Smooth muscle cells (SMCs) and the extracellular matrix (ECM) are intimately associated in the aortic wall. Fbln4SMKO mice with a smooth muscle cell-specific deletion of the Fbln4 gene, which encodes the vascular ECM component fibulin-4, develop ascending aortic aneurysms that have increased abundance of angiotensin converting enzyme (ACE); inhibiting angiotensin II signaling within the first month of life prevents aneurysm development. We used comparative proteomics analysis of Fbln4SMKO aortas from postnatal day (P) 1 to P30 mice to identify key molecules involved in aneurysm initiation and expansion. At P14, the actin depolymerizing factor cofilin was dephosphorylated and thus activated, and at P7, the abundance of slingshot-1 phosphatase (SSH1), an activator of cofilin, was increased, leading to actin cytoskeletal remodeling. Also by P7, biomechanical changes and underdeveloped elastic lamina-SMC connections were evident and the abundance of early growth response-1 (Egr1), a mechanosensitive transcription factor that stimulates ACE expression, was increased, which was before the increases in ACE abundance and cofilin activation. Postnatal deletion of Fbln4 in SMCs at P7 prevented cofilin activation and aneurysm formation, suggesting that these processes required disruption of elastic lamina-SMC connections. Phosphoinsitide 3-kinase (PI3K) is involved in the angiotensin II-mediated activation of SSH1 and administration of PI3K inhibitors from P7 to P30 decreased SSH1 abundance and prevented aneurysms. These results suggest that aneurysm formation arises from abnormal mechanosensing of SMCs resulting from the loss of elastic lamina-SMC connections and from increased SSH1 and cofilin activity, which may be potential therapeutic targets for treating ascending aortic aneurysms.

AimThoracic aortic aneurysms are a life-threatening condition often diagnosed too late. To discover novel robust biomarkers, we aimed to better understand the molecular mechanisms underlying aneurysm formation.Methods and resultsIn Fibulin-4R/R mice, the extracellular matrix protein Fibulin-4 is 4-fold reduced, resulting in progressive ascending aneurysm formation and early death around 3 months of age. We performed proteomics and genomics studies on Fibulin-4R/R mouse aortas. Intriguingly, we observed alterations in mitochondrial protein composition in Fibulin-4R/R aortas. Consistently, functional studies in Fibulin-4R/R vascular smooth muscle cells (VSMCs) revealed lower oxygen consumption rates, but increased acidification rates. Yet, mitochondria in Fibulin-4R/R VSMCs showed no aberrant cytoplasmic localization. We found similar reduced mitochondrial respiration in Tgfbr-1M318R/+ VSMCs, a mouse model for Loeys-Dietz syndrome (LDS). Interestingly, also human fibroblasts from Marfan (FBN1) and LDS (TGFBR2 and SMAD3) patients showed lower oxygen consumption. While individual mitochondrial Complexes I–V activities were unaltered in Fibulin-4R/R heart and muscle, these tissues showed similar decreased oxygen consumption. Furthermore, aortas of aneurysmal Fibulin-4R/R mice displayed increased reactive oxygen species (ROS) levels. Consistent with these findings, gene expression analyses revealed dysregulation of metabolic pathways. Accordingly, blood ketone levels of Fibulin-4R/R mice were reduced and liver fatty acids were decreased, while liver glycogen was increased, indicating dysregulated metabolism at the organismal level. As predicted by gene expression analysis, the activity of PGC1α, a key regulator between mitochondrial function and organismal metabolism, was downregulated in Fibulin-4R/R VSMCs. Increased TGFβ reduced PGC1α levels, indicating involvement of TGFβ signalling in PGC1α regulation. Activation of PGC1α restored the decreased oxygen consumption in Fibulin-4R/R VSMCs and improved their reduced growth potential, emphasizing the importance of this key regulator.ConclusionOur data indicate altered mitochondrial function and metabolic dysregulation, leading to increased ROS levels and altered energy production, as a novel mechanism, which may contribute to thoracic aortic aneurysm formation.

