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1.
Int J Mol Sci ; 22(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34445652

RESUMO

Hereditary Hemorrhagic Telangiectasia type 1 (HHT1) is an autosomal dominant inherited disease characterized by arteriovenous malformations and hemorrhage. HHT1 is caused by mutations in ENDOGLIN, which encodes an ancillary receptor for Transforming Growth Factor-ß/Bone Morphogenetic Protein-9 expressed in all vascular endothelial cells. Haploinsufficiency is widely accepted as the underlying mechanism for HHT1. However, it remains intriguing that only some, but not all, vascular beds are affected, as these causal gene mutations are present in vasculature throughout the body. Here, we have examined the endoglin expression levels in the blood vessels of multiple organs in mice and in humans. We found a positive correlation between low basal levels of endoglin and the general prevalence of clinical manifestations in selected organs. Endoglin was found to be particularly low in the skin, the earliest site of vascular lesions in HHT1, and even undetectable in the arteries and capillaries of heterozygous endoglin mice. Endoglin levels did not appear to be associated with organ-specific vascular functions. Instead, our data revealed a critical endoglin threshold compatible with the haploinsufficiency model, below which endothelial cells independent of their tissue of origin exhibited abnormal responses to Vascular Endothelial Growth Factor. Our results support the development of drugs promoting endoglin expression as potentially protective.


Assuntos
Endoglina/fisiologia , Endotélio Vascular/patologia , Mutação , Telangiectasia Hemorrágica Hereditária/complicações , Doenças Vasculares/patologia , Animais , Endotélio Vascular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Doenças Vasculares/etiologia , Doenças Vasculares/metabolismo
2.
J Am Soc Nephrol ; 30(10): 1886-1897, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31308073

RESUMO

BACKGROUND: A glycocalyx envelope consisting of proteoglycans and adhering proteins covers endothelial cells, both the luminal and abluminal surface. We previously demonstrated that short-term loss of integrity of the luminal glycocalyx layer resulted in perturbed glomerular filtration barrier function. METHODS: To explore the role of the glycocalyx layer of the endothelial extracellular matrix in renal function, we generated mice with an endothelium-specific and inducible deletion of hyaluronan synthase 2 (Has2), the enzyme that produces hyaluronan, the main structural component of the endothelial glycocalyx layer. We also investigated the presence of endothelial hyaluronan in human kidney tissue from patients with varying degrees of diabetic nephropathy. RESULTS: Endothelial deletion of Has2 in adult mice led to substantial loss of the glycocalyx structure, and analysis of their kidneys and kidney function showed vascular destabilization, characterized by mesangiolysis, capillary ballooning, and albuminuria. This process develops over time into glomerular capillary rarefaction and glomerulosclerosis, recapitulating the phenotype of progressive human diabetic nephropathy. Using a hyaluronan-specific probe, we found loss of glomerular endothelial hyaluronan in association with lesion formation in tissue from patients with diabetic nephropathy. We also demonstrated that loss of hyaluronan, which harbors a specific binding site for angiopoietin and a key regulator of endothelial quiescence and maintenance of EC barrier function results in disturbed angiopoietin 1 Tie2. CONCLUSIONS: Endothelial loss of hyaluronan results in disturbed glomerular endothelial stabilization. Glomerular endothelial hyaluronan is a previously unrecognized key component of the extracelluar matrix that is required for glomerular structure and function and lost in diabetic nephropathy.


Assuntos
Ácido Hialurônico/biossíntese , Glomérulos Renais/anatomia & histologia , Glomérulos Renais/fisiologia , Animais , Endotélio/metabolismo , Humanos , Glomérulos Renais/metabolismo , Camundongos , Urotélio
3.
Circulation ; 138(23): 2698-2712, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30571259

