RESUMO
Angiocrine signals derived from endothelial cells are an important component of intercellular communication and have a key role in organ growth, regeneration and disease1-4. These signals have been identified and studied in multiple organs, including the liver, pancreas, lung, heart, bone, bone marrow, central nervous system, retina and some cancers1-4. Here we use the developing liver as a model organ to study angiocrine signals5,6, and show that the growth rate of the liver correlates both spatially and temporally with blood perfusion to this organ. By manipulating blood flow through the liver vasculature, we demonstrate that vessel perfusion activates ß1 integrin and vascular endothelial growth factor receptor 3 (VEGFR3). Notably, both ß1 integrin and VEGFR3 are strictly required for normal production of hepatocyte growth factor, survival of hepatocytes and liver growth. Ex vivo perfusion of adult mouse liver and in vitro mechanical stretching of human hepatic endothelial cells illustrate that mechanotransduction alone is sufficient to turn on angiocrine signals. When the endothelial cells are mechanically stretched, angiocrine signals trigger in vitro proliferation and survival of primary human hepatocytes. Our findings uncover a signalling pathway in vascular endothelial cells that translates blood perfusion and mechanotransduction into organ growth and maintenance.
Assuntos
Comunicação Autócrina , Integrina beta1/metabolismo , Fígado/crescimento & desenvolvimento , Fígado/fisiologia , Mecanotransdução Celular/fisiologia , Transdução de Sinais , Animais , Células Cultivadas , Células Endoteliais/fisiologia , Feminino , Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos/citologia , Hepatócitos/fisiologia , Humanos , Fígado/irrigação sanguínea , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) stem cell niche, which provides a vital source of HSC regulatory signals. Radiation and chemotherapy disrupt the HSC niche, including its sinusoidal vessels and perivascular cells, contributing to delayed hematopoietic recovery. Thus, identification of factors that can protect the HSC niche during an injury could offer a significant therapeutic opportunity to improve hematopoietic regeneration. In this study, we identified a critical function for vascular endothelial growth factor-C (VEGF-C), that of maintaining the integrity of the BM perivascular niche and improving BM niche recovery after irradiation-induced injury. Both global and conditional deletion of Vegfc in endothelial or leptin receptor-positive (LepR+) cells led to a disruption of the BM perivascular niche. Furthermore, deletion of Vegfc from the microenvironment delayed hematopoietic recovery after transplantation by decreasing endothelial proliferation and LepR+ cell regeneration. Exogenous administration of VEGF-C via an adenoassociated viral vector improved hematopoietic recovery after irradiation by accelerating endothelial and LepR+ cell regeneration and by increasing the expression of hematopoietic regenerative factors. Our results suggest that preservation of the integrity of the perivascular niche via VEGF-C signaling could be exploited therapeutically to enhance hematopoietic regeneration.
Assuntos
Células da Medula Óssea/metabolismo , Medula Óssea/metabolismo , Células Endoteliais/metabolismo , Nicho de Células-Tronco , Fator C de Crescimento do Endotélio Vascular/genética , Animais , Biomarcadores , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos da radiação , Expressão Gênica , Hematopoese/genética , Hematopoese/efeitos da radiação , Imunofenotipagem , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Ligação Proteica , RNA Mensageiro , Receptores para Leptina/metabolismo , Nicho de Células-Tronco/genética , Nicho de Células-Tronco/efeitos da radiação , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
Vascular endothelial growth factor C (VEGF-C) is a major driver of lymphangiogenesis in embryos and adults. Vegfc gene deletion in mouse embryos results in failure of lymphangiogenesis, fluid accumulation in tissues, and lethality. The VEGF-C receptors VEGFR3 and VEGFR2 are required for embryonic blood vessel formation. The related VEGF is essential for both blood vessel formation and embryonic hematopoiesis, whereas the possible involvement of VEGF-C in hematopoiesis is unknown. Here we unveil a novel hematopoietic function of VEGF-C in fetal erythropoiesis. Deletion of Vegfc in embryonic day 7.5 (E7.5) embryos in the C57BL6 mouse genetic background led to defective fetal erythropoiesis, characterized by anemia and lack of enucleated red blood cells in blood circulation. Macrophages and erythroid cells in the fetal liver (FL) were also decreased after midgestation because of decreased cell proliferation and increased apoptosis. However, the Lin(-)Sca-1(+)c-Kit(+) stem cell compartment in E14.5 FL was not affected by Vegfc deletion. VEGF-C loss did not disrupt the generation of primitive erythroid cells or erythro-myeloid progenitors (EMPs) in the yolk sac, but it decreased the expression of α4-integrin on EMPs and compromised EMP colonization of the FL. The distribution, maturation, and enucleation of primitive erythroblasts were also impaired by Vegfc deletion. In contrast, Vegfc deletion from E10.5 onward did not compromise definitive hematopoiesis in the liver, and Vegfc deletion in adult mice did not cause anemia. These results reveal an unexpected role for VEGF-C, a major lymphangiogenic growth factor, in the transition to FL erythropoiesis.
