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Protein Phosphatase 1 Beta is Modulated by Chronic Hypoxia and Involved in the Angiogenic Endothelial Cell Migration

机译:蛋白磷酸酶1 Beta是由慢性缺氧调节,并参与血管内皮细胞迁移。

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Background/Aim: Endothelial cell migration is required for physiological angiogenesis, but also contributes to various pathological conditions, including tumour vascularization. The mRNA expression of PP1cβ, the beta isoform of the catalytic PP1 subunit, was shown to be upregulated in chronic hypoxia. Since hypoxia is a major regulator of angiogenesis, the potential role of PP1cβ in angiogenesis was investigated. Methods: We examined PP1cβ protein level in pediatric heart following chronic hypoxia and found PP1cβ upregulation in cyanotic compared with acyanotic myocardium. By treating HUVEC cells with hypoxia mimicking agent, PP1cβ protein level increased with maximum at 8 hours. The effect of PP1cβ pharmacological inhibition, knockdown and overexpression, on endothelial cell migration and morphogenesis, was examined using in vitro wound healing scratch assay and endothelial tube formation assay. The PP1cβ knockdown effects on F-actin reorganization (phalloidin staining), focal adhesion formation (vinculin) and focal adhesion kinases (FAK) activation, were evaluated by immunocytochemical staining and immunoblotting with specific antibodies. Results: PP1cβ knockdown significantly reduces endothelial cell migration, but does not have any significant effect on endothelial tube formation. Endothelial cell migration in the knockdown group is restored to the control level upon consecutive transfection with PP1cβ cDNA. PP1cβ overexpression does not significantly affect endothelial cell migration. Furthermore, PP1cβ knockdown induces profound cytoskeletal reorganization, loss of focal adhesion sites and impairment of focal adhesion kinases (FAK) activation. Conclusions: PP1cβ is regulator of endothelial cell migration, which is critical in the angiogenic process. PP1cβ inhibition reduces endothelial cell migration through focal adhesion turnover and actin polymerization pathways.
机译:背景/目的:内皮细胞迁移是生理性血管生成所必需的,但也会导致多种病理状况,包括肿瘤血管生成。在慢性缺氧中,PP1cβ(催化性PP1亚基的β亚型)的mRNA表达被上调。由于缺氧是血管生成的主要调节因子,因此研究了PP1cβ在血管生成中的潜在作用。方法:我们检查了慢性缺氧后小儿心脏中PP1cβ的蛋白水平,发现发紫的心肌中PP1cβ的表达高于发紫的心肌。通过用缺氧模​​拟剂处理HUVEC细胞,PP1cβ蛋白水平在8小时达到最高。使用体外伤口愈合刮擦测定法和内皮管形成测定法检查了PP1cβ药理抑制,敲低和过表达对内皮细胞迁移和形态发生的影响。通过免疫细胞化学染色和特异性抗体免疫印迹评估PP1cβ敲低对F-肌动蛋白重组(phalloidin染色),粘着斑形成(vinculin)和粘着斑激酶(FAK)活化的影响。结果:PP1cβ敲低显着减少了内皮细胞的迁移,但对内皮管的形成没有任何显着影响。连续转染PP1cβcDNA后,敲低组的内皮细胞迁移恢复到对照水平。 PP1cβ的过表达不会显着影响内皮细胞迁移。此外,PP1cβ敲低诱导深刻的细胞骨架重组,粘着斑丧失和粘着斑激酶(FAK)激活受损。结论:PP1cβ是内皮细胞迁移的调节剂,在血管生成过程中至关重要。 PP1cβ抑制可通过粘着粘附转换和肌动蛋白聚合途径减少内皮细胞迁移。

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