University of Missouri-Columbia, MO, USA.
Department of Ophthalmology, Institute for Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden.
Exp Cell Res. 2020 Dec 15;397(2):112347. doi: 10.1016/j.yexcr.2020.112347. Epub 2020 Oct 29.
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.
为了研究胎盘生长因子/血管内皮生长因子（PlGF-VEGF）异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 异二聚体和 rhPlGF 处理人视网膜内皮细胞（HRECs），并在正常和高糖条件下进行研究。通过测量跨内皮电阻（TEER）评估 HREC 屏障功能。通过向 C57BL6 小鼠眼内玻璃体内注射腺相关病毒 5（AAV5）载体，使视网膜过表达 PlGF。AAV5-GFP 载体和未经处理的动物被用作对照。免疫荧光（IF）和 Western blot 检测 HREC 和小鼠视网膜中 PlGF-VEGF 异二聚体、VEGF、PlGF、NFκB、p-IκBα、ZO-1 和 VE-钙粘蛋白的蛋白表达。基于 IF 和细胞质和核级分实验，发现 PlGF-VEGF 异二聚体主要存在于 HREC 细胞核中。高糖处理增加了 PlGF-VEGF 的核丰度。斑点免疫印迹显示 5D11D4 抗体对 PlGF-VEGF 异二聚体具有很强的亲和力。rhPlGF-VEGF 破坏了 HREC 的屏障功能，而用 5D11D4 抗体中和 PlGF-VEGF 则可防止这种破坏。rhPlGF 刺激 HRECs 也导致 PlGF-VEGF、p-IκBα 的核信号增加，以及 NFκB p65 和 PlGF-VEGF 在核内的共定位增加。选择性 IKK2 抑制剂 IMD0354 破坏了核共定位。用 IMD0354 处理可恢复 HREC 的屏障功能，如 ZO-1 和 VE-钙粘蛋白的表达所示。在小鼠视网膜中，AAV5 载体过表达 PlGF 可降低 ZO-1 的表达并增加 pIκBα 的丰度。PIGF/VEGF 异二聚体通过经典的 NFκB 激活介导 BRB 破坏。
