KLF11 (Krüppel-Like Factor 11) Inhibits Arterial Thrombosis via Suppression of Tissue Factor in the Vascular Wall

Abstract

Objective- Mutations in Krüppel like factor-11 ( KLF11), a gene also known as maturity-onset diabetes mellitus of the young type 7, contribute to the development of diabetes mellitus. KLF11 has anti-inflammatory effects in endothelial cells and beneficial effects on stroke. However, the function of KLF11 in the cardiovascular system is not fully unraveled. In this study, we investigated the role of KLF11 in vascular smooth muscle cell biology and arterial thrombosis. Approach and Results- Using a ferric chloride-induced thrombosis model, we found that the occlusion time was significantly reduced in conventional Klf11 knockout mice, whereas bone marrow transplantation could not rescue this phenotype, suggesting that vascular KLF11 is critical for inhibition of arterial thrombosis. We further demonstrated that vascular smooth muscle cell-specific Klf11 knockout mice also exhibited significantly reduced occlusion time. The expression of tissue factor (encoded by the F3 gene), a main initiator of the coagulation cascade, was increased in the artery of Klf11 knockout mice, as determined by real-time quantitative polymerase chain reaction and immunofluorescence. Furthermore, vascular smooth muscle cells isolated from Klf11 knockout mouse aortas showed increased tissue factor expression, which was rescued by KLF11 overexpression. In human aortic smooth muscle cells, small interfering RNA-mediated knockdown of KLF11 increased tissue factor expression. Consistent results were observed on adenovirus-mediated overexpression of KLF11. Mechanistically, KLF11 downregulates F3 at the transcriptional level as determined by reporter and chromatin immunoprecipitation assays. Conclusions- Our data demonstrate that KLF11 is a novel transcriptional suppressor of F3 in vascular smooth muscle cells, constituting a potential molecular target for inhibition of arterial thrombosis.

Keywords: Krüppel-like factors; diabetes mellitus; gene; thrombosis; tissue factor; vascular disease.

Figure 1.. KLF11 (Krüppel-like Factor 11) deficiency aggravates arterial thrombosis.

A-C, The left carotid arteries of WT (wild type) and conventional Klf11 KO (knockout) mice were subjected to 10% FeCl₃ to induce arterial thrombosis. A, Representative images of blood flow detected by ultrasound are shown with each division representing 8 seconds (left) and the corresponding occlusion time (right) determined in WT and Klf11 KO male mice (n=8/group). B, Occlusion time in WT and Klf11 KO female mice (n=6–8/group). C, WT male mice transplanted with WT bone marrow were designated as WT BM→WT, Klf11 KO male mice transplanted with WT bone marrow were designated as WT BM→Klf11 KO mice. The carotid artery occlusion time after bone marrow transplantation was recorded as in A (n=8/group). **P<0.01 or *P<0.05 using unpaired Student t-test. D-G, PT (prothrombin time), aPTT (activated partial thromboplastin time), bleeding time and TAT (thrombin-antithrombin) complexes were measured from WT and Klf11 KO male mice (n=5/group). NS, no significance using unpaired Student t-test (D, F, G) or nonparametric Mann-Whitney test (E). H, The left carotid arteries of Sm-Cre/Klf11^fl/fl+TAM (Myh11-CreER^T2/Klf11^fl/fl+tamoxifen) mice and controls: Sm-Cre/Klf11^fl/fl+ Oil (Myh11-CreER^T2/Klf11^fl/fl+ corn oi) and Klf11^fl/fl+TAM (Klf11^fl/fl+ tamoxifen) mice, were subjected to 10% FeCl₃ to induce thrombosis. Representative images of blood flow detected by ultrasound are shown and the occlusion time in control and Sm-Klf11 KO mice was recorded (n=11/group). **P<0.01 using one-way ANOVA followed by Tukey’s test.

Figure 2.. KLF11 (Krüppel-like Factor 11) deficiency induces TF (tissue factor) expression in arterial wall.

A, Activity of microvesicles-associated tissue factor (MV-TF) in the plasma after preincubation with IgG or TF 1H1 antibody (n=6/group). Data are presented by subtracting the amount of FXa generated in the presence of TF 1H1 antibody from the amount of total FXa generated in the presence of IgG. B, F3 mRNA level of carotid arteries from WT and Klf11 KO mice. The mRNA level was normalized by 18S and is presented relative to the WT group set as 1 (n=4/group). C, The aortic TF activity was measured and presented as in A, after preincubation with IgG or TF 1H1 antibody and normalized to the total protein quantity (n=6/group). **P<0.01 using unpaired Student t-test (A-C). D, Expression of TF (Alexa 647, displayed in green) and α-SMA (α-smooth muscle actin, Alexa 568, displayed in red) in mouse aorta at basal level or 4 hours after LPS (30 µg/kg) tail vein injection was visualized by immunofluorescence staining. Respective IgG staining was used as negative control. Scale bars=50 µm. Quantification was performed from 4 mice, randomly selecting 3 different medial regions from each specimen and dividing the TF immunofluorescence intensity by medial area (indicated by α-SMA positive cells). Data are presented relative to the basal level of WT group set as 1. **P<0.01 using two-way ANOVA followed by Bonferroni test.

