Krüppel-Like Factor 4 Regulation of Cholesterol-25-Hydroxylase and Liver X Receptor Mitigates Atherosclerosis Susceptibility

Abstract

Background: Atherosclerosis is a multifaceted inflammatory disease involving cells in the vascular wall (eg, endothelial cells [ECs]), as well as circulating and resident immunogenic cells (eg, monocytes/macrophages). Acting as a ligand for liver X receptor (LXR), but an inhibitor of SREBP2 (sterol regulatory element-binding protein 2), 25-hydroxycholesterol, and its catalyzing enzyme cholesterol-25-hydroxylase (Ch25h) are important in regulating cellular inflammatory status and cholesterol biosynthesis in both ECs and monocytes/macrophages.

Methods: Bioinformatic analyses were used to investigate RNA-sequencing data to identify cholesterol oxidation and efflux genes regulated by Krüppel-like factor 4 (KLF4). In vitro experiments involving cultured ECs and macrophages and in vivo methods involving mice with Ch25h ablation were then used to explore the atheroprotective role of KLF4-Ch25h/LXR.

Results: Vasoprotective stimuli increased the expression of Ch25h and LXR via KLF4. The KLF4-Ch25h/LXR homeostatic axis functions through suppressing inflammation, evidenced by the reduction of inflammasome activity in ECs and the promotion of M1 to M2 phenotypic transition in macrophages. The increased atherosclerosis in apolipoprotein E^-/-/Ch25h^-/- mice further demonstrates the beneficial role of the KLF4-Ch25h/LXR axis in vascular function and disease.

Conclusions: KLF4 transactivates Ch25h and LXR, thereby promoting the synergistic effects between ECs and macrophages to protect against atherosclerosis susceptibility.

Keywords: atherosclerosis; cholesterol; endothelial cells; inflammation; macrophages; shear stress.

Figure 1. KLF4 regulates cholesterol metabolism in ECs

(A–C) HUVECs were infected with Ad-KLF4 or Ad-null for 48 hr prior to RNA isolation followed by RNA-seq analysis. Data presented are results from two biological repeats (Ad-KLF4 divided by Ad-null). (A) Gene ontology (GO) enrichment using Metascape of the top 300 upregulated genes plotted as −log(p-value). (B) Heat map comparison of Ad-null versus Ad-KLF4 using log2 fold changes of the indicated genes (refer to Figure S3 for heat map of scaled Z-score). (C) Network projection of customized sterol metabolism pathway.

Figure 2. KLF4 transactivates Ch25h in ECs

(A, B) HUVECs were infected with Ad-KLF4 or Ad-null for 24 hr. Expression levels of KLF4 and Ch25h mRNA and protein were measured by qPCR and Western blot, respectively. (C) Lung ECs were isolated from EC-specific KLF4 knockout (EC-KLF4-KO) or EC-specific overexpression of KLF4 (EC-KLF4-Tg) mice (n=3 per group). Levels of KLF4 and Ch25h mRNA were measured by qPCR. (D) Depiction of the predicted KLF4 binding sites at −913 to −902 bp (Site 1), 607 to 618 bp (Site 2), 656 to 667 bp (Site 3) and 712 to 723 bp (Site 4) in the human Ch25h gene. (E) HUVECs infected with Ad-KLF4 or Ad-null were analyzed by ChIP assay with the use of anti-KLF4. KLF4 promoter enrichment was quantified by qPCR. IgG was used as an isotype control. (F) Bovine aortic endothelial cells (BAECs) were transfected a luciferase reporter fused with the Ch25h promoter region (Ch25h-Luc) or with a Site 1 deletion (Ch25hΔS1-Luc) together with pRSV-β-gal reporter for 6 hr, followed by infection with Ad-KLF4 for an additional 24 hr. Luciferase activity was measured and normalized to that of β-gal. (G, H) HUVECs were subjected to OS (1±4 dyn/cm²) or PS (12±4 dyn/cm²) for 24 hr. (I, J) HUVECs were transfected with control siRNA or KLF4 siRNA prior to PS stimulation or kept as a static control for 24 hr. KLF4 and Ch25h mRNA and protein expression were measured by qPCR (G, I) and Western blot (H, J), respectively. Error bars represent mean±SEM from three independent experiments. *p<0.05 between the indicated groups.

Figure 3. PS transcriptionally induces Ch25h

(A) WashU EpiGenome Browser depiction of the CpG islands and the binding landscape of H3K27ac, H3K4me1, H3K4me3, and Pol II at the promoter of the human Ch25h gene. The epigenetic landscapes shown were from HUVECs without stimulation. (B–D) HUVECs were subjected to PS or OS for 12 hr. (E–G) HUVECs were infected with Ad-KLF4 or Ad-null for 24 hr. In (B, E), DNA methylation status of the Ch25h promoter was measured by methylation-specific (MSP) qPCR. In (C, D, F, G), the binding of H3K4me3 and H3K27ac at the Ch25h promoter was detected using histone-ChIP qPCR. IgG was used as an isotype control. (H–K) Intima was collected from the AA or TA regions of C57BL/6 mice (n=9). In (H), total mRNA was quantified by qPCR. DNA methylation status is shown in (I). The modifications of H3K4me3 and H3K27ac are revealed in (J, K). Data presented are mean±SEM from three independent experiments. *p<0.05 between the indicated groups.

