Restoration of endothelial autophagic flux in vascular endothelial cells exposed to low shear stress reduces atherosclerotic lesions

Abstract

Atherosclerotic lesions preferentially develop in arterial areas exposed to low shear stress (SS), such as the aortic arch. Here, endothelial cells express a pro-inflammatory and pro-senescent phenotype. Our team has shown that this atheroprone phenotype is in part due to an inhibition of endothelial autophagy under low SS conditions. Previous studies have stated that acetylated microtubules are critical for functional autophagy. The aim of the study is to reduce endothelial inflammation by restoring autophagic flux in low SS conditions, via the inhibition of the α-tubulin deacetylase, Histone Deacetylase 6 (HDAC6). Confluent HUVECs, passage 2–4, were exposed to either low SS (2 dyn/cm 2 ) or high SS (20 dyn/cm 2 ) for 24 hours. Localization and levels of the different proteins involved were assessed by immunostaining and Western blot respectively. Autophagic flux was analyzed using tandem fluorescently tagged LC3. To evaluate atherosclerosis development in vivo, we used hypercholesteremic HDAC6 −/− /ApoE −/− mice. We found that blocking HDAC6 activity, by pharmacological (Tubastatin A) or genetic (shHDAC6) approaches, raised levels of acetylated α-tubulin, as well as autophagic flux. This resulted in a reduced expression of inflammatory markers (ICAM-1, VCAM-1 and MCP-1) in TNF-α-stimulated cells. We observed an increase in the colocalization of LC3 and lysosomal marker, LAMP2, in murine aorta of HDAC6 −/− /ApoE −/− mice, compared to control mice. Finally, atherosclerotic plaque size significantly decreased in the atheroprone areas of chimeric HDAC6 −/− /ApoE −/− mice, transplanted with HDAC6 +/+ /ApoE -/− bone marrow, when compared to HDAC6 +/+ /ApoE −/− littermate controls. These results indicate that targeting α-tubulin acetylation, via HDAC6-inhibition, may be an interesting strategy to restore endothelial autophagy and to promote an atheroprotective endothelial phenotype despite unfavorable shear stress conditions.