Publications by authors named "Nobuyuki Takakura"

135 Publications

Endothelial cell-derived Apelin inhibits tumor growth by altering immune cell localization.

Sci Rep 2021 Jul 7;11(1):14047. Epub 2021 Jul 7.

Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka, 565-0871, Japan.

The Apelin/APJ signalling pathway, involved in multiple physiological and pathological processes, has been attracting increasing interest recently. In our previous study, Apelin overexpression in colon26 tumor cells suppressed tumor growth by inducing vascular maturation. Here, we found that MC38 and LLC tumor growth were greater in the absence of Apelin than in wild-type (WT) mice, suggesting that Apelin acts as a tumor suppressor. Consistent with this, treating WT mice with [Pyr]Apelin-13 inhibited tumor growth. In MC38 tumors, only endothelial cells (ECs) strongly express APJ, a cognate receptor for Apelin, indicating that EC-derived Apelin might regulate tumor formation in an autocrine manner. Comparing with WT mice, larger numbers of vessels with narrower diameters were observed in tumors of Apelin knockout mice and lack of Apelin enhanced tumor hypoxia. Investigating immune cells in the tumor revealed that [Pyr]Apelin-13 infusion induced the accumulation of CD8 and CD4 T cells in central areas. Moreover, RNA-sequencing analysis showed that Apelin induces chemokine CCL8 expression in ECs. Thus, enhancing anti-tumor immunity might be one of the mechanisms by which Apelin is involved in tumor growth. Our result indicated that increased CCL8 expression might induce CD8  T cells infiltration into tumor and tumor inhibition.
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http://dx.doi.org/10.1038/s41598-021-93619-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8263715PMC
July 2021

Indispensable role of Galectin-3 in promoting quiescence of hematopoietic stem cells.

Nat Commun 2021 04 9;12(1):2118. Epub 2021 Apr 9.

Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

Hematopoietic stem cells (HSCs) in adult bone marrow (BM) are usually maintained in a state of quiescence. The cellular mechanism coordinating the balance between HSC quiescence and differentiation is not fully understood. Here, we report that galactose-binding lectin-3 (galectin-3; Gal-3) is upregulated by Tie2 or Mpl activation to maintain quiescence. Conditional overexpression of Gal-3 in mouse HSCs under the transcriptional control of Tie2 or Vav1 promoters (Gal-3 Tg) causes cell cycle retardation via induction of p21. Conversely, the cell cycle of long-term repopulating HSCs (LT-HSCs) in Gal-3-deficient (Gal-3) mice is accelerated, resulting in their exhaustion. Mechanistically, Gal-3 regulates p21 transcription by forming a complex with Sp1, thus blocking cell cycle entry. These results demonstrate that Gal-3 is a negative regulator of cell-cycling in HSCs and plays a crucial role in adult hematopoiesis to prevent HSC exhaustion.
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http://dx.doi.org/10.1038/s41467-021-22346-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035175PMC
April 2021

Reduction in miR-219 expression underlies cellular pathogenesis of oligodendrocytes in a mouse model of Krabbe disease.

Brain Pathol 2021 Sep 6;31(5):e12951. Epub 2021 Apr 6.

Department of Cellular Pathology, Institute for Developmental Research, Aichi Developmental Disability Center, Kasugai, Japan.

Krabbe disease (KD), also known as globoid cell leukodystrophy, is an inherited demyelinating disease caused by the deficiency of lysosomal galactosylceramidase (GALC) activity. Most of the patients are characterized by early-onset cerebral demyelination with apoptotic oligodendrocyte (OL) death and die before 2 years of age. However, the mechanisms of molecular pathogenesis in the developing OLs before death and the exact causes of white matter degeneration remain largely unknown. We have recently reported that OLs of twitcher mouse, an authentic mouse model of KD, exhibit developmental defects and endogenous accumulation of psychosine (galactosylsphingosine), a cytotoxic lyso-derivative of galactosylceramide. Here, we show that attenuated expression of microRNA (miR)-219, a critical regulator of OL differentiation and myelination, mediates cellular pathogenesis of KD OLs. Expression and functional activity of miR-219 were repressed in developing twitcher mouse OLs. By using OL precursor cells (OPCs) isolated from the twitcher mouse brain, we show that exogenously supplemented miR-219 effectively rescued their cell-autonomous developmental defects and apoptotic death. miR-219 also reduced endogenous accumulation of psychosine in twitcher OLs. Collectively, these results highlight the role of the reduced miR-219 expression in KD pathogenesis and suggest that miR-219 has therapeutic potential for treating KD OL pathologies.
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http://dx.doi.org/10.1111/bpa.12951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8412087PMC
September 2021

Vascular reconstitution in the tumor for more effective tumor immunotherapy.

Cancer Sci 2021 Apr 3;112(4):1348-1356. Epub 2021 Mar 3.

Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

It has been widely accepted that the regulation of the tumor microenvironment is an important strategy in cancer treatment. Particularly, control of the tumor vasculature has been suggested to be critical for antitumor immunotherapy. Effectiveness of cancer immunotherapy depends on the quality and quantity of immune cells infiltrating into tumor tissues, which may be affected by the status of the tumor vasculature. Under physiological conditions, immune cells migrate from the intravascular lumen into the parenchyma especially by passing through the vascular wall of venulae. Extravasation of immune cells is induced from venulae where endothelial cells (ECs) are fully covered with pericytes from the basal side. Interaction of pericytes with ECs contributes to immune cell extravasation by several steps, ie, adhesion of immune cells to intraluminal ECs, transmigration, and chemotaxis of immune cells. Blood vessels are structurally immature and non-functional in tumors, and therefore, induction of maturation in the tumor vasculature is a promising strategy for effective cancer therapies and is relevant not only for immune cell migration but also drug delivery.
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http://dx.doi.org/10.1111/cas.14854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8019202PMC
April 2021

An in vivo model allowing continuous observation of human vascular formation in the same animal over time.

Sci Rep 2021 01 12;11(1):745. Epub 2021 Jan 12.

Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka, 565-0871, Japan.

Angiogenesis contributes to numerous pathological conditions. Understanding the molecular mechanisms of angiogenesis will offer new therapeutic opportunities. Several experimental in vivo models that better represent the pathological conditions have been generated for this purpose in mice, but it is difficult to translate results from mouse to human blood vessels. To understand human vascular biology and translate findings into human research, we need human blood vessel models to replicate human vascular physiology. Here, we show that human tumor tissue transplantation into a cranial window enables engraftment of human blood vessels in mice. An in vivo imaging technique using two-photon microscopy allows continuous observation of human blood vessels until at least 49 days after tumor transplantation. These human blood vessels make connections with mouse blood vessels as shown by the finding that lectin injected into the mouse tail vein reaches the human blood vessels. Finally, this model revealed that formation and/or maintenance of human blood vessels depends on VEGFR2 signaling. This approach represents a useful tool to study molecular mechanisms of human blood vessel formation and to test effects of drugs that target human blood vessels in vivo to show proof of concept in a preclinical model.
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http://dx.doi.org/10.1038/s41598-020-80497-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7804448PMC
January 2021

[Immune therapy and tumor angiogenesis].

Rinsho Ketsueki 2020 ;61(9):1440-1445

Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University.

Angiogenesis is the process of new vascular formation from preexisting blood vessels, and the contribution of endothelial stem cell population to this process has been elucidated. Vascular endothelial growth factor (VEGF) promotes not only the proliferation of endothelial cells (ECs) but also the vascular permeability by inducing cell-to-cell dissociation between ECs. Chronic hyper vascular leakage induces intra-tumoral interstitial hypertension resulting in disturbed vascular perfusion. Hypoxia in the nonfunctional vasculatures causes chromosomal instability of cancer cells resulting in the development of malignant cancer cells, such as cancer stem cells. Hypoxia also induces exhaustion of immune cells. Moreover, the blood vessel structures in tumors are abnormal and nonfunctional, and the infiltration of leukocytes are suppressed. Moreover, drug delivery, including anti-cancer drugs and immune checkpoint inhibitors, is limited. A drug inducing normalization or maturation of abnormal blood vessels in tumor is warranted for the improvement of malignant tumor microenvironment.
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http://dx.doi.org/10.11406/rinketsu.61.1440DOI Listing
January 2021

Design and Identification of a GPR40 Full Agonist () Possessing a 2-Carbamoylphenyl Piperidine Moiety.

J Med Chem 2020 09 9;63(18):10352-10379. Epub 2020 Sep 9.

Research Division, SCOHIA PHARMA Inc., Shonan Health Innovation Park, 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan.

GPR40/FFAR1 is a G-protein-coupled receptor expressed in pancreatic β-cells and enteroendocrine cells. GPR40 activation stimulates secretions of insulin and incretin, both of which are the pivotal regulators of glycemic control. Therefore, a GPR40 agonist is an attractive target for the treatment of type 2 diabetes mellitus. Using the reported biaryl derivative , we shifted the hydrophobic moiety to the terminal aryl ring and replaced the central aryl ring with piperidine, generating 2-(4,4-dimethylpentyl)phenyl piperidine , which had improved potency for GPR40 and high lipophilicity. We replaced the hydrophobic moiety with -alkyl--aryl benzamides to lower the lipophilicity and restrict the -alkyl moieties to the presumed lipophilic pocket using the intramolecular π-π stacking of cis-preferential -alkyl--aryl benzamide. Among these, orally available (3)-3-cyclopropyl-3-(2-((1-(2-((2,2-dimethylpropyl)(6-methylpyridin-2-yl)carbamoyl)-5-methoxyphenyl)piperidin-4-yl)methoxy)pyridin-4-yl)propanoic acid () effectively stimulated insulin secretion and GLP-1 release and ameliorated glucose tolerance in diabetic rats via GPR40 full agonism.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00843DOI Listing
September 2020

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

The Bartonella autotransporter BafA activates the host VEGF pathway to drive angiogenesis.

Nat Commun 2020 07 16;11(1):3571. Epub 2020 Jul 16.

Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi, 470-1192, Japan.

Pathogenic bacteria of the genus Bartonella can induce vasoproliferative lesions during infection. The underlying mechanisms are unclear, but involve secretion of an unidentified mitogenic factor. Here, we use functional transposon-mutant screening in Bartonella henselae to identify such factor as a pro-angiogenic autotransporter, called BafA. The passenger domain of BafA induces cell proliferation, tube formation and sprouting of microvessels, and drives angiogenesis in mice. BafA interacts with vascular endothelial growth factor (VEGF) receptor-2 and activates the downstream signaling pathway, suggesting that BafA functions as a VEGF analog. A BafA homolog from a related pathogen, Bartonella quintana, is also functional. Our work unveils the mechanistic basis of vasoproliferative lesions observed in bartonellosis, and we propose BafA as a key pathogenic factor contributing to bacterial spread and host adaptation.
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http://dx.doi.org/10.1038/s41467-020-17391-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366657PMC
July 2020

Zinc Finger Protein St18 Protects against Septic Death by Inhibiting VEGF-A from Macrophages.

