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

Abstract

Atherosclerotic lesions develop in arterial areas exposed to low shear stress (SS) like aortic arch where endothelial cells express a pro-inflammatory and pro-senescent phenotype. Our team has shown that defective endothelial autophagy favours an atheroprone phenotype. Previous studies have stated that stabilised microtubules, a function of various post-translational modifications, facilitate efficient fusion of autophagosomes and lysosomes, a critical step in autophagy. IAlso, a link between SS and tubulin acetylation, has been established. The aim of the study is to restore endothelial autophagy flux by inhibition of tubulin deacetylase, Histone Deacetylase6 (HDAC6), thus stabilising microtubules. 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. Levels of protein expression in HUVECs post SS were assessed by western blot analysis and their localisation was observed by immunostaining. Autophagy flux was analysed by using tandem fluorescent-tagged LC3. Levels of acetylated tubulin in HUVECs exposed to LSS and HDAC6 inhibitor, Tubastatin (3 μM) were 10-fold when compared to LSS. Effect of Tubastatin on autophagy was analysed by LC3-II/I ratio. Although, no significant difference was observed by western blot, IF assays show that Tubastatin treatment doubled the area of LC3 punctae in LSS. Monitoring the progression of autophagy flux, we observed that Tubastatin treatment increased the flux in LSS by 2 times. The initial results indicate that Tubastatin may be a good candidate to restore autophagic flux by stabilising microtubules in HUVECs exposed to low SS. We will further investigate the link between HDAC6 inhibition, SS, endothelial autophagy and atherosclerosis by looking at activity, expression and localisation of HDAC6 in HSS and LSS; localisation of acetylated tubulin, LAMP2 and LC3; and anti-inflammatory and anti-senescent effects of Tubastatin.