Synergistic interactions of PlGF and VEGF contribute to blood-retinal barrier breakdown through canonical NFκB activation.
Synergistic interactions of PlGF and VEGF contribute to blood-retinal barrier breakdown through canonical NFκB activation.
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DOI：
10.1016/j.yexcr.2020.112347
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发表时间：
2020-12-15
影响因子：
3.7
通讯作者：
Huang H
中科院分区：
文献类型：
--
作者：
Lennikov A;Mukwaya A;Fan L;Saddala MS;De Falco S;Huang H
为了研究胎盘生长因子/血管内皮生长因子（PlGF-VEGF）异源二聚体在血-视网膜屏障（BRB）破坏中的作用及其相关机制，用重组人（rh）PlGF-VEGF异源二聚体和rhPlGF处理人视网膜内皮细胞（HREC），并在正常和高糖条件下进行研究。通过测量跨内皮电阻（TEER）评价HREC屏障功能。5型腺相关病毒（AAV 5）载体通过玻璃体内注射到C57 BL 6小鼠眼中在视网膜中过表达PlGF。AAV 5-GFP载体和未处理动物用作对照。免疫荧光（IF）和western印迹检测了HREC和小鼠视网膜中PlGF-VEGF异源二聚体、VEGF、PlGF、NFκB、p-IκBα、ZO-1和VE-钙粘蛋白的蛋白表达。PlGF-VEGF异源二聚体主要在HREC细胞核中检测到的基础上IF和细胞质和核分级分离实验。高糖处理增加PlGF-VEGF核丰度。斑点免疫印迹证明5D 11 D4抗体对PlGF-VEGF异二聚体的强亲和力。rhPlGF-VEGF破坏HREC的屏障功能，这通过5D 11 D4抗体中和PlGF-VEGF来防止。用rhPlGF刺激HREC还导致PlGF-VEGF、p-IκBα的核信号增加，以及NFκB p65和PlGF-VEGF在核中的共定位。选择性IKK 2抑制剂IMD 0354破坏了核共定位。用IMD 0354治疗恢复了HREC的屏障功能，如ZO-1和VE-钙粘蛋白表达所示。在小鼠视网膜中，通过AAV 5载体过表达PlGF降低ZO-1表达并增加pIκBα的丰度。PIGF/VEGF异源二聚体可能通过典型的NFκB活化介导BR B的破坏。
To investigate the role of placental growth factor/vascular endothelial growth factor (PlGF-VEGF) heterodimers are involved in the blood-retinal barrier (BRB) breakdown and the associated mechanism, human retinal endothelial cells (HRECs) were treated with recombinant human (rh)PlGF-VEGF heterodimers and rhPlGF and studied in normal and high-glucose conditions. HREC barrier function was evaluated by the measurement of trans-endothelial electrical resistance (TEER). Adeno-Associated Virus Type 5 (AAV5) vectors overexpressed PlGF in the retina by intravitreal injection into the C57BL6 mouse eye. AAV5-GFP vector and naïve animals were used as controls. Immunofluorescence (IF) and western blots examined the protein expression of PlGF-VEGF heterodimers, VEGF, PlGF, NFκB, p-IκBα, ZO-1, and VE-cadherin in HREC and mouse retina. PlGF-VEGF heterodimers were detected predominantly in the HREC cell nuclei based on IF and cytoplasmic and nuclear fractionation experiments. High glucose treatment increased PlGF-VEGF nuclear abundance. Dot immunoblotting demonstrated a strong affinity of the 5D11D4 antibody to PlGF-VEGF heterodimers. rhPlGF-VEGF disrupted the barrier function of HREC, which was prevented by the neutralization of PlGF-VEGF by the 5D11D4 antibody. Stimulation of HRECs with rhPlGF also led to an increase in the nuclear signals for PlGF-VEGF, p-IκBα, and colocalization of NFκB p65 and PlGF-VEGF in the nuclei. The selective IKK2 inhibitor IMD0354 disrupted the nuclear colocalization. Treatment with IMD0354 restored the barrier function of HREC, as indicated by the ZO-1 and VE-cadherin expression. In the mouse retinas, PlGF overexpression by AAV5 vector reduced ZO-1 expression and increased abundance of pIκBα. PIGF/VEGF heterodimers mediate BRB breakdown potentially through the canonical NFκB activation.
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