RESUMO

BACKGROUND: Hereditary Hemorrhagic Telangiectasia type 2 (HHT2) is an inherited genetic disorder characterized by vascular malformations and hemorrhage. HHT2 results from ACVRL1 haploinsufficiency, the remaining wild-type allele being unable to contribute sufficient protein to sustain endothelial cell function. Blood vessels function normally but are prone to respond to angiogenic stimuli, leading to the development of telangiectasic lesions that can bleed. How ACVRL1 haploinsufficiency leads to pathological angiogenesis is unknown. METHODS: We took advantage of Acvrl1+/- mutant mice that exhibit HHT2 vascular lesions and focused on the neonatal retina and the airway system after Mycoplasma pulmonis infection, as physiological and pathological models of angiogenesis, respectively. We elucidated underlying disease mechanisms in vitro by generating Acvrl1+/- mouse embryonic stem cell lines that underwent sprouting angiogenesis and performed genetic complementation experiments. Finally, HHT2 plasma samples and skin biopsies were analyzed to determine whether the mechanisms evident in mice are conserved in humans. RESULTS: Acvrl1+/- retinas at postnatal day 7 showed excessive angiogenesis and numerous endothelial "tip cells" at the vascular front that displayed migratory defects. Vascular endothelial growth factor receptor 1 (VEGFR1; Flt-1) levels were reduced in Acvrl1+/- mice and HHT2 patients, suggesting similar mechanisms in humans. In sprouting angiogenesis, VEGFR1 is expressed in stalk cells to inhibit VEGFR2 (Flk-1, KDR) signaling and thus limit tip cell formation. Soluble VEGFR1 (sVEGFR1) is also secreted, creating a VEGF gradient that promotes orientated sprout migration. Acvrl1+/- embryonic stem cell lines recapitulated the vascular anomalies in Acvrl1+/- (HHT2) mice. Genetic insertion of either the membrane or soluble form of VEGFR1 into the ROSA26 locus of Acvrl1+/- embryonic stem cell lines prevented the vascular anomalies, suggesting that high VEGFR2 activity in Acvrl1+/- endothelial cells induces HHT2 vascular anomalies. To confirm our hypothesis, Acvrl1+/- mice were infected by Mycoplasma pulmonis to induce sustained airway inflammation. Infected Acvrl1+/- tracheas showed excessive angiogenesis with the formation of multiple telangiectases, vascular defects that were prevented by VEGFR2 blocking antibodies. CONCLUSIONS: Our findings demonstrate a key role of VEGFR1 in HHT2 pathogenesis and provide mechanisms explaining why HHT2 blood vessels respond abnormally to angiogenic signals. This supports the case for using anti-VEGF therapy in HHT2.


Assuntos
Telangiectasia Hemorrágica Hereditária/patologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptores de Ativinas Tipo I/genética , Receptores de Activinas Tipo II , Adulto , Animais , Anticorpos/administração & dosagem , Anticorpos/imunologia , Malformações Arteriovenosas/etiologia , Modelos Animais de Doenças , Feminino , Heterozigoto , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Mycoplasma pulmonis/fisiologia , Neovascularização Fisiológica , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Vasos Retinianos/fisiologia , Transdução de Sinais , Pele/patologia , Telangiectasia Hemorrágica Hereditária/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/imunologia
4.
Adv Exp Med Biol ; 1147: 215-246, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31147880

RESUMO

Hereditary hemorrhagic telangiectasia (HHT) is a genetic disorder characterized by multi-systemic vascular dysplasia affecting 1 in 5000 people worldwide. Individuals with HHT suffer from many complications including nose and gastrointestinal bleeding, anemia, iron deficiency, stroke, abscess, and high-output heart failure. Identification of the causative gene mutations and the generation of animal models have revealed that decreased transforming growth factor-ß (TGF-ß)/bone morphogenetic protein (BMP) signaling and increased vascular endothelial growth factor (VEGF) signaling activity in endothelial cells are responsible for the development of the vascular malformations in HHT. Perturbations in these key pathways are thought to lead to endothelial cell activation resulting in mural cell disengagement from the endothelium. This initial instability state causes the blood vessels to response inadequately when they are exposed to angiogenic triggers resulting in excessive blood vessel growth and the formation of vascular abnormalities that are prone to bleeding. Drugs promoting blood vessel stability have been reported as effective in preclinical models and in clinical trials indicating possible interventional targets based on a normalization approach for treating HHT. Here, we will review how disturbed TGF-ß and VEGF signaling relates to blood vessel destabilization and HHT development and will discuss therapeutic opportunities based on the concept of vessel normalization to treat HHT.


Assuntos
Pericitos , Telangiectasia Hemorrágica Hereditária , Animais , Células Endoteliais , Humanos , Fator de Crescimento Transformador beta , Fator A de Crescimento do Endotélio Vascular
5.
Angiogenesis ; 21(1): 169-181, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29147802

RESUMO

Hereditary hemorrhagic telangiectasia is an autosomal dominant trait affecting approximately 1 in 5000 people. A pathogenic DNA sequence variant in the ENG, ACVRL1 or SMAD4 genes, can be found in the majority of patients. The 12th International Scientific HHT Conference was held on June 8-11, 2017 in Dubrovnik, Croatia to present and discuss the latest scientific achievements, and was attended by over 200 scientific and clinical researchers. In total 174 abstracts were accepted of which 58 were selected for oral presentations. This article covers the basic science and clinical talks, and discussions from three theme-based workshops. We focus on significant emergent themes and unanswered questions. Understanding these topics and answering these questions will help to define the future of HHT research and therapeutics, and ultimately bring us closer to a cure.