Assuntos
Eritropoese , Feto/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Anemia/patologia , Animais , Apoptose , Linhagem da Célula , Proliferação de Células , Embrião de Mamíferos/metabolismo , Células Eritroides/metabolismo , Deleção de Genes , Hepatócitos/metabolismo , Integrina alfa4/metabolismo , Fígado/irrigação sanguínea , Fígado/embriologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Tamanho do ÓrgãoRESUMO
In adults, the growth of blood vessels, a process known as angiogenesis, is essential for organ growth and repair. In many disorders including cancer, angiogenesis becomes excessive. The cellular origin of new vascular endothelial cells (ECs) during blood vessel growth in angiogenic situations has remained unknown. Here, we provide evidence for adult vascular endothelial stem cells (VESCs) that reside in the blood vessel wall endothelium. VESCs constitute a small subpopulation within CD117+ (c-kit+) ECs capable of undergoing clonal expansion while other ECs have a very limited proliferative capacity. Isolated VESCs can produce tens of millions of endothelial daughter cells in vitro. A single transplanted c-kit-expressing VESC by the phenotype lin-CD31+CD105+Sca1+CD117+ can generate in vivo functional blood vessels that connect to host circulation. VESCs also have long-term self-renewal capacity, a defining functional property of adult stem cells. To provide functional verification on the role of c-kit in VESCs, we show that a genetic deficit in endothelial c-kit expression markedly decreases total colony-forming VESCs. In vivo, c-kit expression deficit resulted in impaired EC proliferation and angiogenesis and retardation of tumor growth. Isolated VESCs could be used in cell-based therapies for cardiovascular repair to restore tissue vascularization after ischemic events. VESCs also provide a novel cellular target to block pathological angiogenesis and cancer growth.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Células-Tronco/citologia , Animais , Diferenciação Celular , Células Endoteliais/citologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco/metabolismoRESUMO
Herpes zoster remains an important global health issue and mainly occurs in aged and immunocompromised individuals with an early exposure history to Varicella Zoster Virus (VZV). Although the licensed vaccine Shingrix has remarkably high efficacy, undesired reactogenicity and increasing global demand causing vaccine shortage urged the development of improved or novel VZV vaccines. In this study, we developed a novel VZV mRNA vaccine candidate (named as ZOSAL) containing sequence-optimized mRNAs encoding full-length glycoprotein E encapsulated in an ionizable lipid nanoparticle. In mice and rhesus macaques, ZOSAL demonstrated superior immunogenicity and safety in multiple aspects over Shingrix, especially in the induction of strong T-cell immunity. Transcriptomic analysis revealed that both ZOSAL and Shingrix could robustly activate innate immune compartments, especially Type-I IFN signalling and antigen processing/presentation. Multivariate correlation analysis further identified several early factors of innate compartments that can predict the magnitude of T-cell responses, which further increased our understanding of the mode of action of two different VZV vaccine modalities. Collectively, our data demonstrated the superiority of VZV mRNA vaccine over licensed subunit vaccine. The mRNA platform therefore holds prospects for further investigations in next-generation VZV vaccine development.
