A nonapoptotic endothelial barrier-protective role for caspase-3
A nonapoptotic endothelial barrier-protective role for caspase-3
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DOI：
10.1152/ajplung.00487.2018
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发表时间：
2019-06-01
影响因子：
4.9
通讯作者：
Damarla, Mahendra
中科院分区：
文献类型：
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作者：
Suresh, Karthik;Carino, Kathleen;Damarla, Mahendra
caspase-3的非经典作用正在癌症和发育生物学领域出现。然而，在大多数细胞类型中，对caspase-3的非凋亡功能知之甚少。我们最近已经证明了caspase-3激活和细胞凋亡的执行与伴随的细胞质caspase-3隔离和保存内皮屏障功能之间的分离。因此。我们检验了非凋亡caspase-3激活促进内皮屏障完整性的假设。人肺微血管内皮细胞暴露于凝血酶，非凋亡刺激，和内皮屏障功能进行了评估，使用电细胞基质阻抗传感。肌动蛋白细胞骨架重排和细胞旁间隙的形成进行了评估鬼笔环肽染色。使用磁扭转细胞术评价细胞刚度。此外，收获细胞裂解物用于蛋白质分析。半胱天冬酶-3与泛半胱天冬酶和半胱天冬酶-3特异性抑制剂一起被抑制。使用RNA干扰实现了caspase-3的分子抑制。暴露于凝血酶的细胞表现出细胞质激活的caspase-3与短暂的和非凋亡的内皮屏障功能的下降，测量电阻下降，然后迅速恢复。半胱天冬酶的抑制导致凝血酶诱导的内皮屏障功能更明显和快速下降，伴随着内皮细胞硬度和细胞旁间隙增加。Caspase-3特异性抑制和caspase-3敲低均导致更明显的凝血酶诱导的内皮屏障破坏。加在一起。我们的研究结果表明，细胞质caspase-3在人内皮细胞中具有非凋亡功能，并能促进内皮屏障的完整性。
Noncanonical roles for caspase-3 are emerging in the fields of cancer and developmental biology. However, little is known of nonapoptotic functions of caspase-3 in most cell types. We have recently demonstrated a disassociation between caspase-3 activation and execution of apoptosis with accompanying cytoplasmic caspase-3 sequestration and preserved endothelial barrier function. Therefore. we tested the hypothesis that nonapoptotic caspase-3 activation promotes endothelial barrier integrity. Human lung microvascular endothelial cells were exposed to thrombin, a nonapoptotic stimulus, and endothelial barrier function was assessed using electric cell-substrate impedance sensing. Actin cytoskeletal rearrangement and paracellular gap formation were assessed using phalloidin staining. Cell stiffness was evaluated using magnetic twisting cytometry. In addition, cell lysates were harvested for protein analyses. Caspase-3 was inhibited pharmacologically with pan-caspase and a caspase-3-specific inhibitor. Molecular inhibition of caspase-3 was achieved using RNA interference. Cells exposed to thrombin exhibited a cytoplasmic activation of caspase-3 with transient and nonapoptotic decrease in endothelial barrier function as measured by a drop in electrical resistance followed by a rapid recovery. Inhibition of caspases led to a more pronounced and rapid drop in thrombin-induced endothelial barrier function, accompanied by increased endothelial cell stiffness and paracellular gaps. Caspase-3-specific inhibition and caspase-3 knockdown both resulted in more pronounced thrombin-induced endothelial barrier disruption. Taken together. our results suggest cytoplasmic caspase-3 has nonapoptotic functions in human endothelium and can promote endothelial barrier integrity.