Li Yadi, Deng Aiping, Jiao Kangwei, Yan Jie, Zuo Wandong, Dong Yujie, Xu Wenrong, Li Yuting, Guo Chunming, Chen Maorong, Tian Run, Hu Zhulin
Yunnan University, Kunming, Yunnan, China.
Key Laboratory of Yunnan Province, Yunnan Eye Institute, Affiliated Hospital of Yunnan University, Kunming, Yunnan, China.
PLoS One. 2025 Jun 20;20(6):e0325661. doi: 10.1371/journal.pone.0325661. eCollection 2025.
Choroidal neovascularization (CNV) is a key manifestation of intraocular neovascularization, and it is considered one of the main causes of blindness in ophthalmology. Additionally, multiple anti-vascular endothelial growth factor (VEGF) drugs have been used as first-line treatment for CNV. However, several issues posed challenges to the anti-VEGF drugs, which were mainly composed of short duration of action, requirement for repeated injections, and complications. Thrombospondin-1 (TSP-1) is an endogenous protein that was found to regulate multiple biological processes within the body, and it has been proven to exhibit an inhibitory effect on neovascularization. Besides, the function of TSP-1 during the inhibition of neovascularization was currently considered to mainly focus on its type Ⅰ repeats (TSRs), which was attributed to the large molecular weight, complex structure, and possible unknown functions of TSP-1. Therefore, TSRs can be applied as targets and research directions for the further development and exploration of potential therapeutic drugs.
Based on the type I repeats (TSRs) of thrombospondin-1 (TSP-1), amino acid sequences of different lengths were designed and synthesized in this study, named as VR-9 VR-10、VR-11、VR-12、VR-13. The objective was to explore the effects of the above five peptides on angiogenesis in Chori-retinal neovascularization, alongside the screening of the best peptides and the deep exploration into the underlying mechanism, aimed to provide a basis for the development and application of peptide drugs in the treatment of CNV.
Wound healing, CCK-8, and 5-ethynyl-2'-deoxyuridine (EdU) assays were employed to evaluate the proliferation and migration ability of cells. CRISPR-Cas9 technology was utilized to establish CD36 knockdown cell lines, alongside the conduction of qPCR to verify the efficiency of gene knockdown. The expression levels of VEGF and CD31 in RF/6A cells and rats were assessed by Western blot. Additionally, Hematoxylin and eosin (HE) staining was performed to examine the structural integrity of the rat retina, while Fluorescein Isothiocyanate-Dextran Cardiac Perfusion (FITC) labeling was used to observe the occurrence and development of choroidal neovascularization (CNV).
According to the wound-healing and CCK-8 assays, VR-13 was the most effective in inhibiting the proliferation and migration of endothelial cells. Furthermore, VR-13 peptide effectively inhibited the pathological development of CNV without the detection of retinal toxicity in the rat CNV model.
Overall, it was found that VR-13 exhibit significant effects on the inducing of apoptosis and the inhibition of the progression of angiogenesis by regulating the expression of VEGF and CD31 via CD36 signaling pathway.
脉络膜新生血管（CNV）是眼内新生血管形成的关键表现，被认为是眼科致盲的主要原因之一。此外，多种抗血管内皮生长因子（VEGF）药物已被用作CNV的一线治疗药物。然而，抗VEGF药物面临几个问题，主要包括作用持续时间短、需要重复注射以及并发症。血小板反应蛋白-1（TSP-1）是一种内源性蛋白质，被发现可调节体内多种生物学过程，并且已被证明对新生血管形成具有抑制作用。此外，目前认为TSP-1在抑制新生血管形成过程中的功能主要集中在其Ⅰ型重复序列（TSRs）上，这归因于TSP-1分子量较大、结构复杂以及可能存在的未知功能。因此，TSRs可作为进一步开发和探索潜在治疗药物的靶点和研究方向。
本研究基于血小板反应蛋白-1（TSP-1）的Ⅰ型重复序列（TSRs）设计并合成了不同长度的氨基酸序列，命名为VR-9、VR-10、VR-11、VR-12、VR-13。目的是探讨上述五种肽对脉络膜视网膜新生血管形成中血管生成的影响，并筛选最佳肽段并深入探究其潜在机制，旨在为肽类药物治疗CNV的开发和应用提供依据。
采用伤口愈合实验、CCK-8实验和5-乙炔基-2'-脱氧尿苷（EdU）实验评估细胞的增殖和迁移能力。利用CRISPR-Cas9技术建立CD36基因敲低细胞系，并通过qPCR验证基因敲低效率。通过蛋白质免疫印迹法评估RF/6A细胞和大鼠中VEGF和CDH31的表达水平。此外，进行苏木精-伊红（HE）染色以检查大鼠视网膜的结构完整性，同时使用异硫氰酸荧光素-葡聚糖心脏灌注（FITC）标记观察脉络膜新生血管（CNV）的发生和发展。
根据伤口愈合实验和CCK-8实验，VR-13在抑制内皮细胞增殖和迁移方面最为有效。此外，VR-13肽在大鼠CNV模型中有效抑制了CNV的病理发展，且未检测到视网膜毒性。
总体而言，发现VR-13通过CD36信号通路调节VEGF和CD31的表达，对诱导细胞凋亡和抑制血管生成进程具有显著作用。