Assuntos
Vacina contra Herpes Zoster , Herpes Zoster , Animais , Camundongos , Macaca mulatta , Vacinas de mRNA , Herpes Zoster/prevenção & controle , Herpesvirus Humano 3RESUMO
Solid tumors often contain hypoxic and necrotic areas that can be targeted by attenuated Salmonella typhimurium VNP20009 (VNP). We sought to develop a hypoxia- inducible promoter system based on the tumor-specific delivered strain VNP to confine expression of therapeutic gene specifically or selectively within the tumor microenvironment. A hypoxia-inducible promoter - adhE promoter was screened from the hypoxia-regulated endogenous proteins of Salmonella through two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight/time-of-flight MS-based proteomics approaches. The efficiency and specificity of the selected adhE promoter were validated first in both bacteria and animal tumor models. The adhE promoter could specifically drive GFP gene expression under hypoxia, but not under normoxia. Furthermore, luciferase reporter expression controlled by the system was also confined to the tumors. Finally, we investigated the anticancer efficacy of VNP delivering human endostatin controlled by our adhE promoter system in both murine melanoma and Lewis lung carcinoma models. Our results demonstrated that by the dual effects of tumoricidal and anti-angiogenic activities, the recombinant Salmonella strain could generate enhanced antitumor effects compared with those of unarmed VNP treatment or untreated control. The recombinant VNP could retard tumor growth significantly and extend survival of tumor-bearing mice by inducing more apoptosis and more severe necrosis as well as inhibiting blood vessel density within tumors. Therefore, VNP carrying the endostatin gene under our tumor-targeted expression system holds promise for the treatment of solid tumors.
Assuntos
Inibidores da Angiogênese/metabolismo , Terapia Biológica/métodos , Endostatinas/metabolismo , Regiões Promotoras Genéticas , Proteoma/genética , Salmonella typhimurium/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Anaerobiose , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Carcinoma Pulmonar de Lewis/patologia , Carcinoma Pulmonar de Lewis/terapia , Morte Celular , Hipóxia Celular , Linhagem Celular Tumoral , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Endostatinas/genética , Feminino , Estimativa de Kaplan-Meier , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Organismos Geneticamente Modificados , Proteoma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Salmonella typhimurium/genética , Carga Tumoral , Regulação para CimaRESUMO
The recent discovery of lymphatic vessels (LVs) in the dura mater, the outermost layer of meninges around the central nervous system (CNS), has opened a possibility for the development of alternative therapeutics for CNS disorders. The vascular endothelial growth factor C (VEGF-C)/VEGF receptor 3 (VEGFR3) signaling pathway is essential for the development and maintenance of dural LVs. However, its significance in mediating dural lymphatic function in CNS autoimmunity is unclear. We show that inhibition of the VEGF-C/VEGFR3 signaling pathway using a monoclonal VEGFR3-blocking antibody, a soluble VEGF-C/D trap, or deletion of the Vegfr3 gene in adult lymphatic endothelium causes notable regression and functional impairment of dural LVs but has no effect on the development of CNS autoimmunity in mice. During autoimmune neuroinflammation, the dura mater was only minimally affected, and neuroinflammation-induced helper T (TH) cell recruitment, activation, and polarization were significantly less pronounced in the dura mater than in the CNS. In support of this notion, during autoimmune neuroinflammation, blood vascular endothelial cells in the cranial and spinal dura expressed lower levels of cell adhesion molecules and chemokines, and antigen-presenting cells (i.e., macrophages and dendritic cells) had lower expression of chemokines, MHC class II-associated molecules, and costimulatory molecules than their counterparts in the brain and spinal cord, respectively. The significantly weaker TH cell responses in the dura mater may explain why dural LVs do not contribute directly to CNS autoimmunity.
Assuntos
Vasos Linfáticos , Fator C de Crescimento do Endotélio Vascular , Animais , Camundongos , Células Endoteliais/metabolismo , Linfangiogênese , Doenças Neuroinflamatórias , Transdução de Sinais , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/farmacologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The bone marrow contains peripheral nerves that promote haematopoietic regeneration after irradiation or chemotherapy (myeloablation), but little is known about how this is regulated. Here we found that nerve growth factor (NGF) produced by leptin receptor-expressing (LepR+) stromal cells is required to maintain nerve fibres in adult bone marrow. In nerveless bone marrow, steady-state haematopoiesis was normal but haematopoietic and vascular regeneration were impaired after myeloablation. LepR+ cells, and the adipocytes they gave rise to, increased NGF production after myeloablation, promoting nerve sprouting in the bone marrow and haematopoietic and vascular regeneration. Nerves promoted regeneration by activating ß2 and ß3 adrenergic receptor signalling in LepR+ cells, and potentially in adipocytes, increasing their production of multiple haematopoietic and vascular regeneration growth factors. Peripheral nerves and LepR+ cells thus promote bone marrow regeneration through a reciprocal relationship in which LepR+ cells sustain nerves by synthesizing NGF and nerves increase regeneration by promoting the production of growth factors by LepR+ cells.
