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1.
Circ Res ; 127(9): 1122-1137, 2020 10 09.
Artículo en Inglés | MEDLINE | ID: mdl-32762495

RESUMEN

RATIONALE: Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease caused by mutations in ENG, ALK1, or SMAD4. Since proteins from all 3 HHT genes are components of signal transduction of TGF-ß (transforming growth factor ß) family members, it has been hypothesized that HHT is a disease caused by defects in the ENG-ALK1-SMAD4 linear signaling. However, in vivo evidence supporting this hypothesis is scarce. OBJECTIVE: We tested this hypothesis and investigated the therapeutic effects and potential risks of induced-ALK1 or -ENG overexpression (OE) for HHT. METHODS AND RESULTS: We generated a novel mouse allele (ROSA26Alk1) in which HA (human influenza hemagglutinin)-tagged ALK1 and bicistronic eGFP expression are induced by Cre activity. We examined whether ALK1-OE using the ROSA26Alk1 allele could suppress the development of arteriovenous malformations (AVMs) in wounded adult skin and developing retinas of Alk1- and Eng-inducible knockout (iKO) mice. We also used a similar approach to investigate whether ENG-OE could rescue AVMs. Biochemical and immunofluorescence analyses confirmed the Cre-dependent OE of the ALK1-HA transgene. We could not detect any pathological signs in ALK1-OE mice up to 3 months after induction. ALK1-OE prevented the development of retinal AVMs and wound-induced skin AVMs in Eng-iKO as well as Alk1-iKO mice. ALK1-OE normalized expression of SMAD and NOTCH target genes in ENG-deficient endothelial cells (ECs) and restored the effect of BMP9 (bone morphogenetic protein 9) on suppression of phosphor-AKT levels in these endothelial cells. On the other hand, ENG-OE could not inhibit the AVM development in Alk1-iKO models. CONCLUSIONS: These data support the notion that ENG and ALK1 form a linear signaling pathway for the formation of a proper arteriovenous network during angiogenesis. We suggest that ALK1 OE or activation can be an effective therapeutic strategy for HHT. Further research is required to study whether this therapy could be translated into treatment for humans.


Asunto(s)
Receptores de Activinas Tipo II/metabolismo , Malformaciones Arteriovenosas/prevención & control , Células Endoteliales/metabolismo , Telangiectasia Hemorrágica Hereditaria/metabolismo , Receptores de Activinas Tipo II/deficiencia , Receptores de Activinas Tipo II/genética , Alelos , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Malformaciones Arteriovenosas/genética , Modelos Animales de Enfermedad , Endoglina/deficiencia , Endoglina/genética , Endoglina/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Factor 2 de Diferenciación de Crecimiento/metabolismo , Ratones , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , ARN no Traducido , Receptores Notch/genética , Receptores Notch/metabolismo , Vasos Retinianos/anomalías , Transducción de Señal , Piel/irrigación sanguínea , Piel/lesiones , Proteína Smad4/genética , Proteína Smad4/metabolismo , Telangiectasia Hemorrágica Hereditaria/genética , Factor de Crecimiento Transformador beta
2.
Circulation ; 138(5): 513-526, 2018 07 31.
Artículo en Inglés | MEDLINE | ID: mdl-29487140

