RESUMEN
Chronic obstructive pulmonary disease (COPD) is marked by airway inflammation and airspace enlargement (emphysema) leading to airflow obstruction and eventual respiratory failure. Microvasculature dysfunction is associated with COPD/emphysema. However, it is not known if abnormal endothelium drives COPD/emphysema pathology and/or if correcting endothelial dysfunction has therapeutic potential. Here, we show the centrality of endothelial cells to the pathogenesis of COPD/emphysema in human tissue and using an elastase-induced murine model of emphysema. Airspace disease showed significant endothelial cell loss, and transcriptional profiling suggested an apoptotic, angiogenic, and inflammatory state. This alveolar destruction was rescued by intravenous delivery of healthy lung endothelial cells. Leucine-rich α-2-glycoprotein-1 (LRG1) was a driver of emphysema, and deletion of Lrg1 from endothelial cells rescued vascular rarefaction and alveolar regression. Hence, targeting endothelial cell biology through regenerative methods and/or inhibition of the LRG1 pathway may represent strategies of immense potential for the treatment of COPD/emphysema.
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Células Endoteliales/patología , Pulmón/patología , Enfisema Pulmonar/patología , Administración Intravenosa , Animales , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/trasplante , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Glicoproteínas/metabolismo , Humanos , Pulmón/irrigación sanguínea , Pulmón/fisiopatología , Ratones Endogámicos C57BL , Neovascularización Fisiológica , Elastasa Pancreática/metabolismo , Fenotipo , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/patología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Enfisema Pulmonar/genética , Enfisema Pulmonar/fisiopatología , Índice de Severidad de la Enfermedad , Fumar , Transcriptoma/genéticaRESUMEN
Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease associated with cigarette smoking. Alterations in local lung and systemic iron regulation are associated with disease progression and pathogenesis. Hepcidin, an iron regulatory peptide hormone, is altered in subjects with COPD; however, the molecular role of hepcidin in COPD pathogenesis remains to be determined. In this study, using a murine model of smoke-induced COPD, we demonstrate that lung and circulating hepcidin levels are inhibited by cigarette smoke. We show that cigarette smoke exposure increases erythropoietin and bone marrow-derived erythroferrone and leads to expanded but inefficient erythropoiesis in murine bone marrow and an increase in ferroportin on alveolar macrophages (AMs). AMs from smokers and subjects with COPD display increased expression of ferroportin as well as hepcidin. Notably, murine AMs exposed to smoke fail to increase hepcidin in response to Gram-negative or Gram-positive infection. Loss of hepcidin in vivo results in blunted functional responses of AMs and exaggerated responses to Streptococcus pneumoniae infection.
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Hepcidinas/metabolismo , Macrófagos Alveolares/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Fumar/metabolismo , Animales , Médula Ósea/metabolismo , Proteínas de Transporte de Catión/metabolismo , Fumar Cigarrillos/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Eritropoyetina/metabolismo , Humanos , Hierro/metabolismo , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Péptidos/metabolismo , HumoRESUMEN
Erythropoietin (EPO) provides the major survival signal to maturing erythroid precursors (EPs) and is essential for terminal erythropoiesis. Nonetheless, progenitor cells can irreversibly commit to an erythroid fate well before EPO acts, risking inefficiency if these progenitors are unneeded to maintain red blood cell (RBC) counts. We identified a new modular organization of erythropoiesis and, for the first time, demonstrate that the pre-EPO module is coupled to late EPO-dependent erythropoiesis by megakaryocyte (Mk) signals. Disrupting megakaryocytic transforming growth factor ß1 (Tgfb1) disorganized hematopoiesis by expanding the pre-EPO pool of progenitor cells and consequently triggering significant apoptosis of EPO-dependent EPs. Similarly, pharmacologic blockade of TGFß signaling in normal mice boosted the pre-EPO module, leading to apoptosis of EPO-sensitive EPs. Subsequent treatment with low-dose EPO triggered robust RBC production in both models. This work reveals modular regulation of erythropoiesis and offers a new strategy for overcoming chronic anemias.