Assuntos
Medula Óssea , Receptores para Leptina , Medula Óssea/metabolismo , Receptores para Leptina/genética , Receptores para Leptina/metabolismo , Células da Medula Óssea/metabolismo , Fator de Crescimento Neural/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Regeneração NervosaRESUMO
Leukocytes and resident cells in the arterial wall contribute to atherosclerosis, especially at sites of disturbed blood flow. Expression of endothelial Tie1 receptor tyrosine kinase is enhanced at these sites, and attenuation of its expression reduces atherosclerotic burden and decreases inflammation. However, Tie2 tyrosine kinase function in atherosclerosis is unknown. Here we provide genetic evidence from humans and from an atherosclerotic mouse model to show that TIE2 is associated with protection from coronary artery disease. We show that deletion of Tie2, or both Tie2 and Tie1, in the arterial endothelium promotes atherosclerosis by increasing Foxo1 nuclear localization, endothelial adhesion molecule expression and accumulation of immune cells. We also show that Tie2 is expressed in a subset of aortic fibroblasts, and its silencing in these cells increases expression of inflammation-related genes. Our findings indicate that unlike Tie1, the Tie2 receptor functions as the dominant endothelial angiopoietin receptor that protects from atherosclerosis.
RESUMO
The role of Transforming growth factor ß (TGF-ß) as a regulator of blood vessel endothelium is complicated and controversial, and the mechanisms by which TGF-ß is able to induce angiogenesis in vivo are not well understood. Here we show that TGF-ß causes in vivo a massive recruitment of tissue infiltrating hematopoietic cells. Concurrently, TGF-ß induces strong vascular endothelial growth factor (VEGF) production in the recruited hematopoietic cells, resulting in activated angiogenesis and vascular remodeling. TGF-ß also promoted abnormalities of α-smooth muscle actin-expressing pericytes on angiogenic capillaries. TGF-ß-induced angiogenic effect was inhibited by a systemic treatment with VEGF-neutralizing antibodies. When studied in isolated human hematopoietic cells, physiological concentrations of TGF-ß stimulated VEGF mRNA and protein expression in a dose- and time-dependent manner. This induction was p38 and p44/p42 mitogen activated kinase dependent. p38 and p44/p42 activation was also observed in vivo in TGF-ß-treated angiogenic murine tissues. Taken together, our results provide a dual action mechanism by which TGF-ß promotes angiogenesis in vivo via recruitment of paracrine VEGF-expressing hematopoietic effector cells. This mechanism may activate vascular growth and remodeling during inflammatory conditions and tumor growth when TGF-ß activity is upregulated.
Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Células da Medula Óssea/metabolismo , Neovascularização Fisiológica/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Células da Medula Óssea/citologia , Transplante de Medula Óssea , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismoRESUMO
Contribution from diverse tissue-specific stem cell types is required to create the cell populations necessary for the activation of angiogenesis and neovascular growth in cancer. Bone marrow (BM)-derived circulating endothelial progenitors (EPCs) that would differentiate to bona fide endothelial cells (ECs) were previously believed to be necessary for tumor angiogenesis. However, numerous recent studies demonstrate that EPCs are not needed for tumor angiogenesis and indicate EPCs to be artifactual rather than physiological. It is evident that tumor infiltrating hematopoietic cells produced by BM-residing hematopoietic stem cells (HSCs) may contribute to tumor angiogenesis in a paracrine manner by stimulating ECs or by remodeling the extracellular matrix. Therefore, identification of the various hematopoietic cell subpopulations that are critical for tumor angiogenesis and better understanding of their proangiogenic functions and mechanisms of action have potential therapeutic significance. Stem and progenitor cell subsets for also other vascular or perivascular cell types such as pericytes or mesenchymal/stromal cells may provide critical contributions to the growing neovasculature. Furthermore, we hypothesize that the existence of a yet undiscovered-and largely unsearched-tissue-specific adult vascular endothelial stem cell (VESC) would provide completely novel targeted approaches to block pathological angiogenesis and cancer growth. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".
