Abstract Body: Background: Thoracic Aortic Aneurysm (TAA) is a progressive dilation of aortic wall associated with degradation of the extracellular matrix (ECM), cystic medial degeneration (CMD), and smooth muscle cell (SMC) dysfunction and rarefaction. Loss of ECM integrity or familial mutations in genes encoding proteins of the ECM, actomyosin cytoskeleton, or focal adhesion (FA) suggest that alteration of mechanical force propagation and mechanotransduction may contribute to SMC dysfunction in TAA. While progressive aortic dilation increases tensile force and force propagation, advanced ECM degradation and CMD can lead to disruption of mechanotransduction. As such, there is a need to determine the exact contribution of FA disruption in SMC in TAA.
Methods: This project combines in vivo and in vitro loss- and gain-of function studies. We generated SMC-specific (Myh11-ERT2Cre YFP) Vcl (Vinculin), Ilk (Integrin-linked kinase), and Ptk2 (Focal Adhesion Kinase) KO mouse lines and treated them with b-Aminopropionitrile (BAPN) to induce TAA. Mouse survival, aorta morphology, cell composition, and apoptosis (TUNEL) were analyzed. In vitro, we tested effect of shRNA-mediated FAK knockdown (KD) or FAK overexpression (full length and kinase-dead (DK) FAK constructs) on SMC propensity to gliotoxin-induced anoikis (cell apoptosis due to detachment).
Results: Analysis of scRNAseq data from Marfan mouse (Fbn1^+/C1041G) aorta revealed a complex expression profile of FA pathway genes with upregulation of ECM and integrin genes and downregulation of intracellular FA components including Vcl, Ilk, Tes, Limd1 suggesting a defect in propagation of mechanotransduction signaling. Similarly, ILK and VCL protein expression was decreased in the aorta of TAA patients. In vivo, SMC-specific KO of Vcl, Ilk, and Ptk2 (FAK) exacerbates BAPN-induced increasing mortality, aortic dilation, ECM degradation and SMC apoptosis compared to wildtype littermates. In vitro, FAK KD increase gliotoxin-induced anoikis through promoting SMC detachment and apoptosis while overexpressing full length and KD FAK in shFAK SMCs reestablished anoikis resistance. This data demonstrate a kinase-independent protective effect of FAK against anoikis.
Conclusions: Maintenance of FA protein expression (Vcl, Ilk, and FAK) confers protection against TAA progression, and SMC anoikis. FA beneficial effects are independent of FAK kinase activity and are likely due structural integrity necessary for proper force propagation.