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Células Precursoras Eritroides/citología , Eritropoyesis/fisiología , Megacariocitos/citología , Factor de Crecimiento Transformador beta1/fisiología , Animales , Apoptosis/efectos de los fármacos , Médula Ósea/patología , Células Precursoras Eritroides/metabolismo , Eritropoyetina/farmacología , Técnicas de Inactivación de Genes , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Inmunofenotipificación , Células Progenitoras de Megacariocitos y Eritrocitos/citología , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Megacariocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Quimera por Radiación , Proteínas Recombinantes/farmacología , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/farmacologíaAsunto(s)
Encéfalo/metabolismo , Janus Quinasa 2/metabolismo , Leptina/metabolismo , Inhibidores de Proteínas Quinasas/efectos adversos , Pirazoles/efectos adversos , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Humanos , Nitrilos , Pirimidinas , Aumento de Peso/efectos de los fármacosRESUMEN
The actions of BDNF (Brain-derived Neurotrophic Factor) in regulating neuronal development and modulating synaptic activity have been extensively studied and well established. Equally important roles for this growth factor have been uncovered in the cardiovascular system, through the examination of gene targeted animals to define critical actions in development, and to the unexpected roles of BDNF in modulating the response of the heart and vasculature to injury. Here we review the compartmentally distinct realm of cardiac myocytes, vascular smooth muscle cells, endothelial cells, and hematopoietic cells, focusing upon the actions of BDNF to modulate contractility, migration, neoangiogenesis, apoptosis and survival. These studies indicate that BDNF is an important growth factor which directs the response of the cardiovascular system to acute and chronic injury.
RESUMEN
Recent studies have identified a specialized subset of CD31hiendomucinhi (CD31hiEMCNhi) vascular endothelium that positively regulates bone formation. However, it remains unclear how CD31hiEMCNhi endothelium levels are coupled to anabolic bone formation. Mice with an osteoblast-specific deletion of Shn3, which have markedly elevated bone formation, demonstrated an increase in CD31hiEMCNhi endothelium. Transcriptomic analysis identified SLIT3 as an osteoblast-derived, SHN3-regulated proangiogenic factor. Genetic deletion of Slit3 reduced skeletal CD31hiEMCNhi endothelium, resulted in low bone mass because of impaired bone formation and partially reversed the high bone mass phenotype of Shn3-/- mice. This coupling between osteoblasts and CD31hiEMCNhi endothelium is essential for bone healing, as shown by defective fracture repair in SLIT3-mutant mice and enhanced fracture repair in SHN3-mutant mice. Finally, administration of recombinant SLIT3 both enhanced bone fracture healing and counteracted bone loss in a mouse model of postmenopausal osteoporosis. Thus, drugs that target the SLIT3 pathway may represent a new approach for vascular-targeted osteoanabolic therapy to treat bone loss.
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Resorción Ósea/patología , Huesos/patología , Endotelio/patología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Resorción Ósea/diagnóstico por imagen , Huesos/diagnóstico por imagen , Huesos/efectos de los fármacos , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Endotelio/efectos de los fármacos , Curación de Fractura/efectos de los fármacos , Humanos , Proteínas de la Membrana/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neovascularización Fisiológica/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/patología , Osteogénesis/efectos de los fármacos , Osteoporosis Posmenopáusica/tratamiento farmacológico , Osteoporosis Posmenopáusica/patología , Ovariectomía , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Sialoglicoproteínas/metabolismo , Proteínas RoundaboutRESUMEN
Purpose: Epigenetic silencing of tumor suppressor genes (TSG) is an acquired abnormality observed in cancer and is prototypically linked to DNA methylation. We postulated that pretreatment (priming) with 5-azacitidine would increase the efficacy of chemotherapy by reactivating TSGs. This study was conducted to identify a tolerable dose of 5-azacitidine prior to EOX (epirubicin, oxaliplatin, capecitabine) neoadjuvant chemotherapy in patients with locally advanced esophageal/gastric adenocarcinoma (EGC).Experimental Design: Eligible patients had untreated, locally advanced, resectable EGC, ECOG 0-2, and adequate organ function. 