Assuntos
Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Diferenciação Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Comunicação Parácrina , Pericitos/metabolismo , Pericitos/patologiaRESUMO
Aging, obesity, hypertension, and physical inactivity are major risk factors for endothelial dysfunction and cardiovascular disease (CVD). We applied fluorescence-activated cell sorting (FACS), RNA sequencing, and bioinformatic methods to investigate the common effects of CVD risk factors in mouse cardiac endothelial cells (ECs). Aging, obesity, and pressure overload all upregulated pathways related to TGF-ß signaling and mesenchymal gene expression, inflammation, vascular permeability, oxidative stress, collagen synthesis, and cellular senescence, whereas exercise training attenuated most of the same pathways. We identified collagen chaperone Serpinh1 (also called as Hsp47) to be significantly increased by aging and obesity and repressed by exercise training. Mechanistic studies demonstrated that increased SERPINH1 in human ECs induced mesenchymal properties, while its silencing inhibited collagen deposition. Our data demonstrate that CVD risk factors significantly remodel the transcriptomic landscape of cardiac ECs inducing inflammatory, senescence, and mesenchymal features. SERPINH1 was identified as a potential therapeutic target in ECs.
Cardiovascular diseases are the number one cause of death in the western world. Endothelial cells that line the blood vessels of the heart play a central role in the development of these diseases. In addition to helping transport blood, these cells support the normal running of the heart, and help it to grow and regenerate. Over time as the body ages and experiences stress, endothelial cells start to deteriorate. This can cause the cells to undergo senescence and stop dividing, and lay down scar-like tissue via a process called fibrosis. As a result, the blood vessels start to stiffen and become less susceptible to repair. Ageing, obesity, high blood pressure, and inactivity all increase the risk of developing cardiovascular diseases, whereas regular exercise has a protective effect. But it was unclear how these different factors affect endothelial cells. To investigate this, Hemanthakumar et al. compared the gene activity of different sets of mice: old vs young, obese vs lean, heart problems vs healthy, and fit vs sedentary. All these risk factors age, weight, inactivity and heart defects caused the mice's endothelial cells to activate mechanisms that lead to stress, senescence and fibrosis. Whereas exercise training had the opposite effect, and turned off the same genes and pathways. All of the at-risk groups also had high levels of a gene called SerpinH1, which helps produce tissue fiber and collagen. Experiments increasing the levels of SerpinH1 in human endothelial cells grown in the laboratory recreated the effects seen in mice, and switched on markers of stress, senescence and fibrosis. According to the World Health Organization, cardiovascular disease now accounts for 10% of the disease burden worldwide. Revealing the affects it has on gene activity could help identify new targets for drug development, such as SerpinH1. Understanding the molecular effects of exercise on blood vessels could also aid in the design of treatments that mimic exercise. This could help people who are unable to follow training programs to reduce their risk of cardiovascular disease.
Assuntos
Senescência Celular , Células Endoteliais/fisiologia , Proteínas de Choque Térmico HSP47/genética , Coração/fisiopatologia , Mesoderma/fisiologia , Animais , Doenças Cardiovasculares/fisiopatologia , Modelos Animais de Doenças , Feminino , Proteínas de Choque Térmico HSP47/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de RiscoRESUMO
Somatic mutations in APC or CTNNB1 genes lead to aberrant Wnt signaling and colorectal cancer (CRC) initiation and progression via-cateninT cell factor/lymphoid enhancer binding factor TCF/LEF transcription factors. We found that Lef1 was expressed exclusively in Apc-mutant, Wnt ligandindependent tumors, but not in ligand-dependent, serrated tumors. To analyze Lef1 function in tumor development, we conditionally deleted Lef1 in intestinal stem cells of Apcfl/fl mice or broadly from the entire intestinal epithelium of Apcfl/fl or ApcMin/+ mice. Loss of Lef1 markedly increased tumor initiation and tumor cell proliferation, reduced the expression of several Wnt antagonists, and increased Myc proto-oncogene expression and formation of ectopic crypts in Apc-mutant adenomas. Our results uncover a previously unknown negative feedback mechanism in CRC, in which ectopic Lef1 expression suppresses intestinal tumorigenesis by restricting adenoma cell dedifferentiation to a crypt-progenitor phenotype and by reducing the formation of cancer stem cell niches.
