A novel method for visualizing and tracking endogenous mRNA in a specific cell
population in pathological neovascularization.
Authors Uddin MI, Kilburn TC, Jamal SZ, Duvall CL, Penn JS
Submitted By Submitted Externally on 2/10/2021
Status Published
Journal Scientific reports
Year 2021
Date Published 1/1/2021
Volume : Pages 11 : 2565
PubMed Reference 33510218
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.

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