Department of Ophthalmology, University of Missouri School of Medicine, Columbia, MO, USA.
Wilmer Bioinformatics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Curr Eye Res. 2023 Mar;48(3):297-311. doi: 10.1080/02713683.2022.2149808. Epub 2022 Dec 2.
Placental growth factor (PlGF) and Angiopoietin (Ang)-1 are two proteins that are involved in the regulation of endothelial cell (EC) growth and vasculature formation. In the retina and endothelial cells, pericytes are the major source of both molecules. The purpose of this study is to examine the association of PlGF and Ang-1 with human EC/pericyte co-cultures and iPSC-derived vascular organoids.
In this study, we used co-cultures of human primary retinal endothelial cells (HREC) and primary human retinal pericytes (HRP), western blotting, immunofluorescent analysis, TUNEL staining, LDH-assays, and RNA seq analysis, as well as human-induced pluripotent stem cells (iPSC), derived organoids (VO) to study the association between PlGF and Ang-1.
Inhibition of PlGF by PlGF neutralizing antibody in HREC-HRP co-cultures resulted in the increased expression of Ang-1 and Tie-2 in a dose-dependent manner. This upregulation was not observed in HREC and HRP monocultures but only in co-cultures suggesting the association of pericytes and endothelial cells. Furthermore, Vascular endothelial growth factor receptor 1 (VEGFR1) inhibition abolished the Ang-1 and Tie-2 upregulation by PlGF inhibition. The pericyte viability in high-glucose conditions was also reduced by VEGFR1 neutralization. Immunofluorescent analysis showed that Ang-1 and Ang-2 were expressed mainly by perivascular cells in the VO. RNA seq analysis of the RNA isolated from VO in high glucose conditions indicated increased PGF and Ang-2 expressions in the VO. PlGF inhibition increased the expression of Ang-1 and Tie-2 in VO, increasing the pericyte coverage of the VO microvascular network.
Combined, these results suggest PlGF's role in the regulation of Ang-1 and Tie-2 expression through VEGFR1. These findings provide new insights into the neovascularization process in diabetic retinopathy and new targets for potential therapeutic intervention.
胎盘生长因子（PlGF）和血管生成素（Ang）-1 是两种参与调节内皮细胞（EC）生长和血管形成的蛋白质。在视网膜和内皮细胞中，周细胞是这两种分子的主要来源。本研究旨在研究 PlGF 和 Ang-1 与人类 EC/周细胞共培养物和 iPSC 衍生的血管类器官的关系。
在这项研究中，我们使用了人原代视网膜内皮细胞（HREC）和人原代视网膜周细胞（HRP）的共培养物、Western blot 分析、免疫荧光分析、TUNEL 染色、LDH 测定和 RNA 测序分析，以及人诱导多能干细胞（iPSC）、衍生的类器官（VO），来研究 PlGF 和 Ang-1 之间的关系。
在 HREC-HRP 共培养物中，用 PlGF 中和抗体抑制 PlGF 导致 Ang-1 和 Tie-2 的表达呈剂量依赖性增加。这种上调在 HREC 和 HRP 单培养物中没有观察到，而只在共培养物中观察到，提示周细胞和内皮细胞的关联。此外，血管内皮生长因子受体 1（VEGFR1）抑制消除了 PlGF 抑制引起的 Ang-1 和 Tie-2 的上调。高葡萄糖条件下周细胞的活力也因 VEGFR1 中和而降低。免疫荧光分析显示，Ang-1 和 Ang-2 主要由 VO 中的血管周细胞表达。高葡萄糖条件下从 VO 中分离的 RNA 的 RNA 测序分析表明，PGF 和 Ang-2 的表达在 VO 中增加。PlGF 抑制增加了 VO 中 Ang-1 和 Tie-2 的表达，增加了 VO 微血管网络中周细胞的覆盖。
综上所述，这些结果表明 PlGF 通过 VEGFR1 调节 Ang-1 和 Tie-2 的表达。这些发现为糖尿病视网膜病变中的新生血管化过程提供了新的见解，并为潜在的治疗干预提供了新的靶点。