5-Azacitidine (V, 75 mg/m2) was given subcutaneously for 3 (dose level, DL 1) or 5 (DL 2) days prior to each 21-day cycle of EOX (E, 50 mg/m2; O, 130 mg/m2; X, 625 mg/m2 twice daily for 21 days). Standard 3+3 methodology guided V dose escalation. DNA methylation at control and biomarker regions was measured by digital droplet, bisulfite qPCR in tumor samples collected at baseline and at resection.Results: All subjects underwent complete resection of residual tumor (R0). Three of the 12 patients (25%) achieved a surgical complete response and 5 had partial responses. The overall response rate was 67%. The most common toxicities were gastrointestinal and hematologic. Hypomethylation of biomarker genes was observed at all dose levels and trended with therapeutic response.Conclusions: Neoadjuvant VEOX was well-tolerated with significant clinical and epigenetic responses, with preliminary evidence that priming with V prior to chemotherapy may augment chemotherapy efficacy. The recommended phase II trial schedule is 5-azacitidine 75 mg/m2 for 5 days followed by EOX chemotherapy every 21 days. Clin Cancer Res; 23(11); 2673-80. ©2016 AACR.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Azacitidina/administración & dosificación , Metilación de ADN/genética , Epigénesis Genética/genética , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Gástricas/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Cisplatino/administración & dosificación , Relación Dosis-Respuesta a Droga , Epirrubicina/administración & dosificación , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Femenino , Fluorouracilo/administración & dosificación , Humanos , Masculino , Persona de Mediana Edad , Terapia Neoadyuvante , Compuestos Organoplatinos/administración & dosificación , Oxaliplatino , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Proteínas Supresoras de Tumor/efectos de los fármacos , Proteínas Supresoras de Tumor/genéticaRESUMEN
Oxygen status and tissue dimensionality are critical determinants of tumor angiogenesis, a hallmark of cancer and an enduring target for therapeutic intervention. However, it is unclear how these microenvironmental conditions interact to promote neovascularization, due in part to a lack of comprehensive, unbiased data sets describing tumor cell gene expression as a function of oxygen levels within three-dimensional (3D) culture. Here, we utilized alginate-based, oxygen-controlled 3D tumor models to study the interdependence of culture context and the hypoxia response. Microarray gene expression analysis of tumor cells cultured in 2D versus 3D under ambient or hypoxic conditions revealed striking interdependence between culture dimensionality and hypoxia response, which was mediated in part by pro-inflammatory signaling pathways. In particular, interleukin-8 (IL-8) emerged as a major player in the microenvironmental regulation of the hypoxia program. Notably, this interaction between dimensionality and oxygen status via IL-8 increased angiogenic sprouting in a 3D endothelial invasion assay. Taken together, our data suggest that pro-inflammatory pathways are critical regulators of tumor hypoxia response within 3D environments that ultimately impact tumor angiogenesis, potentially providing important therapeutic targets. Furthermore, these results highlight the importance of pathologically relevant tissue culture models to study the complex physical and chemical processes by which the cancer microenvironment mediates new vessel formation.
Asunto(s)
Materiales Biocompatibles/química , Técnicas de Cultivo de Célula , Neoplasias/patología , Neovascularización Patológica , Oxígeno/química , Alginatos/química , Hipoxia de la Célula , Endotelio Vascular/patología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hidrogeles/química , Inflamación/patología , Interleucina-8/metabolismo , Invasividad Neoplásica , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal , Ingeniería de Tejidos/métodos , Células Tumorales CultivadasRESUMEN
Pericyte and vascular smooth muscle cell (SMC) recruitment to the developing vasculature is an important step in blood vessel maturation. Brain-derived neurotrophic factor (BDNF), expressed by endothelial cells, activates the receptor tyrosine kinase TrkB to stabilize the cardiac microvasculature in the perinatal period. However, the effects of the BDNF/TrkB signaling on pericytes/SMCs and the mechanisms downstream of TrkB that promote vessel maturation are unknown. To confirm the involvement of TrkB in vessel maturation, we evaluated TrkB deficient (trkb (-/-)) embryos and observed severe cardiac vascular abnormalities leading to lethality in late gestation to early prenatal life. Ultrastructural analysis demonstrates that trkb(-/-) embryos exhibit defects in endothelial cell integrity and perivascular edema. As TrkB is selectively expressed by pericytes and SMCs in the developing cardiac vasculature, we generated mice deficient in TrkB in these cells. Mice with TrkB deficiency in perivascular cells exhibit reduced pericyte/SMC coverage of the cardiac microvasculature, abnormal endothelial cell ultrastructure, and increased vascular permeability. To dissect biological actions and the signaling pathways downstream of TrkB in pericytes/SMCs, human umbilical SMCs were treated with BDNF. This induced membranous protrusions and cell migration, events dependent on myosin light chain phosphorylation. Moreover, inhibition of Rho GTPase and the Rho-associated protein kinase (ROCK) prevented membrane protrusion and myosin light chain phosphorylation in response to BDNF. These results suggest an important role for BDNF in regulating migration of TrkB-expressing pericytes/SMCs to promote cardiac blood vessel ensheathment and functional integrity during development.
