Actin cytoskeleton regulates functional anchorage-migration switch during T-cadherin-induced phenotype modulation of vascular smooth muscle cells

Vascular smooth muscle cell (SMC) switching between differentiated and dedifferentiated phenotypes is reversible and accompanied by morphological and functional alterations that require reconfiguration of cell-cell and cell-matrix adhesion networks. Studies attempting to explore changes in overall composition of the adhesion nexus during SMC phenotype transition are lacking. We have previously demonstrated that T-cadherin knockdown enforces SMC differentiation, whereas T-cadherin upregulation promotes SMC dedifferentiation. This study used human aortic SMCs ectopically modified with respect to T-cadherin expression to characterize phenotype-associated cell-matrix adhesion molecule expression, focal adhesions configuration and migration modes. Compared with dedifferentiated/migratory SMCs (expressing T-cadherin), the differentiated/contractile SMCs (T-cadherin-deficient) exhibited increased adhesion to several extracellular matrix substrata, decreased expression of several integrins, matrix metalloproteinases and collagens, and also distinct focal adhesion, adherens junction and intracellular tension network configurations. Differentiated and dedifferentiated phenotypes displayed distinct migrational velocity and directional persistence. The restricted migration efficiency of the differentiated phenotype was fully overcome by reducing actin polymerization with ROCK inhibitor Y-27632 whereas myosin II inhibitor blebbistatin was less effective. Migration efficiency of the dedifferentiated phenotype was diminished by promoting actin polymerization with lysophosphatidic acid. These findings held true in both 2D-monolayer and 3D-spheroid migration models. Thus, our data suggest that despite global differences in the cell adhesion nexus of the differentiated and dedifferentiated phenotypes, structural actin cytoskeleton characteristics <i>per se</i> play a crucial role in permissive regulation of cell-matrix adhesive interactions and cell migration behavior during T-cadherin-induced SMC phenotype transition.

血管平滑肌细胞（SMC）在分化表型与去分化表型之间的转换是可逆的，该过程伴随细胞形态与功能的改变，且需要重构细胞-细胞及细胞-基质黏附网络。目前尚缺乏针对平滑肌细胞表型转换过程中黏附网络整体组成变化的探索性研究。本团队此前的研究表明，敲低T-钙黏蛋白（T-cadherin）可促进平滑肌细胞分化，而上调T-钙黏蛋白则会推动平滑肌细胞去分化。本研究利用经T-钙黏蛋白表达异位修饰的人主动脉平滑肌细胞，对表型相关的细胞-基质黏附分子表达、黏着斑（focal adhesions）构型及细胞迁移模式进行了系统表征。与表达T-钙黏蛋白的去分化/迁移型平滑肌细胞相比，T-钙黏蛋白缺陷的分化/收缩型平滑肌细胞对多种细胞外基质底物的黏附能力增强，整合素（integrins）、基质金属蛋白酶（matrix metalloproteinases）及胶原蛋白（collagens）的表达水平下调，同时其黏着斑、黏着连接（adherens junction）及细胞内张力网络的构型也存在显著差异。分化型与去分化型平滑肌细胞表型展现出截然不同的迁移速率与定向持久性。通过ROCK抑制剂Y-27632抑制肌动蛋白聚合，可完全逆转分化型表型的迁移效率受限问题；而肌球蛋白II抑制剂blebbistatin的干预效果则相对较弱。利用溶血磷脂酸（lysophosphatidic acid）促进肌动蛋白聚合，则会降低去分化型表型的迁移效率。上述研究结果在二维单层培养与三维球体培养的迁移模型中均得到验证。综上，本研究数据表明，尽管分化型与去分化型平滑肌细胞的细胞黏附网络存在整体差异，但肌动蛋白细胞骨架的结构特征本身，在T-钙黏蛋白诱导的平滑肌细胞表型转换过程中，对细胞-基质黏附相互作用及细胞迁移行为的许可性调控发挥着关键作用。