Assuntos
Telangiectasia Hemorrágica Hereditária , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Malformações Arteriovenosas/genética , Malformações Arteriovenosas/metabolismo , Malformações Arteriovenosas/patologia , Malformações Arteriovenosas/terapia , Croácia , Endoglina/genética , Endoglina/metabolismo , Epistaxe/genética , Epistaxe/metabolismo , Variação Genética , Humanos , Proteína Smad4/genética , Proteína Smad4/metabolismo , Telangiectasia Hemorrágica Hereditária/genética , Telangiectasia Hemorrágica Hereditária/metabolismo , Telangiectasia Hemorrágica Hereditária/patologia , Telangiectasia Hemorrágica Hereditária/terapia
6.
Arterioscler Thromb Vasc Biol ; 36(4): 707-17, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26821948

RESUMO

OBJECTIVE: To determine the role of Gja5 that encodes for the gap junction protein connexin40 in the generation of arteriovenous malformations in the hereditary hemorrhagic telangiectasia type 2 (HHT2) mouse model. APPROACH AND RESULTS: We identified GJA5 as a target gene of the bone morphogenetic protein-9/activin receptor-like kinase 1 signaling pathway in human aortic endothelial cells and importantly found that connexin40 levels were particularly low in a small group of patients with HHT2. We next took advantage of the Acvrl1(+/-) mutant mice that develop lesions similar to those in patients with HHT2 and generated Acvrl1(+/-); Gja5(EGFP/+) mice. Gja5 haploinsufficiency led to vasodilation of the arteries and rarefaction of the capillary bed in Acvrl1(+/-) mice. At the molecular level, we found that reduced Gja5 in Acvrl1(+/-) mice stimulated the production of reactive oxygen species, an important mediator of vessel remodeling. To normalize the altered hemodynamic forces in Acvrl1(+/-); Gja5(EGFP/+) mice, capillaries formed transient arteriovenous shunts that could develop into large malformations when exposed to environmental insults. CONCLUSIONS: We identified GJA5 as a potential modifier gene for HHT2. Our findings demonstrate that Acvrl1 haploinsufficiency combined with the effects of modifier genes that regulate vessel caliber is responsible for the heterogeneity and severity of the disease. The mouse models of HHT have led to the proposal that 3 events-heterozygosity, loss of heterozygosity, and angiogenic stimulation-are necessary for arteriovenous malformation formation. Here, we present a novel 3-step model in which pathological vessel caliber and consequent altered blood flow are necessary events for arteriovenous malformation development.


Assuntos
Receptores de Activinas Tipo II/metabolismo , Receptores de Ativinas Tipo I/metabolismo , Malformações Arteriovenosas/enzimologia , Conexinas/metabolismo , Células Endoteliais/enzimologia , Vasos Retinianos/enzimologia , Telangiectasia Hemorrágica Hereditária/enzimologia , Receptores de Ativinas Tipo I/genética , Receptores de Activinas Tipo II/genética , Animais , Malformações Arteriovenosas/genética , Malformações Arteriovenosas/patologia , Células Cultivadas , Conexinas/genética , Modelos Animais de Doenças , Predisposição Genética para Doença , Haploinsuficiência , Humanos , Camundongos Mutantes , Camundongos Transgênicos , Neovascularização Patológica , Fenótipo , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Vasos Retinianos/patologia , Transdução de Sinais , Telangiectasia Hemorrágica Hereditária/genética , Telangiectasia Hemorrágica Hereditária/patologia , Transfecção , Remodelação Vascular , Proteína alfa-5 de Junções Comunicantes
7.
J Vis Exp ; (209)2024 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-39141523