Cell Rep 2020 07;32(2):107906

Biotechnology Research Institute for Drug Discovery, Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8565, Japan.

Zinc finger protein St18 was initially reported as candidate tumor suppressor gene, and also suggested that fibroblast St18 positively regulates NF-κB activation. Despite the pleiotropic functions of St18, little is known about its roles in macrophages. Here, we report that myeloid St18 is a potent inhibitor of VEGF-A. Mice lacking St18 in myeloid lineages exhibit increased retinal vasculature with enhanced serum VEGF-A concentrations. Despite the normal activation of NF-κB target genes, these mice are highly susceptible to LPS-induced shock, polymicrobial sepsis, and experimental colitis, accompanied by enhanced vascular and intestinal leakage. Pharmacological inhibition of VEGF signaling rescued the high mortality rate of myeloid-specific St18-deficient mice in response to inflammation. Mechanistically, St18 directly binds to Sp1 and attenuates its activity, leading to the suppression of Sp1 target gene VEGF-A. Using mouse genetic and pharmacological models, we reveal myeloid St18 as a critical septic death protector.
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http://dx.doi.org/10.1016/j.celrep.2020.107906DOI Listing
July 2020

High expression of PSF1 promotes drug resistance and cell cycle transit in leukemia cells.

Cancer Sci 2020 Jul 1;111(7):2400-2412. Epub 2020 Jun 1.

Department of Signal Transduction, Research Institute for Microbial Disease, Osaka University, Suita, Osaka, Japan.

Escape of cancer cells from chemotherapy is a problem in the management of cancer patients. Research on chemotherapy resistance has mainly focused on the heterogeneity of cancer cells, multiple gene mutations, and quiescence of malignant cancer cells. However, some studies have indicated that interactions between cancer cells and vascular cells promote resistance to chemotherapy. Here, we established mouse leukemia models using the cell lines THP-1 or MEG-1. These were derived from acute and chronic myeloid leukemias, respectively, and highly expressed DNA replication factor PSF1, a member of the GINS complex. We found that, after anti-cancer drug administration, surviving GFP-positive leukemia cells in the bone marrow were located adjacent to blood vessels, as previously reported in a subcutaneous solid tumor transplantation model. Treating THP-1 and MEG-1 cells with anti-cancer drugs in vitro revealed that those most strongly expressing PSF1 were most chemoresistant, suggesting that PSF1 induces not only cell cycle progression but also facilitates cell survival. Indeed, when PSF1 expression was suppressed by shRNA, the growth rate was reduced and cell death was enhanced in both cell lines. Furthermore, PSF1 knockdown in leukemia cells led to a change in their location at a distance from the blood vessels in a bone marrow transplantation model. These findings potentially reflect a mechanism of escape of leukemic cells from chemotherapy and suggest that PSF1 may be a possible therapeutic target to enhance the effect of chemotherapy.
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http://dx.doi.org/10.1111/cas.14452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385346PMC
July 2020

Japanese clinical practice guidelines for vascular anomalies 2017.

Jpn J Radiol 2020 Apr;38(4):287-342

Division of Radiology, National Center for Child Health and Development, Tokyo, Japan.

The objective was to prepare guidelines to perform the current optimum treatment by organizing effective and efficient treatments of hemangiomas and vascular malformations, confirming the safety, and systematizing treatment, employing evidence-based medicine (EBM) techniques and aimed at improvement of the outcomes. Clinical questions (CQs) were decided based on the important clinical issues. For document retrieval, key words for literature searches were set for each CQ and literature published from 1980 to the end of September 2014 was searched in Pubmed, Cochrane Library, and Japana Centra Revuo Medicina (JCRM). The strengths of evidence and recommendations acquired by systematic reviews were determined following the Medical Information Network Distribution System (MINDS) technique. A total of 33 CQs were used to compile recommendations and the subjects included efficacy of resection, sclerotherapy/embolization, drug therapy, laser therapy, radiotherapy, and other conservative treatment, differences in appropriate treatment due to the location of lesions and among symptoms, appropriate timing of treatment and tests, and pathological diagnosis deciding the diagnosis. Thus, the Japanese Clinical Practice Guidelines for Vascular Anomalies 2017 have been prepared as the evidence-based guidelines for the management of vascular anomalies.
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http://dx.doi.org/10.1007/s11604-019-00885-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150662PMC
April 2020

Japanese clinical practice guidelines for vascular anomalies 2017.

Pediatr Int 2020 Mar 22;62(3):257-304. Epub 2020 Mar 22.

Division of Radiology, National Center for Child Health and Development, Tokyo, Japan.

