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 of the young type 7, contribute to the development of diabetes. 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 (VSMC) 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 (Klf11 KO) mice while bone marrow transplantation could not rescue this phenotype, suggesting that vascular KLF11 is critical for inhibition of arterial thrombosis. We further demonstrated that VSMC specific Klf11 knockout mice also exhibited significantly reduced occlusion time. The expression of tissue factor (TF, encoded by the F3 gene), a main initiator of the coagulation cascade, was increased in the artery of Klf11 KO mice, as determined by real-time quantitative PCR and immunofluorescence. Furthermore, VSMCs isolated from Klf11 KO mouse aortas showed increased TF expression, which was rescued by KLF11 overexpression. In human aortic smooth muscle cells, siRNA-mediated knockdown of KLF11 increased TF expression. Consistent results were observed upon 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 VSMCs, constituting a potential molecular target for inhibition of arterial thrombosis.

Introduction

Thrombosis is a common pathology underlying many cardiovascular diseases: myocardial infarction, stroke and venous thromboembolism, which collectively cause more than one fourth of deaths worldwide.¹ The primary pathogenic process in arterial thrombosis is the rupture of the atherosclerotic plaque, which promotes platelet recruitment, adhesion, aggregation and activation and results in thrombus growth.² Given the importance of the vascular smooth muscle cells (VSMCs) in vessel homeostasis and pathogenesis of vascular diseases,³ it is of great interest to identify molecular signaling pathways that mediate the effects of both physiological and pathophysiological stimuli in VSMCs.

The Krüppel-like factor (KLF) family is a subclass of Cys2/His2 zinc-finger DNA-binding proteins.⁴ The KLF family plays critical roles in the maintenance of vascular homeostasis and further affects multiple vascular diseases.^{5, 6} In fact, emerging data from population genetics studies suggest that KLF11 gene polymorphisms are significantly associated with type 2 diabetes.⁷ Maturity-onset diabetes of the young type 7 (MODY7), an early-onset type 2 diabetes mellitus, is caused by mutations in the KLF11 gene.⁷ In pancreatic β-cells, KLF11 regulates insulin transcription by directly binding or via increasing the expression of another MODY gene, pancreatic-duodenal homeobox-1 (PDX-1).^{8, 9} Moreover, KLF11 reduces hepatic triglyceride levels by increasing fatty acid oxidation.¹⁰ Apart from the metabolic disorder, diabetes accelerates vascular pathology and enhances thrombotic risk,^{11, 12} which account for the major causes of morbidity and mortality in diabetic patients.¹³

As a transcription factor, KLF11 is expressed ubiquitously and is abundant in adipose tissue, testis, breast, artery and lung (GTEx database). RNA sequencing data show that the mRNA level of KLF11 is modest in human endothelium and moderate in human aortic smooth muscle cells (HASMCs).⁶ Prior studies from others and our group demonstrated that KLF11 plays an important role in maintaining vascular homeostasis.^{14, 15} Specifically, KLF11 inhibits endothelial activation in the presence of inflammatory stimuli and functions as a peroxisome proliferator-activated receptor-γ co-regulator to attenuate middle cerebral artery occlusion-induced stroke in mice.¹⁶ These in vitro and in vivo observations form the basis of the current view that KLF11 is a vessel protective factor. However, the role of KLF11 in VSMC biology and thrombosis has not been explored.

In the present study we focused on VSMC KLF11 and, using in vivo VSMC-specific loss-of-function approaches, demonstrated that this factor is an inhibitor of experimental arterial thrombosis through transcriptional repression of tissue factor (TF, encoded by F3 gene) in VSMCs.

Materials and Methods

The data that support the findings of this study are available from the corresponding author upon reasonable request. An extended version of this section is available online as Extended Materials and Methods.