RESUMO

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the microvasculature using Doppler ultrasound methods, imaging modalities employing the concept of ultrafast imaging are preferred. By increasing the frame rate of the ultrasound scanner to thousands of frames per second, it becomes possible to improve the sensitivity of the blood flow down to 2 mm/s and to obtain functional information about the microcirculation in comparison to a sensitivity of around 1 cm/s in conventional Doppler modes. While Ultrafast Doppler ultrasound (UFUS) imaging has become adopted in neuroscience, where it can capture brain activity through neurovascular coupling, it presents greater challenges when imaging the vasculature of abdominal organs due to larger motions linked to breathing. The liver, positioned anatomically under the diaphragm, is particularly susceptible to out-of-plane movement and oscillating respiratory motion. These artifacts not only adversely affect Doppler imaging but also complicate the anatomical analysis of vascular structures and the computation of vascular parameters. Here, we present a qualitative and quantitative imaging analysis of the hepatic vasculature in mice by UFUS. We identify major anatomical vascular structures and provide graphical illustrations of the hepatic macroscopical anatomy, comparing it to an in-depth anatomical assessment of the hepatic vasculature based on Doppler readouts. Additionally, we have developed a quantification protocol for robust measurements of hepatic blood volume of the microvasculature over time. To contemplate further research, qualitative vascular analysis provides a comprehensive overview and suggests a standardized terminology for researchers working with mouse models of liver disease. Furthermore, it offers the opportunity to apply ultrasound as a non-invasive complementary method to inspect hepatic vascular defects in vivo and measure functional microvascular alterations deep within the organ before unraveling blood vessel anomalies at the micron scale levels using ex vivo staining on tissue sections.


Assuntos
Fígado , Ultrassonografia Doppler , Animais , Camundongos , Ultrassonografia Doppler/métodos , Fígado/diagnóstico por imagem , Fígado/irrigação sanguínea , Microvasos/diagnóstico por imagem
8.
J Clin Med ; 11(18)2022 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-36142926

RESUMO

Haploinsufficiency for Endoglin (ENG) and activin A receptor type II-like I (ACVRL1/ALK1) lead to the formation of weak and abnormal vessels in hereditary hemorrhagic telangiectasia (HHT). These cause epistaxis (nosebleeds) and/or gastrointestinal blood loss. In vitro in cultured endothelial cells, tacrolimus has been shown to increase ENG and ALK1 expression. It is, therefore, a potential treatment option. We report here a proof-of-concept study in patients with HHT and severe epistaxis and/or gastrointestinal bleeding who were treated daily with orally-administered tacrolimus for twenty weeks. Twenty-five patients with HHT (11 females (44%)) and median age of 59 years were enrolled. Five patients (20%) stopped the trial prematurely-four due to (serious) adverse events ((S)AE). Twenty patients were included in further analyses. Hemoglobin levels increased during tacrolimus treatment from 6.1 (IQR 5.2-6.9) mmol/L at baseline (9.8 g/dL) to 6.7 (6.5-7.1) mmol/L (10.8 g/dL), p = 0.003. The number of blood transfusions over the twenty weeks decreased from a mean of 5.0 (±9.2) to 1.9 (±3.5), p = 0.04. In 64% of the patients, at least one AE occurred. Oral tacrolimus, thus, significantly increased hemoglobin levels and decreased blood transfusion needs, epistaxis and/or gastrointestinal bleeding in patients with HHT. However, side-effects were common. Further investigation of the potential therapeutic benefit is justified by the outcome of the study.

9.
Stem Cell Reports ; 17(7): 1536-1545, 2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35777360

RESUMO

Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease characterized by weak blood vessels. HHT1 is caused by mutations in the ENDOGLIN (ENG) gene. Here, we generated induced pluripotent stem cells (hiPSCs) from a patient with rare mosaic HHT1 with tissues containing both mutant (ENGc.1678C>T) and normal cells, enabling derivation of isogenic diseased and healthy hiPSCs, respectively. We showed reduced ENG expression in HHT1 endothelial cells (HHT1-hiPSC-ECs), reflecting haploinsufficiency. HHT1c.1678C>T-hiPSC-ECs and the healthy isogenic control behaved similarly in two-dimensional (2D) culture, forming functionally indistinguishable vascular networks. However, when grown in 3D organ-on-chip devices under microfluidic flow, lumenized vessels formed in which defective vascular organization was evident: interaction between inner ECs and surrounding pericytes was decreased, and there was evidence for vascular leakage. Organs on chip thus revealed features of HHT in hiPSC-derived blood vessels that were not evident in conventional 2D assays.


