Abstract
Purpose :
Bone marrow-derived progenitor cells and macrophages migrate to the retina in response to neovascularization. The exact role of these migratory cells in the pathogenesis of neovascularization remains largely unknown. Here, we describe a novel method to visualize and track unmodified endogenous mRNA in a specific population of bone marrow-derived cells that are associated with pathological neovascularization in a mouse model of oxygen-induced retinopathy (OIR).
Methods :
Fluorescence imaging was performed to detect mRNA in a specific population of migratory cells in the living retina in response to OIR. The imaging probes (AS-shRNA-lipid) were synthesized from diacyl-lipids conjugated to anti-sense short hairpin RNA with an anti-sense sequence complimentary to endoglin mRNA and a fluorophore that is quenched by a black-hole quencher (BHQ). The short hairpin RNA probes were stabilized using 2’-O-methyl-protected ribonucleotides allowing in vivo delivery of AS-shRNA-lipid to the target cells that are associated with neovascularization.
Results :
Endoglin (CD105) is a transmembrane auxiliary receptor for transforming growth factor-beta (TGF-β) that is predominantly expressed in proliferating vascular endothelial cells and bone marrow-derived progenitor cells. In vivo molecular imaging of endoglin mRNA was achieved in bone marrow-derived progenitor cells of living mice using the novel in vivo molecular imaging method. We have identified these bone marrow-derived cells with distinct gene expression profiles in OIR animal model using single cell RNA sequencing analysis.
Conclusions :
In summary, we have developed a novel method to detect endoglin mRNA in a specific population of cells in living ocular tissues using diacyl-lipid-conjugates of antisense short hairpin RNA (AS-shRNA-lipid) designed to target endoglin mRNA. These probes are readily internalized to a specific cell population that is associated with neovascularization, and they bind to endoglin mRNA in vivo, allowing molecular imaging of vascular disease of the living retina. These findings may provide a framework for a new strategy to detect and manage retinal neovascularization.
Keywords: molecular imaging, mRNA, diabetic retinopathy, neovascularization.
This is a 2020 ARVO Annual Meeting abstract.