RESUMO
Anticancer vaccines have recently received renewed attention for immunotherapy of at least a subset of cancer-types. Such vaccines mostly involve either killed cancer or tumor cells alone, or combinations thereof with specific (co-incubated) innate immune cells. In recent years, the immunogenic characteristics of the dead or dying cancer cells have emerged as decisive factors behind the success of anticancer vaccines. This has amplified the importance of accounting for immunology of cell death while preparing anticancer vaccines. This, in turn, has increased the emphasis on the immune reactions at the site-of-vaccination since the therapeutic efficacy of the killed cancer/tumor cell vaccines is contingent upon the nature and characteristics of these reactions at the site-of-injection. In this article, we present a systematic methodology that exploits the murine ear pinna model to study differential immune cell recruitment by dead/dying cancer cells injected in vivo, thereby modeling the site-of-injection relevant for anticancer vaccines.
Assuntos
Vacinas Anticâncer , Neoplasias , Animais , Morte Celular , Imunoterapia , Camundongos , Neoplasias/terapia , VacinaçãoRESUMO
Soybean seed ferritin is essential for human iron supplementation and iron deficiency anemia prevention because it contains abundant bioavailable iron and is frequently consumed in the human diet. However, it is poorly understood in regards its several properties, such as iron mineralization, subunit assembly, and protein folding. To address these issues, we decided to prepare the soybean seed ferritin complex via a recombinant DNA approach. In this paper, we report a rapid and simple Escherichia coli expression system to produce the soybean seed ferritin complex. In this system, two subunits of soybean seed ferritin, H-2 and H-1, were encoded in a single plasmid, and optimal expression was achieved by additionally coexpressing a team of molecular chaperones, trigger factor and GroEL-GroES. The His-tagged ferritin complex was purified by Ni2+ affinity chromatography, and an intact ferritin complex was obtained following His-tagged enterokinase (His-EK) digestion. The purified ferritin complex synthesized in E. coli demonstrated some reported features of its native counterpart from soybean seed, including an apparent molecular weight, multimeric assembly, and iron uptake activity. We believe that the strategy described in this paper may be of general utility in producing other recombinant plant ferritins built up from two types of subunits.
Assuntos
Escherichia coli/metabolismo , Ferritinas/química , Ferritinas/isolamento & purificação , Expressão Gênica , Escherichia coli/genética , Ferritinas/genética , Ferritinas/metabolismo , Engenharia Genética , Ferro/metabolismo , Cinética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sementes/metabolismo , Solubilidade , Proteínas de Soja/química , Proteínas de Soja/genética , Proteínas de Soja/isolamento & purificação , Proteínas de Soja/metabolismoRESUMO
Innate immune sensing of dying cells is modulated by several signals. Inflammatory chemokines-guided early recruitment, and pathogen-associated molecular patterns-triggered activation, of major anti-pathogenic innate immune cells like neutrophils distinguishes pathogen-infected stressed/dying cells from sterile dying cells. However, whether certain sterile dying cells stimulate innate immunity by partially mimicking pathogen response-like recruitment/activation of neutrophils remains poorly understood. We reveal that sterile immunogenic dying cancer cells trigger (a cell autonomous) pathogen response-like chemokine (PARC) signature, hallmarked by co-release of CXCL1, CCL2 and CXCL10 (similar to cells infected with bacteria or viruses). This PARC signature recruits preferentially neutrophils as first innate immune responders in vivo (in a cross-species, evolutionarily conserved manner; in mice and zebrafish). Furthermore, key danger signals emanating from these dying cells, that is, surface calreticulin, ATP and nucleic acids stimulate phagocytosis, purinergic receptors and toll-like receptors (TLR) i.e. TLR7/8/9-MyD88 signaling on neutrophil level, respectively. Engagement of purinergic receptors and TLR7/8/9-MyD88 signaling evokes neutrophil activation, which culminates into H2O2 and NO-driven respiratory burst-mediated killing of viable residual cancer cells. Thus sterile immunogenic dying cells perform 'altered-self mimicry' in certain contexts to exploit neutrophils for phagocytic targeting of dead/dying cancer cells and cytotoxic targeting of residual cancer cells.
