RESUMEN
RATIONALE: The BMPs (bone morphogenetic proteins) are essential morphogens in angiogenesis and vascular development. Disruption of BMP signaling can trigger cardiovascular diseases, such as arteriovenous malformations. OBJECTIVE: A computational model predicted that BMP4 and BMP9 and their inhibitors MGP (matrix gamma-carboxyglutamic acid [Gla] protein) and CV2 (crossveinless-2) would form a regulatory system consisting of negative feedback loops with time delays and that BMP9 would trigger oscillatory expression of the 2 inhibitors. The goal was to investigate this regulatory system in endothelial differentiation and vascular growth. METHODS AND RESULTS: Oscillations in the expression of MGP and CV2 were detected in endothelial cells in vitro, using quantitative real-time polymerase chain reaction and immunoblotting. These organized temporally downstream BMP-related activities, including expression of stalk-cell markers and cell proliferation, consistent with an integral role of BMP9 in vessel maturation. In vivo, the inhibitors were located in distinct zones in relation to the front of the expanding retinal network, as determined by immunofluorescence. Time-dependent changes of the CV2 location in the retina and the existence of an endothelial population with signs of oscillatory MGP expression in developing vasculature supported the in vitro findings. Loss of MGP or its BMP4-binding capacity disrupted the retinal vasculature, resulting in poorly formed networks, especially in the venous drainage areas, and arteriovenous malformations as determined by increased cell coverage and functional testing. CONCLUSIONS: Our results suggest a previously unknown mechanism of temporal orchestration of BMP4 and BMP9 activities that utilize the tandem actions of the extracellular antagonists MGP and CV2. Disruption of this mechanism may contribute to vascular malformations and disease.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Modelos Cardiovasculares , Neovascularización Fisiológica , Animales , Vasos Sanguíneos/crecimiento & desarrollo , Vasos Sanguíneos/metabolismo , Proteínas Morfogenéticas Óseas/genética , Humanos , Proteína Gla de la MatrizRESUMEN
Morbidity in patients with single-ventricle Fontan circulation is common and includes arrhythmias, edema, and pulmonary arteriovenous malformations (PAVM) among others. We sought to identify biomarkers that may predict such complications. Twenty-five patients with Fontan physiology and 12 control patients with atrial septal defects (ASD) that underwent cardiac catheterization were included. Plasma was collected from the hepatic vein and superior vena cava and underwent protein profiling for a panel of 20 analytes involved in angiogenesis and endothelial dysfunction. Ten (40%) of Fontan patients had evidence of PAVM, eighteen (72%) had a history of arrhythmia, and five (20%) were actively in arrhythmia or had a recent arrhythmia. Angiopoietin-2 (Ang-2) was higher in Fontan patients (8,875.4 ± 3,336.9 pg/mL) versus the ASD group (1,663.6 ± 587.3 pg/mL, p < 0.0001). Ang-2 was higher in Fontan patients with active or recent arrhythmia (11,396.0 ± 3,457.7 vs 8,118.2 ± 2,795.1 pg/mL, p < 0.05). A threshold of 8,500 pg/mL gives Ang-2 a negative predictive value of 100% and positive predictive value of 42% in diagnosing recent arrhythmia. Ang-2 is elevated among adults with Fontan physiology. Ang-2 level is associated with active or recent arrhythmia, but was not found to be associated with PAVM.
Asunto(s)
Angiopoyetina 2/sangre , Arritmias Cardíacas/sangre , Proteínas Sanguíneas/genética , Edema/sangre , Procedimiento de Fontan , Adulto , Angiopoyetina 2/genética , Arritmias Cardíacas/patología , Fístula Arteriovenosa/sangre , Malformaciones Arteriovenosas/sangre , Malformaciones Arteriovenosas/fisiopatología , Biomarcadores/sangre , Cateterismo Cardíaco , Edema/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Defectos del Tabique Interatrial/sangre , Defectos del Tabique Interatrial/patología , Humanos , Masculino , Neovascularización Fisiológica , Arteria Pulmonar/anomalías , Venas Pulmonares/anomalíasRESUMEN
