A novel method for visualizing and tracking endogenous mRNA in a specific cell population in pathological neovascularization

Abstract Diabetic retinopathy, retinopathy of prematurity and retinal vein occlusion are potentially blinding conditions largely due to their respective neovascular components. The development of real-time in vivo molecular imaging methods, to assess levels of retinal neovascularization (NV), would...

Descripción completa

Guardado en:
Detalles Bibliográficos
Autores principales: Md Imam Uddin, Tyler C. Kilburn, Sara Z. Jamal, Craig L. Duvall, John S. Penn
Formato: article
Lenguaje:EN
Publicado: Nature Portfolio 2021
Materias:
R
Q
Acceso en línea:https://doaj.org/article/bf1a0d78122540099a116195b39301a3
Etiquetas: Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
id oai:doaj.org-article:bf1a0d78122540099a116195b39301a3
record_format dspace
spelling oai:doaj.org-article:bf1a0d78122540099a116195b39301a32021-12-02T14:16:58ZA novel method for visualizing and tracking endogenous mRNA in a specific cell population in pathological neovascularization10.1038/s41598-021-81367-52045-2322https://doaj.org/article/bf1a0d78122540099a116195b39301a32021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-81367-5https://doaj.org/toc/2045-2322Abstract Diabetic retinopathy, retinopathy of prematurity and retinal vein occlusion are potentially blinding conditions largely due to their respective neovascular components. The development of real-time in vivo molecular imaging methods, to assess levels of retinal neovascularization (NV), would greatly benefit patients afflicted with these conditions. mRNA hybridization techniques offer a potential method to image retinal NV. The success of these techniques hinges on the selection of a target mRNA whose tissue levels and spatial expression patterns correlate closely with disease burden. Using a model of oxygen-induced retinopathy (OIR), we previously observed dramatic increases in retinal endoglin that localized to neovascular structures (NV), directly correlating with levels of neovascular pathology. Based on these findings, we have investigated Endoglin mRNA as a potential marker for imaging retinal NV in OIR mice. Also of critical importance, is the application of innovative technologies capable of detecting mRNAs in living systems with high sensitivity and specificity. To detect and visualize endoglin mRNA in OIR mice, we have designed and synthesized a novel imaging probe composed of short-hairpin anti-sense (AS) endoglin RNA coupled to a fluorophore and black hole quencher (AS-Eng shRNA). This assembly allows highly sensitive fluorescence emission upon hybridization of the AS-Eng shRNA to cellular endoglin mRNA. The AS-Eng shRNA is further conjugated to a diacyl-lipid (AS-Eng shRNA–lipid referred to as probe). The lipid moiety binds to serum albumin facilitating enhanced systemic circulation of the probe. OIR mice received intraperitoneal injections of AS-Eng shRNA–lipid. Ex vivo imaging of their retinas revealed specific endoglin mRNA dependent fluorescence superimposed on neovascular structures. Room air mice receiving AS-Eng shRNA–lipid and OIR mice receiving a non-sense control probe showed little fluorescence activity. In addition, we found that cells in neovascular lesions labelled with endoglin mRNA dependent fluorescence, co-labelled with the macrophage/microglia-associated marker IBA1. Others have shown that cells expressing macrophage/microglia markers associate with retinal neovascular structures in proportion to disease burden. Hence we propose that our probe may be used to image and to estimate the levels of retinal neovascular disease in real-time in living systems.Md Imam UddinTyler C. KilburnSara Z. JamalCraig L. DuvallJohn S. PennNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-16 (2021)
institution DOAJ
collection DOAJ
language EN
topic Medicine
R
Science
Q
spellingShingle Medicine
R
Science
Q
Md Imam Uddin
Tyler C. Kilburn
Sara Z. Jamal
Craig L. Duvall
John S. Penn
A novel method for visualizing and tracking endogenous mRNA in a specific cell population in pathological neovascularization