Assuntos
Quimiocina CCL2/genética , Quimiocina CXCL10/genética , Quimiocina CXCL1/genética , Quimiocinas CC/genética , Neutrófilos/imunologia , Animais , Animais Geneticamente Modificados , Apoptose , Linhagem Celular Tumoral , Quimiocina CCL2/imunologia , Quimiocina CXCL1/imunologia , Quimiocina CXCL10/imunologia , Quimiocinas CC/imunologia , Técnicas de Cocultura , Citotoxicidade Imunológica , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Masculino , Melanócitos/imunologia , Melanócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Neuroglia/imunologia , Neuroglia/patologia , Neutrófilos/citologia , Transdução de Sinais , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/imunologia , Peixe-ZebraRESUMO
The recent discovery of meningeal lymphatic vessels (LVs) has raised interest in their possible involvement in neuropathological processes, yet little is known about their development or maintenance. We show here that meningeal LVs develop postnatally, appearing first around the foramina in the basal parts of the skull and spinal canal, sprouting along the blood vessels and cranial and spinal nerves to various parts of the meninges surrounding the central nervous system (CNS). VEGF-C, expressed mainly in vascular smooth muscle cells, and VEGFR3 in lymphatic endothelial cells were essential for their development, whereas VEGF-D deletion had no effect. Surprisingly, in adult mice, the LVs showed regression after VEGF-C or VEGFR3 deletion, administration of the tyrosine kinase inhibitor sunitinib, or expression of VEGF-C/D trap, which also compromised the lymphatic drainage function. Conversely, an excess of VEGF-C induced meningeal lymphangiogenesis. The plasticity and regenerative potential of meningeal LVs should allow manipulation of cerebrospinal fluid drainage and neuropathological processes in the CNS.
Assuntos
Vasos Linfáticos/fisiologia , Meninges/fisiologia , Animais , Animais Recém-Nascidos , Transporte Biológico/efeitos dos fármacos , Líquido Cefalorraquidiano/metabolismo , Dependovirus/metabolismo , Deleção de Genes , Humanos , Indóis/farmacologia , Injeções Intraventriculares , Linfonodos/efeitos dos fármacos , Linfonodos/metabolismo , Linfangiogênese/efeitos dos fármacos , Vasos Linfáticos/efeitos dos fármacos , Masculino , Meninges/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Microesferas , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Transdução de Sinais , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiologia , Sunitinibe , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator D de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The angiopoietin/Tie (ANG/Tie) receptor system controls developmental and tumor angiogenesis, inflammatory vascular remodeling, and vessel leakage. ANG1 is a Tie2 agonist that promotes vascular stabilization in inflammation and sepsis, whereas ANG2 is a context-dependent Tie2 agonist or antagonist. A limited understanding of ANG signaling mechanisms and the orphan receptor Tie1 has hindered development of ANG/Tie-targeted therapeutics. Here, we determined that both ANG1 and ANG2 binding to Tie2 increases Tie1-Tie2 interactions in a ß1 integrin-dependent manner and that Tie1 regulates ANG-induced Tie2 trafficking in endothelial cells. Endothelial Tie1 was essential for the agonist activity of ANG1 and autocrine ANG2. Deletion of endothelial Tie1 in mice reduced Tie2 phosphorylation and downstream Akt activation, increased FOXO1 nuclear localization and transcriptional activation, and prevented ANG1- and ANG2-induced capillary-to-venous remodeling. However, in acute endotoxemia, the Tie1 ectodomain that is responsible for interaction with Tie2 was rapidly cleaved, ANG1 agonist activity was decreased, and autocrine ANG2 agonist activity was lost, which led to suppression of Tie2 signaling. Tie1 cleavage also occurred in patients with hantavirus infection. These results support a model in which Tie1 directly interacts with Tie2 to promote ANG-induced vascular responses under noninflammatory conditions, whereas in inflammation, Tie1 cleavage contributes to loss of ANG2 agonist activity and vascular stability.