Bone morphogenetic protein (BMP) 10, a cardiac-restricted BMP family member, is essential in cardiomyogenesis, especially during trabeculation. Crossveinless-2 (CV2, also known as BMP endothelial cell precursor derived regulator [BMPER]) is a BMP-binding protein that modulates the activity of several BMPs. The objective of this study was to examine the combined effects of BMP10 and CV2 on cardiomyocyte differentiation using mouse dedifferentiated fat (mDFAT) cells, which spontaneously differentiate into cardiomyocyte-like cells, as a model. Our results revealed that CV2 binds directly to BMP10, as determined by co-immunoprecipitation, and inhibits BMP10 from initiating SMAD signaling, as determined by luciferase reporter gene assays. BMP10 treatment induced mDFAT cell proliferation, whereas CV2 modulated the BMP10-induced proliferation. Differentiation of cardiomyocyte-like cells proceeded in a reproducible fashion in mDFAT cells, starting with small round Nkx2.5-positive progenitor cells that progressively formed myotubes of increasing length that assembled into beating colonies and stained strongly for Troponin I and sarcomeric alpha-actinin. BMP10 enhanced proliferation of the small progenitor cells, thereby securing sufficient numbers to support formation of myotubes. CV2, on the other hand, enhanced formation and maturation of large myotubes and myotube-colonies and was expressed by endothelial-like cells in the mDFAT cultures. Thus BMP10 and CV2 have important roles in coordinating cardiomyogenesis in progenitor cells.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Portadoras/metabolismo , Diferenciación Celular/fisiología , Miocitos Cardíacos/citología , Células Madre/citología , Actinina/metabolismo , Adipocitos/citología , Animales , Proliferación Celular , Células Cultivadas , Proteína Homeótica Nkx-2.5/metabolismo , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/fisiología , Proteínas Smad/metabolismo , Troponina I/metabolismoRESUMEN
The vascular endothelium is critical for induction of appropriate lineage differentiation in organogenesis. In this study, we report that dysfunctional pulmonary endothelium, resulting from the loss of matrix Gla protein (MGP), causes ectopic hepatic differentiation in the pulmonary epithelium. We demonstrate uncontrolled induction of the hepatic growth factor (HGF) caused by dysregulated cross talk between pulmonary endothelium and epithelium in Mgp-null lungs. Elevated HGF induced hepatocyte nuclear factor 4 α (Hnf4a), which competed with NK2 homeobox 1 (Nkx2.1) for binding to forkhead box A2 (Foxa2) to drive hepatic differentiation in Mgp-null airway progenitor cells. Limiting endothelial HGF reduced Hnf4a, abolished interference of Hnf4a with Foxa2, and reduced hepatic differentiation in Mgp-null lungs. Together, our results suggest that endothelial-epithelial interactions, maintained by MGP, are essential in pulmonary cell differentiation.
Asunto(s)
Comunicación Celular , Diferenciación Celular , Endotelio Vascular/metabolismo , Células Epiteliales/metabolismo , Mucosa Respiratoria/metabolismo , Animales , Endotelio Vascular/citología , Células Epiteliales/citología , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Ratones , Ratones Noqueados , Proteínas/genética , Proteínas/metabolismo , Mucosa Respiratoria/citologíaRESUMEN
Endothelial-mesenchymal transition (EndMT) drives endothelium to contribute to normal development and disease processes. Here, we report that EndMTs occur in the diabetic endothelium of Ins2Akita/wt mouse, and show that induction of sex determining region Y-box 2 (Sox2) is a mediator of excess BMP signaling that results in activation of EndMTs and increased vascular calcification. We also find an induction of a complex of serine proteases in the diabetic endothelium, required for the up-regulation of Sox2. Our results suggest that EndMTs contribute to vascular calcification in diabetic arteries.
Asunto(s)
Vasos Sanguíneos/patología , Calcinosis/patología , Endotelio Vascular/patología , Insulina/genética , Mesodermo/patología , Animales , Diabetes Mellitus Experimental/genética , Endotelio Vascular/enzimología , Ratones , Ratones Transgénicos , Factores de Transcripción SOXB1/fisiología , Serina Proteasas/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND AND AIMS: Endothelial-mesenchymal transitions (EndMTs) in endothelial cells (ECs) contribute to vascular disease. METHODS: We used ApoE-/- mice fed a high-fat/high-cholesterol diet. RESULTS: We reported evidence of EndMT in atherosclerotic lesions contributing to calcification. Stem cell and mesenchymal markers, including sex-determining region Y-box 2 (Sox2), were upregulated in aortic ECs of fat-fed ApoE-/- mice. Limiting Sox2 decreased marker expression and calcification in ApoE-/- aortas. Furthermore, a complex of serine proteases was upregulated in ApoE-/- aortic ECs. Blockade of these proteases reduced expression of Sox2 and atherosclerotic lesion calcification. CONCLUSIONS: Together, our data suggest that EndMTs contribute to atherosclerotic lesion calcification.