The objective was to prepare guidelines to perform the current optimum treatment by organizing effective and efficient treatments of hemangiomas and vascular malformations, confirming the safety, and systematizing treatment, employing evidence-based medicine (EBM) techniques and aimed at improvement of the outcomes. Clinical questions (CQs) were decided based on the important clinical issues. For document retrieval, key words for literature searches were set for each CQ and literature published from 1980 to the end of September 2014 was searched in Pubmed, Cochrane Library, and Japana Centra Revuo Medicina (JCRM). The strengths of evidence and recommendations acquired by systematic reviews were determined following the Medical Information Network Distribution System (MINDS) technique. A total of 33 CQs were used to compile recommendations and the subjects included efficacy of resection, sclerotherapy/embolization, drug therapy, laser therapy, radiotherapy, and other conservative treatment, differences in appropriate treatment due to the location of lesions and among symptoms, appropriate timing of treatment and tests, and pathological diagnosis deciding the diagnosis. Thus, the Japanese Clinical Practice Guidelines for Vascular Anomalies 2017 have been prepared as the evidence-based guidelines for the management of vascular anomalies.
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http://dx.doi.org/10.1111/ped.14077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232443PMC
March 2020

Japanese Clinical Practice Guidelines for Vascular Anomalies 2017.

J Dermatol 2020 May 22;47(5):e138-e183. Epub 2020 Mar 22.

Division of Radiology, National Center for Child Health and Development, Tokyo, Japan.

The objective was to prepare guidelines to perform the current optimum treatment by organizing effective and efficient treatments of hemangiomas and vascular malformations, confirming the safety and systematizing treatment, employing evidence-based medicine techniques and aimed at improvement of the outcomes. Clinical questions (CQ) were decided based on the important clinical issues. For document retrieval, key words for published work searches were set for each CQ, and work published from 1980 to the end of September 2014 was searched in PubMed, Cochrane Library and Japana Centra Revuo Medicina databases. The strengths of evidence and recommendations acquired by systematic reviews were determined following the Medical Information Network Distribution System technique. A total of 33 CQ were used to compile recommendations and the subjects included efficacy of resection, sclerotherapy/embolization, drug therapy, laser therapy, radiotherapy and other conservative treatment, differences in appropriate treatment due to the location of lesions and among symptoms, appropriate timing of treatment and tests, and pathological diagnosis deciding the diagnosis. Thus, the Japanese Clinical Practice Guidelines for Vascular Anomalies 2017 have been prepared as the evidence-based guidelines for the management of vascular anomalies.
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http://dx.doi.org/10.1111/1346-8138.15189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317503PMC
May 2020

Isolation of tissue-resident vascular endothelial stem cells from mouse liver.

Nat Protoc 2020 03 31;15(3):1066-1081. Epub 2020 Jan 31.

Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

Endothelial cells (ECs) are fundamental components of the blood vessels that comprise the vascular system; facilitate blood flow; and regulate permeability, angiogenesis, inflammatory responses and homeostatic tissue maintenance. Accumulating evidence suggests there is EC heterogeneity in vivo. However, isolation of fresh ECs from adult mice to investigate this further is challenging. Here, we describe an easy and reproducible protocol for isolation of different types of ECs and CD157 vascular-resident endothelial stem cells (VESCs) by mechano-enzymatic tissue digestion followed by fluorescence-activated cell sorting. The procedure was established on liver tissue but can be used to isolate ECs from other organs with minimal modification. Preparation of single-cell suspensions can be completed in 2.5 h. We also describe assays for EC clonal and network formation, as well as transcriptomic analysis of isolated ECs. The protocol enables isolation of primary ECs and VESCs that can be used for a wide range of downstream analyses in vascular research.
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http://dx.doi.org/10.1038/s41596-019-0276-xDOI Listing
March 2020

Mechanisms of new blood-vessel formation and proliferative heterogeneity of endothelial cells.

Int Immunol 2020 05;32(5):295-305

Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

The vast blood-vessel network of the circulatory system is crucial for maintaining bodily homeostasis, delivering essential molecules and blood cells, and removing waste products. Blood-vessel dysfunction and dysregulation of new blood-vessel formation are related to the onset and progression of many diseases including cancer, ischemic disease, inflammation and immune disorders. Endothelial cells (ECs) are fundamental components of blood vessels and their proliferation is essential for new vessel formation, making them good therapeutic targets for regulating the latter. New blood-vessel formation occurs by vasculogenesis and angiogenesis during development. Induction of ECs termed tip, stalk and phalanx cells by interactions between vascular endothelial growth factor A (VEGF-A) and its receptors (VEGFR1-3) and between Notch and Delta-like Notch ligands (DLLs) is crucial for regulation of angiogenesis. Although the importance of angiogenesis is unequivocal in the adult, vasculogenesis effected by endothelial progenitor cells (EPCs) may also contribute to post-natal vessel formation. However, the definition of these cells is ambiguous and they include several distinct cell types under the simple classification of 'EPC'. Furthermore, recent evidence indicates that ECs within the intima show clonal expansion in some situations and that they may harbor vascular-resident endothelial stem cells. In this article, we summarize recent knowledge on vascular development and new blood-vessel formation in the adult. We also introduce concepts of EC heterogeneity and EC clonal expansion, referring to our own recent findings.
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http://dx.doi.org/10.1093/intimm/dxaa008DOI Listing
May 2020

Spatiotemporally Dependent Vascularization Is Differently Utilized among Neural Progenitor Subtypes during Neocortical Development.