Animals

Conventional KLF11 knockout mice (Klf11 KO) and wild type (WT) mice in the C57BL/6J background were previously described¹⁴ and the KO was confirmed (Supplemental Figure I). The floxed-KLF11 (Klf11^fl/fl) mice were generated by injecting blastocysts developed from Klf11 targeted ES cells (Klf11^{tm2a(KOMP)Wtsi}, UCDavis, KOMP) into C57BL/6J mice. Exon 3 of the Klf11 gene was flanked by LoxP elements. The genotype was determined from mouse tail clippings and a pair of PCR primers flanking the downstream LoxP region (Supplemental Figure IIIA). The inducible smooth muscle cell specific Klf11 knockout (Sm-Klf11 KO) mice were generated by cross-breeding Klf11^fl/fl mice with Myh11-CreER^T2 mice (019079, Jackson Laboratory).¹⁷ The primer sequences for mouse genotyping are shown (Supplemental Table I). The Myh11-CreER^T2-(+)/Klf11^fl/fl mice (Sm-Cre/Klf11^fl/fl +TAM) and Myh11-CreER^T2-(−)/Klf11^fl/fl mice (Klf11^fl/fl +TAM) were treated with tamoxifen (intraperitoneal injections, 80mg/kg per day for 5 consecutive days, indicated as +TAM). Myh11-CreER^T2-(+)/Klf11^fl/fl mice treated with the same volume of vehicle corn oil (Sm-Cre/Klf11^fl/fl + Oil) were used as the control group. Fourteen days after tamoxifen or corn oil treatment, the reduction of KLF11 expression in the aorta was confirmed by real-time quantitative PCR and western blot (Supplemental Figure IIIB-C). Since the Myh11-Cre/ER^T2 transgene is inserted on the Y chromosome,¹⁸ only male Sm-Cre/Klf11^fl/fl mice can be generated using this approach. All animal care and experimental procedures were approved by the University of Michigan Animal Care and Use Committee.

Ferric Chloride-Induced Carotid Artery Thrombosis Model

Ferric chloride (FeCl₃)-induced carotid artery thrombosis was conducted as previously described ¹⁹ and detailed in the “Extended Materials and Methods”.

Bone Marrow Transplantation (BMT)

The protocol for syngeneic BMT was previously described^{20, 21} and detailed in the “Extended Materials and Methods”.

Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT) Measurements

Platelet-poor-plasma (PPP) was collected as previously described ²² and detailed in the “Extended Materials and Methods”.

Tail-Bleeding Assay

Conventional Klf11 KO mice and WT mice (8–10-week-old male) were used in this experiment. As previously described,²³ a transverse incision at the 5 mm distal end of the tail was performed and the tail was immersed in saline at 37°C. Bleeding time was recorded as the time to cessation of bleeding.

Reagents and Antibodies

Reagents and Antibodies are listed in the “Extended Materials and Methods”.

Cell Culture

Human aortic smooth muscle cells (HASMCs, CC-2571, Lonza) were cultured in SmGM™−2 medium containing 5% FBS (CC-3182, Lonza) and used within 10 passages. Before thrombin stimulation, the HASMCs were made quiescent with DMEM/F12 with 0.5% fetal bovine serum (FBS) for 48 hours. A7r5 cells (CRL-1444™, ATCC) were cultured in DMEM/F12 supplemented with 10% FBS) and 50mg/ml of a penicillin/streptomycin mix. Mouse aortic smooth muscle cells (MASMCs) were isolated from the conventional Klf11 KO mice and WT mice (3–4-week-old male) as previously described.^{17, 24} Details are described in the “Extended Materials and Methods”. The purity of MASMCs was validated by immunostaining for α-smooth muscle actin (α-SMA). All cells were cultured in a 5% CO₂ humidified incubator at 37°C.

Preparation of Washed Murine Platelets and Platelet Aggregation Assay

The collection of washed murine platelets and platelet aggregation assay were performed as previously described²³ and detailed in the “Extended Materials and Methods”.

Protein Extracts and Western Blot

All protein extracts and western blot were performed as previously described.²⁵

Adenoviral Constructs

Adenoviral vectors overexpressing human KLF11 or LacZ control were generated as previously described.²⁶ The procedures are detailed in the “Extended Materials and Methods”.

Statistical Analysis

All quantitative data are presented as mean ± SEM. Statistical analysis were performed using the GraphPad Prism 7. All data were first subjected to Shapiro-Wilk normality test and F test to evaluate homogeneity of variances. For normally distributed data with similar variances among groups, unpaired Student t test with Welch’s correction was used for two-group comparisons and one-way analysis of variance (ANOVA) followed by Tukey’s test was used for more than two groups’ comparisons. Two-way ANOVA followed by Bonferroni test was applied for comparisons of grouped data under different conditions. Nonparametric Mann-Whitney test was used for data not normally distributed. All results were representative from at least four independent experiments.