Assuntos
Células-Tronco Pluripotentes Induzidas , Telangiectasia Hemorrágica Hereditária , Receptores de Activinas Tipo II/genética , Endoglina/genética , Endoglina/metabolismo , Células Endoteliais/metabolismo , Humanos , Mutação , Telangiectasia Hemorrágica Hereditária/complicações , Telangiectasia Hemorrágica Hereditária/genética , Telangiectasia Hemorrágica Hereditária/metabolismo
10.
Cardiovasc Res ; 118(5): 1262-1275, 2022 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33909875

RESUMO

AIMS: Hepatic capillaries are lined with specialized liver sinusoidal endothelial cells (LSECs) which support macromolecule passage to hepatocytes and prevent fibrosis by keeping hepatic stellate cells (HSCs) quiescent. LSEC specialization is co-determined by transcription factors. The zinc-finger E-box-binding homeobox (Zeb)2 transcription factor is enriched in LSECs. Here, we aimed to elucidate the endothelium-specific role of Zeb2 during maintenance of the liver and in liver fibrosis. METHODS AND RESULTS: To study the role of Zeb2 in liver endothelium we generated EC-specific Zeb2 knock-out (ECKO) mice. Sequencing of liver EC RNA revealed that deficiency of Zeb2 results in prominent expression changes in angiogenesis-related genes. Accordingly, the vascular area was expanded and the presence of pillars inside ECKO liver vessels indicated that this was likely due to increased intussusceptive angiogenesis. LSEC marker expression was not profoundly affected and fenestrations were preserved upon Zeb2 deficiency. However, an increase in continuous EC markers suggested that Zeb2-deficient LSECs are more prone to dedifferentiation, a process called 'capillarization'. Changes in the endothelial expression of ligands that may be involved in HSC quiescence together with significant changes in the expression profile of HSCs showed that Zeb2 regulates LSEC-HSC communication and HSC activation. Accordingly, upon exposure to the hepatotoxin carbon tetrachloride (CCl4), livers of ECKO mice showed increased capillarization, HSC activation, and fibrosis compared to livers from wild-type littermates. The vascular maintenance and anti-fibrotic role of endothelial Zeb2 was confirmed in mice with EC-specific overexpression of Zeb2, as the latter resulted in reduced vascularity and attenuated CCl4-induced liver fibrosis. CONCLUSION: Endothelial Zeb2 preserves liver angioarchitecture and protects against liver fibrosis. Zeb2 and Zeb2-dependent genes in liver ECs may be exploited to design novel therapeutic strategies to attenuate hepatic fibrosis.


Assuntos
Células Endoteliais , Cirrose Hepática , Animais , Biomarcadores/metabolismo , Células Endoteliais/metabolismo , Endotélio , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Fígado/metabolismo , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/prevenção & controle , Camundongos
11.
Cells ; 10(5)2021 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-34063200

RESUMO

Renal microvascular rarefaction plays a pivotal role in progressive kidney disease. Therefore, modalities to visualize the microcirculation of the kidney will increase our understanding of disease mechanisms and consequently may provide new approaches for evaluating cell-based therapy. At the moment, however, clinical practice is lacking non-invasive, safe, and efficient imaging modalities to monitor renal microvascular changes over time in patients suffering from renal disease. To emphasize the importance, we summarize current knowledge of the renal microcirculation and discussed the involvement in progressive kidney disease. Moreover, an overview of available imaging techniques to uncover renal microvascular morphology, function, and behavior is presented with the associated benefits and limitations. Ultimately, the necessity to assess and investigate renal disease based on in vivo readouts with a resolution up to capillary level may provide a paradigm shift for diagnosis and therapy in the field of nephrology.


Assuntos
Diagnóstico por Imagem/métodos , Rim/irrigação sanguínea , Rim/diagnóstico por imagem , Microcirculação , Capilares , Terapia Baseada em Transplante de Células e Tecidos , Progressão da Doença , Endotélio Vascular/patologia , Fibroblastos/metabolismo , Humanos , Nefropatias , Imageamento por Ressonância Magnética , Pericitos , Transdução de Sinais , Ultrassonografia , Microtomografia por Raio-X
12.
J Vis Exp ; (171)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-34057453

RESUMO

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.