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Vasos Coronarios/enzimología , Miocardio/enzimología , Miocitos del Músculo Liso/enzimología , Pericitos/enzimología , Proteínas Quinasas/metabolismo , Transducción de Señal/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Permeabilidad Capilar/fisiología , Vasos Coronarios/citología , Vasos Coronarios/embriología , Cardiopatías Congénitas/embriología , Cardiopatías Congénitas/enzimología , Cardiopatías Congénitas/genética , Humanos , Glicoproteínas de Membrana , Ratones , Ratones Mutantes , Pericitos/citología , Proteínas Quinasas/genética , Proteínas Tirosina Quinasas , Receptor trkB , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
Brain-derived neurotrophic factor (BDNF) enhances periodontal tissue regeneration. Tissue regeneration is characterized by inflammation that directs the quality of tissue repair. In this study, we investigated the anti-apoptotic effect of BDNF against the toxicity of tumor necrosis factor α (TNFα), which is known for its pro-apoptotic action in human microvascular endothelial cells (HMVECs). We demonstrate that BDNF attenuates TNFα-increased Annexin V-positive cells, lactic dehydrogenase (LDH) release, and intercellular adhesion molecule 1 (ICAM-1) mRNA and cleaved caspase-3 expression. In addition, biochemical analyses indicate that TNFα increases phosphatase and tensin homolog (PTEN) expression; however, it decreases phosphorylated PTEN. BDNF did not affect PTEN expression, but it did increase the phosphorylation of PTEN. BDNF-induced Akt phosphorylation was inhibited by TNFα. Terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay showed that the PTEN inhibitor bpV(pic) rescues HMVECs from TNFα-induced apoptosis. In conclusion, BDNF protects HMVECs from toxicity of TNFα through the regulation of the PTEN/Akt pathway.
Asunto(s)
Apoptosis/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/farmacología , Citoprotección , Células Endoteliales/efectos de los fármacos , Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Anexina A5/metabolismo , Caspasa 3/biosíntesis , Caspasa 3/metabolismo , Línea Celular , Humanos , Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/genética , L-Lactato Deshidrogenasa/metabolismo , Compuestos Organometálicos/farmacología , Fosfohidrolasa PTEN/antagonistas & inhibidores , Fosfohidrolasa PTEN/biosíntesis , Fosforilación , ARN Mensajero/biosíntesis , Regeneración/fisiología , Transducción de SeñalRESUMEN
This protocol describes how to form a 3D cell culture with explicit, endothelialized microvessels. The approach leads to fully enclosed, perfusable vessels in a bioremodelable hydrogel (type I collagen). The protocol uses microfabrication to enable user-defined geometries of the vascular network and microfluidic perfusion to control mass transfer and hemodynamic forces. These microvascular networks (µVNs) allow for multiweek cultures of endothelial cells or cocultures with parenchymal or tissue cells in the extra-lumen space. The platform enables real-time fluorescence imaging of living engineered tissues, in situ confocal fluorescence of fixed cultures and transmission electron microscopy (TEM) imaging of histological sections. This protocol enables studies of basic vascular and blood biology, provides a model for diseases such as tumor angiogenesis or thrombosis and serves as a starting point for constructing prevascularized tissues for regenerative medicine. After one-time microfabrication steps, the system can be assembled in less than 1 d and experiments can run for weeks.