Asunto(s)
Aorta/citología , Aterosclerosis/sangre , Calcinosis/sangre , Células Endoteliales/citología , Transición Epitelial-Mesenquimal , Factores de Transcripción SOXB1/genética , Animales , Aorta/metabolismo , Colesterol/metabolismo , Citocinas/metabolismo , Endotelio Vascular/metabolismo , Humanos , Ratones , Ratones Noqueados para ApoE , Placa Aterosclerótica/patología , Factores de Transcripción SOXB1/metabolismo , Serina Endopeptidasas/metabolismo , Células Madre/citología , Regulación hacia ArribaRESUMEN
Matrix Gla protein (MGP) is an antagonist of bone morphogenetic proteins and expressed in vascular endothelial cells. Lack of MGP causes vascular abnormalities in multiple organs in mice. The objective of this study is to define the role of MGP in early endothelial differentiation. We find that expression of endothelial markers is highly induced in Mgp null organs, which, in wild type, contain high MGP expression. Furthermore, Mgp null embryonic stem cells express higher levels of endothelial markers than wild-type controls and an abnormal temporal pattern of expression. We also find that the Mgp-deficient endothelial cells adopt characteristics of mesenchymal stem cells. We conclude that loss of MGP causes dysregulation of early endothelial differentiation.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Diferenciación Celular , Células Endoteliales/citología , Células Endoteliales/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Células Madre Embrionarias de Ratones/citología , Animales , Biomarcadores/metabolismo , Proteínas de Unión al Calcio/deficiencia , Recuento de Células , Proteínas de la Matriz Extracelular/deficiencia , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína Gla de la MatrizRESUMEN
RATIONALE: Endothelial cells have the ability to undergo endothelial-mesenchymal transitions (EndMTs), by which they acquire a mesenchymal phenotype and stem cell-like characteristics. We previously found that EndMTs occurred in the endothelium deficient in matrix γ-carboxyglutamic acid protein enabling endothelial cells to contribute cells to vascular calcification. However, the mechanism responsible for initiating EndMTs is not fully understood. OBJECTIVE: To determine the role of specific serine proteases and sex determining region Y-box 2 (Sox2) in the initiation of EndMTs. METHODS AND RESULTS: In this study, we used in vivo and in vitro models of vascular calcification to demonstrate that serine proteases and Sox2 are essential for the initiation of EndMTs in matrix γ-carboxyglutamic acid protein-deficient endothelium. We showed that expression of a group of specific serine proteases was highly induced in endothelial cells at sites of vascular calcification in Mgp null aortas. Treatment with serine protease inhibitors decreased both stem cell marker expression and vascular calcification. In human aortic endothelial cells, this group of serine proteases also induced EndMTs, and the activation of proteases was mediated by Sox2. Knockdown of the serine proteases or Sox2 diminished EndMTs and calcification. Endothelial-specific deletion of Sox2 decreased expression of stem cell markers and aortic calcification in matrix γ-carboxyglutamic acid protein-deficient mice. CONCLUSIONS: Our results suggest that Sox2-mediated activation of specific serine proteases is essential for initiating EndMTs, and thus, may provide new therapeutic targets for treating vascular calcification.
Asunto(s)
Calcinosis , Endotelio Vascular/metabolismo , Mesodermo/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Células Cultivadas , Endotelio Vascular/patología , Endotelio Vascular/ultraestructura , Activación Enzimática , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica/métodos , Humanos , Immunoblotting , Calicreínas/genética , Calicreínas/metabolismo , Mesodermo/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Elastasa Pancreática/genética , Elastasa Pancreática/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Serina Endopeptidasas/genética , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismo , Proteína Gla de la MatrizRESUMEN
Mutations in ABCC6 (ATP-binding cassette, subfamily C, member 6), an orphan transporter expressed in the liver, are the cause of pseudoxanthoma elasticum. Since ABCC6 was reported to affect matrix Gla protein (MGP), an inhibitor of bone morphogenetic proteins (BMPs), we studied BMP signaling and expression in various tissues of mice with and without functional ABCC. Enhanced BMP signaling was found in all examined tissues in the absence of ABCC6. Despite this, the expression of particular BMP proteins varied widely between tissues. Interestingly, the expression of most BMP proteins in the liver moved in the opposite direction to the same BMP proteins in kidneys in response to ABCC6 alterations. Thus, ABCC6 deficiency stimulates BMP signaling by acting on the expression of multiple BMPs.
RESUMEN
Cerebral arteriovenous malformations (AVMs) are common vascular malformations, which may result in hemorrhagic strokes and neurological deficits. Bone morphogenetic protein (BMP) and Notch signaling are both involved in the development of cerebral AVMs, but the cross-talk between the two signaling pathways is poorly understood. Here, we show that deficiency of matrix Gla protein (MGP), a BMP inhibitor, causes induction of Notch ligands, dysregulation of endothelial differentiation, and the development of cerebral AVMs in MGP null (Mgp(-/-)) mice. Increased BMP activity due to the lack of MGP induces expression of the activin receptor-like kinase 1, a BMP type I receptor, in cerebrovascular endothelium. Subsequent activation of activin receptor-like kinase 1 enhances expression of Notch ligands Jagged 1 and 2, which increases Notch activity and alters the expression of Ephrin B2 and Ephrin receptor B4, arterial and venous endothelial markers, respectively. Reducing the expression of Jagged 1 and 2 in the Mgp(-/-) mice by crossing them with Jagged 1 or 2 deficient mice reduces Notch activity, normalizes endothelial differentiation, and prevents cerebral AVMs, but not pulmonary or renal AVMs. Our results suggest that Notch signaling mediates and can modulate changes in BMP signaling that lead to cerebral AVMs.