RESUMEN
Our 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.
Asunto(s)
Neovascularización Fisiológica/efectos de los fármacos , Proteínas Gestacionales/antagonistas & inhibidores , Proteínas Gestacionales/metabolismo , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Anticuerpos Monoclonales/uso terapéutico , Carcinoma Hepatocelular/irrigación sanguínea , Carcinoma Hepatocelular/prevención & control , Coroides/irrigación sanguínea , Modelos Animales de Enfermedad , Oftalmopatías/patología , Humanos , Neoplasias Hepáticas Experimentales/irrigación sanguínea , Neoplasias Hepáticas Experimentales/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Papiloma/irrigación sanguínea , Papiloma/inducido químicamente , Papiloma/prevención & control , Factor de Crecimiento Placentario , Neoplasias Cutáneas/irrigación sanguínea , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/prevención & controlRESUMEN
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 PDGFRß-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.
Asunto(s)
Neovascularización Patológica , Pericitos , Animales , Encéfalo/irrigación sanguínea , Hemorragia Cerebral , Humanos , Recién Nacido , Recien Nacido Prematuro , Ratones , MorfogénesisRESUMEN
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.
Asunto(s)
Microambiente Celular , Células Endoteliales/fisiología , Receptor Notch1/metabolismo , Receptor TIE-2/metabolismo , Regeneración , Transducción de Señal , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Microambiente Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Fluorouracilo/farmacología , Rayos gamma/efectos adversos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Ratones Noqueados , Pancitopenia/etiología , Pancitopenia/metabolismo , Pancitopenia/patología , Transducción de Señal/efectos de los fármacosRESUMEN
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 ß promoter: PDGFRß-P2A-CreER T2 . We evaluated the expression of the PDGFRß-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+, PDGFRß+ 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 PDGFRß-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.
Asunto(s)
Técnicas Genéticas , Neovascularización Fisiológica , Pericitos/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Femenino , Genes Reporteros , Integrasas/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Recombinación Genética/genética , Retina/metabolismo , Tamoxifeno/farmacología , Factores de TiempoRESUMEN
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 TGFß 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.
Asunto(s)
Malformaciones Arteriovenosas/genética , Malformaciones Arteriovenosas/patología , Endotelio Vascular/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/deficiencia , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Animales , Eliminación de Gen , Perfilación de la Expresión Génica , Procesamiento de Imagen Asistido por Computador , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Ratones , Microscopía Fluorescente , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor EphB4/metabolismoRESUMEN
UNLABELLED: 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 from the endothelium of postnatal mice. We investigated the hepatic vascular defects in these mutants. The liver was severely affected within 2 weeks of 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 with 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 observed in mice with deletion from birth. Similar to deletion of Rbpj, loss of endothelial Notch1 from birth led to similar hepatic vascular malformations within 2 weeks. CONCLUSIONS: Endothelial Notch signaling is essential for the development and maintenance of proper hepatic vascular architecture and function. These findings may elucidate the molecular pathogenesis of hepatic vascular malformation and the safety of therapeutics inhibiting Notch. (Hepatology 2016;64:1302-1316).
Asunto(s)
Hígado/irrigación sanguínea , Receptor Notch1/fisiología , Malformaciones Vasculares/etiología , Animales , Endotelio Vascular , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/fisiología , Ratones , Transducción de SeñalRESUMEN
Angiogenesis is regulated by complex interactions between endothelial cells and support cells of the vascular microenvironment, such as tissue myeloid cells and vascular mural cells. Multicellular interactions during angiogenesis are difficult to study in animals and challenging in a reductive setting. We incorporated stromal cells into an established bead-based capillary sprouting assay to develop assays that faithfully reproduce major steps of vessel sprouting and maturation. We observed that macrophages enhance angiogenesis, increasing the number and length of endothelial sprouts, a property we have dubbed "angiotrophism." We found that polarizing macrophages toward a pro-inflammatory profile further increased their angiotrophic stimulation of vessel sprouting, and this increase was dependent on macrophage Notch signaling. To study endothelial/pericyte interactions, we added vascular pericytes directly to the bead-bound endothelial monolayer. These pericytes formed close associations with the endothelial sprouts, causing increased sprout number and vessel caliber. We found that Jagged1 expression and Notch signaling are essential for the growth of both endothelial cells and pericytes and may function in their interaction. We observed that combining endothelial cells with both macrophages and pericytes in the same sprouting assay has multiplicative effects on sprouting. These results significantly improve bead-capillary sprouting assays and provide an enhanced method for modeling interactions between the endothelium and the vascular microenvironment. Achieving this in a reductive in vitro setting represents a significant step toward a better understanding of the cellular elements that contribute to the formation of mature vasculature.