Results

KLF11 Deficiency Aggravates Arterial Thrombosis In Vivo

To assess the role of KLF11 in arterial thrombosis, we used the conventional Klf11 KO mice previously reported¹⁴ and applied a FeCl₃-induced thrombosis model for studying arterial thrombosis.¹⁹ The occlusion time in the Klf11 KO male mice was significantly reduced to an average of 62% of that in WT C57BL/6J male mice (Figure 1A). A similar pro-thrombotic phenotype was also observed in Klf11 KO female mice, with the occlusion time reducing to 56% of that in the WT C57BL/6J female mice (Figure 1B). To exclude the effects of Klf11 KO in blood cells (platelets, neutrophils, macrophages, etc.) in this pro-thrombotic phenotype, we performed bone marrow transplantation (BMT) in Klf11 KO and WT mice. The reduced occlusion time in the Klf11 KO group transplanted with WT bone marrow was not rescued when compared with WT mice transplanted with WT bone marrow (Figure 1C).

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.

The hemostatic status in the Klf11 KO male mice was evaluated by measuring the function of coagulation factors and platelets. Our results showed that KLF11 did not alter the prothrombin time (PT) and activated partial thromboplastin time (aPTT), which reflect the function of extrinsic/common or intrinsic coagulation pathways, respectively (Figure 1D-E). Bleeding time and thrombin-antithrombin (TAT) complexes were also unchanged in the Klf11 KO mice, indicating that the general hemostatic status was normal in the conventional Klf11-deficient mice (Figure 1F-G). To evaluate whether the deficiency of KLF11 in megakaryocytes can affect the platelet function, we isolated washed platelets and performed thrombin induced platelet aggregation assay. The maximum platelet aggregation induced by thrombin did not show significant differences between WT and conventional Klf11 KO mice (Supplemental Figure II).

Smooth Muscle Cell Specific KLF11 Deficiency Aggravates FeCl₃-Induced Arterial Thrombosis

Taking into account that the hemostatic status in the circulation was not affected and the bone marrow transplantation cannot rescue the occlusion time in the conventional Klf11 KO mice, the phenotype in Klf11 KO mice might result from molecular changes specific to the vascular wall. To further identify the role of vascular smooth muscle cell (VSMC) KLF11 in thrombosis, we generated tamoxifen-inducible Sm-Klf11 KO (Myh11-CreER^T2/Klf11^fl/fl + TAM) mice (Supplemental Figure IIIA). Klf11^fl/fl mice treated with tamoxifen (Klf11^fl/fl + TAM) and Myh11-CreER^T2/Klf11^fl/fl mice treated with corn oil (Sm-Cre/Klf11^fl/fl + Oil) were used as controls. KLF11 deficiency in the aortic media of Sm-Klf11 KO mice was confirmed at the mRNA and protein levels (Supplemental Figure IIIB-C). The specificity of the Myh11-CreER^T2 in the aorta had been confirmed previously, as no expression was detected in cells from the blood and bone marrow.²⁷ In the FeCl₃ thrombosis model, Sm-Klf11 KO mice exhibited a similar pro-thrombotic phenotype as that observed in the conventional Klf11 KO mice. The occlusion time in the Sm-Klf11 KO mice was significantly reduced to an average of 67% of that in the two control mouse groups (Figure 1H). Our data suggest that VSMC KLF11 protects against arterial thrombosis.

TF Expression is Increased in the Vascular Wall of Conventional Klf11 KO Mice

TF is an important initiator of the coagulation cascade, which can generate insoluble fibrins and form a thrombus. The vascular wall TF contributes to arterial thrombosis in cardiovascular diseases such as atherosclerotic plaque rupture and myocardial infarction.²⁸ VSMC TF is critical for arterial thrombus formation in the mouse FeCl₃ thrombosis model.¹⁹ To determine whether TF is an effector mediating the enhanced thrombosis resulting from KLF11 deficiency, we measured the expression of TF in aorta of conventional Klf11 KO mice and WT mice in basal conditions. First, the activity of circulating microvesicle-associated TF (MV-TF) showed no significant differences between Klf11 KO and WT mice (Figure 2A). We applied anti-mouse TF 1H1 antibody²⁹ or rat IgG to validate the specificity of this assay. Next, we observed increased F3 mRNA level and TF activity in the isolated carotid artery of conventional Klf11 KO mice (Figure 2B-C). The elevated aortic TF activity can also be blocked by TF 1H1 antibody. Furthermore, the immunofluorescence staining data showed that TF protein was consistently upregulated in the vascular wall, and co-localized with α-smooth muscle actin (α-SMA), a specific marker of VSMCs. Moreover, the Klf11 KO mice also showed increased TF expression under lipopolysaccharide (LPS)-induced inflammatory conditions (Figure 2D). The relative TF intensity in the vascular wall was quantified and statistically analyzed. Collectively, our data suggest that KLF11 negatively regulates TF levels in VSMCs in vivo. Similar to the data in Figure 1G, no significant difference in TAT complexes was observed between WT and Klf11 KO mice at basal conditions. However, there was a higher TAT complexes in Klf11 KO mice after LPS treatment (Supplemental Figure IV).