Cell Rep 2019 10;29(5):1113-1129.e5

Laboratory of Stem Cell Biology, Graduate School of Pharmaceutical Sciences, Kobe Gakuin University, Kobe 650-8586, Japan; Graduate School of Brain Science, Doshisha University, Kyoto 619-0225, Japan; PRESTO, Japan Science and Technology Agency, Saitama 332-0012, Japan. Electronic address:

To facilitate efficient oxygen and nutrient delivery, blood vessels in the brain form three-dimensional patterns. However, little is known about how blood vessels develop stereographically in the neocortex and how they control the expansion and differentiation of neural progenitors during neocortical development. We show that highly vascularized and avascular regions are strictly controlled in a spatially and temporally restricted manner and are associated with distinct cell populations. Dividing basal progenitors and oligodendrocyte precursors preferentially contact honeycomb vessels, but dividing apical progenitors are localized in avascular regions without Flt1-positive endothelial cells but directly contact with sprouting neovascular tip cells. Therefore, not all blood vessels are associated equally with neural progenitors. Furthermore, a disruption of normal vascular patterning can induce abnormalities in neural development, whereas the impaired features of neural progenitors influenced angiogenesis patterning. These results indicate that close association between the nervous and vascular systems is essential for neocortex assembly.
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http://dx.doi.org/10.1016/j.celrep.2019.09.048DOI Listing
October 2019

TAK1 safeguards endothelial cells from gut microbes and inflammation.

Mol Cell Oncol 2019 13;6(3):1588657. Epub 2019 Mar 13.

Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

How endothelial cells (ECs) survive in inflammatory environments remains unclear. We recently showed that TAK1 protects ECs against apoptosis induced by TNFα under physiological conditions in the intestine and liver, and under inflammatory conditions in other organs. Our results document that a single gene can affect cell fate decisions in mature ECs.
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http://dx.doi.org/10.1080/23723556.2019.1588657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6512928PMC
March 2019

Embryonic expression of GINS members in the development of the mammalian nervous system.

Neurochem Int 2019 10 13;129:104465. Epub 2019 May 13.

Department of Signal Transduction, Research Institute for Microbial Disease, Osaka University, 3-1 Yamada-oka, Suita, Osaka, 565-0871, Japan. Electronic address:

The GINS (Go, Ichi, Nii, and San) complex contains four protein subunits (PSF1, PSF2, PSF3, and SLD5) and has been identified as a factor essential for the initiation and elongation stages of the DNA replication process. A previous study indicated that PSF2 participated in the developing central nervous system (CNS) of Xenopus laevis. However, the expression and function of GINS members in the mammalian developing nervous system remains unclear. Here, we examined the expression of GINS members in mice during nervous system development via immunofluorescence staining. At the beginning of neural development, PSF1 and SLD5 were highly expressed in neuroepithelial stem cells (NSCs) of the inner surface of neural tube (NT) and overlapped with proliferation marker Ki67. After entering the mid- and late-phase of neural development, PSF1 and SLD5 changed their regions of expression. These genes were highly expressed in dorsal root ganglion (DRG) progenitors, but they showed no overlap with Ki67 positive cells. Instead, a reduction of SLD5 expression promoted neuronal differentiation and maturation in the late-phase. PSF2 and PSF3 showed no tissue-specificity. PSF2 was constitutively and highly expressed whereas PSF3 was expressed at very low levels during neural development. In this study, we demonstrated variations in proteins and expression regions of the GINS members during mammalian CNS development and revealed a correlation between GINS expression and cell proliferation. Furthermore, we have suggested a novel function of GINS member SLD5, which regulates the differentiation of neural stem/progenitors.
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http://dx.doi.org/10.1016/j.neuint.2019.104465DOI Listing
October 2019

Isolation of tissue-resident endothelial stem cells and their use in regenerative medicine.

Inflamm Regen 2019 8;39. Epub 2019 May 8.

1Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871 Japan.

Background: During sprouting angiogenesis, stalk cells, localized behind tip cells, generate endothelial cells (ECs) for the elongation of new vessels. We hypothesized that stalk cells may have endothelial progenitor cell properties because of their highly proliferative ability. We conducted Hoechst dye DNA staining in ECs of preexisting blood vessels from hind limb muscle and found that endothelial-side population (E-SP) cells, which efflux Hoechst rapidly with abundant ABC transporters, show highly producing ability of ECs. We previously showed the existence of E-SP cells in hind limb muscle, retina, and liver, but not in other tissues such as adipose tissue, skin, and placenta.

Methods: We investigated the existence of E-SP cells and analyzed their proliferative ability among CD31CD45 ECs from adipose tissue, skin, and placenta of adult mice. We also analyzed the neovascular formation of E-SP cells from adipose tissue in vivo.

Results: We detected E-SP cells in all tissues examined. However, by in vitro colony formation analysis on OP9 cells, we found that E-SP cells from adipose tissue and skin, but not from placenta, have highly proliferative ability. Moreover, E-SP cells from adipose tissue could contribute to the neovascular formation in hind limb ischemia model.

Conclusion: The adipose tissue and skin are available sources to obtain endothelial stem cells for conducting therapeutic angiogenesis in regenerative medicine.
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http://dx.doi.org/10.1186/s41232-019-0098-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6505211PMC
May 2019

Powerful Homeostatic Control of Oligodendroglial Lineage by PDGFRα in Adult Brain.