Asunto(s)
Comunicación Celular , Microambiente Celular , Células Endoteliales de la Vena Umbilical Humana/citología , Macrófagos/citología , Modelos Biológicos , Neovascularización Fisiológica , Pericitos/citología , Receptores Notch/metabolismo , Animales , Línea Celular , Polaridad Celular , Supervivencia Celular , Técnicas de Cocultivo , Técnicas de Silenciamiento del Gen , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inflamación/patología , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Ratones , Células Mieloides/citología , Células Mieloides/metabolismo , Pericitos/metabolismo , Proteínas Serrate-Jagged/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: 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 ß-galactoside-containing glycoconjugates, is upregulated upon infection, and it has been recently involved in the pathophysiology of heart failure. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
Asunto(s)
Enfermedad de Chagas/inmunología , Galectina 3/inmunología , Inmunidad Innata/inmunología , Miocardio/patología , Trypanosoma cruzi/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Cardiomiopatía Chagásica/inmunología , Cardiomiopatía Chagásica/prevención & control , Enfermedad de Chagas/parasitología , Chlorocebus aethiops , Fibrosis/inmunología , Fibrosis/prevención & control , Galactósidos/inmunología , Galectina 3/metabolismo , Humanos , Ratones , Ratones Noqueados , Miocardio/inmunología , Parasitemia , Receptores de Superficie Celular/inmunología , Receptores Toll-Like/inmunología , Células VeroRESUMEN
Notch signaling guides vascular development and function by regulating diverse endothelial cell behaviors, including migration, proliferation, vascular density, endothelial junctions, and polarization in response to flow. Notch proteins form transcriptional activation complexes that regulate endothelial gene expression, but few of the downstream effectors that enable these phenotypic changes have been characterized in endothelial cells, limiting our understanding of vascular Notch activities. Using an unbiased screen of translated mRNA rapidly regulated by Notch signaling, we identified novel in vivo targets of Notch signaling in neonatal mouse brain endothelium, including UNC5B, a member of the netrin family of angiogenic-regulatory receptors. Endothelial Notch signaling rapidly upregulates UNC5B in multiple endothelial cell types. Loss or gain of UNC5B recapitulated specific Notch-regulated phenotypes. UNC5B expression inhibited endothelial migration and proliferation and was required for stabilization of endothelial junctions in response to shear stress. Loss of UNC5B partially or wholly blocked the ability of Notch activation to regulate these endothelial cell behaviors. In the developing mouse retina, endothelial-specific loss of UNC5B led to excessive vascularization, including increased vascular outgrowth, density, and branchpoint count. These data indicate that Notch signaling upregulates UNC5B as an effector protein to control specific endothelial cell behaviors and inhibit angiogenic growth.
Asunto(s)
Movimiento Celular , Proliferación Celular , Células Endoteliales , Receptores de Netrina , Receptores Notch , Retina , Transducción de Señal , Animales , Receptores de Netrina/metabolismo , Receptores Notch/metabolismo , Ratones , Células Endoteliales/metabolismo , Retina/metabolismo , Humanos , Vasos Retinianos/metabolismo , Neovascularización FisiológicaRESUMEN
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 â¼70% 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.
Asunto(s)
Cardiomiopatía Chagásica/metabolismo , Cardiomiopatía Chagásica/patología , Células Mieloides/metabolismo , Animales , Arginasa/metabolismo , Arginina/sangre , Antígeno CD11b/biosíntesis , Separación Celular , Cardiomiopatía Chagásica/inmunología , Citometría de Flujo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células Mieloides/inmunología , Óxido Nítrico Sintasa de Tipo II/metabolismo , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trypanosoma cruzi/inmunologíaRESUMEN
Vessel formation and differentiation to a proper hierarchical vasculature requires a coordinated effort from endothelial and mural cells. Over the last decade Notch was identified as a key player in this process by promoting vascular arterialization and modulating endothelial tip-stalk phenotypes. Recent work has identified that Notch fine-tunes the diverse endothelial phenotypes through regulation of canonical cell-cycle and metabolism regulators, such as ERK and Myc. During arterialization, Notch signaling inhibits the cell-cycle and metabolism of endothelial cells which coincides with the acquisition of arterial identity. During angiogenesis, the same molecular machinery prevents the hypermitogenic arrest and excessive sprouting of vessels. Notch also signals in pericytes and smooth muscle cells promoting vascular coverage and maturation. Here, we will review the latest findings on how Notch signals regulate the differentiation and interactions among vascular cells during organ development and homeostasis.
