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Figure S1 shows that N11-13 decoy blocks activation of Notch1 by DLL1 but not JAG2 whereas N110-24 decoy blocks activation of Notch1 by JAG2 but not DLL1. Figure S2 shows that purified N11-13 decoy preferentially inhibited DLL4-mediated Notch1 activation, while purified N11-24 decoy inhibited both DLL4 and JAG1-mediated Notch1 activation. Figure S3 shows using co-immunoprecipitation of N1 decoys and soluble versions of DLL4 and JAG1 lacking transmembrane domains that similar ligand specificity of the decoys was observed as when using membrane bound DLL4 or JAG1. Figure S4 shows serum levels of Notch1 decoys in P5 neonatal mice. Figure S5 shows N1 decoys that block JAG disrupted vascular smooth muscle cell association in the actively remodeling neonatal retinal vasculature, but not the mature retinal vasculature. Figure S6 shows that N11-13, N110-24, and N11-24 decoys block soft agar growth of Mm5MT-FGF4 tumor cells but do not affect growth of KP1-VEGF, LLC or B16-F10 tumor lines. Figure S7 shows N1 decoys detected in mouse serum after adenovirus infection and quantification of these levels using ELISA. Figure S8 shows that JAG- inhibiting decoys disrupt vascular smooth muscle coverage in tumors. Figure S9 shows that N1 decoys expressed in mice cause a modest increase of intestinal goblet cells but no significant change in murine weight. Figure S10 shows N1 decoys had no effect on serum markers of liver damage. Figure S11 shows N1 decoys did not affect liver and kidney histopathology.

SUMMARYGlioma cells hijack developmental transcriptional programs to control cell state. During neural development, lineage trajectories rely on specialized metabolic pathways. However, the link between tumor cell state and metabolic programs is poorly understood in glioma. Here we uncover a glioma cell state-specific metabolic liability that can be leveraged therapeutically. To model cell state diversity, we generated genetically engineered murine gliomas, induced by deletion of p53 alone (p53) or with constitutively active Notch signaling (N1IC), a pathway critical in controlling cellular fate. N1IC tumors harbored quiescent astrocyte-like transformed cell states while p53 tumors were predominantly comprised of proliferating progenitor-like cell states. N1IC cells exhibit distinct metabolic alterations, with mitochondrial uncoupling and increased ROS production rendering them more sensitive to inhibition of the lipid hydroperoxidase GPX4 and induction of ferroptosis. Importantly, treating patient-derived organotypic slices with a GPX4 inhibitor induced selective depletion of quiescent astrocyte-like glioma cell populations with similar metabolic profiles.

Understanding how coronary vessels develop is important for designing better strategies to repair ischemic hearts. In this issue of Developmental Cell, D'Amato et al. report that BMP2 and CXCL12/CXCR4 act sequentially on endocardial cells to drive coronary angiogenesis and artery morphogenesis.

Hematopoietic stem cell (HSC) generation in the aorta-gonad-mesonephros region requiresHSC specification signals from the surrounding microenvironment. In zebrafish, PDGFB/PDGFRβ signaling controls hematopoietic stem/progenitor cell (HSPC) generation and isrequired in the HSC specification niche. Little is known about murine HSPC specification invivo and whether PDGF-B/PDGFRβ is involved. Here we show that PDGFRβ is expressed indistinct perivascular stromal cell layers surrounding the mid-gestation dorsal aorta, and itsdeletion impairs hematopoiesis. We demonstrate that PDGFRβ+ cells play a dual role in murinehematopoiesis. They act in the aortic niche to support HSPCs, and in addition, PDGFRβ+embryonic precursors give rise to a subset of HSPCs that persist into adulthood. These findingsprovide crucial information for the controlled production of HSPCs in vitro.

Germinal matrix-intraventricular hemorrhage (GM-IVH) is the most devastating neurological complication in premature infants. GM-IVH usually begins in the GM, a highly vascularized region of the developing brain where glial and neuronal precursors reside underneath the lateral ventricular ependyma. Previous studies using human fetal tissue have suggested increased angiogenesis and paucity of pericytes as key factors contributing to GM-IVH pathogenesis. Yet, despite its relevance, the mechanisms underlying the GM vasculature's susceptibility to hemorrhage remain poorly understood. To gain better understanding on the vascular dynamics of the GM, we performed a comprehensive analysis of the mouse GM vascular endothelium and pericytes during development. We hypothesize that vascular development of the mouse GM will provide a good model for studies of human GM vascularization and provide insights into the role of pericytes in GM-IVH pathogenesis. Our findings show that the mouse GM presents significantly greater vascular area and vascular branching compared to the developing cortex (CTX). Analysis of pericyte coverage showed abundance in PDGFRβ-positive and NG2-positive pericyte coverage in the GM similar to the developing CTX. However, we found a paucity in Desmin-positive pericyte coverage of the GM vasculature. Our results underscore the highly angiogenic nature of the GM and reveal that pericytes in the developing mouse GM exhibit distinct phenotypical and likely functional characteristics compared to other brain regions which might contribute to the high susceptibility of the GM vasculature to hemorrhage.

