RESUMEN
Objective: Very few clinical predictors of descending thoracic aorta dissection have been determined. Although aneurysms can dissect in a size-dependent process, most descending dissections will occur without prior enlargement. We compared the proteomic profiles of normal, dissected, aneurysm, and both aneurysm and dissected descending thoracic aortas to identify novel biomarkers and further understand the molecular pathways that lead to tissue at risk of dissection. Methods: We performed proteomic profiling of descending thoracic aortas with four phenotypes: normal (n = 46), aneurysm (n = 22), dissected (n = 12), and combined aneurysm and dissection (n = 8). Pairwise differential protein expression analyses using a Bayesian approach were then performed to identify common proteins that were dysregulated between each diseased tissue type and control aorta and to uncover unique proteins between aneurysmal and dissected aortas. Network and Markov cluster algorithms of differentially expressed proteins were used to find enriched ontology processes. A convex analysis of mixtures was also performed to identify the molecular subtypes within the different tissue types. Results: The diseased aortas had 71 common differentially expressed proteins compared with the control, including higher amounts of the protein thrombospondin 1. We found 42 differentially expressed proteins between the aneurysm and dissected tissue, with an abundance of apolipoproteins in the former and higher quantities of extracellular matrix proteins in the latter. The convex analysis of mixtures showed enhancement of a molecular subtype enriched in contractile proteins within the control tissue compared with the diseased tissue, in addition to increased proportions of molecular subtypes enriched in inflammation and red blood cell expression in the aneurysmal compared with the dissected tissue. Conclusions: We found some overlapping differentially expressed proteins in aneurysmal and nonaneurysmal descending thoracic aortas at risk of dissection compared with normal aortas. However, we also found uniquely altered molecular pathways that might uncover mechanisms for dissection.
RESUMEN
Mitochondria are the major source of cellular energy (ATP), as well as critical mediators of widespread functions such as cellular redox balance, apoptosis, and metabolic flux. The organelles play an especially important role in the maintenance of cardiac homeostasis; their inability to generate ATP following impairment due to ischemic damage has been directly linked to organ failure. Methods to quantify mitochondrial content are limited to low throughput immunoassays, measurement of mitochondrial DNA, or relative quantification by untargeted mass spectrometry. Here, we present a high throughput, reproducible and quantitative mass spectrometry multiple reaction monitoring based assay of 37 proteins critical to central carbon chain metabolism and overall mitochondrial function termed 'MitoPlex'. We coupled this protein multiplex with a parallel analysis of the central carbon chain metabolites (219 metabolite assay) extracted in tandem from the same sample, be it cells or tissue. In tests of its biological applicability in cells and tissues, "MitoPlex plus metabolites" indicated profound effects of HMG-CoA Reductase inhibition (e.g., statin treatment) on mitochondria of i) differentiating C2C12 skeletal myoblasts, as well as a clear opposite trend of statins to promote mitochondrial protein expression and metabolism in heart and liver, while suppressing mitochondrial protein and ii) aspects of metabolism in the skeletal muscle obtained from C57Bl6 mice. Our results not only reveal new insights into the metabolic effect of statins in skeletal muscle, but present a new high throughput, reliable MS-based tool to study mitochondrial dynamics in both cell culture and in vivo models.
Asunto(s)
Espectrometría de Masas , Metabolómica/métodos , Proteínas Mitocondriales/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Cromatografía Liquida/métodos , Ciclo del Ácido Cítrico/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Espectrometría de Masas/métodos , Espectrometría de Masas/normas , Metabolómica/normas , Ratones , Mitocondrias Musculares/efectos de los fármacos , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Reproducibilidad de los Resultados , Simvastatina/farmacología , Ubiquinona/farmacologíaRESUMEN
The objective of the present study was to 1) analyze the ascending aortic proteome within a mouse model of Marfan syndrome (MFS; Fbn1C1041G/+) at early and late stages of aneurysm and 2) subsequently test a novel hypothesis formulated on the basis of this unbiased proteomic screen that links changes in integrin composition to transforming growth factor (TGF)-ß-dependent activation of the rapamycin-independent component of mammalian target of rapamycin (Rictor) signaling pathway. Ingenuity Pathway Analysis of over 1,000 proteins quantified from the in vivo MFS mouse aorta by data-independent acquisition mass spectrometry revealed a predicted upstream regulator, Rictor, that was selectively activated in aged MFS mice. We validated this pattern of Rictor activation in vivo by Western blot analysis for phosphorylation on Thr1135 in a separate cohort of mice and showed in vitro that TGF-ß activates Rictor in an integrin-linked kinase-dependent manner in cultured aortic vascular smooth muscle cells. Expression of ß3-integrin was upregulated in the aged MFS aorta relative to young MFS mice and wild-type mice. We showed that ß3-integrin expression and activation modulated TGF-ß-induced Rictor phosphorylation in vitro, and this signaling effect was associated with an altered vascular smooth muscle cell proliferative-migratory and metabolic in vitro phenotype that parallels the in vivo aneurysm phenotype in MFS. These results reveal that Rictor is a novel, context-dependent, noncanonical TGF-ß signaling effector with potential pathogenic implications in aortic aneurysm. NEW & NOTEWORTHY We present the most comprehensive quantitative analysis of the ascending aortic aneurysm proteome in Marfan syndrome to date resulting in novel and potentially wide-reaching findings that expression and signaling by ß3-integrin constitute a modulator of transforming growth factor-ß-induced rapamycin-independent component of mammalian target of rapamycin (Rictor) signaling and physiology in aortic vascular smooth muscle cells.