Dataset Information

A Novel Therapeutic Approach to Corneal Alkaline Burn Model by Targeting Fidgetin-Like 2, a Microtubule Regulator.

ABSTRACT:

Purpose

The purpose of this study was to determine the efficacy of nanoparticle-encapsulated Fidgetin-like 2 (FL2) siRNA (FL2-NPsi), a novel therapeutic agent targeting the FL2 gene, for the treatment of corneal alkaline chemical injury.

Methods

Eighty 12-week-old, male Sprague-Dawley rats were divided evenly into 8 treatment groups: prednisolone, empty nanoparticles, control-NPsi (1 µM, 10 µM, and 20 µM) and FL2-NPsi (1 µM, 10 µM, and 20 µM). An alkaline burn was induced onto the cornea of each rat, which was then treated for 14 days according to group assignment. Clinical, histopathologic, and immunohistochemical analyses were conducted to assess for wound healing. FL2-NPsi-mediated knockdown of FL2 was confirmed by in vitro quantitative polymerase chain reaction (qPCR). Toxicity assays were performed to assess for apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling [TUNEL] assay) and nerve damage (whole mount immunochemical staining). Statistical analyses were performed using Student's t-test and ANOVA.

Results

Compared with controls, FL2-NPsi-treated groups demonstrated enhanced corneal wound healing, with the 10 and 20 µM FL2-NPsi-treated groups demonstrating maximum rates of corneal re-epithelialization as assessed by ImageJ software, enhanced corneal transparency, and improved stromal organization on histology. Immunohistochemical analysis of vascular endothelial cells, macrophages, and neutrophils did not show significant differences between treatment groups. FL2-NPsi was not found to be toxic to nerves or induce apoptosis (p = 0.917).

Conclusions

Dose-response studies found both 10 and 20 µM FL2-NPsi to be efficacious in this rat model. FL2-NPsi may offer a novel treatment for corneal alkaline chemical injuries.

Translational relevance

Basic cell biology findings about the microtubule cytoskeleton were used to design a therapeutic to enhance corneal cell migration, highlighting the promise of targeting microtubules to regulate corneal wound healing.

SUBMITTER: Wang J

PROVIDER: S-EPMC7804583 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Publications

A Novel Therapeutic Approach to Corneal Alkaline Burn Model by Targeting Fidgetin-Like 2, a Microtubule Regulator.

Wang Jessie J Dey Abhinav A Kramer Adam H AH Miao Yuan Y Liu Juan J Baker Lisa L Friedman Joel M JM Nacharaju Parimala P Chuck Roy S RS Zhang Cheng C Sharp David J DJ

Translational vision science & technology 20210108 1

<h4>Purpose</h4>The purpose of this study was to determine the efficacy of nanoparticle-encapsulated Fidgetin-like 2 (FL2) siRNA (FL2-NPsi), a novel therapeutic agent targeting the <i>FL2</i> gene, for the treatment of corneal alkaline chemical injury.<h4>Methods</h4>Eighty 12-week-old, male Sprague-Dawley rats were divided evenly into 8 treatment groups: prednisolone, empty nanoparticles, control-NPsi (1 µM, 10 µM, and 20 µM) and FL2-NPsi (1 µM, 10 µM, and 20 µM). An alkaline burn was induced o ...[more]

PMID: 33510956

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Project description:PurposeCorneal neovascularization (CNV) impairs corneal transparency and visual acuity. The study aims to deepen our understanding of the molecules involved in CNV induced by alkali burns, facilitate a better grasp of CNV mechanisms, and uncover potential therapeutic targets.MethodsEighty-four mice were selected for establishing CNV models via alkali burns. On days 3, 7, and 14 after the burns, corneal observations and histological investigations were conducted. An integrated analysis of RNA sequencing (RNA-seq)-based transcriptomics and label-free quantitative proteomics was performed in both normal and burned corneas. Bioinformatics approaches, encompassing Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, were applied to discern differentially expressed genes (DEGs) and crucial signaling pathways. Four potentially CNV-related genes were validated using quantitative real-time PCR (qRT-PCR) and Western blot.ResultsSignificant CNV was observed on the seventh day. Forty-one genes were differentially expressed in neovascularized corneas, with 15 upregulated and 26 downregulated at both mRNA and protein levels. Bioinformatics analysis revealed that these DEGs participated in diverse biological processes, encompassing retinol and retinoic acid metabolism, neutrophil chemotaxis, and actin filament assembly, along with significant enrichment pathways like cytochrome P450, tyrosine, and phenylalanine metabolism. The upregulation of lymphocyte cytosolic protein 1 (LCP1) and cysteine and glycine-rich protein 2 (CSRP2) genes and the downregulation of transglutaminase 2 (TGM2) and transforming growth factor-beta-induced (TGFBI) genes were confirmed.ConclusionsWe analyzed gene expression differences in mouse corneas 7 days after alkali burns, finding 41 genes with altered expression. The exact role of these genes in CNV is not fully understood, but exploring angiogenesis-related molecules offers potential for CNV treatment or prevention.

Project description:Lymphangiogenesis is one of the major causes of corneal graft rejection. Among the lymphangiogenic factors, vascular endothelial growth factor (VEGF)-C and -D are considered to be the most potent. Both bind to VEGF receptor 3 (VEGFR3) to activate Prospero homeobox 1 (Prox1), a transcription factor essential for the development and maintenance of lymphatic vasculature. MicroRNAs (miRNAs) bind to the 3' untranslated regions (3' UTRs) of target genes in a sequence-specific manner and suppress gene expression. In the current study, we searched for miRNAs that target the pro-lymphangiogenic factor Prox1.Among the miRNAs predicted by the bioinformatic analysis to seed match with the 3' UTR of Prox-1, we chose 3 (miR-466, miR-4305, and miR-4795-5p) for further investigation. Both the miR-466 and miR-4305 mimics, but not the miR-4795-5p mimic, significantly reduced the luciferase activity of the Prox-1 3' UTR reporter vector. In primary lymphatic endothelial cells (HDLEC), miR-466 mimic transfection suppressed Prox1 mRNA and protein expression, while miR-4305 mimic transfection did not. Experiments using mutated reporter constructs of the two possible seed match sites on the 3' UTR of Prox1 suggested that the target site 2 directly bound miR-466. HDLEC transfected with the miR-466 mimic suppressed tube formation as compared to the scrambled control. Furthermore, HDLEC transfected with a miR-466 inhibitor showed enhanced tube formation as compared to control inhibitor transfected cells, and this inhibitory effect was counteracted by Prox1 siRNA. The miR-466 mimic reduced angiogenesis and lymphangiogenesis resulting in clearer corneas in an cornea injury rat model compared to the scrambled control.Our data suggest that miR-446 may have a protective effect on transplanted corneas by suppressing Prox1 expression at the post-transcriptional level. The results of the current study may provide insights into the mechanisms of lymphangiogenesis resulting from corneal graft rejection and alkali-burn injuries, as well as into the development of new treatments for lymphangiogenic eye diseases.

Dataset Information

A Novel Therapeutic Approach to Corneal Alkaline Burn Model by Targeting Fidgetin-Like 2, a Microtubule Regulator.

Purpose

Methods

Results

Conclusions

Translational relevance

Publications

A Novel Therapeutic Approach to Corneal Alkaline Burn Model by Targeting Fidgetin-Like 2, a Microtubule Regulator.

Similar Datasets

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