AbstractDiabetic retinopathy (DR) is a multifactorial disease displaying vascular-associated pathologies, including vascular leakage and neovascularization, ultimately leading to visual impairment. However, animal models accurately reflecting these pathologies are lacking. Vascular endothelial growth factor A (VEGF-A) is an important factor in the development of micro- and macro-vascular pathology in DR.

摘要糖尿病视网膜病变（DR）是一种多因素疾病，表现为血管相关病变，包括血管渗漏和新血管形成，最终导致视力障碍。然而，缺乏准确反映这些病理的动物模型。血管内皮生长因子A（VEGF-A）是DR微观和宏观血管病理学发展的重要因素。

In this study, we evaluated the feasibility of using a cumate-inducible lentivirus (LV) mediated expression of vegf-a to understand DR pathology in vitro and in vivo. Retinal pigment epithelial cells (ARPE-19) were transduced with cumate-inducible LV expressing vegf-a, with subsequent analysis of vegf-a expression and its impact on cell proliferation, viability, motility, and permeability.

在这项研究中，我们评估了使用累积诱导型慢病毒（LV）介导的vegf-a表达来了解体外和体内DR病理学的可行性。用表达vegf-a的cumate诱导型LV转导视网膜色素上皮细胞（ARPE-19），随后分析vegf-a的表达及其对细胞增殖，活力，运动性和通透性的影响。

Cumate tolerability in adult Wistar rat eyes was assessed as an initial step towards a potential DR animal model development, by administering cumate via intravitreal injections (IVT) and evaluating consequent effects by spectral domain optical coherence tomography (SD-OCT), flash electroretinography (fERG), ophthalmic examination (OE), and immunohistochemistry.

通过玻璃体内注射（IVT）给予孜酸盐并通过光谱域光学相干断层扫描（SD-OCT），闪光视网膜电图（fERG）评估随后的效果，评估成年Wistar大鼠眼睛的孜酸盐耐受性，作为潜在DR动物模型开发的第一步，眼科检查（OE）和免疫组织化学。

Transduction of ARPE-19 cells with cumate-inducible LV resulted in ~ 2.5-fold increase in vegf-a mRNA and ~ threefold increase in VEGF-A protein secretion. Transduced cells displayed enhanced cell proliferation, viability, permeability, and migration in tube-like structures. However, IVT cumate injections led to apparent retinal toxicity, manifesting as retinal layer abnormalities, haemorrhage, vitreous opacities, and significant reductions in a- and b-wave amplitudes, along with increased microglial activation and reactive gliosis.

用累积诱导型LV转导ARPE-19细胞导致vegf-a mRNA增加约2.5倍，vegf-a蛋白分泌增加约三倍。转导的细胞在管状结构中显示出增强的细胞增殖，活力，通透性和迁移。然而，IVT-cumate注射导致明显的视网膜毒性，表现为视网膜层异常，出血，玻璃体混浊，a波和b波振幅显着降低，以及小胶质细胞活化和反应性神经胶质增生增加。

In summary, while cumate-inducible LV-mediated vegf-a expression is valuable for in vitro mecha.

总之，虽然cumate诱导的LV介导的vegf-a表达对于体外机制是有价值的。

IntroductionDiabetic retinopathy (DR) is the leading cause of preventable blindness in the world and the primary cause of vision loss among working-age adults1. DR primarily results in microvascular changes, including pericyte loss2, increased vascular permeability3 and formation of acellular capillaries4.

引言糖尿病视网膜病变（DR）是世界上可预防失明的主要原因，也是工作年龄成年人视力丧失的主要原因1。DR主要导致微血管改变，包括周细胞丢失2，血管通透性增加3和无细胞毛细血管形成4。

Prolonged duration of diabetes results in the appearance of microaneurysms and haemorrhages, associated with vascular leakage and neovascularization5. In the absence of intervention, diabetic retinopathy (DR) may progress to diabetic macular edema (DME) and proliferative diabetic retinopathy (PDR), ultimately culminating in complete vision loss.

