Sub-lethal HHcy-induced adaptive UPR controls endothelial migration defect. (A) Representative western blots showing protein levels of UPR markers GRP78, IRE1p and ATF4 are upregulated upon 2 mM Hcy treatment for 24 h. Terminal UPR marker CHOP remained unaltered post-sub-lethal Hcy treatment. As a loading control β-actin was used. Corresponding bar graph showing densitometric analysis (normalized to β-actin) of the blots. (B,C) Respective quantifications of scratch wound assay at 24 h revealing that chemical chaperone 4-PBA (1 mM) and TUDCA (1 mM) can significantly improve sub-lethal HHcy-induced endothelial migration defect. (D) Representative confocal images of rhodamine-phalloidin stained endothelial cells showing that sub-lethal Hcy-induced abnormally elongated cell morphology as well as actin stress fiber (white arrows) disappearance are rescued by 4-PBA pre-treatment. Scale bar, 5 μm. (E) ImageJ based analysis demonstrating that 4-PBA pre-treatment reversed the aberrant reduction in cellular aspect ratio (major axis/minor axis), induced by 24 h treatment of 2 mM Hcy. (F) Quantification by ImageJ suggesting that exposure to sub-lethal Hcy significantly decreased the surface area of endothelial cells which was rescued by 4-PBA. (G) Bar plot showing no beneficial effect of chemical chaperone TUDCA on impairment of endothelial proliferation caused by sub-lethal Hcy treatment. Data are shown as Mean ± SEM with n ≥ 3. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, and ns is non-significant (p > 0.05).
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