Selective inhibition of PKR improves vascular inflammation and remodelling in high fructose treated primary vascular smooth muscle cells

Abstract

Background and objectiveDouble-stranded RNA dependent protein kinase (PKR) is reported to play a critical role in the pathogenesis of diabetes and associated vascular complications. Increased PKR activity is observed in metabolic disorders. Increased PKR activity is reported to induce inflammation and oxidative stress. Inflammation and oxidative stress are implicated in the pathogenesis of vascular disease. There are no studies done so far about the role of PKR in vascular smooth muscle cells (VSMCs) and the underlying molecular mechanism. Thus the aim of the present study is to investigate the role of PKR in high fructose treated VSMCs. Moreover, a selective PKR inhibitor, imoxin (C16) was used to investigate the underlying molecular mechanism. MethodsVSMCs were isolated by enzymatic digestion method from thoracic aorta of rats and incubated with high fructose (HF) and PKR inhibitor. Immunocytochemistry and Western blotting were performed for PKR and its downstream markers of inflammation, apoptosis and phenotypic transition (AGEs, MMP-9, and ERK1/2). Oxidative stress was measured using flow cytometry. Cellular hypertrophy and proliferative index were determined by haematoxylin and eosin staining, MTT assay, BrdU labelling assay and agarose gel electrophoresis. Scratch test was done for migratory behaviour. Alizarin red staining was performed for assessing vascular calcification. Mitochondrial membrane potential and chromatin condensation was determined by rhodamine 6G and DAPI staining. ResultsPKR expression was significantly increased in HF treated VSMCs which was accompanied by increase in levels of gene markers of inflammation, oxidative stress and apoptosis. Moreover, increase in cellular proliferation, phenotypic switch and decrease in membrane potential was observed in HF treated VSMCs. All these effects of HF were attenuated by selective PKR inhibitor, imoxin (C16). ConclusionIn conclusion PKR activation plays an important role in the pathogenesis of vascular inflammation and remodelling, and therapeutically targeting PKR could be an effective approach to treat the abnormalities associated with vascular complications.