Loss-of-function studies have determined that Notch signaling is essential for hematopoietic and endothelial development. By deleting a single allele of the Notch1 transcriptional activation domain we generated viable, post-natal mice exhibiting hypomorphic Notch signaling. These heterozygous mice, which lack only one copy of the transcriptional activation domain, appear normal and have no endothelial or hematopoietic phenotype, apart from an inherent, cell-autonomous defect in T-cell lineage development. Following chemotherapy, these hypomorphs exhibited severe pancytopenia, weight loss and morbidity. This phenotype was confirmed in an endothelial-specific, loss-of-function Notch1 model system. Ang1, secreted by hematopoietic progenitors after damage, activated endothelial Tie2 signaling, which in turn enhanced expression of Notch ligands and potentiated Notch1 receptor activation. In our heterozygous, hypomorphic model system, the mutant protein that lacks the Notch1 transcriptional activation domain accumulated in endothelial cells and interfered with optimal activity of the wildtype Notch1 transcriptional complex. Failure of the hypomorphic mutant to efficiently drive transcription of key gene targets such as Hes1 and Myc prolonged apoptosis and limited regeneration of the bone marrow niche. Thus, basal Notch1 signaling is sufficient for niche development, but robust Notch activity is required for regeneration of the bone marrow endothelial niche and hematopoietic recovery.

Gliogenesis in the adult mouse brain Neural stem cells in the adult mouse brain can generate both neurons and glia. Exactly where each stem cell is positioned can determine what type of neurons it generates. Delgado et al. show that neural stem cells are also choosy about what sorts of glia they make and when (see the Perspective by Baldwin and Silver). Injury or selective deletion of platelet-derived growth factor receptor β (PDGFRβ) from the stem cells kicked them into overdrive and revealed their selectivity with respect to gliogenesis. An unusual type of glial progenitor cell, intraventricular oligodendrocyte progenitors, are found nestled between the cilia of ependymal cells derived from tight clusters of PDGFRβ-expressing stem cells. Science , abg8467, this issue p. 1205 ; see also abj1139, p. 1151

Hematopoietic stem cell (HSC) generation in the aorta-gonad-mesonephros region requires HSC specification signals from the surrounding microenvironment. In zebrafish, PDGF-B/PDGFRβ signaling controls hematopoietic stem/progenitor cell (HSPC) generation and is required in the HSC specification niche. Little is known about murine HSPC specification in vivo and whether PDGF-B/PDGFRβ is involved. Here, we show that PDGFRβ is expressed in distinct perivascular stromal cell layers surrounding the mid-gestation dorsal aorta, and its deletion impairs hematopoiesis. We demonstrate that PDGFRβ

SUMMARY Mitochondrial dynamics are tightly controlled by fusion and fission, and their dysregulation and excess reactive oxygen species (ROS) contribute to endothelial cell (EC) dysfunction. How redox signals regulate coupling between mitochondrial dynamics and endothelial (dys)function remains unknown. Here, we identify protein disulfide isomerase A1 (PDIA1) as a thiol reductase for the mitochondrial fission protein Drp1. A biotin-labeled Cys-OH trapping probe and rescue experiments reveal that PDIA1 depletion in ECs induces sulfenylation of Drp1 at Cys644, promoting mitochondrial fragmentation and ROS elevation without inducing ER stress, which drives EC senescence. Mechanistically, PDIA1 associates with Drp1 to reduce its redox status and activity. Defective wound healing and angiogenesis in diabetic or PDIA1+/− mice are restored by EC-targeted PDIA1 or the Cys oxidation-defective mutant Drp1. Thus, this study uncovers a molecular link between PDIA1 and Drp1 oxidoreduction, which maintains normal mitochondrial dynamics and limits endothelial senescence with potential translational implications for vascular diseases associated with diabetes or aging., In Brief Kim et al. demonstrate a molecular link between PDIA1 and Drp1 oxidoreduction, which protects against mitochondrial fragmentation and ROS elevation, limiting endothelial senescence. This study provides insights into restoring endothelial PDIA1 function or targeting Drp1 Cys oxidation as potential therapeutic strategies for treating diabetes-associated vascular and metabolic diseases., Graphical Abstract

Pericytes are essential mural cells distinguished by their association with small caliber blood vessels and the presence of a basement membrane shared with endothelial cells. Pericyte interaction with the endothelium plays an important role in angiogenesis; however, very few tools are currently available that allow for the targeting of pericytes in mouse models, limiting our ability to understand their biology. We have generated a novel mouse line expressing tamoxifen-inducible Cre-recombinase under the control of the platelet-derived growth factor receptor β promoter: PDGFRβ-P2A-CreER T2 . We evaluated the expression of the PDGFRβ-P2A-CreER T2 line by crossing it with fluorescent reporter lines and analyzed reporter signal in the angiogenic retina and brain at different time points after tamoxifen administration. Reporter lines showed labeling of NG2+, desmin+, PDGFRβ+ perivascular cells in the retina and the brain, indicating successful targeting of pericytes; however, signal from reporter lines was also observed in a small subset of glial cells both in the retina and the brain. We also evaluated recombination in tumors and found efficient recombination in perivascular cells associated with tumor vasculature. As a proof of principle, we used our newly generated driver to delete Notch signaling in perivascular cells and observed a loss of smooth muscle cells in retinal arteries, consistent with previously published studies evaluating Notch3 null mice. We conclude that the PDGFRβ-P2A-CreER T2 line is a powerful new tool to target pericytes and will aid the field in gaining a deeper understanding of the role of these cells in physiological and pathological settings.

