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1.
J Vasc Res ; 59(5): 261-274, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35797968

RESUMEN

INTRODUCTION: We previously identified Notch2 in smooth muscle cells (SMC) in human atherosclerosis and found that signaling via Notch2 suppressed human SMC proliferation. Thus, we tested whether loss of Notch2 in SMC would alter atherosclerotic plaque progression using a mouse model. METHODS: Atherogenesis was examined at the brachiocephalic artery and aortic root in a vascular SMC null (inducible smooth muscle myosin heavy chain Cre) Notch2 strain on the ApoE-/- background. We measured plaque morphology and size, as well as lipid, inflammation, and smooth muscle actin content after Western diet. RESULTS: We generated an inducible SMC Notch2 null on the ApoE-/- background. We observed ∼90% recombination efficiency with no detectable Notch2 in the SMC. Loss of SMC Notch2 did not significantly change plaque size, lipid content, necrotic core, or medial area. However, loss of SMC Notch2 reduced the contractile SMC in brachiocephalic artery lesions and increased inflammatory content in aortic root lesions after 6 weeks of Western diet. These changes were not present with loss of SMC Notch2 after 14 weeks of Western diet. CONCLUSIONS: Our data show that loss of SMC Notch2 does not significantly reduce atherosclerotic lesion formation, although in early stages of plaque formation there are changes in SMC and inflammation.


Asunto(s)
Aterosclerosis , Miocitos del Músculo Liso , Placa Aterosclerótica , Receptor Notch2 , Animales , Ratones , Actinas , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Receptor Notch2/genética , Receptor Notch2/metabolismo , Miosinas del Músculo Liso
2.
Arterioscler Thromb Vasc Biol ; 40(9): 2227-2243, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32640901

RESUMEN

OBJECTIVE: Perivascular adipose tissue (PVAT) surrounding arteries supports healthy vascular function. During obesity, PVAT loses its vasoprotective effect. We study pathological conversion of PVAT, which involves molecular changes in protein profiles and functional changes in adipocytes. Approach and Results: C57BL6/J mice were fed a 60% high-fat diet for 12 weeks or a cardioprotective 30% calorie-restricted diet for 5 weeks. Proteomic analysis identified PVAT as a molecularly distinct adipose depot, and novel markers for thermogenic adipocytes, such as GRP75 (stress-70 protein, mitochondrial), were identified. High-fat diet increased the similarity of protein signatures in PVAT and brown adipose, suggesting activation of a conserved whitening pathway. The whitening phenotype was characterized by suppression of UCP1 (uncoupling protein 1) and increased lipid deposition, leptin, and inflammation, and specifically in PVAT, elevated Notch signaling. Conversely, PVAT from calorie-restricted mice had decreased Notch signaling and less lipid. Using the Adipoq-Cre strain, we constitutively activated Notch1 signaling in adipocytes, which phenocopied the changes in PVAT caused by a high-fat diet, even on a standard diet. Preadipocytes from mouse PVAT expressed Sca1, CD140a, Notch1, and Notch2, but not CD105, showing differences compared with preadipocytes from other depots. Inhibition of Notch signaling during differentiation of PVAT-derived preadipocytes reduced lipid deposition and adipocyte marker expression. CONCLUSIONS: PVAT shares features with other adipose depots, but has a unique protein signature that is regulated by dietary stress. Increased Notch signaling in PVAT is sufficient to initiate the pathological conversion of PVAT by promoting adipogenesis and lipid accumulation and may thus prime the microenvironment for vascular disease.


Asunto(s)
Adipocitos Blancos/metabolismo , Adipogénesis , Tejido Adiposo Blanco/metabolismo , Lipogénesis , Obesidad/metabolismo , Receptores Notch/metabolismo , Adipocitos Blancos/patología , Tejido Adiposo Blanco/patología , Adiposidad , Animales , Ataxina-1/metabolismo , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Restricción Calórica , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Endoglina/metabolismo , Femenino , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Obesidad/genética , Obesidad/patología , Fenotipo , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/metabolismo , Proteómica , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores Notch/genética , Transducción de Señal
3.
Lab Invest ; 99(3): 290-304, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-29795127

