Abstract 1012: Single-Cell RNA Sequencing Defines Focal Adhesion-Matrix Interactions In Smooth Muscle Cells In Thoracic Aortic Disease Triggered By BAPN Inhibiting Lysyl Oxidase

Background: Thoracic aortic aneurysms and/or dissections (TAD) are associated with pathogenic variants in genes that involved in either smooth muscle cell (SMC) contraction or extracellular matrix (ECM). The inability to maintain homeostasis between SMCs and ECM is pivotal in TAD pathogenesis. Focal adhesions (FA) act as mechanosensors in cells, transferring mechanical signals from the ECM to the intracellular contractile filaments and regulating downstream Rho/ROCK signaling. However, the landscape of SMC-ECM interactions, has not been fully explored at the early stages of TAD. Methods: C57BL/6J wild-type mice aged 21 days postnatal (P21) were administered the ECM cross-link blocker, BAPN, for up to four weeks to induce TAD. Proximal aortas were harvested for single-cell RNA sequencing at P35. Immunofluorescence and immunoblots were used to validate findings. Results: Single-cell RNA sequencing classified SMCs into 5 clusters marked by Rgs5 (SMC Rgs5 + ), high levels of Palld (SMC Palld + ), Tnnt2 (SMC Tnnt2 + , marker of second heart field-derived cells), Atf3 (SMC Atf3+ cluster), or no clear marker (SMC NOS ). The analysis of how BAPN exposure affects different cell types, based on the Jensen-Shannon divergence in the probability distribution between groups, demonstrated that SMCs, and specifically SMC Atf3 + and SMC NOS , are the most perturbed cell cluster by BAPN. Gene set enrichment analysis identified significantly dysregulated pathways of FA and Rho/ROCK signaling. The expression of Itga5 and Fn1 (fibronectin) were increased and Itga8 was decreased. An SMC-SMC interaction analysis found Fn1 interactions were the most upregulated interaction in 4 SMC clusters. When analyzing downstream pathways due to the changes in interaction, 80% of the top 25 interactions were related to integrins, including increased Fn1 interactions with Itga5-Itgb1 and Itgav-Itgb3. In SMC Rgs5 + cluster, the most affected interactions are related to TGF-β signaling. Conclusions: These findings suggest that, in the majority of aortic SMCs, mechanosensing through FAs, driven by fibronectin interactions with specific integrin receptor pairs, is activated with BAPN treatment, potentially contributing to the pathogenesis of TAD.