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.

KLF11 Overexpression Rescues the TF Upregulation in KLF11-Deficient MASMCs

As a complementary and necessary approach, mouse aortic smooth muscle cells (MASMCs) were isolated from male and female conventional Klf11 KO mice and WT mice. The isolated MASMCs were characterized by immunostaining for α-SMA (Supplemental Figure V). The Klf11 deficiency was confirmed by real-time quantitative PCR (Figure 3A, E). A higher TF expression was observed in the MASMCs from both male and female Klf11 KO mice compared with WT mice (Figure 3B-D, F-H, Ad-LacZ). In both genders, restoration of KLF11 can rescue the phenotype in Klf11 KO MASMCs. As expected, the upregulated TF expression was significantly alleviated after KLF11 overexpression, at both mRNA and protein levels (Figure 3B-D, F-H, Ad-KLF11). These results indicate that endogenous KLF11 is required to prevent excessive TF upregulation, a hallmark of VSMC involvement in thrombosis.¹⁹

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).

KLF11 Inhibits TF Expression in HASMCs

To determine whether KLF11 is essential to regulate TF in VSMCs, we measured TF expression upon KLF11 knockdown in HASMCs. The efficiency of siRNA-mediated KLF11 knockdown was confirmed at the mRNA and protein levels (Figure 4A). The KLF11 knockdown in HASMCs increased both the basal and thrombin-induced expression of TF at mRNA and protein levels (Figure 4B-D). Similar effects were observed in HASMCs stimulated with 10% FBS-containing culture medium (Figure 4E-G).

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).

Apart from TF, other factors can also affect the formation of arterial thrombus. In HASMCs, the expression of other thrombosis related factors such as tissue factor pathway inhibitor (TFPI) and protease activated receptor-1 (PAR-1) and inflammatory genes such as monocyte chemoattractant protein-1 (MCP-1) and interleukin 1 beta (IL-1β) was not significantly changed in the KLF11-deficient HASMCs (Supplemental Figure VI).

Further, we upregulated KLF11 in primary HASMCs to determine whether KLF11 regulates TF in vitro. In HASMCs, adenovirus-mediated overexpression of KLF11 (Figure 5A) significantly inhibited the thrombin-induced TF expression at both mRNA and protein levels (Figure 5B-D). Similarly, KLF11 also suppressed TF expression in HASMCs stimulated with 10% FBS-containing culture medium (Figure 5E-G). Thus, our data suggest that KLF11 potently inhibits TF in human VSMCs under either thrombin stimulation or normal serum conditions.

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).

Interestingly, KLF11 overexpression also decreased tumor necrosis factor alpha (TNF-α)-induced TF expression in human umbilical vein endothelial cells (HUVECs) at both mRNA and protein levels (Supplemental Figure VII), implicating that endothelial KLF11 also may have an important role in thrombosis.

KLF11 Inhibits F3 Transcription

Next, we determined the mechanism that mediates the regulation of F3by KLF11. KLF11 was originally identified as the transforming growth factor-β-inducible early gene 2 (TIEG2), with a preference to bind at GC-rich sequences (GGGTG).³⁰ Transcription factor binding site analysis of the human F3 gene (Genomatix) revealed a relatively conserved TIEG binding site (−177 to −161 base pairs) upstream of the F3 transcription start site (Figure 6A). The TIEG binding site is conserved among human, mouse, rat and rabbit (Figure 6B). To determine whether this TIEG binding site is a functional KLF11 binding region, we performed chromatin immunoprecipitation (ChIP) assay in the HASMCs infected with Ad-flag-KLF11 or Ad-LacZ. Our data suggest that KLF11 can bind to the region containing this TIEG binding site (Figure 6C). To determine whether KLF11 regulates F3 at the transcriptional level, we generated luciferase reporter constructs, which were under the control of different lengths (−906/+162 and −556/+162) of the human F3 promoter. In A7R5 cells, a rat aortic smooth muscle cell line, transfected with different reporter constructs, KLF11 overexpression significantly reduced the luciferase activity (Figure 6D). Next, we deleted the TIEG bind site (−177 to −161) in the F3 promoter-driven luciferase construct. As expected, the deletion of the TIEG binding site significantly attenuated the KLF11 inhibition of F3 luciferase activity (Figure 6E) from the reporter plasmid. In conclusion, we identified that KLF11 inhibits F3 expression at transcription level through direct binding to the F3 promoter (Figure 6F).