Quiescent neural stem cells (NSCs) in the adult ventricular-subventricular zone (V-SVZ) have a regional identity and undergo activation to generate neurons. The domains for gliogenesis are less explored. Here we show that Platelet-Derived Growth Factor Receptor beta (PDGFRβ) is expressed by adult V-SVZ NSCs that generate olfactory bulb interneurons and glia with slow baseline kinetics. Selective deletion of PDGFRβ in adult V-SVZ NSCs leads to their release from quiescence uncovering multiple domains in the septal wall for oligodendrocyte and astrocyte formation. Unexpectedly, we identify a novel intraventricular oligodendrocyte progenitor inside the brain ventricles. Together our findings reveal different NSC spatial domains for gliogenesis in the adult V-SVZ that are largely quiescent under homeostasis and may have key functions for brain plasticity.

Chagas disease is caused by the protozoan Trypanosoma cruzi, affecting millions of people worldwide. One of the major causes of mortality in the disease is the cardiomyopathy observed in chronic patients, despite the low number of parasites detected in cardiac tissue. Galectin-3, a carbohydrate-binding protein with affinity for β-galactoside-containing glycoconjugates, is upregulated upon infection, and it has been recently involved in the pathophysiology of heart failure.We investigated the role of galectin-3 in systemic and local responses in a murine model of T. cruzi infection, using knockout animals. Molecular mechanisms underlying galectin-3-dependent inflammatory responses were further assessed in cultured dendritic cells in vitro.Mice deficient for galectin-3 have elevated blood parasitemia levels and impaired cytokine production during infection. Remarkably, galectin-3 promotes cellular infiltration in the heart of infected mice and subsequent collagen deposition and cardiac fibrosis. Furthermore, we show that an unbalanced Toll-like receptor expression on antigen-presenting cells may be the cause of the impaired immune response observed in galectin-3-deficient mice in vivo.These results suggest that galectin-3 is strongly involved in Chagas disease, not only in the immune response against T. cruzi, but also in mediating cardiac tissue damage.

A proangiogenic role for Jagged (JAG)-dependent activation of NOTCH signaling in the endothelium has yet to be described. Using proteins that encoded different NOTCH1 EGF-like repeats, we identified unique regions of Delta-like ligand (DLL)–class and JAG-class ligand–receptor interactions, and developed NOTCH decoys that function as ligand-specific NOTCH inhibitors. N110–24 decoy blocked JAG1/JAG2–mediated NOTCH1 signaling, angiogenic sprouting in vitro, and retinal angiogenesis, demonstrating that JAG-dependent NOTCH signal activation promotes angiogenesis. In tumors, N110–24 decoy reduced angiogenic sprouting, vessel perfusion, pericyte coverage, and tumor growth. JAG–NOTCH signaling uniquely inhibited expression of antiangiogenic soluble (s) VEGFR1/sFLT1. N11–13 decoy interfered with DLL1–DLL4-mediated NOTCH1 signaling and caused endothelial hypersprouting in vitro, in retinal angiogenesis, and in tumors. Thus, blockade of JAG- or DLL-mediated NOTCH signaling inhibits angiogenesis by distinct mechanisms. JAG–NOTCH signaling positively regulates angiogenesis by suppressing sVEGFR1–sFLT1 and promoting mural–endothelial cell interactions. Blockade of JAG-class ligands represents a novel, viable therapeutic approach to block tumor angiogenesis and growth. Significance: This is the first report identifying unique regions of the NOTCH1 extracellular domain that interact with JAG-class and DLL-class ligands. Using this knowledge, we developed therapeutic agents that block JAG-dependent NOTCH signaling and demonstrate for the first time that JAG blockade inhibits experimental tumor growth by targeting tumor angiogenesis. Cancer Discov; 5(2); 182–97. ©2014 AACR. See related commentary by Briot and Iruela-Arispe, p. 112 This article is highlighted in the In This Issue feature, p. 97

