Suberanilohydroxamic Acid as a Pharmacological Kruppel-Like Factor 2 Activator That Represses Vascular Inflammation and Atherosclerosis

Abstract

Background: Kruppel-like factor 2 (KLF2) is an important zinc-finger transcription factor that maintains endothelial homeostasis by its anti-inflammatory, -thrombotic, -oxidative, and -proliferative effects in endothelial cells. In light of the potent vasoprotective effects of KLF2, modulating KLF2 expression or function could give rise to new therapeutic strategies to treat cardiovascular diseases.

Methods and results: High-throughput drug screening based on KLF2 promoter luciferase reporter assay was performed to screen KLF2 activators. Real-time PCR and western blot were used to detect gene and protein expression. Identified KLF2 activator was orally administered to ApoE^-/- mice to evaluate anti-atherosclerotic efficacy. By screening 2400 compounds in the Spectrum library, we identified suberanilohydroxamic (SAHA) acid, also known as vorinostat as a pharmacological KLF2 activator through myocyte enhancer factor 2. We found that SAHA exhibited anti-inflammatory effects and attenuated monocyte adhesion to endothelial cells inflamed with tumor necrosis factor alpha. We further showed that the inhibitory effect of SAHA on endothelial inflammation and ensuing monocyte adhesion was KLF2 dependent using KLF2-deficient mouse lung endothelial cells or KLF2 small interfering RNA- depleted human endothelial cells. Importantly, we observed that oral administration of SAHA reduced diet-induced atherosclerotic lesion development in ApoE^-/- mice without significant effect on serum lipid levels.

Conclusions: These results demonstrate that SAHA has KLF2-dependent anti-inflammatory effects in endothelial cells and provide the proof of concept that KLF2 activation could be a promising therapeutic strategy for treating atherosclerosis.

Keywords: SAHA; atherosclerosis; endothelial cell; inflammation.

Figure 1

Identification of SAHA as a pharmacological KLF2 activator. A, Results of KLF2‐luc‐based drug screening. B, The chemical structure of SAHA. C, SAHA upregulates KLF2 mRNA expression in HCAECs. HCAECs were treated with vehicle (0.1% DMSO) or indicated concentration of SAHA (0.1, 1, and 10 μmol/L), and then the levels of KLF2 mRNA expression were detected by qPCR using GADPH as the loading control. *P<0.05 (SAHA vs vehicle), nonparametric Wilcoxon rank‐sum test, 2‐tailed, n=4. D, SAHA upregulates KLF2 promoter activity by MEF2. COS7 cells were transfected with KLF2‐wild type (−221 bp, KLF2‐221 WT) or KLF2‐mutant (−221 bp, mutation of MEF2 binding site, KLF2‐221 mutant) before stimulation with indicated concentration of SAHA for 24 hours. Then, luciferase activity was read and expressed as fold over control, nonparametric Wilcoxon rank‐sum test, 2‐tailed, n=4, *P<0.05. KLF2 indicates Kruppel‐like factor 2; SAHA, suberoylanilide hydroxamic acid; WT, wild type.

Figure 2

SAHA attenuates THP‐1 monocyte adhesion to HCAECs. HCAECs were plated in 6‐well plates the day before pretreatment with DMSO (vehicle) or SAHA (10 μmol/L) for 12 hours. Then, TNFα (10 ng/mL, final concentration) or vehicle (PBS) was added for an additional 6 hours. Then, THP‐1 monocytes (0.5 mL/well) were added for 30 minutes and then nonadherent monocytes were washed out. A, Representative images of monocyte adhesion assay. Cobblestone‐shaped cells are endothelial cells whereas small and round cells are adherent THP‐1 monocytes. B, Quantification of (A). Adherent THP‐1 cells were counted from 6 independent experiments. One‐way ANOVA with post‐hoc Bonferroni test; **P<0.01. SAHA indicates suberoylanilide hydroxamic acid; TNFα, tumor necrosis factor alpha.

Figure 3

SAHA decreases TNFα‐stimulated vascular inflammation in HCAECs. A, HCAECs were plated in 6‐well plates the day before pretreatment with DMSO (vehicle) or SAHA (10 μmol/L) for 12 hours. Then, TNFα (10 ng/mL, final concentration) or vehicle (PBS) was added for an additional 3 hours, and then qPCR was performed to assess VCAM1 and ICAM1 mRNA expression. Nonparametric Kruskal–Wallis test; *P<0.05; **P<0.01, n=6. B, HCAECs were treated as described above, and then western blot assays were performed to examine VCAM1 and ICAM1 protein expression. Representative images are shown, and the protein levels of VCAM1 and ICAM1 were normalized to GAPDH. One‐way ANOVA with post‐hoc Bonferroni test; *P<0.05; **P<0.01, n=5. GAPDH indicates glyceraldehyde 3‐phosphate dehydrogenase; ICAM1, intercellular adhesion molecule 1; KLF2, Kruppel‐like factor 2; SAHA, suberoylanilide hydroxamic acid; TNFα, tumor necrosis factor alpha; VCAM1, vascular cell adhesion molecule 1.

Figure 4

SAHA inhibits vascular inflammation by KLF2. A, Representative genotyping results of KLF2^+/+ and KLF2^+/− mouse tail DNA. B and C, Lung ECs from KLF2^+/+ and KLF2^+/− mice were treated with or without SAHA for 12 hours, and then TNFα was added for an additional 3 hours. qPCR was performed to examine VCAM1 and ICAM1 mRNA expression. One‐way ANOVA with post‐hoc Bonferroni test; *P<0.05; **P<0.01, n=4. D, HUVECs were treated with control siRNA or KLF2 siRNA (siKLF2) for 48 hours, then treated with SAHA for 12 hours before stimulation with TNFα for monocyte adhesion assay. One‐way ANOVA with post‐hoc Bonferroni test; *P<0.05; **P<0.01, n=6. ECs indicates endothelial cells; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; ICAM1, intercellular adhesion molecule 1; KLF2, Kruppel‐like factor 2; SAHA, suberoylanilide hydroxamic acid; TNFα, tumor necrosis factor alpha; VCAM1, vascular cell adhesion molecule 1.

Figure 5

SAHA attenuates atherosclerosis development in ApoE^−/− mice. A, 6‐week‐old male ApoE^−/− mice were fed a high‐fat diet, concurrent with the treatment of vehicle (CMC‐Na) or SAHA (50 mg/kg/day, oral gavage) for 12 weeks. Representative gross images of aortic arch of each group of mice were shown. B, Representative images (composite of 5‐6 images of the same aorta) of en face Oil Red O (ORO) staining for the lesions of each group mice are shown. C, Quantitative analysis of en face ORO staining lesions. Student t test, n=9 to 10. **P<0.01. D, Effect of SAHA on serum lipid profile. P>0.05 for all comparisons, Student t test, n=8. LDL‐C indicates low‐density lipoprotein cholesterol; SAHA, suberoylanilide hydroxamic acid; TC, total cholesterol; TG, triglyceride; Veh, vehicle; VLDL‐C, very‐low‐density lipoprotein cholesterol.