Asunto(s)
Células Endoteliales , Receptores Notch , Células Endoteliales/metabolismo , Receptores Notch/metabolismo , Comunicación Celular , Transducción de Señal/fisiología , Diferenciación Celular , Neovascularización Fisiológica/fisiologíaRESUMEN
Previously considered passive support cells, mural cells-pericytes and vascular smooth muscle cells-have started to garner more attention in disease research, as more subclassifications, based on morphology, gene expression, and function, have been discovered. Central nervous system (CNS) arteriovenous malformations (AVMs) represent a neurovascular disorder in which mural cells have been shown to be affected, both in animal models and in human patients. To study consequences to mural cells in the context of AVMs, various animal models have been developed to mimic and predict human AVM pathologies. A key takeaway from recently published work is that AVMs and mural cells are heterogeneous in their molecular, cellular, and functional characteristics. In this review, we summarize the observed perturbations to mural cells in human CNS AVM samples and CNS AVM animal models, and we discuss various potential mechanisms relating mural cell pathologies to AVMs.
RESUMEN
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.
Asunto(s)
Vasos Coronarios , Endocardio , Humanos , Neovascularización Patológica , Corazón , MorfogénesisRESUMEN
Hematopoietic stem cell (HSC) generation in the aorta-gonad-mesonephros region requires HSC specification signals from the surrounding microenvironment. In zebrafish, PDGF-B/PDGFRß 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/PDGFRß is involved. Here, we show that PDGFRß is expressed in distinct perivascular stromal cell layers surrounding the mid-gestation dorsal aorta, and its deletion impairs hematopoiesis. We demonstrate that PDGFRß+ cells play a dual role in murine hematopoiesis. They act in the aortic niche to support HSPCs, and in addition, PDGFRß+ embryonic precursors give rise to a subset of HSPCs that persist into adulthood. These findings provide crucial information for the controlled production of HSPCs in vitro.
Asunto(s)
Mesonefro , Pez Cebra , Animales , Hematopoyesis , Células Madre Hematopoyéticas , Ratones , Receptor beta de Factor de Crecimiento Derivado de Plaquetas , Células del EstromaRESUMEN
Quiescent neural stem cells (NSCs) in the adult mouse ventricular-subventricular zone (V-SVZ) undergo activation to generate neurons and some glia. Here we show that platelet-derived growth factor receptor beta (PDGFRß) is expressed by adult V-SVZ NSCs that generate olfactory bulb interneurons and glia. Selective deletion of PDGFRß in adult V-SVZ NSCs leads to their release from quiescence, uncovering gliogenic domains for different glial cell types. These domains are also recruited upon injury. We identify an intraventricular oligodendrocyte progenitor derived from NSCs inside the brain ventricles that contacts supraependymal axons. Together, our findings reveal that the adult V-SVZ contains spatial domains for gliogenesis, in addition to those for neurogenesis. These gliogenic NSC domains tend to be quiescent under homeostasis and may contribute to brain plasticity.