Cell Rep 2019 04;27(4):1073-1089.e5

Department of Pathology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan. Electronic address:

Oligodendrocyte progenitor cells (OPCs) are widely distributed cells of ramified morphology in adult brain that express PDGFRα and NG2. They retain mitotic activities in adulthood and contribute to oligodendrogenesis and myelin turnover; however, the regulatory mechanisms of their cell dynamics in adult brain largely remain unknown. Here, we found that global Pdgfra inactivation in adult mice rapidly led to elimination of OPCs due to synchronous maturation toward oligodendrocytes. Surprisingly, OPC densities were robustly reconstituted by the active expansion of Nestin immature cells activated in meninges and brain parenchyma, as well as a few OPCs that escaped from Pdgfra inactivation. The multipotent immature cells were induced in the meninges of Pdgfra-inactivated mice, but not of control mice. Our findings revealed powerful homeostatic control of adult OPCs, engaging dual cellular sources of adult OPC formation. These properties of the adult oligodendrocyte lineage and the alternative OPC source may be exploited in regenerative medicine.
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http://dx.doi.org/10.1016/j.celrep.2019.03.084DOI Listing
April 2019

Endothelial Cells Regulate Physiological Cardiomyocyte Growth via VEGFR2-Mediated Paracrine Signaling.

Circulation 2019 05 29;139(22):2570-2584. Epub 2019 Mar 29.

Wihuri Research Institute, Helsinki, Finland and Translational Cancer Biology Program, Research Programs Unit, Faculty of Medicine, University of Helsinki, Finland (R.K., K.A.H., M.R., K.A.).

Background: Heart failure, which is a major global health problem, is often preceded by pathological cardiac hypertrophy. The expansion of the cardiac vasculature, to maintain adequate supply of oxygen and nutrients, is a key determinant of whether the heart grows in a physiological compensated manner or a pathological decompensated manner. Bidirectional endothelial cell (EC)-cardiomyocyte (CMC) cross talk via cardiokine and angiocrine signaling plays an essential role in the regulation of cardiac growth and homeostasis. Currently, the mechanisms involved in the EC-CMC interaction are not fully understood, and very little is known about the EC-derived signals involved. Understanding how an excess of angiogenesis induces cardiac hypertrophy and how ECs regulate CMC homeostasis could provide novel therapeutic targets for heart failure.

Methods: Genetic mouse models were used to delete vascular endothelial growth factor (VEGF) receptors, adeno-associated viral vectors to transduce the myocardium, and pharmacological inhibitors to block VEGF and ErbB signaling in vivo. Cell culture experiments were used for mechanistic studies, and quantitative polymerase chain reaction, microarrays, ELISA, and immunohistochemistry were used to analyze the cardiac phenotypes.

Results: Both EC deletion of VEGF receptor (VEGFR)-1 and adeno-associated viral vector-mediated delivery of the VEGFR1-specific ligands VEGF-B or placental growth factor into the myocardium increased the coronary vasculature and induced CMC hypertrophy in adult mice. The resulting cardiac hypertrophy was physiological, as indicated by preserved cardiac function and exercise capacity and lack of pathological gene activation. These changes were mediated by increased VEGF signaling via endothelial VEGFR2, because the effects of VEGF-B and placental growth factor on both angiogenesis and CMC growth were fully inhibited by treatment with antibodies blocking VEGFR2 or by endothelial deletion of VEGFR2. To identify activated pathways downstream of VEGFR2, whole-genome transcriptomics and secretome analyses were performed, and the Notch and ErbB pathways were shown to be involved in transducing signals for EC-CMC cross talk in response to angiogenesis. Pharmacological or genetic blocking of ErbB signaling also inhibited part of the VEGF-B-induced effects in the heart.

Conclusions: This study reveals that cross talk between the EC VEGFR2 and CMC ErbB signaling pathways coordinates CMC hypertrophy with angiogenesis, contributing to physiological cardiac growth.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.118.036099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6553980PMC
May 2019

Regnase-1-mediated post-transcriptional regulation is essential for hematopoietic stem and progenitor cell homeostasis.

Nat Commun 2019 03 6;10(1):1072. Epub 2019 Mar 6.

Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Osaka, Suita,, 565-0871, Japan.

The balance between self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) maintains hematopoietic homeostasis, failure of which can lead to hematopoietic disorder. HSPC fate is controlled by signals from the bone marrow niche resulting in alteration of the stem cell transcription network. Regnase-1, a member of the CCCH zinc finger protein family possessing RNAse activity, mediates post-transcriptional regulatory activity through degradation of target mRNAs. The precise function of Regnase-1 has been explored in inflammation-related cytokine expression but its function in hematopoiesis has not been elucidated. Here, we show that Regnase-1 regulates self-renewal of HSPCs through modulating the stability of Gata2 and Tal1 mRNA. In addition, we found that dysfunction of Regnase-1 leads to the rapid onset of abnormal hematopoiesis. Thus, our data reveal that Regnase-1-mediated post-transcriptional regulation is required for HSPC maintenance and suggest that it represents a leukemia tumor suppressor.
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http://dx.doi.org/10.1038/s41467-019-09028-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6403248PMC
March 2019

Galectin-3 Inhibits Cancer Metastasis by Negatively Regulating Integrin β3 Expression.

Am J Pathol 2019 04 14;189(4):900-910. Epub 2019 Jan 14.

Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Suita, Japan; Division of Signal Transduction, Immunology Frontier Research Center, Osaka University, Suita, Japan. Electronic address:

Galectin-3 (Gal-3; gene LGALS3) is a member of the β-galactose-binding lectin family. Previous studies showed that Gal-3 is expressed in several tissues across species and functions as a regulator of cell proliferation, apoptosis, adhesion, and migration, thus affecting many aspects of events, such as angiogenesis and tumorigenesis. Although several reports have suggested that the level of Gal-3 expression correlates positively with tumor progression, herein we show that highly metastatic mouse melanoma B16/BL6 cells express less Gal-3 than B16 cells with a lower metastatic potential. It was found that overexpression of Gal-3 in melanoma cells in fact suppresses metastasis. In contrast, knocking out Gal-3 expression in cancer cells promoted cell aggregation mediated through interactions with platelets and fibrinogen in vitro and increased the number of metastatic foci in vivo. Thus, reduced Gal-3 expression results in the up-regulation of β3 integrin expression, and this contributes to metastatic potential. These findings indicate that changes of Gal-3 expression in cancer cells during tumor progression influence the characteristics of metastatic cells.
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http://dx.doi.org/10.1016/j.ajpath.2018.12.005DOI Listing
April 2019

TAK1 Prevents Endothelial Apoptosis and Maintains Vascular Integrity.

Dev Cell 2019 01 10;48(2):151-166.e7. Epub 2019 Jan 10.

Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan; Laboratory of Signal Transduction, World Premier Institute Immunology Frontier Research Center, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan. Electronic address:

TNF-α is a pleiotropic cytokine that has the potential to induce apoptosis under inflammation. How endothelial cells (ECs) are spared from this fate in inflammatory environments where TNF-α is present is not known. Here, we show that TGF-β-activated kinase 1 (TAK1) ensures EC survival and maintains vascular integrity upon TNF-α stimulation. Endothelial-specific TAK1 knockout mice exhibit intestinal and liver hemorrhage due to EC apoptosis, leading to vascular destruction and rapid death. This EC apoptosis was induced by TNF-α from myeloid cells responding to intestinal microbiota. TNF-α secretion associated with inflammation also induced vascular defects in inflamed organs. Additionally, we determined that TAK1 deletion in tumor ECs resulted in blood vessel and hence tumor regression. Our results illuminate mechanisms ensuring survival of intestinal and liver ECs under physiological conditions and ECs of other organs under inflammatory conditions that could be exploited for anti-angiogenic therapy to treat cancer.
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http://dx.doi.org/10.1016/j.devcel.2018.12.002DOI Listing
January 2019

Discovery of a Vascular Endothelial Stem Cell (VESC) Population Required for Vascular Regeneration and Tissue Maintenance.

Circ J 2018 12 28;83(1):12-17. Epub 2018 Nov 28.

Department of Signal Transduction, Research Institute for Microbial Deseases, Osaka University.

The roles that blood vessels play in the maintenance of organs and tissues in addition to the delivery of oxygen and nutrients are being gradually clarified. The maintenance of tissue-specific organ stem cells, such as hematopoietic and neuronal stem cells, is supported by endothelial cells (ECs), which represent an important component of the stem cell niche. The maintenance of organogenesis, for example, osteogenesis and liver generation/regeneration, is supported by molecules referred to as "angiocrine signals" secreted by EC. The mechanisms responsible for the well-known functions of blood vessels, such as thermoregulation and metabolism, especially removal of local metabolites, have now been determined at the molecular level. Following the development of single-cell genetic analysis, blood cell heterogeneity, especially of mural cell populations, has been established and tissue-specific blood vessel formation and function are now also understood at the molecular level. Among the heterogeneous populations of ECs, it seems that a stem cell population with the ability to maintain the production of ECs long-term is present in pre-existing blood vessels. Neovascularization by therapeutic angiogenesis yields benefits in many diseases, not only ischemic disease but also metabolic disease and other vascular diseases. Therefore, vascular endothelial stem cells should be considered to use in vascular regeneration therapy.
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http://dx.doi.org/10.1253/circj.CJ-18-1180DOI Listing
December 2018

Induced expression of GINS complex is an essential step for reactivation of quiescent stem-like tumor cells within the peri-necrotic niche in human glioblastoma.

J Cancer Res Clin Oncol 2019 Feb 21;145(2):363-371. Epub 2018 Nov 21.

Department of Pathology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi, 755-8505, Japan.

Purpose: Glioblastoma is still intractable despite the progress in therapies, and the intractability is attributable to a minor population of stem-like tumor cells. As a niche harboring quiescent stem-like tumor cells with potentially high tumorigenicity, we have specified an area around large ischemic necrosis, termed 'peri-necrotic niche', in glioblastoma. In this study, the behavior of tumor cells inside and outside the peri-necrotic niche was analyzed to find out molecules responsible for reactivation of quiescent stem-like tumor cells to proliferate outside the niche.

Methods: Expression of Ki-67 and GINS complex composed of SLD5, PSF1, PSF2 and PSF3 was analyzed by immunohistochemistry in human glioblastoma tissue samples. Proliferation assays, immunoblotting and siRNA experiments were performed using a glioblastoma cell line.

