RESUMO
Endothelial-to-mesenchymal transition (EndMT), a process initiated by activation of endothelial TGF-ß signaling, underlies numerous chronic vascular diseases and fibrotic states. Once induced, EndMT leads to a further increase in TGF-ß signaling, thus establishing a positive-feedback loop with EndMT leading to more EndMT. Although EndMT is understood at the cellular level, the molecular basis of TGF-ß-driven EndMT induction and persistence remains largely unknown. Here, we show that metabolic modulation of the endothelium, triggered by atypical production of acetate from glucose, underlies TGF-ß-driven EndMT. Induction of EndMT suppresses the expression of the enzyme PDK4, which leads to an increase in ACSS2-dependent Ac-CoA synthesis from pyruvate-derived acetate. This increased Ac-CoA production results in acetylation of the TGF-ß receptor ALK5 and SMADs 2 and 4 leading to activation and long-term stabilization of TGF-ß signaling. Our results establish the metabolic basis of EndMT persistence and unveil novel targets, such as ACSS2, for the potential treatment of chronic vascular diseases.
Assuntos
Células Endoteliais , Doenças Vasculares , Humanos , Células Endoteliais/metabolismo , Transdução de Sinais , Endotélio/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Doenças Vasculares/metabolismoRESUMO
Thoracic aortic aneurysms (TAA) in Marfan syndrome, caused by fibrillin-1 mutations, are characterized by elevated cytokines and fragmentated elastic laminae in the aortic wall. This study explored whether and how specific fibrillin-1-regulated miRNAs mediate inflammatory cytokine expression and elastic laminae degradation in TAA. miRNA expression profiling at early and late TAA stages using a severe Marfan mouse model (Fbn1mgR/mgR) revealed a spectrum of differentially regulated miRNAs. Bioinformatic analyses predicted the involvement of these miRNAs in inflammatory and extracellular matrix-related pathways. We demonstrate that upregulation of pro-inflammatory cytokines and matrix metalloproteinases is a common characteristic of mouse and human TAA tissues. miR-122, the most downregulated miRNA in the aortae of 10-week-old Fbn1mgR/mgR mice, post-transcriptionally upregulated CCL2, IL-1ß and MMP12. Similar data were obtained at 70 weeks of age using Fbn1C1041G/+ mice. Deficient fibrillin-1-smooth muscle cell interaction suppressed miR-122 levels. The marker for tissue hypoxia HIF-1α was upregulated in the aortic wall of Fbn1mgR/mgR mice, and miR-122 was reduced under hypoxic conditions in cell and organ cultures. Reduced miR-122 was partially rescued by HIF-1α inhibitors, digoxin and 2-methoxyestradiol in aortic smooth muscle cells. Digoxin-treated Fbn1mgR/mgR mice demonstrated elevated miR-122 and suppressed CCL2 and MMP12 levels in the ascending aortae, with reduced elastin fragmentation and aortic dilation. In summary, this study demonstrates that miR-122 in the aortic wall inhibits inflammatory responses and matrix remodeling, which is suppressed by deficient fibrillin-1-cell interaction and hypoxia in TAA.
Assuntos
Aneurisma da Aorta Torácica , Síndrome de Marfan , MicroRNAs , Animais , Aneurisma da Aorta Torácica/genética , Aneurisma da Aorta Torácica/metabolismo , Citocinas , Digoxina , Modelos Animais de Doenças , Fibrilina-1/genética , Fibrilina-1/metabolismo , Humanos , Hipóxia/genética , Síndrome de Marfan/complicações , Síndrome de Marfan/genética , Metaloproteinase 12 da Matriz , MicroRNAs/genéticaRESUMO
A 4 month-old, 14.8 kg, male Newfoundland dog was presented for cardiovascular evaluation following detection of a heart murmur. Echocardiography revealed enlargement of the sinuses of Valsalva and marked, diffuse dilation of the ascending aorta (annuloaortic ectasia, AAE), with mild/equivocal subaortic stenosis (SAS). The dog was monitored over the duration of its lifetime, with serial echocardiograms performed at 5, 6, and 8 months and 1, 2, 3, 4, 8, and 10 years demonstrating persistent, diffuse dilation of the ascending aorta. The dog lived until it was 10 years old and died of metastatic carcinoma. Postmortem examination confirmed AAE and mild SAS. Hematoxylin and eosin and Weigert van Gieson stains were used to compare the ascending aorta to the descending aorta and left subclavian artery, and to compare aortic samples to those of three control dogs. Histopathologic evaluation revealed mild medial degeneration in the ascending aorta of all four dogs. Immunofluorescent microscopy was used for determining the deposition of proteins known to play a role in aortic aneurysms in humans: fibrillin-1 (FBN1), latent transforming growth factor beta binding protein 4 (LTBP4) and fibronectin. The ascending aorta of the AAE case demonstrated reduced deposition of FBN1, indicating that its loss may have contributed to aortic dilation. Diffuse, primary ascending aortic dilation is uncommonly reported in dogs; when it is, it carries a poor prognosis. This case provides an important example of marked dilation of the ascending aorta in a dog that lived with no associated adverse effects for 10 years.