Assuntos
Células-Tronco Pluripotentes Induzidas , Neovascularização Fisiológica , Preparações Farmacêuticas , Doenças Vasculares , Animais , Diferenciação Celular , Humanos , Camundongos , Células-Tronco Embrionárias Murinas , Neovascularização Patológica
13.
Circ Res ; 103(4): 432-40, 2008 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-18617693

RESUMO

Apelin has been identified as the endogenous ligand of the human orphan G protein-coupled receptor APJ. This peptide exerts a variety of cardiovascular effects and particularly acts as an activator of angiogenesis. Importantly, hypoxia has been reported to regulate apelin expression but the molecular mechanism underlying hypoxia-induced apelin expression and the relationship with the physiological response of the apelin/APJ system are still not established. Here, we demonstrate that apelin expression is induced by hypoxia in cultured endothelial and vascular smooth muscle cells as well as in lung from mice exposed to acute hypoxia. Transient transfection experiments show that hypoxia-inducible transcriptional activation of apelin requires an intact hypoxia-responsive element (+813/+826) located within the first intron of the human apelin gene. Chromatin immunoprecipitation assay reveals that hypoxia-inducible factor-1alpha binds to the endogenous hypoxia-responsive element site of the apelin gene. Moreover, overexpression of hypoxia-inducible factor-1alpha increases the transcriptional activity of a reporter construct containing this hypoxia-responsive element, whereas small interfering RNA-mediated hypoxia-inducible factor-1alpha knockdown abolishes hypoxia-induced apelin expression. Finally, microinterfering RNA-mediated apelin or APJ receptor knockdown inhibits both hypoxia-induced endothelial cell proliferation in vitro and hypoxia-induced vessel regeneration in the caudal fin regeneration of Fli-1 transgenic zebrafish. The hypoxia-induced apelin expression may, thus, provide a new mechanism involved in adaptive physiological and pathophysiological response of vascular cells to low oxygen level.


Assuntos
Proteínas de Transporte/metabolismo , Proliferação de Células , Endotélio Vascular/metabolismo , Hipóxia/metabolismo , Músculo Liso Vascular/metabolismo , Neovascularização Fisiológica/fisiologia , Regeneração/imunologia , Adipocinas , Sequência de Aminoácidos , Animais , Apelina , Proteínas de Transporte/genética , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Endotélio Vascular/citologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Músculo Liso Vascular/citologia , Transfecção , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
14.
Nat Biotechnol ; 25(7): 803-16, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17572666

RESUMO

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.


Assuntos
Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Fosfatase Alcalina/metabolismo , Antígenos CD/biossíntese , Biotecnologia/métodos , Diferenciação Celular , Linhagem da Célula , Membrana Celular/metabolismo , Células Cultivadas , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Genótipo , Glicolipídeos/química , Humanos , Glicoproteínas de Membrana/biossíntese , Tetraspanina 29
15.
Stroke ; 40(2): 439-47, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19074480

RESUMO

BACKGROUND AND PURPOSE: Unstable atherosclerotic plaques are characterized by increased macrophages and reduced smooth muscle cells (SMCs) and collagen content. Endoglin, an accessory transforming growth factor-beta (TGFbeta) receptor, is a modulator of TGFbeta signaling recently found to be expressed on SMCs in atherosclerotic plaques. Its function in plaque SMCs and plaque development is unknown. Early growth response-1 (EGR-1), a transcription factor downstream of TGFbeta, stimulates SMC proliferation and collagen synthesis. In atherosclerotic lesions, it is mainly expressed by SMCs. Therefore, we studied the TGFbeta, endoglin, and EGR-1 pathway in advanced atherosclerotic plaques in relation to plaque phenotype. METHODS: Human carotid atherosclerotic plaques (n=103) were collected from patients undergoing carotid endarterectomy. Histologically, plaques were analyzed for plaque characteristics, ie, collagen, macrophage and SMC content, and intraplaque thrombus. Intraplaque endoglin, pSmad (indicative for TGFbeta signaling), EGR-1, and TGFbeta levels were analyzed using Western blots and enzyme-linked immunosorbent assays, respectively. RESULTS: Higher endoglin and EGR-1 protein levels correlated positively with increased plaque collagen levels, increased smooth muscle cell content, and decreased intraplaque thrombi as well as TGFbeta signaling (pSmad). Although EGR-1 overexpression in vitro stimulated collagen synthesis, inhibiting endoglin resulted in lower EGR-1 levels, decreased SMC proliferation, and decreased collagen content. CONCLUSIONS: TGFbeta in human atherosclerotic plaques is active and signals through the TGFbeta/Smad pathway. For the first time, we show a strong association between endoglin and EGR-1, increased collagen and SMCs expression, decreased levels of intraplaque thrombosis, and a stable plaque phenotype.


