis is not fully understood and the treatment of pulmonary hypertension is still unresolved.

Objective: The purpose of this study was to investigate whether genistein, a phytoestrogen derived from soybean, would prevent the development of monocrotaline (MCT)-induced pulmonary hypertension in rats. Hemodynamic parameters of catheterized rats and morphological feature of lungs were evaluated among MCT-treated rats receiving or not receiving genistein. Furthermore, examination of expression in endothelial nitric oxide synthase and endothelin-1 peptide level was performed.

Methods: Daily supplementation with either genistein (0.2 mg/kg) or vehicle was started 2 days prior to a single-dose injection of MCT (60 mg/kg). On day 28, rats underwent catheterization, and right ventricular hypertrophy and morphological features were assessed. Furthermore, endothelial nitric oxide synthase and endothelin-1 were examined by Western blot analysis and radioimmunoassay, respectively, in homogenated lungs.

Results: In rats that received daily supplementation of genistein, mean pulmonary arterial pressure was significantly reduced, whereas mean systemic arterial pressure and heart rate were unaltered compared with MCT control rats on day 28 after MCT injection. Right ventricular hypertrophy, medial wall thickness of pulmonary arteries corresponding to the terminal bronchioles, and the degree of neo-muscularization of more distal arteries were less severe in genistein-treated rats. Genistein supplementation improved MCT-induced downregulation of expression of endothelial nitric oxide synthase in the lungs. However, endothelin-1 peptide levels did not differ among all groups of lungs.

Conclusions: We conclude that daily supplementation of genistein potently attenuates MCT-induced pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular remodeling in rats. The underlying mechanism responsible for this effect may be partly related to the restoration of a decreased expression of endothelial nitric oxide synthase.
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http://dx.doi.org/10.1159/000088946DOI Listing
June 2006

Downregulation of type II bone morphogenetic protein receptor in hypoxic pulmonary hypertension.

Am J Physiol Lung Cell Mol Physiol 2006 Mar 16;290(3):L450-8. Epub 2005 Dec 16.

Department of Respiratory Medicine, Juntendo University School of Medicine, Tokyo Metropolitan Geriatric Hospital, 35-2 Sakae-Machi, Itabashi-Ku, Tokyo 173-0015, Japan.

Heterozygous mutations in the type II receptor for bone morphogenetic protein (BMPR-II) and dysfunction of BMPR-II have been implicated in patients with primary pulmonary hypertension (PH). To clarify the possible involvement of BMP and BMPR-II in the development of hypoxic PH, the expression of BMP-2, BMPR-II, and their downstream signals were investigated in rat lung under normal and hypoxic conditions by RT-PCR, immunoblot, and immunohistochemical methods. In rats under normal conditions, BMP-2 is localized in the endothelium of the pulmonary artery, whereas BMPR-II is abundantly expressed in the endothelium, smooth muscle cells, and adventitial fibroblasts. After 0.5 and 3 days of exposure to hypoxia, upregulation of BMP-2 was observed in the intrapulmonary arteries. The change was accompanied by activation of its downstream signaling, p38 MAPK, and Erk1/2 MAPK, and the apoptotic process, measured by caspase-3 activity and TdT-mediated dUTP nick end labeling-positive cells. In contrast, a significant decrease in the expression of BMPR-II and inactivation of p38 MAPK and caspase-3 were observed in the pulmonary vasculature after 7-21 days of hypoxia exposure. Because BMP-2 is known to inhibit proliferation of vascular smooth muscle cells and promote cellular apoptosis, disruption of BMP signaling pathway through downregulation of BMPR-II in chronic hypoxia may result in pulmonary vascular remodeling due to the failure of critical antiproliferative/differentiation programs in the pulmonary vasculature. These results suggest abrogation of BMP signaling may be a common molecular pathogenesis in the development of PH with various pathophysiological events, including primary and hypoxic PH.
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http://dx.doi.org/10.1152/ajplung.00206.2005DOI Listing
March 2006

[An elderly case of sarcoidosis with multiple pulmonary cysts].

Nihon Kokyuki Gakkai Zasshi 2005 Jun;43(6):370-4

Department of Respiratory Medicine, Juntendo University School of Medicine.

We report an elderly case of sarcoidosis with multiple pulmonary cysts. An 80-year-old woman was admitted to our hospital for detailed examinations of multiple cysts in both lungs. Chest radiography and chest CT revealed multiple cystic lesions with thin walls which were clearly separated from normal lung. A transbronchial lung biopsy revealed epitheloid cell granuloma, indicating sarcoidosis. Although elderly cases of sarcoidosis with multiple pulmonary cysts are very rare, sarcoidosis should be considered in the differential diagnosis.
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June 2005

[A case of pulmonary squamous cell carcinoma coexisting with pulmonary actinomycosis].

Nihon Kokyuki Gakkai Zasshi 2002 Jun;40(6):525-9

Department of Respiratory Medicine, Juntendo University School of Medicine.

A 71-year-old man was referred to our hospital complaining of cough. Chest radiography revealed a mass opacity in the right upper lung field. A transbronchial biopsy specimen revealed non-specific inflammatory changes. Percutaneous lung aspiration biopsy under ultrasound guidance demonstrated gram-positive rods, suggesting actinomyces. On the diagnosis of pulmonary actinomycosis, the patient was treated with penicillin-G and his symptoms were relieved. In a three-month follow-up, the mass shadow in the right upper lung field was found to have increased in size. Squamous cell lung cancer was diagnosed on the basis of repeated transbronchial tumor biopsies, and right upper lobectomy was performed. Most cases of pulmonary actinomycosis have been diagnosed from post-surgical tumor specimens taken on suspicion of the presence of lung cancer. However, the lung cancer in this case was difficult to diagnose because the lung cancer was co-existent with pulmonary actinomycosis.
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June 2002
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