Endothelial cell RpL17-dependent translational control mediates intima-media thickening in response to disturbed flow
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Background
Carotid intima-media thickening (IMT) is a major risk factor for cardiovascular disease (CVD). The large ribosomal subunit protein 17 (Rpl17) was recently reported as a CVD-associated gene; however, ribosomal mutations generally are not associated with vascular dysfunction. We have created a novel genetic model of decreased RpL17 in endothelial cells (EC) to determine how changes in endothelial ribosome expression cause IMT.
Methods
EC-restricted RpL17 heterozygous mice (Cdh5-Cre; RpL17 fl/wt , or Rpl17-Het), were generated and subjected to sham or partial carotid ligation (PCL) surgery of the left artery to induce acute disturbed (d)-flow in vivo . Carotids were harvested on day 14 for quantitative tissue immunostaining. Purified mouse and human EC in vitro were exposed to steady (s)-flow or d-flow using cone viscometry, and collected for flow cytometry, protein expression, electron microscopy, or purification of ribosomes. Human carotid samples from healthy and endarterectomy patients were used for tissue analysis.
Results
Carotids from RpL17-Het mice with PCL-induced d-flow showed increased IMT relative to RpL17-WT controls. In addition, RpL17 protein levels were decreased in regions of d-flow compared to s-flow. Increased levels of ER stress markers were observed by carotid immunostaining, as well as activation of the integrated stress response (ISR) in RpL17-Het EC. Analysis of mRNAs bound to polysomes vs. monosomes in EC-RpL17-Het revealed increased translational efficiency of key regulators of glycolysis, redox, inflammation, matrix, and endothelial-to-mesenchymal transition (EndMT). Metabolic profiling by Seahorse assay showed enhanced anaerobic glycolysis and decreased oxidative respiration in RpL17-Het EC, consistent with the translational efficiency data. Immunostaining of carotids identified upregulated EC inflammation and EndMT.
Conclusions
Our data support RpL17 as a key mediator of EC phenotypic modulation that causes IMT in response to d-flow. We show a novel pathway for d-flow-mediated IMT: endoplasmic reticulum stress and activation of the ISR. These changes alter translational efficiency and reprogram EC cell cycle, metabolism, and redox state in the presence of d-flow to cause IMT, a precursor to cardiovascular pathology.
