Genetic Dissection of the Impact of miR-33a and miR-33b during the Progression of Atherosclerosis
As an important regulator of macrophage cholesterol efflux and HDL biogenesis, miR-33 is a promising target for treatment of atherosclerosis, and numerous studies demonstrate that inhibition of miR-33 increases HDL levels and reduces plaque burden. However, important questions remain about how miR-33 impacts atherogenesis, including whether this protection is primarily due to direct effects on plaque macrophages, or regulation of lipid metabolism in the liver. We demonstrate that miR-33 deficiency in Ldlr−/− mice promotes obesity, insulin-resistance and hyperlipidemia, but does not impact plaque development. We further assess how loss of miR-33 or addition of miR-33b in macrophages and other hematopoietic cells impact atherogenesis. Macrophage-specific loss of miR-33 decreases lipid accumulation and inflammation under hyperlipidemic conditions, leading to reduced plaque burden. Therefore, the pro-atherogenic effects observed in miR-33 deficient mice are likely counterbalanced by protective effects in macrophages, which may be the primary mechanism through which anti-miR-33 therapies reduce atherosclerosis.To address some of the primary unresolved questions surrounding how miR-33 regulates atherosclerosis, we utilized CRISPR/Cas9 technology to excise the intronic region of the Srebp-2 gene encoding miR-33, thus generating a new miR-33 knockout mouse model (miR-33−/−). This excision of roughly 200bp can be readily confirmed by PCR analysis and has been used to detect effective excision of the miR-33 sequence in all tissues examined (Figure 1A). Consistent with previous studies, Western blot analysis of the liver of miR-33−/− mice reveals increased expression of miR-33 target genes ABCA1 and HADHβ compared to control animals, while other miR-33 targets including CROT were unaffected (Figure 1B). Consistent with the observed increase in ABCA1 expression, plasma HDL-C levels were found to be elevated in miR-33−/− mice as visualized by FPLC fractionation (Figure 1C) and confirmed by quantification of circulating HDL-C levels (Figure 1D).This work was supported by grants from the National Institutes of Health (R35HL135820 to CF-H; R01HL105945 and R01HL135012 to YS; R01HL107794 to AB and F32DK10348902 to NP), the American Heart Association (16EIA27550005 to CF-H; 16GRNT26420047 to YS and 17SDG33110002 to NR), the American Diabetes Association (1-16-PMF-002 to AC-D), and the Foundation Leducq Transatlantic Network of Excellence in Cardiovascular Research MIRVAD (to CF-H). We would like to thank Sameet Mehta, and Rolando Garcia Milian for their assistance with the processing and analysis of RNA-seq data.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.