RESUMEN

Atherosclerosis is the most common cause of heart disease and stroke. The use of animal models has advanced our understanding of the molecular signaling that contributes to atherosclerosis. Further understanding of this degenerative process in humans will require human tissue. Plaque removed during endarterectomy procedures to relieve arterial obstructions is usually discarded, but can be an important source of diseased cells. Resected tissue from carotid and femoral endarterectomy procedures were compared with carotid arteries from donors with no known cardiovascular disease. Vascular smooth muscle cells (SMC) contribute to plaque formation and may determine susceptibility to rupture. Notch signaling is implicated in the progression of atherosclerosis, and plays a receptor-specific regulatory role in SMC. We defined protein localization of Notch2 and Notch3 within medial and plaque SMC using immunostaining, and compared Notch2 and Notch3 levels in total plaques with whole normal arteries using immunoblot. We successfully derived SMC populations from multiple endarterectomy specimens for molecular analysis. To better define the protein signature of diseased SMC, we utilized sequential window acquisition of all theoretical spectra (SWATH) proteomic analysis to compare normal carotid artery SMC with endarterectomy-derived SMC. Similarities in protein profile and differentiation markers validated the SMC identity of our explants. We identified a subset of differentially expressed proteins that are candidates as functional markers of diseased SMC. To understand how Notch signaling may affect diseased SMC, we performed Jagged1 stimulation of primary cultures. In populations that displayed significant growth, Jagged1 signaling through Notch2 suppressed proliferation; cultures with low growth potential were non-responsive to Jagged1. In addition, Jagged1 did not promote contractile smooth muscle actin nor have a significant effect on the mature differentiated phenotype. Thus, SMC derived from atherosclerotic lesions show distinct proteomic profiles and have altered Notch signaling in response to Jagged1 as a differentiation stimulus, compared with normal SMC.


Asunto(s)
Aterosclerosis/metabolismo , Aterosclerosis/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Receptores Notch/metabolismo , Anciano , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Proliferación Celular , Células Cultivadas , Endarterectomía , Femenino , Humanos , Inmunohistoquímica , Proteína Jagged-1/metabolismo , Masculino , Persona de Mediana Edad , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología , Receptor Notch2/metabolismo , Receptor Notch3/metabolismo , Transducción de Señal
4.
Cardiovasc Drugs Ther ; 32(5): 519-530, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30105417

RESUMEN

PURPOSE: Perivascular adipose tissue (PVAT) surrounds blood vessels and regulates vascular tone through paracrine secretion of cytokines. During conditions promoting cardiometabolic dysfunction, such as obesity, cytokine secretion is altered towards a proinflammatory and proatherogenic profile. Despite the clinical implications for cardiovascular disease, studies addressing the biology of human PVAT remain limited. We are interested in characterizing the resident adipose progenitor cells (APCs) because of their potential role in PVAT expansion during obesity. We also focused on proteins regulating paracrine interactions, including the small GTPase Rab27a, which regulates protein trafficking and secretion. METHODS: PVAT from the ascending aorta was collected from patients with severe cardiovascular disease undergoing coronary artery bypass grafting (CABG). Freshly-isolated PVAT was digested and APC expanded in culture for characterizing progenitor markers, evaluating adipogenic potential and assessing the function(s) of Rab27a. RESULTS: Using flow cytometry, RT-PCR, and immunoblot, we characterized APC from human PVAT as negative for CD45 and CD31 and expressing CD73, CD105, and CD140A. These APCs differentiate into multilocular, UCP1-producing adipocytes in vitro. Rab27a was detected in interstitial cells of human PVAT in vivo and along F-actin tracks of PVAT-APC in vitro. Knockdown of Rab27a using siRNA in PVAT-APC prior to induction resulted in a marked reduction in lipid accumulation and reduced expression of adipogenic differentiation markers. CONCLUSIONS: PVAT-APC from CABG donors express common adipocyte progenitor markers and differentiate into UCP1-containing adipocytes. Rab27a has an endogenous role in promoting the maturation of adipocytes from human PVAT-derived APC.


Asunto(s)
Adipogénesis , Tejido Adiposo/enzimología , Células Madre/enzimología , Proteínas rab27 de Unión a GTP/metabolismo , Tejido Adiposo/citología , Adulto , Anciano , Aorta , Biomarcadores/metabolismo , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Humanos , Metabolismo de los Lípidos , Masculino , Persona de Mediana Edad , Fenotipo , Transducción de Señal , Proteínas rab27 de Unión a GTP/genética
5.
Arterioscler Thromb Vasc Biol ; 38(7): 1576-1593, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29853569

