Endothelial monocarboxylate transporter 1 drives atherosclerosis via a lactate/NADH/CtBP‐mediated transrepression pathway

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  FIGURE 1. Lactate promotes vascular inflammation in atherosclerosis. (A) Representative images and quantification data of en face IF staining for Icam‐1 (green) in the aortic endothelium from wild‐type (WT) and Apoe −/− mice treated with WD for 1 month. The endothelium was visualized by CD31 staining (red). Nuclei were counterstained with DAPI (blue) (n = 10 mice per group). (B and C) Western blot analysis and quantification of protein levels of Vcam‐1 and Icam‐1 in MAECs isolated from WT and Apoe −/− mice (n = 5 mice per group). (D) Real‐time PCR analysis of the mRNA levels of Vcam‐1 and Icam‐1 genes in endothelial RNA obtained from aortas of 1‐month WD‐fed WT and Apoe −/− mice through flushing with TRIzol reagent (n = 6 mice per group). (E) The lactate levels in blood and aortas from 1‐month WD‐fed WT and Apoe −/− mice were measured by Lactate Assay Kit (n = 6 mice per group). (F) Western blot detection of VCAM‐1 and ICAM‐1 in HUVECs treated with lactate (10 mM) or vehicle for 0–24 h. (G) Real‐time PCR analysis of the mRNA levels of Vcam‐1 and Icam‐1 genes in endothelial RNA obtained from 1‐month WD‐fed Apoe −/− mice treated with lactate (0.5 g/kg body weight) or vehicle via i.p.…

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  FIGURE 2. MCT1 facilitates lactate uptake into ECs for vascular inflammation. (A and B) Representative IF staining and quantification of MCT1 (green) in normal (upper panels) and stenotic (lower panels) human carotid artery specimens. The endothelium was visualized by CD31 staining (red). Nuclei were counterstained with DAPI (blue) (n = 6). (C) Real‐time PCR analysis of the mRNA levels of Mct1 genes in endothelial RNA obtained from aortas of 1‐month WD‐fed WT and Apoe −/− mice through flushing with TRIzol reagent (n = 6 mice per group). (D) Western blot detection of VCAM‐1 and ICAM‐1 in HUVECs pretreated with the MCT1 inhibitor AZD3965 (500 nM) for 24 h and then stimulated with lactate (10 mM) or vehicle for 6 h (n = 3). (E) Real‐time PCR analysis of the mRNA levels of VCAM‐1 and ICAM‐1 genes in HUVECs pretreated with the MCT1 inhibitor AZD3965 (500 nM) for 24 h and then stimulated with lactate (10 mM) or vehicle for 3 h (n = 6). (F and G) Western blot detection and quantification of protein levels of VCAM‐1 and ICAM‐1 in MCT1 KD or control HUVECs with lactate (10 mM) or vehicle stimulation for 6 h (n = 5). (H) Real‐time PCR analysis of the mRNA levels of VCAM‐1 and ICAM‐1 in MCT1 KD or…

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  FIGURE 3. Endothelial‐specific targeting ability of DSPE–PEG–PEI/Pep nanoparticles. (A) Representative confocal image of the intracellular 5‐FAM fluorescence in HUVECs treated with FAM‐labeled DSPE–PEG–PEI/Pep (FAM‐siCtrl NP) and DSPE–PEG–PEI/Pep (FAM‐siCtrl/Pep NP) nanoparticles. HUVECs were treated with vehicle or 10 mM lactate for 6 h, and then subjected to FAM‐labeled DSPE–PEG–PEI/Pep (FAM‐siCtrl NP) and DSPE–PEG–PEI/Pep (FAM‐siCtrl/Pep NP) nanoparticles treatment for 3 h. (B and C) Representative confocal image of intracellular Cy5 fluorescence in endothelium layer from Apoe −/− mice treated with Cy5‐labeled DSPE–PEG–PEI/Pep (Cy5‐siCtrl NP) and DSPE–PEG–PEI/Pep (Cy5‐siCtrl/Pep NP) nanoparticles. (D) En face IF staining and quantification of Mct1 (green) in the aortic endothelium of 3‐month HFD‐fed Apoe −/− mice treated with siCtrl/Pep NP, siMct1/Pep NP. The endothelium was visualized by CD31 staining (red). Nuclei were counterstained with DAPI (blue) (n = 10 mice per group). For all bar graphs, data are the mean ± SEM, ****p < 0.0001 (unpaired, two‐tailed Student's t‐test). (E) Real‐time PCR analysis of the mRNA levels of Vcam‐1 and Icam‐1 genes in endothelial RNA obtained from…