Assuntos
Aneurisma da Aorta Torácica , Aneurisma Aórtico , Doenças do Cão , Síndrome de Marfan , Animais , Aneurisma Aórtico/veterinária , Aneurisma da Aorta Torácica/veterinária , Doenças do Cão/diagnóstico por imagem , Cães , Seguimentos , Masculino , Síndrome de Marfan/veterináriaRESUMO
Fibrillin-1 is an extracellular matrix protein which contains one conserved RGD integrin-binding motif. It constitutes the backbone of microfibrils in many tissues, and mutations in fibrillin-1 cause various connective tissue disorders. Although it is well established that fibrillin-1 interacts with several RGD-dependent integrins, very little is known about the associated intracellular signaling pathways. Recent published evidence identified a subset of miRNAs regulated by fibrillin-1 RGD-cell adhesion, with miR-1208 among the most downregulated. The present study shows that the downregulated miR-1208 controls fibroblast proliferation. Inhibitor experiments revealed that fibrillin-1 RGD suppressed miR-1208 expression via c-Src kinase and the downstream JNK signaling. Bioinformatic prediction and experimental target sequence validation demonstrated four miR-1208 binding sites on the ERK2 mRNA and one on the MEK1 mRNA. ERK2 and MEK1 are critical proliferation-promoting kinases. Decreased miR-1208 levels elevated the total and phosphorylated ERK1/2 and MEK1/2 protein levels and the phosphorylated to total ERK1/2 ratio. Together, the data demonstrate a novel outside-in signaling mechanism explaining how fibrillin-1 RGD-cell binding regulates fibroblast proliferation.
Assuntos
Fibrilina-1/metabolismo , Fibroblastos/citologia , MicroRNAs/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Oligopeptídeos/metabolismo , Processamento Pós-Transcricional do RNA , Proliferação de Células , Células Cultivadas , Fibrilina-1/genética , Fibroblastos/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Oligopeptídeos/genéticaRESUMO
ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs) proteins regulate tissue homeostasis and extracellular matrix (ECM)-related pathogenesis. Some ADAMTS proteins interact with or process multiple ECM proteins, including fibrillins, fibronectin, and collagens. Therefore, characterization and quantification of these ECM fiber systems is essential to understand their functional relationship with ADAMTS proteins. Here we describe unbiased methods to quantify various aspects of ADAMTS-related ECM fiber systems in cell culture and in tissues. We focus on cell counting, overall fiber intensity, fiber length, and focal adhesion analysis in cell culture, and on the quantification of immunohistochemical and immunofluorescent tissue sections. We use ImageJ/Fiji, a widely used Java-based open source software which provides efficient and customizable quantification methods for microscopy images.
Assuntos
Proteínas ADAMTS/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Animais , Contagem de Células , Células Cultivadas , Matriz Extracelular/metabolismo , Humanos , Camundongos , SoftwareRESUMO
Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and non-elastic tissues. Fibrillin-1 contains one evolutionarily conserved RGD sequence that mediates cell-matrix interactions through cell-surface integrins. Here, we present a novel paradigm how extracellular fibrillin-1 controls cellular function through integrin-mediated microRNA regulation. Comparative mRNA studies by global microarray analysis identified growth factor activity, actin binding and integrin binding as the most important functional groups that are regulated upon fibrillin-1 binding to dermal fibroblasts. Many of these mRNAs are targets of miRNAs that were identified when RNA from the fibrillin-1-ligated fibroblasts was analyzed by a miRNA microarray. The expression profile was specific to fibrillin-1 since interaction with fibronectin displayed a partially distinct profile. The importance of selected miRNAs for the regulation of the identified mRNAs was suggested by bioinformatics prediction and the interactions between miRNAs and mRNAs were experimentally validated. Functionally, we show that miR-503 controls p-Smad2-dependent TGF-ß signaling, and that miR-612 and miR-3185 are involved in the focal adhesion formation regulated by fibrillin-1. In conclusion, we demonstrate that fibrillin-1 interaction with fibroblasts regulates miRNA expression profiles which in turn control critical cell functions.