Pericytes are essential mural cells distinguished by their association with small caliber vessels and the presence of a basement membrane shared with endothelial cells. Pericyte interaction with the endothelium plays an important role in angiogenesis, however very few tools are currently available that allow for the targeting of pericytes in mouse models, limiting our ability to understand their biology. We have generated a novel mouse line expressing tamoxifen-inducible Cre-recombinase under the control of the platelet derived growth factor receptor β promoter: PDGFRβ-P2A-CreERT2. We evaluated the expression of the PDGFRβ-P2A-CreERT2 line by crossing it with fluorescent reporter lines and analyzed reporter signal in the angiogenic retina and brain at different time points after tamoxifen administration. Reporter lines showed labeling of NG2+, desmin+, PDGFRβ+ perivascular cells in the retina and the brain, indicating successful targeting of pericytes; however, signal from reporter lines was also observed in a small subset of glial cells both in the retina and the brain. We also evaluated recombination in tumors and found efficient recombination in perivascular cells associated with tumor vasculature. As a proof of principle, we used our newly generated driver to delete Notch signaling in perivascular cells and observed a loss of smooth muscle cells in retinal arteries, consistent with previously published studies evaluating Notch3 null mice. We conclude that the PDGFRβ-P2A-CreERT2 line is a powerful new tool to target pericytes and will aid the field in gaining a deeper understanding of the role of these cells in physiological and pathological settings.

Arteriovenous malformations (AVMs) are tortuous vessels characterized by arteriovenous (AV) shunts, which displace capillaries and shunt blood directly from artery to vein. Notch signaling regulates embryonic AV specification by promoting arterial, as opposed to venous, endothelial cell (EC) fate. To understand the essential role of endothelial Notch signaling in postnatal AV organization, we used inducible Cre-loxP recombination to delete Rbpj, a mediator of canonical Notch signaling, from postnatal ECs in mice. Deletion of endothelial Rbpj from birth resulted in features of AVMs by P14, including abnormal AV shunting and tortuous vessels in the brain, intestine and heart. We further analyzed brain AVMs, as they pose particular health risks. Consistent with AVM pathology, we found cerebral hemorrhage, hypoxia and necrosis, and neurological deficits. AV shunts originated from capillaries (and possibly venules), with the earliest detectable morphological abnormalities in AV connections by P8. Prior to AV shunt formation, alterations in EC gene expression were detected, including decreased Efnb2 and increased Pai1, which encodes a downstream effector of TGFβ signaling. After AV shunts had formed, whole-mount immunostaining showed decreased Efnb2 and increased Ephb4 expression within AV shunts, suggesting that ECs were reprogrammed from arterial to venous identity. Deletion of Rbpj from adult ECs led to tortuosities in gastrointestinal, uterine and skin vascular beds, but had mild effects in the brain. Our results demonstrate a temporal requirement for Rbpj in postnatal ECs to maintain proper artery, capillary and vein organization and to prevent abnormal AV shunting and AVM pathogenesis.

Liver vasculature is crucial for adequate hepatic functions. Global deletion of Notch signaling in mice results in liver vascular pathologies. However, whether Notch in endothelium is essential for hepatic vascular structure and function remains unknown. To uncover the function of endothelial Notch in the liver, we deleted Rbpj, a transcription factor mediating all canonical Notch signaling, or Notch1, specifically from the endothelium of postnatal mice. We investigated the hepatic vascular defects in these mutants. The liver was severely affected within two weeks following endothelial deletion of Rbpj from birth. Two-week old mutant mice had enlarged vessels on the liver surface, abnormal vascular architecture, and dilated sinusoids. Vascular casting and fluorosphere passage experiments indicated the presence of porto-systemic shunts. These mutant mice presented severely necrotic liver parenchyma and significantly larger hypoxic areas, likely resulting from vascular shunts. We also found elevated levels of VEGF receptor 3 together with reduced levels of ephrin-B2, suggesting a possible contribution of these factors to the generation of hepatic vascular abnormalities. Deletion of Rbpj from the adult endothelium also led to dilated sinusoids, vascular shunts, and necrosis albeit milder than that in mice with deletion from birth. Similar to deletion of Rbpj, loss of endothelial Notch1 from birth led to similar hepatic vascular malformations within two weeks. Conclusions: Endothelial Notch signaling is essential for the development and maintenance of proper hepatic vascular architecture and function. Our findings may help understand the molecular pathogenesis of hepatic vascular malformation and the safety of therapeutics inhibiting Notch. This article is protected by copyright. All rights reserved.

Multiple Sclerosis (MS) is a demyelinating/inflammatory disease of the central nervous system. Relapsing-remitting MS is characterized by a relapsing phase with clinical symptoms and the production of inflammatory cell infiltrates, and a period of remission during which patients recover partially. Myeloid-derived suppressor cells (MDSCs) are immature cells capable of suppressing the inflammatory response through Arginase-I (Arg-I) activity, among other mechanisms. Here, we have identified Arg-I(+) -MDSCs in the spinal cord during experimental autoimmune encephalomyelitis (EAE), cells that were largely restricted to the demyelinating plaque and that always exhibited the characteristic MDSC surface markers Arg-I/CD11b/Gr-1/M-CSF1R. The presence and density of Arg-I(+) -cells, and the proportion of apoptotic but not proliferative T cells, were correlated with the EAE time course: peaked in parallel with the clinical score, decreased significantly during the remitting phase and completely disappeared during the chronic phase. Spinal cord-isolated MDSCs of EAE animals augmented the cell death when co-cultured with stimulated control splenic CD3 T cells. These data point to an important role for MDSCs in limiting inflammatory damage in MS, favoring the relative recovery in the remitting phase of the disease. Thus, the MDSC population should be considered as a potential therapeutic target to accelerate the recovery of MS patients.