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Microvasos , Ingeniería de Tejidos/métodos , Técnicas de Cultivo de Célula , Células Cultivadas , Técnicas de Cocultivo , Colágeno Tipo I/química , Células Endoteliales , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Microscopía Electrónica de Transmisión , Microtecnología , Neovascularización Fisiológica , Imagen Óptica , Ingeniería de Tejidos/instrumentaciónRESUMEN
Myelosuppression is a life-threatening complication of antineoplastic therapy, but treatment is restricted to a few cytokines with unilineage hematopoietic activity. Although hematopoietic stem cells (HSCs) are predominantly quiescent during homeostasis, they are rapidly recruited into cell cycle by stresses, including myelosuppressive chemotherapy. Factors that induce HSCs to proliferate during stress have been characterized, but it is not known how HSC quiescence is then reestablished. In this study, we show that TGFß signaling is transiently activated in hematopoietic stem and progenitor cells (HSPCs) during hematopoietic regeneration. Blockade of TGFß signaling after chemotherapy accelerates hematopoietic reconstitution and delays the return of cycling HSCs to quiescence. In contrast, TGFß blockade during homeostasis fails to induce cycling of HSPCs. We identified the cyclin-dependent kinase inhibitor Cdkn1c (p57) as a key downstream mediator of TGFß during regeneration because the recovery of chimeric mice, incapable of expressing p57 in HSPCs, phenocopies blockade of TGFß signaling after chemotherapy. This study demonstrates that context-dependent activation of TGFß signaling is central to an unrecognized counterregulatory mechanism that promotes homeostasis once hematopoiesis has sufficiently recovered from myelosuppressive chemotherapy. These results open the door to new, potentially superior, approaches to promote multilineage hematopoietic recovery by blocking the TGFß signaling that dampens regeneration.
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Antineoplásicos/toxicidad , Médula Ósea/efectos de los fármacos , Hematopoyesis/efectos de los fármacos , Homeostasis , Factor de Crecimiento Transformador beta/fisiología , Animales , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/análisis , Células Madre Hematopoyéticas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Factor de Crecimiento Transformador beta/antagonistas & inhibidoresRESUMEN
Treatment of acute cardiac ischemia focuses on reestablishment of blood flow in coronary arteries. However, impaired microvascular perfusion damages peri-infarct tissue, despite arterial patency. Identification of cytokines that induce microvascular dysfunction would provide new targets to limit microvascular damage. Pro-nerve growth factor (NGF), the precursor of NGF, is a well characterized cytokine in the brain induced by injury. ProNGF activates p75 neurotrophin receptor (p75(NTR)) and sortilin receptors to mediate proapoptotic responses. We describe induction of proNGF by cardiomyocytes, and p75(NTR) in human arterioles after fatal myocardial infarction, but not with unrelated pathologies. After mouse cardiac ischemia-reperfusion (I-R) injury, rapid up-regulation of proNGF by cardiomyocytes and p75(NTR) by microvascular pericytes is observed. To identify proNGF actions, we generated a mouse expressing a mutant Ngf allele with impaired processing of proNGF to mature NGF. The proNGF-expressing mouse exhibits cardiac microvascular endothelial activation, a decrease in pericyte process length, and increased vascular permeability, leading to lethal cardiomyopathy in adulthood. Deletion of p75(NTR) in proNGF-expressing mice rescues the phenotype, confirming the importance of p75(NTR)-expressing pericytes in the development of microvascular injury. Furthermore, deficiency in p75(NTR) limits infarct size after I-R. These studies identify novel, nonneuronal actions for proNGF and suggest that proNGF represents a new target to limit microvascular dysfunction.