RESUMEN

BACKGROUND: Heart failure is a growing cause of morbidity and mortality worldwide. Transforming growth factor beta (TGF-ß1) promotes cardiac fibrosis, but also activates counterregulatory pathways that serve to regulate TGF-ß1 activity in heart failure. Bone morphogenetic protein 9 (BMP9) is a member of the TGFß family of cytokines and signals via the downstream effector protein Smad1. Endoglin is a TGFß coreceptor that promotes TGF-ß1 signaling via Smad3 and binds BMP9 with high affinity. We hypothesized that BMP9 limits cardiac fibrosis by activating Smad1 and attenuating Smad3, and, furthermore, that neutralizing endoglin activity promotes BMP9 activity. METHODS: We examined BMP9 expression and signaling in human cardiac fibroblasts and human subjects with heart failure. We used the transverse aortic constriction-induced model of heart failure to evaluate the functional effect of BMP9 signaling on cardiac remodeling. RESULTS: BMP9 expression is increased in the circulation and left ventricle (LV) of human subjects with heart failure and is expressed by cardiac fibroblasts. Next, we observed that BMP9 attenuates type I collagen synthesis in human cardiac fibroblasts using recombinant human BMP9 and a small interfering RNA approach. In BMP9-/- mice subjected to transverse aortic constriction, loss of BMP9 activity promotes cardiac fibrosis, impairs LV function, and increases LV levels of phosphorylated Smad3 (pSmad3), not pSmad1. In contrast, treatment of wild-type mice subjected to transverse aortic constriction with recombinant BMP9 limits progression of cardiac fibrosis, improves LV function, enhances myocardial capillary density, and increases LV levels of pSmad1, not pSmad3 in comparison with vehicle-treated controls. Because endoglin binds BMP9 with high affinity, we explored the effect of reduced endoglin activity on BMP9 activity. Neutralizing endoglin activity in human cardiac fibroblasts or in wild-type mice subjected to transverse aortic constriction-induced heart failure limits collagen production, increases BMP9 protein levels, and increases levels of pSmad1, not pSmad3. CONCLUSIONS: Our results identify a novel functional role for BMP9 as an endogenous inhibitor of cardiac fibrosis attributable to LV pressure overload and further show that treatment with either recombinant BMP9 or disruption of endoglin activity promotes BMP9 activity and limits cardiac fibrosis in heart failure, thereby providing potentially novel therapeutic approaches for patients with heart failure.


Asunto(s)
Factor 2 de Diferenciación de Crecimiento/metabolismo , Factores de Diferenciación de Crecimiento/metabolismo , Insuficiencia Cardíaca/metabolismo , Miocardio/metabolismo , Función Ventricular Izquierda , Remodelación Ventricular , Animales , Modelos Animales de Enfermedad , Endoglina/deficiencia , Endoglina/genética , Endoglina/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Factor 2 de Diferenciación de Crecimiento/deficiencia , Factor 2 de Diferenciación de Crecimiento/genética , Factores de Diferenciación de Crecimiento/genética , Haploinsuficiencia , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/patología , Fosforilación , Recuperación de la Función , Transducción de Señal , Proteína Smad1/metabolismo , Proteína smad3/metabolismo
3.
Mol Cell Endocrinol ; 474: 184-193, 2018 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-29574003

RESUMEN

Beige adipocytes are thermogenic adipocytes with developmental and anatomical properties distinct from those of classical brown adipocytes. Recent studies have revealed several key molecular regulators of beige adipocyte development. CD105, also called endoglin, is a membrane protein composed of TGF-ß receptor complex. It regulates TGF-ß-family signal transduction and vascular formation in vivo. We report here that CD105 maintains the thermogenic gene program of beige adipocytes by regulating Smad2 signaling. Cd105-/- adipocyte precursors showed augmented Smad2 activation and decreased expression of thermogenic genes such as Ucp1 and Prdm16-which encodes a transcriptional regulatory protein for thermogenesis-after adipogenic differentiation. Smad2 signaling augmentation by the constitutively active form of Smad2 decreased the expression of thermogenic genes in beige adipocytes. Loss of thermogenic activity in Cd105-/- beige adipocytes was rescued by Prdm16 expression. These data reveal a novel function of CD105 in beige adipocytes: maintaining their thermogenic program by regulating Smad2 signaling.