Figure 3.. KLF11 (Krüppel-like Factor 11) overexpression rescues the TF (tissue factor) upregulation in KLF11-deficient MASMCs (mouse aortic smooth muscle cells).

MASMCs were isolated from male (A-D) and female (E-H) WT or Klf11 KO mice. A and E, Klf11 mRNA level of MASMCs from WT and Klf11 KO mice. The mRNA level was normalized by 18S and is presented relative to the WT group set as 1 (n=4/group). **P<0.01 using unpaired Student t-test. B-H, MASMCs isolated from WT or Klf11 KO mice were infected with Ad-LacZ or Ad-KLF11 (50 MOI). B and F, F3 mRNA level of MASMCs was normalized by 18S and is presented relative to the WT infected with Ad-LacZ group set as 1 (n=4/group). C and G, Representative western blot of TF protein level. D and H, Band density from 4 independent western blots was quantitatively analyzed and normalized against β-actin. The WT infected with Ad-LacZ group was set as 1. **P<0.01 or NS, no significance using two-way ANOVA followed by Bonferroni test (B, D, F, H).

Figure 4.. TF (Tissue factor) expression is increased upon KLF11 (Krüppel-like Factor 11) knockdown in HASMCs (human aortic smooth muscle cells).

HASMCs were transfected with si-Control or si-KLF11 (40 nM) and 24 hours later serum starved with 0.5% FBS (fetal bovine serum) for 48 hours. Three days after transfection, HASMCs were exposed to thrombin (3.24 µg/ml) (B-D) or 10% FBS (E-G) for 4 hours. A, The knockdown efficiency of KLF11 was determined by real-time quantitative PCR and western blot. The mRNA level was normalized by GAPDH and is presented relative to HASMCs transfected with si-Control group set as 1 (n=4/group). **P<0.01 using unpaired Student’s t-test. B and E, F3 mRNA level of HASMCs was normalized by GAPDH and is presented relative to HASMCs transfected with si-Control group set as 1 (n=4/group). C and F, Representative western blot showing the protein level of TF. D and G, Band density from 4 independent western blots was quantitatively analyzed and normalized against GAPDH. HASMCs transfected with si-Control group was set as 1. *P<0.05, **P<0.01 using two-way ANOVA followed by Bonferroni test (B, D, E, G).

Figure 5.. TF (Tissue factor) expression is decreased upon KLF11 (Krüppel-like Factor 11) overexpression in HASMCs (human aortic smooth muscle cells).

HASMCs were infected with Ad-LacZ or Ad-KLF11 (50 MOI). Twelve hours after infection, HASMCs were serum starved with 0.5% FBS (fetal bovine serum) for 48 hours and then stimulated to thrombin (3.24 µg/ml) (B-D) or 10% FBS (E-G) for 4 hours. A, The overexpression of KLF11 was determined by western blot. B and E, F3 mRNA level of HASMCs from each group was normalized by GAPDH and is presented relative to HASMCs infected with Ad-LacZ group set as 1 (n=4/group). C and F, Representative western blot showed the protein level of TF. D and G, Band density from 4 independent western blots was quantitatively analyzed and normalized against GAPDH. HASMCs infected with Ad-LacZ group was set as 1. **P<0.01 using two-way ANOVA followed by Bonferroni test (B, D, E, G).

Figure 6.. KLF11 (Krüppel-like Factor 11) inhibits F3 (coagulation factor III) transcription.

A, A diagram showing the simplified structure of the human F3 promoter region with an illustration of the TIEG (transforming growth factor-beta-inducible early gene) binding site. KLF binding region is shown in bold. B, The bold bases indicate the conservation of the TIEG binding site among species. C, HASMCs were infected with Ad-LacZ or Ad-Flag KLF11. Forty-eight hours after infection, the binding of KLF11 to the F3 promoter was determined by ChIP assays using an antibody against Flag (n=4/group). D-E, A7r5 cells were transfected with two different length (D), or wt (wild type) or del (region deleted) (E) luciferase reporter driven by the F3 promoter and then infected with Ad-LacZ or Ad-KLF11 (50 MOI). Two days later, the luciferase activity was measured and normalized by Renilla activity. The results are presented relative to A7r5 transfected with pF3 (−906/+162) (D) or wt (E) and infected with Ad-LacZ group set as 1 (n=4/group). **P<0.01 using two-way ANOVA followed by Bonferroni test (C, D, E). F, Schematic summary: KLF11 inhibits F3 transcription by directly binding to the F3 promoter region.