糖尿病持续时间延长会导致微动脉瘤和出血的出现，并伴有血管渗漏和新血管形成5。在没有干预的情况下，糖尿病视网膜病变（DR）可能会发展为糖尿病性黄斑水肿（DME）和增殖性糖尿病视网膜病变（PDR），最终导致完全视力丧失。

Animal models which recapitulate vascular pathophysiology of DR are essential for studying and understanding the underlying molecular mechanisms, as well as developing and testing novel therapies to treat the disease.Vascular endothelial growth factor-A (VEGF-A) is highly upregulated in diabetic retinopathy and induces the progression of various events characterizing the disease, including vascular leakage, neovascularization, and DME6.

概括DR血管病理生理学的动物模型对于研究和理解潜在的分子机制以及开发和测试治疗该疾病的新疗法至关重要。血管内皮生长因子-A（VEGF-A）在糖尿病视网膜病变中高度上调，并诱导表征该疾病的各种事件的进展，包括血管渗漏，新血管形成和DME6。

VEGF antagonists are currently in clinical use for treating vascular abnormalities6, but such therapies require re-peated intraocular injections. To study the role of VEGF-A and develop novel anti-VEGF-A treatments, VEGF-A-induced animal models have been generated. Intravitreal injections of recombinant VEGF-A have been shown to mimic many of the complex DR mechanisms, including vascular leakage and neovascularization in rabbits7,8.

VEGF拮抗剂目前正在临床上用于治疗血管异常6，但这种疗法需要重复眼内注射。为了研究VEGF-A的作用并开发新的抗VEGF-A治疗方法，已经产生了VEGF-A诱导的动物模型。玻璃体内注射重组VEGF-A已被证明可以模拟许多复杂的DR机制，包括兔子的血管渗漏和新血管形成7,8。

However, VEGF-A-induced effects are transient due to the short half-life of injected peptides. To overcome this drawback, gene transfer models which mediate long-term expression of VEGF-A have been established. Adeno-associated virus (AAV) based VEGF-A delivery in m.

然而，由于注射肽的半衰期短，VEGF-A诱导的作用是短暂的。为了克服这个缺点，已经建立了介导VEGF-A长期表达的基因转移模型。基于腺相关病毒（AAV）的VEGF-A在m中的递送。

Data availability

数据可用性

All fERG a-wave and b-wave absolute values can be found in Supplementary Table S1. Other datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

所有fERG a波和b波绝对值均可在补充表S1中找到。在本研究中使用和/或分析的其他数据集可根据合理要求从通讯作者处获得。

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Download referencesAcknowledgementsThis work was funded, in part, by the European Union’s Horizon 2020 Research and Innovation Programme under the Mari Sklodowska-Curie Actions (grant agreement—No 813440). Additional funding from the Dr. John P. and Therese E. Mulcahy Endowed Professorship in Ophthalmology (to SK) and the Richard A.

下载参考文献致谢这项工作部分由Mari Sklodowska-Curie Actions（第813440号赠款协议）下的欧盟地平线2020研究与创新计划资助。John P.博士和Therese E.Mulcahy博士额外资助眼科教授（SK）和Richard A。

Perritt M.D. Charitable Foundation (to SK) is gratefully acknowledged.FundingEuropean Union’s Horizon 2020 Research and Innovation Programme under the Mari Sklodowska-Curie Actions,813440,813440,813440,813440Author informationAuthors and AffiliationsInstitute of Inflammation and Ageing, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UKInesa Lelyte & Zubair AhmedR&D Division, Experimentica Ltd., 10243, Vilnius, LithuaniaInesa Lelyte, Giedrius Kalesnykas & Symantas RagauskasDepartment of Ophthalmology, Loyola University Chicago, Maywood, IL, 60153, USAInesa Lelyte, Vidhya R.

感谢Perritt M.D.慈善基金会（致SK）。资助Mari Sklodowska-Curie Actions下的欧盟地平线2020研究与创新计划，813440813440813440813440813440813440813440作者信息作者和附属机构伯明翰大学炎症与衰老研究所，Edgbaston，伯明翰，B15 2TT，UKInesa Lelyte＆Zubair AhmedR＆D部门，实验有限公司，10243，维尔纽斯，立陶宛Lelyte，Giedrius Kalesnykas＆Symantas Ragauskas芝加哥洛约拉大学眼科，梅伍德，伊利诺伊州，60153，USAInesa Lelyte，Vidhya R。

Rao & Simon KajaR&D Division, Experimentica Ltd., Kuopio, FinlandGiedrius KalesnykasExperimentica Inc., Fort Worth, TX, USAGiedrius KalesnykasDepartment of Molecular Pharmacology and Neuroscience, Loyola University Chicago, Maywood, IL, 60153, USASimon KajaCentre for Trauma Sciences Research, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UKZubair AhmedAuthorsInesa LelyteView author publicationsYou can also search for this author in.

