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...

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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:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/bf1a0d78122540099a116195b39301a3
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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
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A novel method for visualizing and tracking endogenous mRNA in a specific cell population in pathological neovascularization

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