Abstract
The cardiovascular disease of atherosclerosis is characterised by aged vascular smooth muscle cells and compromised cell survival. Analysis of human and murine plaques highlights markers of DNA damage such as p53, Ataxia telangiectasia mutated (ATM), and defects in mitochondrial oxidative metabolism as significant observations. The antiageing protein Klotho could prolong VSMC survival in the atherosclerotic plaque and delay the consequences of plaque rupture by improving VSMC phenotype to delay heart attacks and stroke. Comparing wild-type VSMCs from an ApoE model of atherosclerosis with a flox’d Pink1 knockout of inducible mitochondrial dysfunction we show WT Pink1 is essential for normal cell viability, while Klotho mediates energetic switching which may preserve cell survival. Methods: Wild-type ApoE VSMCs were screened to identify potential drug candidates that could improve longevity without inducing cytotoxicity. The central regulator of cell metabolism AMP Kinase was used as a readout of energy homeostasis. Functional energetic switching between oxidative and glycolytic metabolism was assessed using XF24 technology. Live cell imaging was then used as a functional readout for the WT drug response, compared with Pink1 (phosphatase-and-tensin-homolog (PTEN)-induced kinase-1) knockout cells. Results: Candidate drugs were assessed to induce pACC, pAMPK, and pLKB1 before selecting Klotho for its improved ability to perform energetic switching. Klotho mediated an inverse dose-dependent effect and was able to switch between oxidative and glycolytic metabolism. Klotho mediated improved glycolytic energetics in wild-type cells which were not present in Pink1 knockout cells that model mitochondrial dysfunction. Klotho improved WT cell survival and migration, increasing proliferation and decreasing necrosis independent of effects on apoptosis. Conclusions: Klotho plays an important role in VSMC energetics which requires Pink1 to mediate energetic switching between oxidative and glycolytic metabolism. Klotho improved VSMC phenotype and, if targeted to the plaque early in the disease, could be a useful strategy to delay the effects of plaque ageing and improve VSMC survival.
Highlights
Cardiovascular diseases such as atherosclerosis exhibit profound ageing of the vessel wall that is linked to excess DNA damage and mitochondrial dysfunction [1]
As we have previously shown, Pink1 is an important regulator of VSMC energetic switching from oxidative phosphorylation to glycolytic metabolism, and we determined if the known energetic switching effects of Klotho were relevant in VSMCs and influenced by Pink1
We identified 10 compounds from the literature that have been shown to play important roles in cell survival, either through activation of the energy sensor AMPK or by evidence of inducing energetic switching between oxidative and glycolytic metabolism
Summary
Cardiovascular diseases such as atherosclerosis exhibit profound ageing of the vessel wall that is linked to excess DNA damage and mitochondrial dysfunction [1]. Vascular smooth muscle cells found in disease-prone regions are characterised by defects in DNA repair, oxidative metabolism, and shortened telomeres. These activate signalling pathways to mediate cell cycle arrest and apoptosis which promote cell senescence and inflammation [2,3]. Intervening in oxidative metabolism required for normal cellular homeostasis is an attractive candidate to improve plaque cellular energetics. Our previous work has shown that diseased VSMCs retain their ability to energetically switch between oxidative and glycolytic metabolism. If a regulatable switch to aerobic glycolysis could be achieved pharmacologically, improving VSMC survival could be a realistic goal
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