description Abstract Diabetic retinopathy, retinopathy of prematurity and retinal vein occlusion are potentially blinding conditions largely due to their respective neovascular components. The development of real-time in vivo molecular imaging methods, to assess levels of retinal neovascularization (NV), would greatly benefit patients afflicted with these conditions. mRNA hybridization techniques offer a potential method to image retinal NV. The success of these techniques hinges on the selection of a target mRNA whose tissue levels and spatial expression patterns correlate closely with disease burden. Using a model of oxygen-induced retinopathy (OIR), we previously observed dramatic increases in retinal endoglin that localized to neovascular structures (NV), directly correlating with levels of neovascular pathology. Based on these findings, we have investigated Endoglin mRNA as a potential marker for imaging retinal NV in OIR mice. Also of critical importance, is the application of innovative technologies capable of detecting mRNAs in living systems with high sensitivity and specificity. To detect and visualize endoglin mRNA in OIR mice, we have designed and synthesized a novel imaging probe composed of short-hairpin anti-sense (AS) endoglin RNA coupled to a fluorophore and black hole quencher (AS-Eng shRNA). This assembly allows highly sensitive fluorescence emission upon hybridization of the AS-Eng shRNA to cellular endoglin mRNA. The AS-Eng shRNA is further conjugated to a diacyl-lipid (AS-Eng shRNA–lipid referred to as probe). The lipid moiety binds to serum albumin facilitating enhanced systemic circulation of the probe. OIR mice received intraperitoneal injections of AS-Eng shRNA–lipid. Ex vivo imaging of their retinas revealed specific endoglin mRNA dependent fluorescence superimposed on neovascular structures. Room air mice receiving AS-Eng shRNA–lipid and OIR mice receiving a non-sense control probe showed little fluorescence activity. In addition, we found that cells in neovascular lesions labelled with endoglin mRNA dependent fluorescence, co-labelled with the macrophage/microglia-associated marker IBA1. Others have shown that cells expressing macrophage/microglia markers associate with retinal neovascular structures in proportion to disease burden. Hence we propose that our probe may be used to image and to estimate the levels of retinal neovascular disease in real-time in living systems.
format article
author Md Imam Uddin
Tyler C. Kilburn
Sara Z. Jamal
Craig L. Duvall
John S. Penn
author_facet Md Imam Uddin
Tyler C. Kilburn
Sara Z. Jamal
Craig L. Duvall
John S. Penn
author_sort Md Imam Uddin
title A novel method for visualizing and tracking endogenous mRNA in a specific cell population in pathological neovascularization
title_short A novel method for visualizing and tracking endogenous mRNA in a specific cell population in pathological neovascularization
title_full A novel method for visualizing and tracking endogenous mRNA in a specific cell population in pathological neovascularization
title_fullStr A novel method for visualizing and tracking endogenous mRNA in a specific cell population in pathological neovascularization
title_full_unstemmed A novel method for visualizing and tracking endogenous mRNA in a specific cell population in pathological neovascularization
title_sort novel method for visualizing and tracking endogenous mrna in a specific cell population in pathological neovascularization
publisher Nature Portfolio
publishDate 2021
url https://doaj.org/article/bf1a0d78122540099a116195b39301a3
work_keys_str_mv AT mdimamuddin anovelmethodforvisualizingandtrackingendogenousmrnainaspecificcellpopulationinpathologicalneovascularization
AT tylerckilburn anovelmethodforvisualizingandtrackingendogenousmrnainaspecificcellpopulationinpathologicalneovascularization
AT sarazjamal anovelmethodforvisualizingandtrackingendogenousmrnainaspecificcellpopulationinpathologicalneovascularization
AT craiglduvall anovelmethodforvisualizingandtrackingendogenousmrnainaspecificcellpopulationinpathologicalneovascularization
AT johnspenn anovelmethodforvisualizingandtrackingendogenousmrnainaspecificcellpopulationinpathologicalneovascularization
AT mdimamuddin novelmethodforvisualizingandtrackingendogenousmrnainaspecificcellpopulationinpathologicalneovascularization
AT tylerckilburn novelmethodforvisualizingandtrackingendogenousmrnainaspecificcellpopulationinpathologicalneovascularization
AT sarazjamal novelmethodforvisualizingandtrackingendogenousmrnainaspecificcellpopulationinpathologicalneovascularization
AT craiglduvall novelmethodforvisualizingandtrackingendogenousmrnainaspecificcellpopulationinpathologicalneovascularization
AT johnspenn novelmethodforvisualizingandtrackingendogenousmrnainaspecificcellpopulationinpathologicalneovascularization
_version_ 1718391629963853824