Asunto(s)
Adipocitos Beige/metabolismo , Endoglina/metabolismo , Transducción de Señal , Proteína Smad2/metabolismo , Termogénesis , Adipocitos Beige/citología , Animales , Diferenciación Celular/genética , Células Cultivadas , Citoprotección , Proteínas de Unión al ADN/metabolismo , Endoglina/deficiencia , Regulación de la Expresión Génica , Masculino , Ratones Endogámicos C57BL , Fosforilación , Termogénesis/genética , Factores de Transcripción/metabolismo , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo
4.
Nat Cell Biol ; 19(6): 653-665, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28530658

RESUMEN

The hierarchical organization of properly sized blood vessels ensures the correct distribution of blood to all organs of the body, and is controlled via haemodynamic cues. In current concepts, an endothelium-dependent shear stress set point causes blood vessel enlargement in response to higher flow rates, while lower flow would lead to blood vessel narrowing, thereby establishing homeostasis. We show that during zebrafish embryonic development increases in flow, after an initial expansion of blood vessel diameters, eventually lead to vessel contraction. This is mediated via endothelial cell shape changes. We identify the transforming growth factor beta co-receptor endoglin as an important player in this process. Endoglin mutant cells and blood vessels continue to enlarge in response to flow increases, thus exacerbating pre-existing embryonic arterial-venous shunts. Together, our data suggest that cell shape changes in response to biophysical cues act as an underlying principle allowing for the ordered patterning of tubular organs.


Asunto(s)
Forma de la Célula , Endoglina/metabolismo , Células Endoteliales/metabolismo , Hemodinámica , Mecanotransducción Celular , Proteínas de Pez Cebra/metabolismo , Animales , Malformaciones Arteriovenosas/genética , Malformaciones Arteriovenosas/metabolismo , Malformaciones Arteriovenosas/fisiopatología , Endoglina/deficiencia , Endoglina/genética , Predisposición Genética a la Enfermedad , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones Noqueados , Mutación , Neovascularización Fisiológica , Fenotipo , Flujo Sanguíneo Regional , Estrés Mecánico , Factores de Tiempo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética
5.
Nat Cell Biol ; 19(6): 639-652, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28530660

RESUMEN

Loss-of-function (LOF) mutations in the endothelial cell (EC)-enriched gene endoglin (ENG) cause the human disease hereditary haemorrhagic telangiectasia-1, characterized by vascular malformations promoted by vascular endothelial growth factor A (VEGFA). How ENG deficiency alters EC behaviour to trigger these anomalies is not understood. Mosaic ENG deletion in the postnatal mouse rendered Eng LOF ECs insensitive to flow-mediated venous to arterial migration. Eng LOF ECs retained within arterioles acquired venous characteristics and secondary ENG-independent proliferation resulting in arteriovenous malformation (AVM). Analysis following simultaneous Eng LOF and overexpression (OE) revealed that ENG OE ECs dominate tip-cell positions and home preferentially to arteries. ENG knockdown altered VEGFA-mediated VEGFR2 kinetics and promoted AKT signalling. Blockage of PI(3)K/AKT partly normalized flow-directed migration of ENG LOF ECs in vitro and reduced the severity of AVM in vivo. This demonstrates the requirement of ENG in flow-mediated migration and modulation of VEGFR2 signalling in vascular patterning.


Asunto(s)
Malformaciones Arteriovenosas/prevención & control , Endoglina/metabolismo , Células Endoteliales/metabolismo , Neovascularización Patológica , Neovascularización Fisiológica , Transducción de Señal , Telangiectasia Hemorrágica Hereditaria/prevención & control , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Malformaciones Arteriovenosas/genética , Malformaciones Arteriovenosas/metabolismo , Malformaciones Arteriovenosas/patología , Linaje de la Célula , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Endoglina/deficiencia , Endoglina/genética , Células Endoteliales/patología , Predisposición Genética a la Enfermedad , Humanos , Cinética , Ratones Noqueados , Fenotipo , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Estrés Mecánico , Telangiectasia Hemorrágica Hereditaria/genética , Telangiectasia Hemorrágica Hereditaria/metabolismo , Telangiectasia Hemorrágica Hereditaria/patología , Técnicas de Cultivo de Tejidos , Transfección , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética
6.
Oncotarget ; 7(51): 84314-84325, 2016 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-27741515