RESUMEN

OBJECTIVE: Vascular remodeling is associated with complex molecular changes, including increased Notch2, which promotes quiescence in human smooth muscle cells. We used unbiased protein profiling to understand molecular signatures related to neointimal lesion formation in the presence or absence of Notch2 and to test the hypothesis that loss of Notch2 would increase neointimal lesion formation because of a hyperproliferative injury response. APPROACH AND RESULTS: Murine carotid arteries isolated at 6 or 14 days after ligation injury were analyzed by mass spectrometry using a data-independent acquisition strategy in comparison to uninjured or sham injured arteries. We used a tamoxifen-inducible, cell-specific Cre recombinase strain to delete the Notch2 gene in smooth muscle cells. Vessel morphometric analysis and immunohistochemical staining were used to characterize lesion formation, assess vascular smooth muscle cell proliferation, and validate proteomic findings. Loss of Notch2 in smooth muscle cells leads to protein profile changes in the vessel wall during remodeling but does not alter overall lesion morphology or cell proliferation. Loss of smooth muscle Notch2 also decreases the expression of enhancer of rudimentary homolog, plectin, and annexin A2 in vascular remodeling. CONCLUSIONS: We identified unique protein signatures that represent temporal changes in the vessel wall during neointimal lesion formation in the presence and absence of Notch2. Overall lesion formation was not affected with loss of smooth muscle Notch2, suggesting compensatory pathways. We also validated the regulation of known injury- or Notch-related targets identified in other vascular contexts, providing additional insight into conserved pathways involved in vascular remodeling.


Asunto(s)
Traumatismos de las Arterias Carótidas/metabolismo , Espectrometría de Masas , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Neointima , Proteómica/métodos , Receptor Notch2/metabolismo , Remodelación Vascular , Anciano , Anciano de 80 o más Años , Animales , Anexina A2/metabolismo , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/patología , Arteria Carótida Común/metabolismo , Arteria Carótida Común/patología , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Plectina/metabolismo , Receptor Notch2/deficiencia , Receptor Notch2/genética , Transducción de Señal , Factores de Transcripción/metabolismo
6.
Arterioscler Thromb Vasc Biol ; 35(12): 2626-37, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26471266

RESUMEN

OBJECTIVE: Bone morphogenetic protein-9 (BMP9)/activin-like kinase-1 and delta-like 4 (DLL4)/Notch promote endothelial quiescence, and we aim to understand mechanistic interactions between the 2 pathways. We identify new targets that contribute to endothelial quiescence and test whether loss of Dll4(+/-) in adult vasculature alters BMP signaling. APPROACH AND RESULTS: Human endothelial cells respond synergistically to BMP9 and DLL4 stimulation, showing complete quiescence and induction of HEY1 and HEY2. Canonical BMP9 signaling via activin-like kinase-1-Smad1/5/9 was disrupted by inhibition of Notch signaling, even in the absence of exogenous DLL4. Similarly, DLL4 activity was suppressed when the basal activin-like kinase-1-Smad1/5/9 pathway was inhibited, showing that these pathways are interdependent. BMP9/DLL4 required induction of P27(KIP1) for quiescence, although multiple factors are involved. To understand these mechanisms, we used proteomics data to identify upregulation of thrombospondin-1, which contributes to the quiescence phenotype. To test whether Dll4 regulates BMP/Smad pathways and endothelial cell phenotype in vivo, we characterized the vasculature of Dll4(+/-) mice, analyzing endothelial cells in the lung, heart, and aorta. Together with changes in endothelial structure and vascular morphogenesis, we found that loss of Dll4 was associated with a significant upregulation of pSmad1/5/9 signaling in lung endothelial cells. Because steady-state endothelial cell proliferation rates were not different in the Dll4(+/-) mice, we propose that the upregulation of pSmad1/5/9 signaling compensates to maintain endothelial cell quiescence in these mice. CONCLUSIONS: DLL4/Notch and BMP9/activin-like kinase-1 signaling rely on each other's pathways for full activity. This represents an important mechanism of cross talk that enhances endothelial quiescence and sensitively coordinates cellular responsiveness to soluble and cell-tethered ligands.


Asunto(s)
Senescencia Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Células Endoteliales/metabolismo , Factores de Diferenciación de Crecimiento/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Receptor Notch1/metabolismo , Trombospondina 1/metabolismo , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Aorta/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al Calcio , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Células Cultivadas , Vasos Coronarios/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Genotipo , Factor 2 de Diferenciación de Crecimiento , Humanos , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Pulmón/irrigación sanguínea , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Interferencia de ARN , Receptor Notch1/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Proteínas Smad Reguladas por Receptores/genética , Proteínas Smad Reguladas por Receptores/metabolismo , Trombospondina 1/genética , Transfección
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