Human primary AoSMC treated by Y27632 and not to investigate the role of RhoA/ROCK1/YAP/F-actin in aortic dissection formation

Rho-kinase signaling and YAP signaling are related to aortic dissection (AD) formation. However, as important biomechanical signaling pathways, their relationships with aortic smooth muscle cell (AoSMC) mechanics have not been investigated in AD formation. This study aims to explore the correlation between RhoA/ROCK1 and YAP, as well as their relationship with intrinsic AoSMC stiffness during AD formation. The expression of RhoA/ROCK1 and YAP as well as F-actin polymerization were analyzed in AoSMCs isolated from normal and AD human aortas. The intrinsic cell stiffness of normal and AD AoSMCs was measured using atomic force microscopy (AFM). The correlations among RhoA/ROCK1, YAP, and intrinsic AoSMC stiffness were explored by manipulating the activity of RhoA and ROCK1, and YAP expression. Finally, the role of RhoA/ROCK1/YAP/F-actin and AoSMC stiffness during AD formation was studied in pharmaceutically induced AD mouse models.Compared to normal human AoSMCs, the expression of RhoA and ROCK1 was downregulated, while the phosphorylation of YAP was increased in AD human AoSMCs, coupled with impaired F-actin polymerization and decreased intrinsic cell stiffness. Pharmaceutical inhibition of RhoA or ROCK1 activities and depletion of YAP all led to impaired F-actin polymerization and decreased intrinsic AoSMC stiffness. Moreover, abnormal collagen deposition and impaired cell-ECM interactions were found when RhoA/ROCK1/YAP/F-actin signaling was inhibited in normal human AoSMCs. We further analyzed the normal human AoSMC treated by Y27632 or not using RNA sequencing to investigate the impact of RhoA/ROCK1/YAP/F-actin on AoSMCs. GO analysis showed that differentially expressed genes (DEGs) related to cAMP-mediated signaling, cytoskeleton organization, cell-matrix adhesion, and extracellular matrix (ECM) organization were affected when ROCK1 activity was inhibited by Y27632 in normal AoSMCs. KEGG analysis showed differences in PI3K-Akt signaling, focal adhesion, MAPK, actin cytoskeleton, and ECM-receptor interaction. Consistently, cAMP, actin cytoskeleton organization, and ECM structural constituents were all downregulated in AoSMCs treated with Y27632 according to GSEA. We also analyzed the downregulated genes in AoSMCs treated with Y27632. Results demonstrated that the downregulated genes were mainly related to the cell-ECM unit, PI3K, MAPK, focal adhesion, and cAMP signaling pathways.