RESUMEN

Angiogenesis occurs early in tumor development, sustains primary tumor growth and provides a route for metastatic escape. The TGF-ß family receptors modulate angiogenesis via endothelial-cell specific pathways. Here we investigate the interaction of two such receptors, ALK1 and endoglin, in pancreatic neuroendocrine tumors (PanNET). Independently, ALK1 and endoglin deficiencies exhibited genetically divergent phenotypes, while both highly correlate to an endothelial metagene in human and mouse PanNETs. A concurrent deficiency of both receptors synergistically decreased tumor burden to a greater extent than either individual knockdown. Furthermore, the knockout of Gdf2 (BMP9), the primary ligand for ALK1 and endoglin, exhibited a mixed phenotype from each of ALK1 and endoglin deficiencies; overall primary tumor burden decreased, but hepatic metastases increased. Tumors lacking BMP9 display a hyperbranching vasculature, and an increase in vascular mesenchymal-marker expression, which may be implicit in the increase in metastases. Taken together, our work cautions against singular blockade of BMP9 and instead demonstrates the utility of dual blockade of ALK1 and endoglin as a strategy for anti-angiogenic therapy in PanNET.


Asunto(s)
Receptores de Activinas Tipo I/genética , Endoglina/genética , Neovascularización Patológica/genética , Tumores Neuroendocrinos/genética , Neoplasias Pancreáticas/genética , Factor de Crecimiento Transformador beta/genética , Receptores de Activinas Tipo I/deficiencia , Receptores de Activinas Tipo II , Animales , Endoglina/deficiencia , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Factor 2 de Diferenciación de Crecimiento/deficiencia , Factor 2 de Diferenciación de Crecimiento/genética , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neovascularización Patológica/metabolismo , Tumores Neuroendocrinos/metabolismo , Tumores Neuroendocrinos/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Transducción de Señal/genética , Factor de Crecimiento Transformador beta/metabolismo , Carga Tumoral/genética
7.
Angiogenesis ; 19(4): 451-461, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27325285

RESUMEN

An abnormally high number of macrophages are present in human brain arteriovenous malformations (bAVM) with or without evidence of prior hemorrhage, causing unresolved inflammation that may enhance abnormal vascular remodeling and exacerbate the bAVM phenotype. The reasons for macrophage accumulation at the bAVM sites are not known. We tested the hypothesis that persistent infiltration and pro-inflammatory differentiation of monocytes in angiogenic tissues increase the macrophage burden in bAVM using two mouse models and human monocytes. Mouse bAVM was induced through deletion of AVM causative genes, Endoglin (Eng) globally or Alk1 focally, plus brain focal angiogenic stimulation. An endothelial cell and vascular smooth muscle cell co-culture system was used to analyze monocyte differentiation in the angiogenic niche. After angiogenic stimulation, the Eng-deleted mice had fewer CD68(+) cells at 2 weeks (P = 0.02), similar numbers at 4 weeks (P = 0.97), and more at 8 weeks (P = 0.01) in the brain angiogenic region compared with wild-type (WT) mice. Alk1-deficient mice also had a trend toward more macrophages/microglia 8 weeks (P = 0.064) after angiogenic stimulation and more RFP(+) bone marrow-derived macrophages than WT mice (P = 0.01). More CD34(+) cells isolated from peripheral blood of patients with ENG or ALK1 gene mutation differentiated into macrophages than those from healthy controls (P < 0.001). These data indicate that persistent infiltration and pro-inflammatory differentiation of monocytes might contribute to macrophage accumulation in bAVM. Blocking macrophage homing to bAVM lesions should be tested as a strategy to reduce the severity of bAVM.


Asunto(s)
Malformaciones Arteriovenosas Intracraneales/patología , Monocitos/patología , Receptores de Activinas Tipo I/deficiencia , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo II , Animales , Diferenciación Celular , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Endoglina/deficiencia , Endoglina/genética , Células Endoteliales/patología , Humanos , Malformaciones Arteriovenosas Intracraneales/genética , Malformaciones Arteriovenosas Intracraneales/metabolismo , Macrófagos/patología , Ratones , Ratones Noqueados , Ratones Transgénicos , Miocitos del Músculo Liso/patología , Neovascularización Patológica/genética
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