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  FIGURE 4. The production of NADH due to lactate oxidation are critical in endothelial inflammation. (A) Real‐time PCR analysis of the mRNA levels of VCAM‐1 and ICAM‐1 genes in HUVECs pretreated with the LDH‐B selective inhibitor AXKO‐0046 dihydrochloride (50 nM) for 30 min and then stimulated with lactate (10 mM) or vehicle for 3 h (n = 6). (B and C) The ratio of NADH/NAD+ (B) and the content of NADH (C) measured using a NADH/NAD+ Quantitation Kit in lactate (10 mM for 3 h)‐treated HUVECs pretreated with the MCT1 inhibitor AZD3965 (500 nM) for 24 h (n = 6). (D and E) The ratio of NADH/NAD+ (D) and the content of NADH (E) in lactate‐treated MCT1 KD or control HUVECs (n = 6). (F and G) The ratio of NADH/NAD+ (F) and the content of NADH (G) in lactate‐treated HUVECs pretreated with the NADH depletor MTOB (2.5 mM) for 24 h (n = 6). (H and I) Western blot detection and quantification of the protein levels of VCAM‐1 and ICAM‐1 in HUVECs pretreated with the NADH depletor MTOB (2.5 mM) for 24 h and then stimulated with 10 mM lactate or vehicle for 6 h (n = 5). (J) Real‐time PCR analysis of the mRNA levels of VCAM‐1 and ICAM‐1 genes in HUVECs pretreated with the NADH depletor MTOB (2.5 mM) for 24 h…

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  FIGURE 5. Oligomerization of CtBP1 is critical for lactate‐induced endothelial inflammation. (A) Co‐IP of CtBP1–FLAG and CtBP1–HIS in FLAG/HIS‐tagged CtBP1‐overexpressed HUVECs pretreated with the NADH depletor MTOB (2.5 mM) for 24 h and then treated with vehicle or 10 mM lactate for 3 h. Two hundred micrograms of protein lysates was precipitated with rabbit anti‐FLAG antibody followed by immunoblotting with mouse anti‐HIS antibody (n = 4). (B) Change of CtBP1 oligomer states in FLAG‐tagged CtBP1‐overexpressed HUVECs pretreated with the NADH depletor MTOB (2.5 mM) for 24 h and then treated with vehicle or 10 mM lactate for 3 h. The whole‐cell lysates were treated with 0.25‰ glutaraldehyde (GA) for 5 min and then analyzed by Western blot with the Flag antibody (n = 3). (C) Co‐IP of CtBP1–FLAG and CtBP1–HIS in FLAG/HIS‐tagged CtBP1‐overexpressed HUVECs transfected with control or siMCT1 for 48 h and then challenged with vehicle or 10 mM lactate for 3 h (n = 4). (D) Change of CtBP1 oligomer states in FLAG‐tagged CtBP1‐overexpressed HUVECs transfected with control or siMCT1 for 48 h and then challenged with vehicle or 10 mM lactate for 3 h. The whole‐cell lysates were treated with 0.25‰…