Introduction: Gliomas are highly infiltrative tumors rendering complete resection impossible. As such, prognosis remains dismal. Recent studies have identified both proneural and mesenchymal cell populations. Crucially, recurring gliomas bear a mesenchymal phenotype. Understanding mechanisms driving mesenchymal transformation is important in developing tailored, more efficient therapeutic approaches. Methods: Using targeted biopsies from a mouse glioma model and human glioma databases, we applied bioinformatics and identified Notch as an additional master regulator (MR) protein driving mesenchymal transformation. To better understand the role of Notch, we overexpressed active forms of Notch1 (N1IC) or Notch3 (N3IC) in primary cells, orthotopic xenotransplants, and transgenic mouse models. We performed mouse survival studies and examined Notch-induced phenotypic alterations in tumor cells and the microenvironment using molecular and immunohistochemical approaches. Finally, we performed high-throughput drug and radiation screening studies to assess responses to treatment. Results: In silico analyses on RNAseq of biopsies from spatially distinct tumor regions in a mouse glioma model identified Notch as a highly active pathway in the cores of tumors undergoing mesenchymal transformation. Importantly, MR analysis of TCGA samples and single cell transcriptomic data identified the Notch pathway as a driver of proneural to mesenchymal transformation. Immunophenotyping and RNAseq profiling of human GBMs identified Notch receptors on tumor cells and Notch ligands on astrocytes and microglia suggesting potential microenvironment-mediated plasticity since Notch proteins are not frequently mutated in GBM. Overexpression of N1IC or N3IC in proneural cells induced mesenchymal transformation with slow proliferation and a highly invasive phenotype. Orthotransplantation of N3IC-expressing cells in mice led to slower growing tumors, perivascular invasion, increased recruitment of reactive phagocytes and slightly prolonged survival. A novel transgenic mouse model in which N1IC was overexpressed also led to prolonged survival compared to a control population. Importantly, high-throughput screening studies showed that N1IC and N3IC rendered tumor cells significantly more resistant to standard and targeted therapies, including irradiation and etoposide. Conclusions: Our study identifies Notch signaling as an important, likely microenvironment-driven pathway driving proneural to mesenchymal transformation in glioma. While Notch was shown to slow tumor growth and prolong survival, cells were more invasive and less sensitive to treatment. These findings suggest that Notch plays a role in the mesenchymal transformation and therapeutic resistance seen in post-treatment recurrence. Targeting Notch may therefore be important in improving therapeutic responses against glioma. Citation Format: Matei A. Banu, Athanassios Dovas, Henar Cuervo, Brianna Pereira, Aayushi Mahajan, Hongxu Ding, Mukesh Bansal, Sergey Pampou, Charles Karan, Andrea Califano, Peter A. Sims, Jan Kitajewski, Jeffrey N. Bruce, Peter Canoll. Notch is a master regulator of mesenchymal transformation and therapeutic resistance in glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5103.

In Chagas disease, which is caused by Trypanosoma cruzi, macrophages and cardiomyocytes are the main targets of infection. Classical activation of macrophages during infection is protective, whereas alternative activation of macrophages is involved in the survival of host cells and parasites. We studied the expression of inducible nitric oxide synthase (iNOS) and arginase as markers of classical and alternative activation, respectively, in heart tissue during in vivo infection of BALB/c and C57BL/6 mice. We found that expression of arginase I and II, as well as that of ornithine decarboxylase, was much higher in BALB/c mice than in C57BL/6 mice and that it was associated with the parasite burden in heart tissue. iNOS and arginase II were expressed by cardiomyocytes. Interestingly, heart-infiltrated CD68 + macrophages were the major cell type expressing arginase I. T helper (Th) 1 and Th2 cytokines were expressed in heart tissue in both infected mouse strains; however, at the peak of parasite infection, the balance between Th1 and Th2 predominantly favored Th1 in C57BL/6 mice and Th2 in BALB/c mice. The results of the present study suggest that Th2 cytokines induce arginase expression, which may influence host and parasite cell survival but which might also down-regulate the counterproductive effects triggered by iNOS in the heart during infection.

Chagas disease, caused by Trypanosoma cruzi, affects several million people in Central and South America. About 30% of chronic patients develop cardiomyopathy probably caused by parasite persistence and/or autoimmunity. While several cross-reactive antibodies generated during mammal T. cruzi infection have been described, very few cross-reactive T cells have been identified. We performed adoptive transfer experiments of T cells isolated from chronically infected mice. The results showed the generation of cardiac pathology in the absence of parasites. We also transferred cross-reactive SAPA-specific T cells and observed unspecific alterations in heart repolarization, cardiac inflammatory infiltration, and tissue damage.

