利用腺相关病毒1型-Rac1T17N预防实验性增殖性玻璃体视网膜病变。

Leveraging AAV1-Rac1T17N to prevent experimental proliferative vitreoretinopathy.

作者信息

Li Duo, Linghu Minli, Tang Jisen, Yang Gukun, Li Chuanwu, Yao Hang, Lei Hetian, Huang Yikeng, Huang Xionggao

机构信息

Department of Ophthalmology, The First Affiliated Hospital of Hainan Medical University, Haikou, 570102, China.

Key Laboratory of Emergency and Trauma of Ministry of Education, Department of Emergency Surgery, Key Laboratory of Hainan Trauma and Disaster Rescue, The First Affiliated Hospital, Hainan Medical University, Haikou, 570102, China.

出版信息

J Transl Med. 2025 Mar 26;23(1):374. doi: 10.1186/s12967-025-06391-9.

DOI:10.1186/s12967-025-06391-9

PMID:40140939

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11948691/

Abstract

BACKGROUND

Platelet-derived growth factor receptor β (PDGFRβ) is the principal PDGFR isoform in retinal pigment epithelial (RPE) cells from the epiretinal membranes of patients with proliferative vitreoretinopathy (PVR). Ras-related C3 Botulinum toxin substrate 1 (Rac1), a member of the Rho family, is a crucial factor in the cell migration and contraction processes that are inherent to the pathogenesis of PVR. The mutants Rac1T17N and Rac1Q61L can block and promote Rac1 activation, respectively. The major objective of this research was to ascertain whether PDGFRβ mediates vitreous-induced Rac1 activation and whether Rac1T17N could be leveraged for the prevention of PVR pathogenesis in a rabbit model.

METHODS

A pull-down assay was used to examine GTP Rac1 levels, which are indicative of Rac1 activation, and western blotting was used to assess cellular protein expression. A CCK8 assay, a wound healing assay, a transwell invasion assay and a collagen contraction assay were employed to analyze cell proliferation, migration, invasion and contraction capacity, respectively. A PVR model was created by injecting platelet-rich plasma and human retinal pigment epithelial cells (ARPE-19) into the vitreous cavities of rabbits, and this model was used to evaluate the severity of PVR impacted by intravitreally injected ARPE-19 cells transduced with adeno-associated virus (AAV)1-Rac1T17N or Rac1Q61L. PVR grade was evaluated by a double-blinded investigator according to the Fastenberg classification; in addition, ultrasound and histological analyses were performed to assess PVR severity.

RESULTS

Vitreous-induced GTP Rac1 is mediated by PDGFRβ. There was a significant decrease in vitreous-induced GTP Rac1 in ARPE-19 cells transduced with AAV1-Rac1T17N compared with those transduced with AAV1-GFP. In addition, the suppression of GTP Rac1 production in human RPE cells by transduction with AAV1-Rac1T17N inhibited vitreous-induced proliferation, migration, invasion, and contractility. Importantly, the results of the animal experiments indicated that although there was a significant increase in PVR potential in rabbits intravitreally injected with ARPE-19 cells infected with AAV1-Rac1Q61L, there was a significant decrease in PVR potential in rabbits intravitreally injected with ARPE-19 cells infected with AAV1-Rac1T17N (P < 0.01).

CONCLUSIONS

AAV1-Rac1T17N has great potential for PVR therapy.

摘要

背景

血小板衍生生长因子受体β（PDGFRβ）是增殖性玻璃体视网膜病变（PVR）患者视网膜前膜中视网膜色素上皮（RPE）细胞的主要PDGFR亚型。Ras相关的C3肉毒杆菌毒素底物1（Rac1）是Rho家族的成员，是PVR发病机制中固有的细胞迁移和收缩过程的关键因素。突变体Rac1T17N和Rac1Q61L可分别阻断和促进Rac1激活。本研究的主要目的是确定PDGFRβ是否介导玻璃体诱导的Rac1激活，以及Rac1T17N是否可用于预防兔模型中的PVR发病机制。

方法

采用下拉试验检测指示Rac1激活的GTP Rac1水平，并用蛋白质印迹法评估细胞蛋白表达。分别采用CCK8试验、伤口愈合试验、transwell侵袭试验和胶原收缩试验分析细胞增殖、迁移、侵袭和收缩能力。通过将富含血小板的血浆和人视网膜色素上皮细胞（ARPE-19）注入兔玻璃体腔建立PVR模型，该模型用于评估经腺相关病毒（AAV）1-Rac1T17N或Rac1Q61L转导的玻璃体内注射ARPE-19细胞对PVR严重程度的影响。由一名双盲研究者根据Fastenberg分类评估PVR分级；此外，进行超声和组织学分析以评估PVR严重程度。

结果

玻璃体诱导的GTP Rac1由PDGFRβ介导。与用AAV1-GFP转导的ARPE-19细胞相比，用AAV1-Rac1T17N转导的ARPE-19细胞中玻璃体诱导的GTP Rac1显著降低。此外，用AAV1-Rac1T17N转导抑制人RPE细胞中GTP Rac1的产生可抑制玻璃体诱导的增殖、迁移、侵袭和收缩性。重要的是，动物实验结果表明，尽管玻璃体内注射感染AAV1-Rac1Q61L的ARPE-19细胞的兔的PVR潜能显著增加，但玻璃体内注射感染AAV1-Rac1T17N的ARPE-19细胞的兔的PVR潜能显著降低（P<0.01）。