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Encéfalo/metabolismo , Microvasos/patología , Infarto del Miocardio/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Pericitos/metabolismo , Precursores de Proteínas/metabolismo , Daño por Reperfusión/metabolismo , Animales , Western Blotting , Cartilla de ADN/genética , Ecocardiografía , Ensayo de Inmunoadsorción Enzimática , Técnicas de Sustitución del Gen , Humanos , Inmunohistoquímica , Ratones , Microscopía Electrónica , Microscopía Fluorescente , Microvasos/metabolismo , Mutagénesis Sitio-Dirigida , Infarto del Miocardio/patología , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/metabolismo , Receptores de Factor de Crecimiento Nervioso/deficiencia , Receptores de Factor de Crecimiento Nervioso/metabolismo , Daño por Reperfusión/patologíaRESUMEN
Microvascular networks support metabolic activity and define microenvironmental conditions within tissues in health and pathology. Recapitulation of functional microvascular structures in vitro could provide a platform for the study of complex vascular phenomena, including angiogenesis and thrombosis. We have engineered living microvascular networks in three-dimensional tissue scaffolds and demonstrated their biofunctionality in vitro. We describe the lithographic technique used to form endothelialized microfluidic vessels within a native collagen matrix; we characterize the morphology, mass transfer processes, and long-term stability of the endothelium; we elucidate the angiogenic activities of the endothelia and differential interactions with perivascular cells seeded in the collagen bulk; and we demonstrate the nonthrombotic nature of the vascular endothelium and its transition to a prothrombotic state during an inflammatory response. The success of these microvascular networks in recapitulating these phenomena points to the broad potential of this platform for the study of cardiovascular biology and pathophysiology.
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Microvasos/crecimiento & desarrollo , Neovascularización Patológica , Trombosis/fisiopatología , Células Cultivadas , Colágeno Tipo I/metabolismo , Humanos , Microvasos/metabolismo , Microvasos/fisiopatologíaRESUMEN
Recent studies indicate that, in addition to its neuropoietic actions, brain derived neurotrophic factor (BDNF) promotes endothelial cell survival and induces neoangiogenesis in ischemic tissues. Unlike many vascular growth factors that act on many vascular beds, BDNF activity is relatively restricted to central arteries, vessels of cardiac and skeletal muscle, and skin. Studies of newly described biologic mediators that act on large-vessel and microvascular beds in these organs will help us to better understand organ-specific vascular development, as well as to develop novel therapeutic strategies to improve the condition of patients with cardiac and peripheral vascular disease. In this review, we summarize dual proangiogenic actions of BDNF, which, through local activation of TrkB receptor, expressed on a subpopulation of endothelial cells and, in addition, by recruitment of bone marrow-derived cells, contribute to neoangiogenesis.
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Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/uso terapéutico , Isquemia/terapia , Neovascularización Patológica/etiología , Inductores de la Angiogénesis/metabolismo , Células de la Médula Ósea , Células Endoteliales/metabolismo , Humanos , Isquemia/etiología , Neovascularización Patológica/terapia , Receptor trkB , RegeneraciónRESUMEN
PURPOSE: Tumor stromal microenvironment promotes neoplastic growth and angiogenesis. We have previously shown that recruitment of marrow-derived vascular endothelial growth factor receptor-1(+) (VEGFR-1(+)) proangiogenic hematopoietic progenitors contributes instructively and structurally to neoangiogenesis in mouse models. Here, we investigated whether stromal incorporation of CD68(+) hemangiogenic cells and alpha-smooth muscle actin(+) (alpha-SMA(+)) stromal cells correlates with neoangiogenesis and progression in human non-Hodgkin's lymphoma subtypes. EXPERIMENTAL DESIGN: Spatial localizations of vascular and stromal cells expressing CD34, VEGFR-1, alpha-SMA, and CD68 were examined by immunohistochemistry in 42 cases of non-Hodgkin's lymphoma, including diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), and compared with benign follicular hyperplasia. RESULTS: Compared with indolent lymphomas, there was a profound increase in recruitment of CD68(+) cells and VEGFR-1(+) neovessels in aggressive subtypes (including those transformed from indolent subtypes), where CD68(+) cells were localized to the perivascular region of neovessels as well as the stromal compartment. The perivascular CD68(+) cells expressed VEGFR-1 and VEGF-A. In contrast, there was a diffuse increase in alpha-SMA incorporation throughout the stromal compartment of indolent subtype of CLL/SLL compared with the scant perivascular pattern in aggressive subtypes. Overall, there was no correlation between CD34(+) microvessel density and lymphoma histologic subtype. CONCLUSIONS: Heightened stromal hemangiogenesis as marked by infiltration of proangiogenic VEGFR-1(+)CD68(+)VEGF-A(+) cells and their paracrine cross-talk with neovasculature appears to be a distinct feature of aggressive lymphoma, providing novel targets for antiangiogenic therapy, whereas alpha-SMA(+) stromal vascular network may be differentially targeted in CLL/SLL.
