| Abstract: | The protocols in this chapter describe methods for identifying the functional roles of retinoic acid receptor (RAR) and retinoid X receptor (RXR) signaling in atherosclerosis and developing RARalpha/RXRalpha-specific agonists as hemodynamics-based therapeutic components for atherosclerosis treatment. In vitro cell culture flow system is used to elucidate the effects of different flow patterns and shear stresses, i.e., atherogenic oscillatory shear stress (OS) vs. atheroprotective pulsatile shear stress (PS), on RAR/RXR signaling and inflammatory responses in vascular endothelial cells (ECs). Western blotting, nuclear and cytoplasmic protein extraction, immunoprecipitation, and in situ proximity ligation assay are used to examine the expression, location, and association of RARs (i.e., RARalpha, RARbeta, and RARgamma) and RXRs (i.e., RXRalpha, RXRbeta, and RXRgamma) in ECs in response to OS vs. PS. Chromatin immunoprecipitation is used to examine the binding activity of RARalpha/RA-responsive elements (RARE). RT-microRNA (miR) quantitative real-time PCR and RT-PCR are used to detect the expressions of miR-10a and pro-inflammatory molecules, respectively. Specific siRNAs of RARalpha and RXRalpha, precursor miR-10a (PreR-10a), and antagomiR-10a (AMR-10a) are used to elucidate the regulatory roles of RARalpha, RXRalpha, and miR-10a in pro-inflammatory signaling in ECs. RARalpha/RXRalpha-specific agonists are used to induce miR-10a expression and inhibit OS-induced pro-inflammatory signaling in ECs in vitro. Apolipoprotein E-deficient (ApoE(-/-)) mice are used as an atherosclerotic animal model. Administration of ApoE(-/-) mice with RARalpha/RXRalpha-specific agonists results in inhibitions in atherosclerotic lesion formation. Co-administration of ApoE(-/-) mice with RARalpha/RXRalpha agonists and AMR-10a is performed to identify the role of miR-10a in RARalpha/RXRalpha agonists-mediated inhibition in atherosclerotic lesions. Oil Red O staining and H&E staining are used to examine the levels of atherosclerotic lesions in the vessel wall. In situ miR hybridization and immunohistochemical staining are used to detect the expression of miR-10a and pro-inflammatory molecules and the infiltration of inflammatory cells in the vessel wall. RARalpha/RXRalpha-specific agonists are used to mimic the atheroprotective effects of PS to induce endothelial miR-10a and hence repress OS-induced pro-inflammatory signaling and atherosclerotic lesion formation in vivo. The results indicate that RAR/RXR-specific agonists have great potential to be developed as hemodynamics-based therapeutic components for atherosclerosis treatment. |