Rationale Mutations in ACTA2, encoding the smooth muscle isoform of α-actin (SM α-actin), cause thoracic aortic aneurysms, acute aortic dissections, and occlusive vascular diseases. Objective We sought to identify the mechanism by which loss of SM α-actin causes aortic disease. Methods and Results Acta2−/− mice have an increased number of elastic lamellae in the ascending aorta and progressive aortic root dilation as assessed by echocardiography that can be attenuated by treatment with losartan, an angiotensin II (AngII) type 1 receptor blocker. AngII levels are not increased in Acta2−/− aortas or kidneys. Aortic tissue and explanted smooth muscle cells (SMCs) from Acta2−/− aortas show increased production of reactive oxygen radicals (ROS) and increased basal NF-κB signaling, leading to an increase in the expression of the AngII receptor type I (Agtr1a) and activation of signaling at 100-fold lower levels of AngII in the mutant compared to wild-type cells. Furthermore, disruption of SM α-actin filaments in wildtype SMCs by various mechanisms activates NF-κB signaling and increases expression of Agtr1a. Conclusions These findings reveal that disruption of SM α-actin filaments in SMCs increases ROS levels, activates NF-κB signaling and increases Agtr1a expression, thus potentiating AngII signaling in vascular SMCs without an increase in the exogenous levels of AngII.

Homozygous recessive mutations in either EFEMP2 (encoding fibulin-4) or FBLN5 (encoding fibulin-5), critical genes for elastogenesis, lead to autosomal recessive cutis laxa types 1B and 1A, respectively. Previously, fibulin-4 was shown to bind lysyl oxidase (LOX), an elastin/collagen cross-linking enzyme, in vitro. Consistently, reported defects in humans with EFEMP2 mutations are more severe and broad in range than those due to FBLN5 mutations and encompass both elastin-rich and collagen-rich tissues. However, the underlying disease mechanism in EFEMP2 mutations has not been fully addressed. Here, we show that fibulin-4 is important for the integrity of aortic collagen in addition to elastin. Smooth muscle-specific Efemp2 loss in mouse (termed SMKO) resulted in altered fibrillar collagen localization with larger, poorly organized fibrils. LOX activity was decreased in Efemp2-null cells, and collagen cross-linking was diminished in SMKO aortas; however, elastin cross-linking was unaffected and the level of mature LOX was maintained to that of wild-type aortas. Proteomic screening identified multiple proteins involved in procollagen processing and maturation as potential fibulin-4-binding partners. We showed that fibulin-4 binds procollagen C-endopeptidase enhancer 1 (Pcolce), which enhances proteolytic cleavage of the procollagen C-terminal propeptide during procollagen processing. Interestingly, however, procollagen cleavage was not affected by the presence or absence of fibulin-4 in vitro. Thus, our data indicate that fibulin-4 serves as a potential scaffolding protein during collagen maturation in the extracellular space. Analysis of collagen in other tissues affected by fibulin-4 loss should further increase our understanding of underlying pathologic mechanisms in patients with EFEMP2 mutations.

Thoracic aortic aneurysms are a life-threatening condition often diagnosed too late. The underlying mechanism is largely unknown. An accurate and early predictive biomarker for aneurysm formation has not yet been identified, although some molecular processes, such as disturbed TGF-β signalling, have been implicated. To discover novel robust biomarkers, we aimed to better understand the molecular mechanisms involved in aneurysm initiation and progression. In Fibulin-4 R/R mice, the extracellular matrix protein Fibulin-4 is 4-fold reduced, resulting in progressive ascending aneurysm formation and early death around 3 months of age. We performed LC-MS/MS proteomics and transcriptomics analyses on the aortas of Fibulin-4 R/R and Fibulin-4 +/+ mice. Protein and gene data sets were separately analysed for genotype specific differences with Ingenuity Pathway analysis tools. Intriguingly, we observed alterations in mitochondrial composition in aortas from Fibulin-4 R/R mice. Consistently, functional studies in Fibulin-4 R/R vascular smooth muscle cells (VSMCs) revealed lower oxygen consumption rates, but increased acidification rates compared to Fibulin-4 +/+ . The mitochondria in VSMCs of Fibulin-4 R/R mice were reduced in size and had increased complex I-IV levels. Furthermore, aortas of aneurysmal Fibulin-4 R/R mice displayed increased levels of ROS. Consistent with these findings, gene expression analyses revealed the dysregulation of metabolic pathways. In accordance, ketone levels in the blood of Fibulin-4 R/R mice were reduced and liver fatty acids were decreased, while liver glycogen was increased. As predicted by these findings, activity of PGC1α, a key regulator between mitochondrial function and organismal metabolism, was downregulated in Fibulin-4 R/R VSMCs. In conclusion, our data indicate altered mitochondrial function and metabolic dysregulation, leading to increased ROS levels and altered energy production, as a novel mechanism, which may contribute to thoracic aortic aneurysm formation. This discovery will not only provide new biomarkers that can be validated in human aortas, but they will also provide the rational for new interventions such as alterations in diet to prevent aneurysm formation.