Abstract
Background and aimsDNA damage and mitochondrial dysfunction are thought to play an essential role in ageing and the energetic decline of vascular smooth muscle cells (VSMCs) essential for maintaining plaque integrity. We aimed to better understand VSMCs and identify potentially useful compensatory pathways that could extend their lifespan. Moreover, we wanted to assess if defects in mitochondrial respiration exist in human atherosclerotic plaques and to identify the appropriate markers that may reflect a switch in VSMC energy metabolism. MethodsHuman plaque tissue and cells were assessed for composition and evidence of DNA damage, repair capacity and mitochondrial dysfunction. Fresh plaque tissue was evaluated using high resolution oxygen respirometry to assess oxidative metabolism. Recruitment and processing of the mitochondrial regulator of autophagy Pink1 kinase was investigated in combination with transcriptional and protein markers associated with a potential switch to a more glycolytic metabolism. ResultsHuman VSMC have increased nuclear (nDNA) and mitochondrial (mtDNA) damage and reduced repair capacity. A subset of VSMCs within plaque cap had decreased oxidative phosphorylation and expression of Pink1 kinase. Plaque cells demonstrated increased glycolytic activity in response to loss of mitochondrial function. A potential compensatory glycolytic program may act as energetic switch via AMP kinase (AMPK) and hexokinase 2 (Hex2). ConclusionsWe have identified a subset of plaque VSMCs required for plaque stability that have increased mitochondrial dysfunction and decreased oxidative phosphorylation. Pink1 kinase may initiate a cellular response to promote a compensatory glycolytic program associated with upregulation of AMPK and Hex2.
Highlights
Human atherosclerotic plaques are fibro-fatty arterial wall lesions which frequently rupture into the circulation and arguably are responsible for the majority of heart attacks and strokes
Using time-lapse microscopy, over 48 h, we found plaque cells proliferated more slowly and underwent apoptosis more localisation, (viii) and after uncoupling. (C) Quantification of Pink1 and Tom20 localisation after uncoupling using the non-potentiometric dye and Pink-1 ± uncoupling with CCCP. (D) Western blot of phospho and total AMP kinase (AMPK) abundance and before and after uncoupling with CCCP and the DNA damaging agent t-BHP (n 1⁄4 3) normalised to beta tubulin. (E) Quantification of p-AMPK protein expression. (ANOVA * p 1⁄4 0.05)
We identified the plaque cap cells in terminal decline, as tentatively having an alternative bioenergetic profile compared to the vessel wall and neighbouring plaque structures
Summary
Human atherosclerotic plaques are fibro-fatty arterial wall lesions which frequently rupture into the circulation and arguably are responsible for the majority of heart attacks and strokes. It is predicted that this need to proliferate and repair generates an ATP (adenosine triphosphate) demand and excess reactive species (RS) and oxygen free radicals that drives further DNA damage, genomic instability and mitochondrial dysfunction [6e8]. We predict that these characteristics accelerate atherosclerotic plaque formation and limit VSMCs regenerative capacity [9,10].
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