Results: Immunohistochemical analysis revealed quiescent and proliferative phenotypes of tumor cells inside and outside the niche, respectively, and the proliferation was spatially correlated with the expression of GINS components in tumor cells. To mimic the tissue microenvironment inside versus outside the niche, glioblastoma cells were cultured under hypoxic versus normoxic conditions, or without versus with serum. Quiescence and proliferation of tumor cells were reversibly determined by the microenvironment inside and outside the niche, respectively, and proliferative activities paralleled the expression levels of GINS components. Furthermore, the reactivation of proliferation after reoxygenation or serum replenishment was suppressed in quiescent tumor cells with PSF1 knockdown.

Conclusions: These findings indicate the essential role of GINS complex in the switch between quiescence and proliferation of tumor cells inside and outside the peri-necrotic niche.
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http://dx.doi.org/10.1007/s00432-018-2797-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6373247PMC
February 2019

LPA4-Mediated Vascular Network Formation Increases the Efficacy of Anti-PD-1 Therapy against Brain Tumors.

Cancer Res 2018 12 9;78(23):6607-6620. Epub 2018 Oct 9.

Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

: The structure and function of tumor blood vessels profoundly affects the tumor microenvironment. Signals mediated through the lysophosphatidic acid receptor 4 (LPA4) promote vascular network formation to restore normal vascular barrier function in subcutaneous tumors and thus improve drug delivery. However, the characteristics of the vasculature vary by organ and tumor types, and how drug delivery and leukocyte trafficking are affected by modification of vascular function by LPA in different cancers is unclear. Here, we show that LPA4 activation promotes the formation of fine vascular structures in brain tumors. RhoA/ROCK signaling contributed to LPA-induced endothelial cell-cell adhesion, and RhoA/ROCK activity following LPA4 stimulation regulated expression of VCAM-1. This resulted in increased lymphocyte infiltration into the tumor. LPA improved delivery of exogenous IgG into brain tumors and enhanced the anticancer effect of anti-programmed cell death-1 antibody therapy. These results indicate the effects of LPA on vascular structure and function apply not only to chemotherapy but also to immunotherapy. SIGNIFICANCE: These findings demonstrate that lysophosphatidic acid, a lipid mediator, promotes development of a fine capillary network in brain tumors by inducing tightening of endothelial cell-to-cell adhesion, facilitating improved drug delivery, and lymphocyte penetration.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-0498DOI Listing
December 2018

Developmental defects and aberrant accumulation of endogenous psychosine in oligodendrocytes in a murine model of Krabbe disease.

Neurobiol Dis 2018 12 31;120:51-62. Epub 2018 Aug 31.

Department of Pathology, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya-cho, Kasugai, Aichi 480-0392, Japan. Electronic address:

Krabbe disease (KD), or globoid cell leukodystrophy, is an inherited lysosomal storage disease with leukodystrophy caused by a mutation in the galactosylceramidase (GALC) gene. The majority of patients show the early onset form of KD dominated by cerebral demyelination with apoptotic oligodendrocyte (OL) death. However, the initial pathophysiological changes in developing OLs remain poorly understood. Here, we show that OLs of twitcher mice, an authentic mouse model of KD, exhibited developmental defects and impaired myelin formation in vivo and in vitro. In twitcher mouse brain, abnormal myelination and reduced expression of myelin genes during the period of most active OL differentiation and myelination preceded subsequent progressive OL death and demyelination. Importantly, twitcher mouse OL precursor cells proliferated normally, but their differentiation and survival were intrinsically defective. These defects were associated with aberrant accumulation of endogenous psychosine (galactosylsphingosine) and reduced activation of the Erk1/2 and Akt/mTOR pathways before apoptotic cell death. Collectively, our results demonstrate that GALC deficiency in developing KD OLs profoundly affects their differentiation and maturation, indicating the critical contribution of OL dysfunction to KD pathogenesis.
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http://dx.doi.org/10.1016/j.nbd.2018.08.023DOI Listing
December 2018

Visualization of Proliferative Vascular Endothelial Cells in Tumors in Vivo by Imaging Their Partner of Sld5-1 Promoter Activity.

Am J Pathol 2018 05 9;188(5):1300-1314. Epub 2018 Apr 9.

Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Suita, Japan. Electronic address:

Vascular endothelial cells (ECs) isolated from tumors characteristically express certain genes. It has recently been suggested that tumor vessel normalization facilitates effective drug delivery into tumors; however, how tumor vessel normalization can be recognized on the basis of the molecules expressed by tumor ECs is not clearly defined. The degree of cell proliferation is an important indicator to characterize the condition of the ECs. Herein, we generated transgenic mice expressing enhanced green fluorescent protein (EGFP) under the transcriptional control of the DNA replication factor partner of Sld5-1 (PSF1; official name GINS1) promoter to assess whether active ECs can be distinguished from dormant ECs. Predictably, ECs in the adult skin exhibited no EGFP signals. However, after s.c. injection of tumor cells, some ECs shifted to EGFP positivity, enabling distinction of EGFP-positive from EGFP-negative cells. We found that only a fraction of the EGFP-negative ECs strongly expressed the glycosylphosphatidylinositol-anchor protein CD109 associated with the phosphatidylinositol 3-kinase pathway. Taken together, these data indicate that areas of vascular normalization in tumors can be detected by CD109 expression, and this provides a window of opportunity for timing chemotherapy.
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http://dx.doi.org/10.1016/j.ajpath.2018.01.015DOI Listing
May 2018
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