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Linfoma no Hodgkin/patología , Neovascularización Patológica/patología , Células del Estroma/patología , Actinas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/metabolismo , Antígenos CD34/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Linfoma Folicular/metabolismo , Linfoma Folicular/patología , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Linfoma no Hodgkin/metabolismo , Masculino , Microcirculación , Persona de Mediana Edad , Músculo Liso/metabolismo , Neovascularización Patológica/metabolismo , Células del Estroma/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The molecular and cellular pathways that support the maintenance and stability of tumor neovessels are not well defined. The efficacy of microtubule-disrupting agents, such as combretastatin A4 phosphate (CA4P), in inducing rapid regression of specific subsets of tumor neovessels has opened up new avenues of research to identify factors that support tumor neoangiogenesis. Herein, we show that CA4P selectively targeted endothelial cells, but not smooth muscle cells, and induced regression of unstable nascent tumor neovessels by rapidly disrupting the molecular engagement of the endothelial cell-specific junctional molecule vascular endothelial-cadherin (VE-cadherin) in vitro and in vivo in mice. CA4P increases endothelial cell permeability, while inhibiting endothelial cell migration and capillary tube formation predominantly through disruption of VE-cadherin/beta-catenin/Akt signaling pathway, thereby leading to rapid vascular collapse and tumor necrosis. Remarkably, stabilization of VE-cadherin signaling in endothelial cells with adenovirus E4 gene or ensheathment with smooth muscle cells confers resistance to CA4P. CA4P synergizes with low and nontoxic doses of neutralizing mAbs to VE-cadherin by blocking assembly of neovessels, thereby inhibiting tumor growth. These data suggest that the microtubule-targeting agent CA4P selectively induces regression of unstable tumor neovessels, in part through disruption of VE-cadherin signaling. Combined treatment with anti-VE-cadherin agents in conjunction with microtubule-disrupting agents provides a novel synergistic strategy to selectively disrupt assembly and induce regression of nascent tumor neovessels, with minimal toxicity and without affecting normal stabilized vasculature.
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Antineoplásicos Fitogénicos/farmacología , Cadherinas/fisiología , Endotelio Vascular/efectos de los fármacos , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Estilbenos/farmacología , Animales , Capilares/crecimiento & desarrollo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Femenino , Humanos , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/metabolismo , beta Catenina/fisiologíaRESUMEN
Brain-derived neurotrophic factor (BDNF) is best characterized for critical roles in neuronal survival, differentiation, and synaptic modulation mediated by the TrkB receptor tyrosine kinase. Developmentally regulated death signaling by BDNF has also been demonstrated via activation of p75NTR. Because recent studies suggest that proNGF, the precursor form of NGF, is more active than mature NGF in inducing apoptosis after binding to p75NTR and a coreceptor, sortilin, we asked whether the precursor of BDNF (proBDNF) is also a proapoptotic ligand in the nervous system. proBDNF is secreted by cultured neurons, and recombinant proBDNF binds to sortilin. In sympathetic neurons coexpressing sortilin and p75NTR, we found that proBDNF is an apoptotic ligand that induces death at subnanomolar concentrations. In contrast, mature BDNF, but not proBDNF, is effective in inducing TrkB phosphorylation. proBDNF effects are dependent on cellular coexpression of both p75NTR and sortilin, because neurons deficient in p75NTR are resistant to proBDNF-induced apoptosis, and competitive antagonists of sortilin block sympathetic neuron death. Moreover, addition of preformed complexes of soluble sortilin and proBDNF failed to induce apoptosis of cells coexpressing both sortilin and p75NTR, suggesting that interaction of proBDNF with both receptors on the cell surface is required to initiate cell death. Together with our past findings, these data suggest that the neurotrophin family is capable of modulating diverse biological processes via differential processing of the proneurotrophins.