Figure 4. KLF4 transactivates LXRα

(A, B) HUVECs were infected with Ad-null or Ad-KLF4. Expression levels of LXRα mRNA (A) and protein (B) were measured by qPCR and Western blot, respectively. (C, D) The mRNA level of LXRα, ABCA1, and ABCG1 in lung ECs isolated from EC-KLF4-KO or EC-KLF4-Tg mice (n=3 per group) was detected by qPCR. (E, F) HUVECs infected with Ad-null or Ad-KLF4 were transfected with control siRNA or Ch25h siRNA. The level of ABCA1, ABCG1, Ch25h, SREBP2, and NLRP3 mRNA was assessed by qPCR. (G) Depiction of the predicted KLF4 binding sites at −3643 to 3632 bp (Site 1), −419 to −408 bp (Site 2), −313 to −302 bp (Site3), −219 to −208 bp (Site 4) and −133 to −122 bp (Site 5) in the human LXRα promoter region. (H) HUVECs were infected with Ad-null or Ad-KLF4 followed by ChIP assays using anti-KLF4. IgG was used as an isotype control. (I) BAECs were transfected with LXRα-Luc together with pRSV-β-gal reporter plasmids followed by infection with Ad-KLF4. Luciferase activity was measured and normalized to that of β-gal. (J) HUVECs were transfected with control siRNA or KLF4 siRNA followed by stimulation with PS or kept under static conditions for an additional 24 hr. (K) Intima was collected from the AA or TA regions of C57BL/6 mice (n=9). The mRNA expression of LXRα, ABCA1, and ABCG1 were measured by qPCR. Error bars represent mean±SEM from three independent experiments. *p<0.05 between the indicated groups.

Figure 5. KLF4 regulation of Ch25h/LXR in macrophages promotes M2 polarization

(A) RAW264.7 cells were infected with Ad-KLF4 or Ad-null prior to qPCR assessment of the indicated mRNA. (B) LC-MS/MS measurement of intracellular 25-HC level of THP1 cells infected with Ad-KLF4 or Ad-null. (C) BMDMs from C57BL/6 mice were treated with 25-HC (5 nM) for the indicated time points. The mRNA expression profiles were analyzed by microarray as described in Shibata et al., 2013. The heat map shown indicate values plotted as relative fold change, compared with those from untreated controls. (D, E) RAW264.7 cells were treated with 25-HC (1 μM) or DMSO for 24 hr (D) or transfected with Ch25h siRNA or control siRNA (E). The mRNA levels of pro-inflammatory genes (NLRP3, IL-1β), M1 marker genes (COX2, Gro1, TNFα, CCL5, iNOS, and IL-6), and M2 marker genes (PPARγ, MRC, Chil3, Arg1, Retnla, and IL-10) were measured by qPCR. (F–I) PMs were isolated from 8-week old Ch25h^−/− mice and their Ch25h^+/+ littermates (n=12 per group). The mRNA levels of the indicated genes were quantified by qPCR (F, I). In (G, H), the total cholesterol content and cholesterol efflux were measured. (J) PMs isolated from 8-week old Ch25h^−/− mice (n=12) were treated with or without 25-HC (1 μM) for 24 hr. The mRNA levels of indicated genes were measured by qPCR. Error bars represent mean±SEM from three independent experiments. *p<0.05 between indicated groups.

Figure 6. Ch25h ablation increases atherosclerosis susceptibility

(A) Two biological replicates of RNA-seq performed on PMs isolated from ApoE^−/−/Ch25h^−/− compared to ApoE^−/−/Ch25h^+/+ mice (n=12 per group). Heat map is plotted as log2 fold change (refer to Figure S11 for heat map of scaled Z-score). (B) Six week-old ApoE^−/−/Ch25h^+/+ (n=13) and ApoE^−/−/Ch25h^−/− (n=13) mice fed a high-fat diet for 12 weeks. Histological sections of aortic roots were stained with Oil Red O, H&E, MAC1, and CD31. Graphs on the right represent quantification of the Oil Red O-positive areas and percentage of macrophages in the lesions. (C) Representative en face Oil Red O staining of the whole aorta from ApoE^−/−/Ch25h^−/− mice and ApoE^−/−/Ch25h^+/+ littermates (n=10 per group). Graphs on the right represent quantification of the lesion area in thoracic aorta (TA) versus aortic arch (AA) as well as total lesion area, summed from that of AA (i.e., aortic root, ascending aorta, and descending thoracic aorta to the 4^th thoracic vertebra), TA (i.e., descending aorta from the 4^th thoracic vertebra to the renal arteries), and abdominal aorta (i.e., below the renal arteries). Error bars represent mean±SEM from three independent experiments. *p<0.05 between the indicated groups.

Figure 7. KLF4 mediates Ch25h and LXR in ECs and macrophages to promote a atheroprotective phenotype

Atheroprotective factors, such as pulsatile shear stress (PS) and statins, induce KLF4 in ECs and macrophages. KLF4 then transactivates Ch25h and LXR thereby promoting the suppression of SREBP2 and NLRP3 inflammasome in ECs as well as promotes cholesterol efflux and M1 to M2 transition in macrophages. The synergism between the anti-inflammatory effects in both ECs and macrophages contributes to the atheroprotective phenotype.