Asunto(s)
Células Madre Adultas/fisiología , Ventrículos Cerebrales/fisiología , Ventrículos Laterales/fisiología , Células-Madre Neurales/fisiología , Neuroglía/fisiología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Astrocitos/citología , Astrocitos/fisiología , Axones/fisiología , Diferenciación Celular , División Celular , Ventrículos Cerebrales/citología , Epéndimo/citología , Epéndimo/fisiología , Femenino , Perfilación de la Expresión Génica , Homeostasis , Ventrículos Laterales/citología , Masculino , Ratones , Neurogénesis , Bulbo Olfatorio/citología , Bulbo Olfatorio/fisiología , Oligodendroglía/citología , Oligodendroglía/fisiología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genéticaRESUMEN
Arteriovenous malformations (AVMs) are high-flow lesions directly connecting arteries and veins. In the brain, AVM rupture can cause seizures, stroke, and death. Patients with AVMs exhibit reduced coverage of the vessels by pericytes, the mural cells of microvascular capillaries; however, the mechanism underlying this pericyte reduction and its association with AVM pathogenesis remains unknown. Notch signaling has been proposed to regulate critical pericyte functions. We hypothesized that Notch signaling in pericytes is crucial to maintain pericyte homeostasis and prevent AVM formation. We inhibited Notch signaling specifically in perivascular cells and analyzed the vasculature of these mice. The retinal vessels of mice with deficient perivascular Notch signaling developed severe AVMs, together with a significant reduction in pericytes and vascular smooth muscle cells (vSMC) in the arteries, while vSMCs were increased in the veins. Vascular malformations and pericyte loss were also observed in the forebrain of embryonic mice deficient for perivascular Notch signaling. Moreover, the loss of Notch signaling in pericytes downregulated Pdgfrb levels and increased pericyte apoptosis, pointing to a critical role for Notch in pericyte survival. Overall, our findings reveal a mechanism of AVM formation and highlight the Notch signaling pathway as an essential mediator in this process.
Asunto(s)
Malformaciones Arteriovenosas/patología , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/fisiología , Neovascularización Patológica/patología , Pericitos/patología , Receptores Notch/fisiología , Retina/patología , Animales , Malformaciones Arteriovenosas/etiología , Malformaciones Arteriovenosas/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Neovascularización Patológica/metabolismo , Pericitos/metabolismo , Retina/metabolismoRESUMEN
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.
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Enfermedad de Chagas/inmunología , Enfermedad de Chagas/patología , Linfocitos T/inmunología , Trypanosoma cruzi/inmunología , Animales , Autoinmunidad/inmunología , Enfermedad de Chagas/parasitología , Epítopos/inmunología , Humanos , Imitación Molecular/inmunologíaRESUMEN
Pericytes regulate vessel stability and pericyte dysfunction contributes to retinopathies, stroke, and cancer. Here we define Notch as a key regulator of pericyte function during angiogenesis. In Notch1(+/-); Notch3(-/-) mice, combined deficiency of Notch1 and Notch3 altered pericyte interaction with the endothelium and reduced pericyte coverage of the retinal vasculature. Notch1 and Notch3 were shown to cooperate to promote proper vascular basement membrane formation and contribute to endothelial cell quiescence. Accordingly, loss of pericyte function due to Notch deficiency exacerbates endothelial cell activation caused by Notch1 haploinsufficiency. Mice mutant for Notch1 and Notch3 develop arteriovenous malformations and display hallmarks of the ischemic stroke disease CADASIL. Thus, Notch deficiency compromises pericyte function and contributes to vascular pathologies.
Asunto(s)
Malformaciones Arteriovenosas/genética , CADASIL/genética , Pericitos/metabolismo , Receptor Notch1/genética , Receptores Notch/genética , Animales , Malformaciones Arteriovenosas/metabolismo , Western Blotting , CADASIL/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales/ultraestructura , Expresión Génica , Células HEK293 , Humanos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica de Transmisión , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Pericitos/patología , Pericitos/ultraestructura , Receptor Notch1/deficiencia , Receptor Notch3 , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores Notch/deficiencia , Vasos Retinianos/metabolismo , Vasos Retinianos/patología , Vasos Retinianos/fisiopatología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
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.
Asunto(s)
Enfermedad de Chagas/inmunología , Ciclooxigenasa 2/inmunología , Dinoprostona/inmunología , Miocarditis/inmunología , Subtipo EP2 de Receptores de Prostaglandina E/inmunología , Trypanosoma cruzi/fisiología , Animales , Enfermedad de Chagas/complicaciones , Enfermedad de Chagas/enzimología , Enfermedad de Chagas/genética , Ciclooxigenasa 2/genética , Citocinas/genética , Citocinas/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Miocarditis/etiología , Miocarditis/genética , Miocardio/enzimología , Miocardio/inmunología , Subtipo EP2 de Receptores de Prostaglandina E/genética , Trypanosoma cruzi/inmunologíaRESUMEN
UNLABELLED: 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.