Abstract
It has been suggested that miR-144 is pro-atherosclerotic via effects on reverse cholesterol transportation targeting the ATP binding cassette protein. This study used proteomic analysis to identify additional cardiovascular targets of miR-144, and subsequently examined the role of a newly identified regulator of atherosclerotic burden in miR-144 knockout mice receiving a high fat diet. To identify affected secretory proteins, miR-144 treated endothelial cell culture medium was subjected to proteomic analysis including two-dimensional gel separation, trypsin digestion, and nanospray liquid chromatography coupled to tandem mass spectrometry. We identified 5 gel spots representing 19 proteins that changed consistently across the biological replicates. One of these spots, was identified as vimentin. Atherosclerosis was induced in miR-144 knockout mice by high fat diet and vascular lesions were quantified by Oil Red-O staining of the serial sectioned aortic root and from en-face views of the aortic tree. Unexpectedly, high fat diet induced extensive atherosclerosis in miR-144 knockout mice and was accompanied by severe fatty liver disease compared with wild type littermates. Vimentin levels were reduced by miR-144 and increased by antagomiR-144 in cultured cardiac endothelial cells. Compared with wild type, ablation of the miR-144/451 cluster increased plasma vimentin, while vimentin levels were decreased in control mice injected with synthetic miR-144. Furthermore, increased vimentin expression was prominent in the commissural regions of the aortic root which are highly susceptible to atherosclerotic plaque formation. We conclude that miR-144 maybe a potential regulator of the development of atherosclerosis via changes in vimentin signaling.
Highlights
It has been suggested that miR-144 is pro-atherosclerotic via effects on reverse cholesterol transportation targeting the ATP binding cassette protein
We took advantage of powerful nanospray liquid chromatography coupled to tandem mass spectrometry proteomics, identified vimentin as a target of miR-144, and employed primary cultured cardiac endothelial cell, miR-144/451 KO fed high fat diet (HFD), and mice injected with synthetized miR-144 as model systems to examine the impact of loss of miR-144 on vimentin expression and atherogenesis
In this study we identified, using proteomic analysis and subsequent in-vitro and in-vivo models, that miR-144 targets vimentin which, in turn, is associated with enhanced atherogenesis in miR-144 KO mice
Summary
It has been suggested that miR-144 is pro-atherosclerotic via effects on reverse cholesterol transportation targeting the ATP binding cassette protein. MiR-144 (transcribed with miR-451 in cluster) has been suggested to be pro-atherosclerotic by targeting ATP binding cassette protein[1] (ABCA1) resulting in interrupted cholesterol metabolism, and in a study performed in ApoE KO mice administered high fat diet (HFD), an agomir (a type of specially labeled and chemically modified double strand miRNA) of miR-144-3p accelerated plaque formation through impairing reverse cholesterol transport and promoting pro-inflammatory cytokine production 9–11. These data, while interesting, do not allow a complete understanding of the role of miR-144 in atherogenesis. We took advantage of powerful nanospray liquid chromatography coupled to tandem mass spectrometry proteomics, identified vimentin as a target of miR-144, and employed primary cultured cardiac endothelial cell, miR-144/451 KO fed HFD, and mice injected with synthetized miR-144 as model systems to examine the impact of loss of miR-144 on vimentin expression and atherogenesis
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