Rao＆Simon KajaR＆D Division，Experistica Ltd.，Kuopio，FinlandGiedrius KalesnykasExperistica Inc.，德克萨斯州沃思堡，USAGiedrius Kalesnykas芝加哥洛约拉大学分子药理学和神经科学系，伊利诺伊州梅伍德，60153，USASimon KajaCentre for Trauma Sciences Research，伯明翰大学埃德巴斯顿，伯明翰，B15 2TT，UKZubair AhmedAuthorsInesa LelyteView作者出版物您也可以在中搜索这位作者。

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PubMed Google ScholarContributionsI.L.: Methodology, Investigation, Statistics, Writing—original draft, Writing—review and editing. V.R.R., and S.R.: Methodology, Investigation, Writing—review and editing. G.K., and S.K.; Conceptualization, Supervision, Funding Acquisition, Writing—review and editing, Supervision.

PubMed谷歌学术贡献。五十、 ：方法论、调查、统计、撰写初稿、撰写评论和编辑。五、 R.R.和S.R.：方法论，调查，写作评论和编辑。G、 K.和S.K。；概念化，监督，资金获取，写作审查和编辑，监督。

Z.A.: Funding acquisition, Writing—review & editing, Supervision.Corresponding authorsCorrespondence to.

Z、 答：资金获取，写作审查和编辑，监督。通讯作者通讯。

Inesa Lelyte or Zubair Ahmed.Ethics declarations

Inesa Lelyte或Zubair Ahmed。道德宣言

Competing interests

相互竞争的利益

Employment: G.K., S.R. and I.L. (Experimentica Ltd.); Stock/equity ownership: G.K. (Experimentica Ltd.) and S.K. (K&P Scientific LLC). S.K. conducts academic research in areas of interest like the business interests of K&P Scientific LLC. The terms of this arrangement have been reviewed and approved by Loyola University Chicago in accordance with its conflict-of-interest policy.

；股票/股权所有权：G.K.（Experiica Ltd.）和S.K.（K＆P Scientific LLC）。S、 K.在K＆P Scientific LLC的商业利益等利益领域进行学术研究。该安排的条款已由芝加哥洛约拉大学根据其利益冲突政策进行了审查和批准。

The funders had no role in the study. Z.A. and V.R.R. declare no conflicts of interest..

资助者在这项研究中没有任何作用。Z、 A.和V.R.R.声明没有利益冲突。。

Additional informationPublisher's noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary InformationSupplementary Table S1.Rights and permissions

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Reprints and permissionsAbout this articleCite this articleLelyte, I., Rao, V.R., Kalesnykas, G. et al. Prospects and limitations of cumate-inducible lentivirus as a tool for investigating VEGF-A-mediated pathology in diabetic retinopathy.

转载和许可本文引用本文Lelyte，I.，Rao，V.R.，Kalesnykas，G。等人。枯草酸盐诱导型慢病毒作为研究糖尿病视网膜病变中VEGF-a介导的病理学的工具的前景和局限性。

Sci Rep 14, 14325 (2024). https://doi.org/10.1038/s41598-024-63590-yDownload citationReceived: 20 November 2023Accepted: 30 May 2024Published: 21 June 2024DOI: https://doi.org/10.1038/s41598-024-63590-yShare this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

科学报告1414325（2024）。https://doi.org/10.1038/s41598-024-63590-yDownload引文接收日期：2023年11月20日接收日期：2024年5月30日发布日期：2024年6月21日OI：https://doi.org/10.1038/s41598-024-63590-yShare本文与您共享以下链接的任何人都可以阅读此内容：获取可共享链接对不起，本文目前没有可共享的链接。复制到剪贴板。

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KeywordsDiabetic retinopathyCumate-inducible lentivirusGene expressionVascular endothelial growth factorRetinal toxicity

关键词糖尿病视网膜病变诱导型慢病毒基因表达血管内皮生长因子视网膜毒性

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