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Rationale Abnormal mechanosensing of smooth muscle cells (SMCs) resulting from the defective elastin-contractile units has been suggested to drive the formation of thoracic aortic aneurysms (TAAs); however, the precise molecular mechanism has not been elucidated. Objective The aim of this study was to identify the crucial mediator(s) involved in abnormal mechanosensing and propagation of biochemical signals during the aneurysm formation and to establish a basis for a novel therapeutic strategy. Methods and Results We used a mouse model of postnatal ascending aortic aneurysms (Fbln4SMKO; termed SMKO), in which deletion of Fbln4 leads to disruption of the elastin-contractile units caused by a loss of elastic lamina-SMC connections. In this mouse, upregulation of early growth response-1 (Egr1) and angiotensin converting enzyme leads to activation of angiotensin II signaling. Here we showed that the matricellular protein, thrombospondin-1 (Thbs1), was highly upregulated in SMKO ascending aortas and in human TAAs. Thbs1 was induced by mechanical stretch and Ang II in SMCs, for which Egr1 was required, and reduction of Fbln4 sensitized the cells to these stimuli and led to higher expression of Egr1 and Thbs1. Deletion of Thbs1 in SMKO mice prevented the aneurysm formation in approximately 80% of SMKO; Thbs1−/− (termed DKO) animals and suppressed slingshot-1 and cofilin de-phosphorylation, leading to the formation of normal actin filaments. Furthermore, elastic lamina-SMC connections were restored in DKO aortas and mechanical testing showed that structural and material properties of DKO aortas were markedly improved. Conclusions Thbs1 is a critical component of mechanotransduction as well as a modulator of elastic fiber organization. Maladaptive upregulation of Thbs1 results in disruption of elastin-contractile units and dysregulation of actin cytoskeletal remodeling, contributing to the development of ascending aortic aneurysms in vivo. Thbs1 may serve as a potential therapeutic target for treating TAAs.

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.

In the large arteries, it is believed that elastin provides the resistance to stretch at low pressure, while collagen provides the resistance to stretch at high pressure. It is also thought that elastin is responsible for the low energy loss observed with cyclic loading. These tenets are supported through experiments that alter component amounts through protease digestion, vessel remodeling, normal growth, or in different artery types. Genetic engineering provides the opportunity to revisit these tenets through the loss of expression of specific wall components. We used newborn mice lacking elastin (Eln−/−) or two key proteins (lysyl oxidase, Lox−/−, or fibulin-4, Fbln4−/−) that are necessary for the assembly of mechanically-functional elastic fibers to investigate the contributions of elastic fibers to large artery mechanics. We determined component content and organization and quantified the nonlinear and viscoelastic mechanical behavior of Eln−/−, Lox−/−, and Fbln4−/− ascending aorta and their respective controls. We confirmed that the lack of elastin, fibulin-4, or lysyl oxidase leads to absent or highly fragmented elastic fibers in the aortic wall and a 56 – 97% decrease in crosslinked elastin amounts. We found that the resistance to stretch at low pressure is decreased only in Eln−/− aorta, confirming the role of elastin in the nonlinear mechanical behavior of the aortic wall. Dissipated energy with cyclic loading and unloading is increased 53 – 387% in Eln−/−, Lox−/−, and Fbln4−/− aorta, indicating that not only elastin, but properly assembled and crosslinked elastic fibers, are necessary for low energy loss in the aorta.