Several questions about the role of the oxygen sensor prolyl-hydroxylase 2 (PHD2) in cancer have not been addressed. First, the role of PHD2 in metastasis has not been studied in a spontaneous tumor model. Here, we show that global PHD2 haplodeficiency reduced metastasis without affecting tumor growth. Second, it is unknown whether PHD2 regulates cancer by affecting cancer-associated fibroblasts (CAFs). We show that PHD2 haplodeficiency reduced metastasis via two mechanisms: (1) by decreasing CAF activation, matrix production, and contraction by CAFs, an effect that surprisingly relied on PHD2 deletion in cancer cells, but not in CAFs; and (2) by improving tumor vessel normalization. Third, the effect of concomitant PHD2 inhibition in malignant and stromal cells (mimicking PHD2 inhibitor treatment) is unknown. We show that global PHD2 haplodeficiency, induced not only before but also after tumor onset, impaired metastasis. These findings warrant investigation of PHD2's therapeutic potential. publisher: Elsevier articletitle: The Cancer Cell Oxygen Sensor PHD2 Promotes Metastasis via Activation of Cancer-Associated Fibroblasts journaltitle: Cell Reports articlelink: http://dx.doi.org/10.1016/j.celrep.2015.07.010 content_type: article copyright: Copyright © 2015 The Authors. Published by Elsevier Inc. ispartof: Cell Reports vol:12 issue:6 pages:992-1005 ispartof: location:United States status: published

Inflammation plays an important role in the pathophysiology of Chagas disease, caused by Trypanosoma cruzi. Prostanoids are regulators of homeostasis and inflammation and are produced mainly by myeloid cells, being cyclooxygenases, COX-1 and COX-2, the key enzymes in their biosynthesis from arachidonic acid (AA). Here, we have investigated the expression of enzymes involved in AA metabolism during T. cruzi infection. Our results show an increase in the expression of several of these enzymes in acute T. cruzi infected heart. Interestingly, COX-2 was expressed by CD68+ myeloid heart-infiltrating cells. In addition, infiltrating myeloid CD11b+Ly6G- cells purified from infected heart tissue express COX-2 and produce prostaglandin E2 (PGE2) ex vivo. T. cruzi infections in COX-2 or PGE2- dependent prostaglandin receptor EP-2 deficient mice indicate that both, COX-2 and EP-2 signaling contribute significantly to the heart leukocyte infiltration and to the release of chemokines and inflammatory cytokines in the heart of T. cruzi infected mice. In conclusion, COX-2 plays a detrimental role in acute Chagas disease myocarditis and points to COX-2 as a potential target for immune intervention., This work was supported by (NG) grants from “Fondo de Investigaciones Sanitarias” (PS09/00538 and PI12/00289); “Universidad Autónoma de Madrid” and “Comunidad de Madrid” (CC08-UAM/SAL-4440/08); by (MF) grants from “Ministerio de Ciencia e Innovación” (SAF2010-17833); “Red de Investigación de Centros de Enfermedades Tropicales” (RICET RD12/0018/0004); European Union (HEALTH-FE-2008-22303, ChagasEpiNet); AECID Cooperation with Argentine (A/025417/09 and A/031735/10), Comunidad de Madrid (S-2010/BMD- 2332) and “Fundación Ramón Areces”. NAG was recipient of a ISCIII Ph.D. fellowship financed by the Spanish “Ministerio de Sanidad”. CCM and HC were recipients of contracts from SAF2010-17833 and PI060388, respectively.