Assuntos
Antígenos CD/biossíntese , Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Arteriosclerose Intracraniana/metabolismo , Receptores de Superfície Celular/biossíntese , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/biossíntese , Idoso , Western Blotting , Linhagem Celular , Proliferação de Células , Colágeno/biossíntese , Colágeno/genética , Colágeno Tipo I , Endoglina , Ensaio de Imunoadsorção Enzimática , Feminino , Perfilação da Expressão Gênica , Genes Reporter/genética , Humanos , Imuno-Histoquímica , Arteriosclerose Intracraniana/fisiopatologia , Masculino , Metaloproteinases da Matriz/biossíntese , Miócitos de Músculo Liso/fisiologia , Fenótipo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
16.
Stem Cells ; 26(9): 2257-65, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18599809

RESUMO

Defined growth conditions are essential for many applications of human embryonic stem cells (hESC). Most defined media are presently used in combination with Matrigel, a partially defined extracellular matrix (ECM) extract from mouse sarcoma. Here, we defined ECM requirements of hESC by analyzing integrin expression and ECM production and determined integrin function using blocking antibodies. hESC expressed all major ECM proteins and corresponding integrins. We then systematically replaced Matrigel with defined medium supplements and ECM proteins. Cells attached efficiently to natural human vitronectin, fibronectin, and Matrigel but poorly to laminin + entactin and collagen IV. Integrin-blocking antibodies demonstrated that alphaVbeta5 integrins mediated adhesion to vitronectin, alpha5beta1 mediated adhesion to fibronectin, and alpha6beta1 mediated adhesion to laminin + entactin. Fibronectin in feeder cell-conditioned medium partially supported growth on all natural matrices, but in defined, nonconditioned medium only Matrigel or (natural and recombinant) vitronectin was effective. Recombinant vitronectin was the only defined functional alternative to Matrigel, supporting sustained self-renewal and pluripotency in three independent hESC lines.


Assuntos
Células-Tronco Embrionárias/citologia , Receptores de Vitronectina/fisiologia , Vitronectina/farmacologia , Animais , Adesão Celular , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Colágeno , Meios de Cultura , Combinação de Medicamentos , Células-Tronco Embrionárias/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Humanos , Laminina , Camundongos , Proteoglicanas , Proteínas Recombinantes/farmacologia
17.
Trends Cardiovasc Med ; 18(1): 25-32, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18206806

RESUMO

Endoglin is emerging as a pivotal component of the gateway for signaling by transforming growth factor-beta (TGF-beta) in vascular endothelial cells. Mutations in endoglin cause a rare vascular disorder in humans known as hereditary hemorrhagic telengiectasia (HHT). Although rare, in-depth analysis of mutant mice and mononuclear cells from the blood of patients with HHT have provided novel and exciting insights into how the vasculature is formed, maintained, and repaired during disease. Here, we review recent data on how endoglin is thought to function in endothelial cells and place it in the broader context of signaling by TGF-beta family members in vascular cells in general. We highlight where the controversies on underlying molecular mechanisms currently lie and indicate areas of present research focus.


Assuntos
Antígenos CD/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Neovascularização Patológica/fisiopatologia , Receptores de Superfície Celular/fisiologia , Telangiectasia Hemorrágica Hereditária/fisiopatologia , Animais , Antígenos CD/genética , Endoglina , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Músculo Liso Vascular/metabolismo , Mutação , Receptores de Superfície Celular/genética , Transdução de Sinais/fisiologia , Telangiectasia Hemorrágica Hereditária/genética , Fator de Crescimento Transformador beta/metabolismo
18.
Circulation ; 114(21): 2288-97, 2006 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-17088457

RESUMO

BACKGROUND: Endoglin, an accessory receptor for transforming growth factor-beta in vascular endothelial cells, is essential for angiogenesis during mouse development. Mutations in the human gene cause hereditary hemorrhagic telangiectasia type 1 (HHT1), a disease characterized by vascular malformations that increase with age. Although haploinsufficiency is the underlying cause of the disease, HHT1 individuals show great heterogeneity in age of onset, clinical manifestations, and severity. METHODS AND RESULTS: In situ hybridization and immunohistochemical analysis of mouse and human hearts revealed that endoglin is upregulated in neoangiogenic vessels formed after myocardial infarction. Microvascularity within the infarct zone was strikingly lower in mice with reduced levels of endoglin (Eng+/-) compared with wild-type mice, which resulted in a greater deterioration in cardiac function as measured by magnetic resonance imaging. This did not appear to be because of defects in host inflammatory cell numbers in the infarct zone, which accumulated to a similar extent in wild-type and heterozygous mice. However, defects in vessel formation and heart function in Eng+/- mice were rescued by injection of mononuclear cells from healthy human donors but not by mononuclear cells from HHT1 patients. CONCLUSIONS: These results establish defective vascular repair as a significant component of the origin of HHT1. Because vascular damage or inflammation occurs randomly, it may also explain disease heterogeneity. More generally, the efficiency of vascular repair may vary between individuals because of intrinsic differences in their mononuclear cells.


