Adducin Is Involved in Endothelial Barrier Stabilization

Adducins tightly regulate actin dynamics which is critical for endothelial barrier function. Adducins were reported to regulate epithelial junctional remodeling by controlling the assembly of actin filaments at areas of cell-cell contact. Here, we investigated the role of α-adducin for endothelial barrier regulation by using microvascular human dermal and myocardial murine endothelial cells. Parallel transendothelial electrical resistance (TER) measurements and immunofluorescence analysis revealed that siRNA-mediated adducin depletion impaired endothelial barrier formation and led to severe fragmentation of VE-cadherin immunostaining at cell-cell borders. To further test whether the peripheral localization of α-adducin is functionally linked with the integrity of endothelial adherens junctions, junctional remodeling was induced by a Ca2+-switch assay. Ca2+-depletion disturbed both linear vascular endothelial (VE)-cadherin and adducin location along cell junctions, whereas their localization was restored following Ca2+-repletion. Similar results were obtained for α-adducin phosphorylated at a site typical for PKA (pSer481). To verify that endothelial barrier properties and junction reorganization can be effectively modulated by altering Ca2+-concentration, TER measurements were performed. Thus, Ca2+-depletion drastically reduced TER, whereas Ca2+-repletion led to recovery of endothelial barrier properties resulting in increased TER. Interestingly, the Ca2+-dependent increase in TER was also significantly reduced after efficient α-adducin downregulation. Finally, we report that inflammatory mediator-induced endothelial barrier breakdown is associated with loss of α-adducin from the cell membrane. Taken together, our results indicate that α-adducin is involved in remodeling of endothelial adhesion junctions and thereby contributes to endothelial barrier regulation.内收蛋白（Adducin）可紧密调控肌动蛋白动力学（actin dynamics），而该过程对内皮屏障功能至关重要。已有研究证实，内收蛋白可通过调控细胞间接触区域的肌动蛋白丝组装，参与上皮连接重塑过程。本研究以人真皮微血管内皮细胞与小鼠心肌微血管内皮细胞为实验模型，探究α-内收蛋白（α-adducin）在内皮屏障调控中的作用。通过同步开展的跨内皮电阻（transendothelial electrical resistance, TER）检测与免疫荧光分析，我们发现经小干扰RNA（small interfering RNA, siRNA）介导的内收蛋白敲低会损伤内皮屏障形成，并导致细胞间边界处的血管内皮钙粘蛋白（VE-cadherin）免疫染色出现严重碎片化。为进一步验证α-内收蛋白的外周定位是否与内皮黏着连接的完整性存在功能关联，本研究通过钙切换（Ca²⁺-switch）实验诱导细胞连接重塑。钙缺失会破坏血管内皮钙粘蛋白与内收蛋白沿细胞连接的线性分布，但在钙复补充后，二者的定位可得以恢复。针对蛋白激酶A（Protein Kinase A, PKA）经典磷酸化位点Ser481的磷酸化α-内收蛋白（pSer481-α-adducin），实验也得到了一致结果。为验证改变胞外钙浓度可有效调控内皮屏障功能与连接重塑，本研究开展了跨内皮电阻检测。结果显示，钙缺失会大幅降低跨内皮电阻值，而钙复补充则可恢复内皮屏障功能，使跨内皮电阻值回升。值得注意的是，经高效敲低α-内收蛋白后，钙依赖的跨内皮电阻升高现象也显著减弱。最后，本研究证实炎症介质诱导的内皮屏障破坏，与细胞膜上α-内收蛋白的缺失存在显著关联。综上，本研究结果表明，α-内收蛋白参与内皮黏着连接的重塑过程，进而在内皮屏障调控中发挥关键作用。