Trypanosoma cruzi, the causative agent of Chagas' disease, induces multiple responses in the heart, a critical organ of infection and pathology in the host. Among diverse factors, eicosanoids and the vasoactive peptide endothelin-1 (ET-1) have been implicated in the pathogenesis of chronic chagasic cardiomyopathy. In the present study, we found that T. cruzi infection in mice induces myocardial gene expression of cyclooxygenase-2 (Cox2) and thromboxane synthase (Tbxas1) as well as endothelin-1 (Edn1) and atrial natriuretic peptide (Nppa). T. cruzi infection and ET-1 cooperatively activated the Ca2+/calcineurin (Cn)/nuclear factor of activated T cells (NFAT) signaling pathway in atrial myocytes, leading to COX-2 protein expression and increased eicosanoid (prostaglandins E2 and F2α, thromboxane A2) release. Moreover, T. cruzi infection of ET-1-stimulated cardiomyocytes resulted in significantly enhanced production of atrial natriuretic peptide (ANP), a prognostic marker for impairment in cardiac function of chagasic patients. Our findings support an important role for the Ca2+/Cn/NFAT cascade in T. cruzi-mediated myocardial production of inflammatory mediators and may help define novel therapeutic targets. © 2013 Corral et al., Ministerio de Ciencia y Tecnología, Spain (SAF2007-61716; SAF2005-02220); Red Temática de Investigación en Enfermedades Cardiovasculares (RECAVA RD06/0014/1013); Red de Investigación de Centros de Enfermedades Tropicales (RICET RD06/0021/0016); European Union (HEALTH-FE-2008-22303, ChagasEpiNet); Universidad Autónoma de Madrid and Comunidad de Madrid (CC08-UAM/SAL-4440/08); Fundación Ramón Areces and Agencia Española de Cooperación Internacional para el Desarrollo (AECID, A/9418/07 and A/017500/08 to M.F. and R.S.C.); and Fondo de Investigaciones Sanitarias (PS09/00538 to N.G.). R.S.C. is a Member of Research Career from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects several million people in Latin America. Myocarditis, observed in the acute and chronic phases of the disease, is characterized by a mononuclear cell inflammatory infiltrate. We previously identified a myeloid cell population in the inflammatory heart infiltrate of infected mice that expressed arginase I. In this study, we purified CD11b(+) myeloid cells from the heart and analyzed their phenotype and function. Those CD11b(+) cells were ∼70% Ly6G(-)Ly6C(+) and 25% Ly6G(+)Ly6C(+). Moreover, purified CD11b(+)Ly6G(-) cells, but not Ly6G(+) cells, showed a predominant monocytic phenotype, expressed arginase I and inducible NO synthase, and suppressed anti-CD3/anti-CD28 Ab-induced T cell proliferation in vitro by an NO-dependent mechanism, activity that best defines myeloid-derived suppressor cells (MDSCs). Contrarily, CD11b(+)Ly6G(+) cells, but not CD11b(+)Ly6G(-) cells, expressed S100A8 and S100A9, proteins known to promote recruitment and differentiation of MDSCs. Together, our results suggest that inducible NO synthase/arginase I-expressing CD11b(+)Ly6G(-) myeloid cells in the hearts of T. cruzi-infected mice are MDSCs. Finally, we found plasma l-arginine depletion in the acute phase of infection that was coincident in time with the appearance of MDSCs, suggesting that in vivo arginase I could be contributing to l-arginine depletion and systemic immunosuppression. Notably, l-arginine supplementation decreased heart tissue parasite load, suggesting that sustained arginase expression through the acute infection is detrimental for the host. This is, to our knowledge, the first time that MDSCs have been found in the heart in the context of myocarditis and also in infection by T. cruzi., This work was supported by Ministerio de Ciencia y Tecnología (SAF2007-61716 and SAF2005-02220); Fondo de Investigaciones Sanitarias (PS09/00538); Red Tematica de Investigación en Enfermedades Cardiovasculares (RECAVA RD06/0014/1013); Red de Investigación de Centros de Enfermedades Tropicales (RICET RD06/0021/0016); European Union (HEALTH-FE-2008-22303, ChagasEpiNet); Universidad Autónoma de Madrid and Comunidad de Madrid (CC08-UAM/SAL-4440/08); Agencia Española de Cooperación Internacional para el Desarrollo cooperation with Argentina (A/025417/09); and Fundación Ramón Areces. H.C. and N.A.G. were financed by Fondo de Investigaciones Sanitarias, Instituto de salud Carlos III contracts. S.C. was a holder of a Formación de personal investigador fellowship. A.B. was funded by the Interuniversity Attraction Pole Program of the Belgian government.

Multiple Sclerosis (MS) is a demyelinating/inflammatory disease of the central nervous system. Relapsing-remitting MS is characterized by a relapsing phase with clinical symptoms and the production of inflammatory cell infiltrates, and a period of remission during which patients recover partially. Myeloid-derived suppressor cells (MDSCs) are immature cells capable of suppressing the inflammatory response through Arginase-I (Arg-I) activity, among other mechanisms. Here, we have identified Arg-I(+) -MDSCs in the spinal cord during experimental autoimmune encephalomyelitis (EAE), cells that were largely restricted to the demyelinating plaque and that always exhibited the characteristic MDSC surface markers Arg-I/CD11b/Gr-1/M-CSF1R. The presence and density of Arg-I(+) -cells, and the proportion of apoptotic but not proliferative T cells, were correlated with the EAE time course: peaked in parallel with the clinical score, decreased significantly during the remitting phase and completely disappeared during the chronic phase. Spinal cord-isolated MDSCs of EAE animals augmented the cell death when co-cultured with stimulated control splenic CD3 T cells. These data point to an important role for MDSCs in limiting inflammatory damage in MS, favoring the relative recovery in the remitting phase of the disease. Thus, the MDSC population should be considered as a potential therapeutic target to accelerate the recovery of MS patients.

SummaryOur findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective. We also provide additional evidence for the specificity of our anti-PlGF antibody and experiments to suggest that anti-PlGF treatment will not be effective for all tumors and why. Further, we show that PlGF blockage inhibits vessel abnormalization rather than density in certain tumors while enhancing VEGF-targeted inhibition in ocular disease. Our findings warrant further testing of anti-PlGF therapies.

