CRISPR/dCas9-induced upregulation of endogenous apolipoprotein A1 and paraoxonase 1 genes reduces the aortic lipid deposits in apoE−/− mice

Figures

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  Fig. 1. APOA1 and PON1 transcriptional activation by CRISPR/dCas9 system in Huh7 cells under standard or pro-inflammatory conditions. Diagram of the in vitro experimental design (a) (created with BioRender), APOA1 mRNA (b), intracellular protein (c) and secreted protein (d) in Huh7 transfected cells in standard conditions or in the presence of tumor necrosis factor α (TNFα) (e, f, g). PON1 mRNA (h), intracellular protein (i) and secreted protein (j) in Huh7 transfected cells in standard conditions or in the presence of TNFα (k, l, m). Levels of cell protein are represented relative to β-actin, while the levels of secreted proteins in the culture medium are normalized to total cell protein. Results are represented as fold change versus cells transfected with Control plasmids and expressed as mean ± SD of 3–5 independent experiments. **p < 0.01, ***p < 0.001 versus Control

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  Fig. 2. Protein expression of cholesterol transporters and sirtuin 1 in Huh7 cells with upregulated APOA1 or PON1 under standard or pro-inflammatory conditions. Protein expression of scavenger receptor class B type 1 (SCARB1) (a), ATP-binding cassette sub-family-G-member-8 (ABCG8) (b), and sirtuin 1 (SIRT-1) (c) in Huh7 transfected cells in standard conditions or under pro-inflammatory conditions (d, e, f) expressed relative to β-actin (blots and their densitometric analysis). Results are represented as fold change compared to cells transfected with Control plasmids and expressed as mean ± SD of 3–5 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus Control

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  Fig. 3. Oxidative and inflammatory stress in TNFα-activated endothelial cells incubated with conditioned media from Huh7 cells with upregulated APOA1. Cellular reactive oxygen species (ROS) production expressed relative to cellular protein (a), THP-1 monocytes adhesion to activated EC (b), and protein expression of monocyte chemoattractant protein-1 (MCP-1) (c) and vascular cell adhesion molecule-1 (VCAM-1) (d) expressed relative to β-actin (blots and their densitometric analysis). Results are represented as fold change compared to cells transfected with Control plasmids and presented as mean ± SD of 3–5 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus Control; ##p < 0.01, ###p < 0.001 versus un-transfected cells not exposed to TNFα; &&&p < 0.001 versus un-transfected cells exposed to TNFα

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  Fig. 4. Gene expression and protein levels of Apoa1 and Pon1 in the liver and sera of apoE−/− mice injected with CRISPR/dCas9 plasmids for their transcriptional activation. Diagram of the in vivo experimental design (a) (created with BioRender), liver Apoa1 mRNA (b), liver APOA1 protein (c), APOA1 level in sera (d), liver Pon1 mRNA (e), liver PON1 protein (f), PON1 protein levels in sera (g), PON1 enzymatic activity in sera (h), and thiobarbituric acid reactive substances (TBARS) levels in the plasma of the mice (i). The data in b, c, e, f and g are expressed as fold change compared to Control group. All data are presented as mean ± SD (n = 5); *p < 0.05, ***p < 0.001 versus mice injected with Control plasmids

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  Fig. 5. The levels of total cholesterol, APOA1 and PON1 in lipoproteins separated by FPLC from plasma of apoE−/− mice injected with CRISPR/dCas9 plasmids for Apoa1 activation. Lipoprotein FPLC profiles analyzed as total cholesterol (a), protein profile measured at 280 nm (b), blots and their densitometric analysis representing the levels of HDL-bound APOA1 (fractions no. 30–33) (c), lipid-free APOA1 (fractions no. 34–36) (d), APOA1 in intermediate-density lipoproteins/low-density lipoproteins (IDL/LDL) (fractions no. 19–25) (e), HDL-bound PON1 (fractions no. 30–33) (f), and free PON1 (fractions no. 34–36) (g). All data are expressed as fold change relative to the equivalent fraction of the Control group and presented as mean ± SD (technical replicates of 3–7 fractions from pool of 5 plasma); *p < 0.05 versus mice injected with Control plasmids

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  Fig. 6. Gene and protein expression of cholesterol transporters SCARB1 and ABCG8, and CYP7A1 in the liver of apoE−/− mice injected with Apoa1 CRISPR/dCas9 plasmids. Scarb1 mRNA (a) and protein (b) levels, Abcg8 mRNA (c) and protein (d) levels, Cyp7a1 mRNA (e) and protein (f) levels, total cholesterol content of the bile (g) and feces (h). The data in a-f are expressed as fold change compared to Control group. All data are presented as mean ± SD (n = 5); *p < 0.05, **p < 0.01, ***p < 0.001 versus mice injected with Control plasmids

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  Fig. 7. Lipid deposits area in the lesions located in the aorta and aortic root of apoE−/− mice injected with Apoa1 or Pon1 CRISPR/dCas9 plasmids. Light microscopy images of the thoracic aorta stained with Oil Red O, scale bar: 2000 μm (a), lipid content of the aortic root lesions evidenced by Nile Red staining (b), sections of the aortic root stained with Trichrome Masson (c), specimens of the aortic root immunostained for CD68 (red fluorescence) and α-SMA (green fluorescence) (d), diagram presenting the ratio between the area of Oil Red O stained lipid deposits and the total area of the thoracic aorta, data presented as mean ± SD (n = 5) (e), diagram presenting the percentage of Nile Red-positive area to the total aortic wall area from 3–5 consecutive sections per animal (n = 3) (f), diagram presenting the size of the aortic root lesions represented as the percentage relative to the total aortic wall area from 3–5 consecutive sections per animal (n = 3) (g), diagram presenting the size of the CD68 (h) and α-SMA (i) positive area represented as percentage of the total area of lesions from 4 consecutive sections per animal (n = 3). All data from the aortic root images are presented as…