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  FIGURE 6. FOXP1 binds with monomeric CtBP to transrepress the expression of adhesion molecules. (A) Sequence analysis showing FOXP1 binding sites (red square) in the proximal promoter of VCAM‐1 and ICAM‐1 genes. (B) Co‐IP of endogenous CtBP1 and FOXP1 in HUVECs. Upper: IP was performed with mouse anti‐CtBP1 followed by immunoblotting with rabbit anti‐FOXP1 and mouse anti‐CtBP1. Lower: IP was performed with rabbit anti‐FOXP1 followed by immunoblotting with mouse anti‐CtBP1 and rabbit anti‐FOXP1 (n = 4). (C) Sequential chromatin immunoprecipitation (SeqChIP; also referred to as Re‐ChIP) assay showing the accumulation of the complex of FOXP1 and CtBP1 on the promoter of VCAM‐1 and ICAM‐1 genes in HUVECs (n = 4). (D) Western blot detection and quantification of the protein levels of VCAM‐1 and ICAM‐1 in FOXP1‐overexpressed or control HUVECs stimulated with 10 mM lactate or vehicle for 6 h (n = 5). S, short exposure; L, long exposure. (E) Western blot detection and quantification of the protein levels of VCAM‐1 and ICAM‐1 in FOXP1 KD or control HUVECs stimulated with 10 mM lactate or vehicle for 6 h (n = 5). (F) Luciferase reporter assay showing the activity of promoter regions of VCAM‐1 and…

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  FIGURE 7. Nanoparticle‐mediated endothelial‐specific MCT1 silencing reduces atherogenesis. (A) Schematic diagram showing the experimental design for treatment of Apoe−/− mice with DSPE–PEG–PEI/Pep/sictl (siCtrl/Pep NP) and DSPE–PEG–PEI/Pep/siMct1 (siMct1/Pep NP) nanoparticles. (B–D) IF staining and quantification of ICAM‐1 and VCAM‐1 in the aortic endothelium of 3‐month HFD‐fed Apoe −/− mice treated with siCtrl/Pep NP and siMct1/Pep NP. The endothelium was visualized by CD31 staining (red). Nuclei were counterstained with DAPI (blue) (n = 10 mice per group). (E and F) Oil Red O‐stained en face aortic preparations from 3‐month HFD‐fed Apoe −/− mice injected with siCtrl/Pep NP and siMct1/Pep NP (E), and quantification of the Oil Red O‐stained areas (F) (n = 10 mice per group). (G and H) Oil Red O staining of aortic roots of the 3‐month HFD‐fed Apoe −/− mice injected with siCtrl/Pep NP and siMct1/Pep NP (G) and quantification of the Oil Red O‐stained areas (H) (n = 10 mice per group). (I and J) IHC staining of F4/80 (I) and quantification of F4/80+ areas (J) in aortic roots of 3‐month‐WD‐fed Apoe −/− mice injected with siCtrl/Pep NP and siMct1/Pep NP (n = 10 mice per group). For all bar…

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  FIGURE 8. Endothelial‐specific Mct1 silencing reduces atherogenesis in a FOXP1‐dependent manner. (A) Schematic diagram showing the experimental design for treatment of Apoe−/− mice with AAV–Cdh5–Vehicle, AAV–Cdh5–shFoxp1, AAV–Cdh5–Vehicle plus siMct1/Pep NP, and AAV–Cdh5–shFoxp1 plus siMct1/Pep NP. (B) Oil Red O‐stained en face aortic preparations from 3‐month HFD‐fed Apoe −/− mice injected with AAV–Cdh5–Vehicle, AAV–Cdh5–shFoxp1, AAV–Cdh5–Vehicle plus siMct1/Pep NP, and AAV–Cdh5–shFoxp1 plus siMct1/Pep NP, and quantification of the Oil Red O‐stained areas (n = 10 mice per group). (C) Oil Red O staining of aortic roots and quantification of the Oil Red O‐stained areas (n = 10 mice per group). (D) IHC staining of F4/80 and quantification of F4/80+ areas in aortic roots (n = 10 mice per group). For all bar graphs, data are the mean ± SEM. The composition of the two groups delineated by horizontal lines, *p < 0.05 and **p < 0.01; one‐way ANOVA with Tukey's post hoc test (B, C, D).