Assuntos
Antígenos CD/metabolismo , Monócitos/transplante , Infarto do Miocárdio/fisiopatologia , Neovascularização Fisiológica , Receptores de Superfície Celular/metabolismo , Cicatrização , Animais , Antígenos CD/genética , Células Cultivadas , Vasos Coronários , Endoglina , Heterozigoto , Humanos , Ligadura , Camundongos , Mutação , Infarto do Miocárdio/etiologia , Miocárdio/metabolismo , Receptores de Superfície Celular/genética , Telangiectasia Hemorrágica Hereditária/genética , Remodelação Ventricular
19.
Stem Cells Dev ; 26(23): 1682-1694, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28922973

RESUMO

Neural crest (NC) cells are a migratory, multipotent population giving rise to numerous lineages in the embryo. Their plasticity renders attractive their use in tissue engineering-based therapies, but further knowledge on their in vivo behavior is required before clinical transfer may be envisioned. We here describe the isolation and characterization of a new mouse embryonic stem (ES) line derived from Wnt1-CRE-R26 RosaTomatoTdv blastocyst and show that it displays the characteristics of typical ES cells. Further, these cells can be efficiently directed toward an NC stem cell-like phenotype as attested by concomitant expression of NC marker genes and Tomato fluorescence. As native NC progenitors, they are capable of differentiating toward typical derivative phenotypes and interacting with embryonic tissues to participate in the formation of neo-structures. Their specific fluorescence allows purification and tracking in vivo. This cellular tool should facilitate a better understanding of the mechanisms driving NC fate specification and help identify the key interactions developed within a tissue after in vivo implantation. Altogether, this novel model may provide important knowledge to optimize NC stem cell graft conditions, which are required for efficient tissue repair.


Assuntos
Células-Tronco Embrionárias/citologia , Crista Neural/citologia , Células-Tronco Neurais/citologia , Neurogênese , Animais , Linhagem Celular , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/transplante , Integrases/genética , Integrases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Crista Neural/embriologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/transplante , Transplante de Células-Tronco/métodos , Proteína Wnt1/genética , Proteína Wnt1/metabolismo
20.
Cardiovasc Res ; 65(3): 599-608, 2005 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-15664386

RESUMO

Genetic studies in mice and humans have revealed the pivotal role of transforming growth factor-beta (TGF-beta) signaling during angiogenesis. Mice deficient for various TGF-beta signaling components present an embryonic lethality due to vascular defects. In patients, mutations in the TGF-beta type I receptor ALK1 or in the accessory TGF-beta receptor endoglin are linked to an autosomal dominant disorder of vascular dysplasia termed Hereditary Haemorrhagic Telangiectasia (HHT). It has puzzled researchers for years to explain the effects of TGF-beta being a stimulator and an inhibitor of angiogenesis in vitro and in vivo. Recently, a model has been proposed in which TGF-beta by binding to the TGF-beta type II receptor can activate two distinct type I receptors in endothelial cells (ECs), i.e., the EC-restricted ALK1 and the broadly expressed ALK-5, which have opposite effects on ECs behavior. ALK1 via Smad1/5 transcription factors stimulates EC proliferation and migration, whereas ALK5 via Smad2/3 inhibits EC proliferation and migration. Here, the new findings are presented concerning the molecular mechanisms that take place in ECs to precisely regulate and even switch between TGF-beta-induced biological responses. In particular, the role of the accessory TGF-beta receptor endoglin in the regulation of EC behavior is addressed and new insights are discussed concerning the possible mechanisms that are implicated in the development of HHT.


Assuntos
Endotélio Vascular/fisiologia , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Animais , Humanos , Camundongos , Neovascularização Fisiológica/fisiologia , Transdução de Sinais/fisiologia , Telangiectasia Hemorrágica Hereditária/fisiopatologia , Fator de Crescimento Transformador beta/fisiologia
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