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Apoptosis , Factor Neurotrófico Derivado del Encéfalo/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Precursores de Proteínas/fisiología , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Animales , Encéfalo/citología , Línea Celular , Humanos , Ligandos , Glicoproteínas de Membrana/antagonistas & inhibidores , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Fosforilación , Ratas , Ratas Sprague-Dawley , Receptor de Factor de Crecimiento Nervioso/genética , Receptor trkB/metabolismo , Receptores de Factores de Crecimiento/genética , Receptores de Factor de Crecimiento Nervioso , Células de Schwann/citología , Células de Schwann/metabolismo , Ganglio Cervical Superior/citologíaRESUMEN
The neurotrophin brain-derived neurotrophic factor (BDNF) is required for the maintenance of cardiac vessel wall stability during embryonic development through direct angiogenic actions on endothelial cells expressing the tropomysin receptor kinase B (TrkB). However, the role of BDNF and a related neurotrophin ligand, neurotrophin-4 (NT-4), in the regulation of revascularization of the adult tissues is unknown. To study the potential angiogenic capacity of BDNF in mediating the neovascularization of ischemic and non-ischemic adult mouse tissues, we utilized a hindlimb ischemia and a subcutaneous Matrigel model. Recruitment of endothelial cells and promotion of channel formation within the Matrigel plug by BDNF and NT-4 was comparable to that induced by VEGF-A. The introduction of BDNF into non-ischemic ears or ischemic limbs induced neoangiogenesis, with a 2-fold increase in the capillary density. Remarkably, treatment with BDNF progressively increased blood flow in the ischemic limb over 21 days, similar to treatment with VEGF-A. The mechanism by which BDNF enhances capillary formation is mediated in part through local activation of the TrkB receptor and also by recruitment of Sca-1+CD11b+ pro-angiogenic hematopoietic cells. BDNF induces a potent direct chemokinetic action on subsets of marrow-derived Sca-1+ hematopoietic cells co-expressing TrkB. These studies suggest that local regional delivery of BDNF may provide a novel mechanism for inducing neoangiogenesis through both direct actions on local TrkB-expressing endothelial cells in skeletal muscle and recruitment of specific subsets of TrkB+ bone marrow-derived hematopoietic cells to provide peri-endothelial support for the newly formed vessels.
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Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Endoteliales/fisiología , Células Madre Hematopoyéticas/fisiología , Neovascularización Fisiológica , Factores de Crecimiento Nervioso/metabolismo , Receptor trkB/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Animales , Biomarcadores/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Línea Celular , Quimiotaxis , Células Endoteliales/citología , Femenino , Vectores Genéticos , Células Madre Hematopoyéticas/citología , Humanos , Isquemia/metabolismo , Isquemia/patología , Masculino , Ratones , Ratones Endogámicos , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: CD133 (AC133) is a surface antigen that defines a broad population of stem cells, including myogenic and endothelial progenitors. CD133+ cells are rare in adult tissues, and the factors that support their differentiation into mature angiomyogenic cells are not known. These hurdles have hampered the use of CD133+ cells for therapeutic purposes. Because human fetal liver is a rich source of CD133+ cells, we sought to identify the growth factors that promote codifferentiation of these cells into angiogenic and myogenic cells. METHODS AND RESULTS: Human fetal liver CD133+ and CD133- cell subpopulations were cultured with 5'-azacytidine or vascular endothelial growth factor (VEGF165) and/or brain-derived nerve growth factor (BDNF). CD133+ but not CD133- cells from human fetal liver codifferentiated into spindle-shaped cells, as well as flat adherent multinucleated cells capable of spontaneous contractions in culture. The resulting spindle-shaped cells were confirmed to be endothelial cells by immunohistochemistry analysis for von Willebrand factor and by acetylated LDL uptake. Multinucleated cells were characterized as striated muscles by electron microscopy and immunohistochemistry analysis for myosin heavy chain. Presence of VEGF165 and BDNF significantly enhanced angiomyogenesis in vitro. Inoculation of cells derived from CD133+ cells, but not CD133- cells, into the ear pinna of NOD/SCID mice resulted in the formation of cardiomyocytes, as identified by immunostaining with cardiac troponin-T antibody. These cells generated electrical action potentials, detectable by ECG tracing. CONCLUSIONS: CD133 defines a population of human fetal liver cells capable of differentiating into both angiogenic and myogenic cells. Preconditioning of these CD133+ cells with VEGF165 and BDNF enhances the angiomyogenesis. CD133+ fetal liver cells ultimately may be used for therapeutic angiomyogenesis.