Assuntos
Angiopoietina-1/metabolismo , Angiopoietina-2/metabolismo , Inflamação , Receptor de TIE-1/metabolismo , Receptor TIE-2/metabolismo , Remodelação Vascular , Adulto , Idoso , Animais , Estudos de Casos e Controles , Estudos de Coortes , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Endotoxemia/metabolismo , Feminino , Deleção de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrina beta1/metabolismo , Lipopolissacarídeos/química , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Fosforilação , Sepse , Transdução de Sinais , Adulto JovemRESUMO
Trans-membrane (or T cell) immunoglobulin and mucin (TIM) molecules are known regulators of immune response whose function in hematopoiesis is unknown. Earlier, we found that tim-1 and tim-4 are expressed by CD45(+) cells in the para-aortic region of chicken embryo. Because the para-aortic region is a known site for hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) differentiation and expansion, we hypothesize that TIM molecules have a role in hematopoiesis. To study this role further, we analyzed TIM expression more precisely in chicken para-aortic region and mouse fetal liver hematopoietic cells. Additionally, we examined the hematopoietic potential of TIM-4(+) mouse fetal liver cells with a colony-forming assay. tim-1 gene expression was detected in chicken and mouse embryos in the aorta-gonads-mesonephros-region at the time of HSC emergence, whereas tim-3 mRNA was widely expressed in different tissues. tim-4 expression was restricted to fetal liver CD45(+)F4/80(+) cells. Moreover, two TIM-4(+) populations were distinguished: F4/80(hi)TIM-4(hi) and F4/80(lo)TIM-4(lo). F4/80(hi)TIM-4(hi) cells had no hematopoietic potential and were morphologically similar to mature macrophages, suggesting that they are yolk sac-derived macrophages. Instead, many of the F4/80(lo)TIM-4(lo) cells were c-kit(+) and Sca-1(+) and had primitive morphology and multilineage colony-forming ability. In addition, F4/80(lo)TIM-4(lo) cells included a considerable population expressing ER-MP12, a known marker for macrophage colony-forming cells and other myeloid progenitors. We conclude that TIM molecules are expressed in embryonic hematopoietic tissues in chicken and mouse and that in fetal liver, TIM-4 is expressed by myeloid progenitor cells.
Assuntos
Proteínas Aviárias/genética , Sistema Hematopoético/metabolismo , Fígado/metabolismo , Proteínas de Membrana/genética , Animais , Antígenos de Diferenciação/metabolismo , Proteínas Aviárias/metabolismo , Embrião de Galinha , Galinhas , Ensaio de Unidades Formadoras de Colônias , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Sistema Hematopoético/embriologia , Antígenos Comuns de Leucócito/metabolismo , Fígado/citologia , Fígado/embriologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/citologia , Células Mieloides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
BACKGROUND: Angiogenin is a potent inducer of angiogenesis. We prospectively evaluated the prognostic significance of serum angiogenin from 204 consecutive non-Hodgkin lymphoma (NHL) patients diagnosed and treated in a single institution. METHODS: Serum angiogenin, VEGF, and bFGF concentrations at diagnosis were determined using a quantitative sandwich enzyme immunoassay technique. Kaplan-Meier survival curves were compared by the log-rank test. Multivariate survival analyses were performed using the parametric model of Weibull and the non-parametric proportional hazards model of Cox. RESULTS: Patients with a high serum angiogenin at diagnosis (>median; 401 ng/ml) had significantly lower 5-year survival rate than those with a low (≤ median) angiogenin (42% versus 63%, respectively; P = 0.0073). Serum angiogenin provided additional information to the International Prognostic Index (IPI) identifying a subgroup (serum angiogenin >median and IPI>1) with very poor prognosis (5-year survival 19%, P < 0.0001). In receiver operating characteristic (ROC) analyses the accuracy of the IPI to correctly classify patients with favourable or poor survival was improved from fair to good by complementing the IPI with serum angiogenin concentration. With patients who initially achieved complete response (CR) after chemotherapy, a high angiogenin at diagnosis (>median; relative risk (RR) 2.38; P = 0.0077) and an advanced tumour stage (III-IV; RR 2.41; P = 0.0087) were the only independent predictors for patients with unfavourable outcome although first responding well to therapy. CONCLUSIONS: We conclude that elevated serum angiogenin surfaced as an independent predictor for failure in long-term treatment response and for poor overall survival in a series of 204 NHL patients, and might thus also complement the IPI in identifying the patients with particularly aggressive and/or treatment resistant disease.