Chagas’ disease, caused by Trypanosoma cruzi, has been considered a paradigm of infection-induced autoimmune disease. Thus, the scarcity of parasites in the chronic phase of the disease contrasts with the severe cardiac pathology observed in approximately 30% of chronic patients and suggested a role for autoimmunity as the origin of the pathology. Antigen-specific and antigen-non-specific mechanisms have been described by which T. cruzi infection might activate T and B cells, leading to autoimmunity. Among the first mechanisms, molecular mimicry has been claimed as the most important mechanism leading to autoimmunity and pathology in the chronic phase of this disease. In this regard, various T. cruzi antigens, such as B13, cruzipain and Cha, cross-react with host antigens at the B or T cell level and their role in pathogenesis has been widely studied. Immunization with those antigens and/or passive transfer of autoreactive T lymphocytes in mice lead to clinical disturbances similar to those found in Chagas’ disease patients. On the other hand, the parasite is becoming increasingly detected in chronically infected hosts and may also be the cause of pathology either directly or through parasite-specific mediated inflammatory responses. Thus, the issue of autoimmunity versus parasite persistence as the cause of Chagas’ disease pathology is hotly debated among many researchers in the field. We critically review here the evidence in favor of and against autoimmunity through molecular mimicry as responsible for Chagas’ disease pathology from clinical, pathological and immunological perspectives.

Background Notch4 is a member of the Notch family of receptors that is primarily expressed in the vascular endothelial cells. Genetic deletion of Notch4 does not result in an overt phenotype in mice, thus the function of Notch4 remains poorly understood. Methods We examined the requirement for Notch4 in the development of breast cancer vasculature. Orthotopic transplantation of mouse mammary tumor cells wild type for Notch4 into Notch4 deficient hosts enabled us to delineate the contribution of host Notch4 independent of its function in the tumor cell compartment. Results Here, we show that Notch4 expression is required for tumor onset and early tumor perfusion in a mouse model of breast cancer. We found that Notch4 expression is upregulated in mouse and human mammary tumor vasculature. Moreover, host Notch4 deficiency delayed the onset of MMTV-PyMT tumors, wild type for Notch4, after transplantation. Vessel perfusion was decreased in tumors established in Notch4-deficient hosts. Unlike in inhibition of Notch1 or Dll4, vessel density and branching in tumors developed in Notch4-deficient mice were unchanged. However, final tumor size was similar between tumors grown in wild type and Notch4 null hosts. Conclusion Our results suggest a novel role for Notch4 in the establishment of tumor colonies and vessel perfusion of transplanted mammary tumors.

To overcome the resistance to VEGF-inhibitor treatment, additional anti-angiogenic agents need to be valorized. Placental growth factor (PlGF), a VEGF homologue, stimulates endothelial, tumor and inflammatory cells. Studies in independently generated PlGF−/− mice by us and Regeneron identified a role for PlGF in inflammatory, ischemic and malignant disease, while the anti-PlGF mAb 5D11D4 blocks growth of implanted tumors. Several clinical studies also show that PlGF levels correlate with poor prognosis in diverse types of cancer, while the humanized anti-human PlGF mAb TB403 has been succesfully tested in a phase I trial in out-treated cancer patients. However, genetic evidence for a disease-candidate role of PlGF or an effect of the anti-PlGF mAb 5D11D4 in spontaneous cancer models has been lacking so far. Here, we show that loss of PlGF inhibited a carcinogen-induced skin tumor model papilloma formation and angiogenesis. Furthermore, silencing of PlGF in a transgenic hepatocellular carcinoma (HCC) model reduced tumor size and incidence. In a carcinogen-induced HCC model, most WT but only a minority of PlGF−/− mice died, while fewer tumor nodules developed in PlGF−/− mice. When WT mice with established HCC were treated with 5D11D4, more control IgG than 5D11D4 treated mice died. PlGF blockage inhibited the characteristic arterialization response in these tumors and blocked vessel abnormalization in HCC leading to reduced levels of hypoxia, known to stimulate HCC growth, however, without affecting microvascular density. These findings indicate that the anti-cancer activity of PlGF-blockage is not reflected by a change in vessel density alone. In the Rip1Tag2 model, no differences between WT and PlGF−/− mice or 5D11D4-treated mice in tumor burden or incidence were found, indicating that 5D11D4 does not have off-target effects. Resistance of this model is likely due to the lack of infiltration by Gr1+ neutrophils by PlGF blockage. These findings and previously published results of a Phase I trial with TB403 warrant furter analysis of anti-PlGF strategies for (non)-oncology indications. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-362.

Glioma cells hijack developmental transcriptional programs to control cell state. During neural development, lineage trajectories rely on specialized metabolic pathways. However, the link between tumor cell state and metabolic programs is poorly understood in glioma. Here we uncover a glioma cell state-specific metabolic liability that can be leveraged therapeutically. To model cell state diversity, we generated genetically engineered murine gliomas, induced by deletion of p53 alone (p53) or with constitutively active Notch signaling (N1IC), a pathway critical in controlling cellular fate. N1IC tumors harbored quiescent astrocyte-like transformed cell states while p53 tumors were predominantly comprised of proliferating progenitor-like cell states. N1IC cells exhibit distinct metabolic alterations, with mitochondrial uncoupling and increased ROS production rendering them more sensitive to inhibition of the lipid hydroperoxidase GPX4 and induction of ferroptosis. Importantly, treating patient-derived organotypic slices with a GPX4 inhibitor induced selective depletion of quiescent astrocyte-like glioma cell populations with similar metabolic profiles., Competing Interests: Declaration of interests: The authors declare no competing interests.