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.

Discussion

In the current study, we observed an increase of arterial thrombosis in both genetically engineered conventional Klf11 KO mice and VSMC-specific Klf11 KO mice. In cultured human aortic smooth muscle cells, we demonstrated that KLF11 inhibits TF expression. Mechanistically, KLF11 directly binds to F3 promoter region and thereby suppresses the transcription of F3. This study demonstrated a potential role for VSMC KLF11 in arterial thrombosis.

The KLF family modulates cardiovascular activity through regulation of metabolism and inflammation in the cardiovascular system.^4–6 Endothelial KLF2 and KLF4 have been reported to inhibit thrombus formation by inhibiting the transcription of pro-thrombotic factors (e.g. plasminogen activator inhibitor 1 and TF) and increasing the expression of anti-thrombotic factors (e.g. thrombomodulin) under inflammatory conditions.^{6, 31–33}

We previously identified that KLF11 inhibits endothelial activation¹⁴ and attenuates endothelial dysfunction in the mouse middle brain artery occlusion-induced stroke model.¹⁶ Our current finding that VSMC KLF11 inhibits arterial thrombosis advances the understanding of the protective role of KLF11 in vascular diseases. Population genetics studies identified that mutations in KLF11 gene are positively associated with type 2 diabetes.⁷ Cardiovascular events are the major causes of death in diabetes.³⁴ Our study points to a potentially beneficial effect of KLF11 on cardiovascular complications in the diabetic patients. Follow-up studies are warranted to determine the role of VSMC KLF11 in diabetes associated cardiovascular diseases such as atherosclerosis, thrombosis, and angiogenesis.

In this study, we used the FeCl₃ thrombosis model, a widely used mouse arterial thrombosis model,³⁵ to study the role of KLF11 in the vascular wall in vivo. The penetration of FeCl₃ from adventitia triggers thrombosis.³⁶ In this study, we found that conventional Klf11 KO mice were more pro-thrombotic. The bone marrow transplantation study and the measurement of MV-TF activity excluded a potential involvement of KLF11 from blood cells and circulation-derived TF. Similar pro-thrombotic phenotype was observed in the Sm-Klf11 KO mice, which further demonstrated the anti-thrombotic effects of KLF11 in the vascular wall under basal conditions.

TF is critical in maintaining the balance between hemostasis and thrombosis.³⁷ VSMCs in human atherosclerotic plaques express high levels of TF.^38–40 Interestingly, a previous study using a low-TF mice demonstrated that the vascular wall derived TF, rather than leukocytes derived TF, is responsible for the macrovascular thrombosis.⁴¹ Therefore, the lower expression of TF in the vascular wall, especially in the VSMCs, can limit the initiation of the TF-dependent coagulation cascade and thus be a potentially protective mechanism for the prolonged occlusion time in vivo. Compared with endothelial cells (ECs), VSMCs have a higher constitutive expression of TF and are considered as the primary source of TF in the arterial wall.⁴² The expression of TF can be rapidly induced in VSMCs after artery injury⁴³ and contributes to thrombosis events after plaque rupture.^{38, 44} The SM22-driven VSMC-specific TF-deficient mice showed an increase in occlusion time in FeCl₃-induced arterial thrombosis, indicating a key role of VSMC-derived TF in arterial thrombosis.¹⁹ Although TF is not the only key factor in thrombosis formation, inhibition of TF activity by a monoclonal antibody⁴⁵ or administration of its counterpart recombinant TFPI⁴⁶ showed beneficial effects, which make inhibition of TF a potential pharmaceutical target for thrombosis. In addition, it has been reported that the deficiency of TFPI in smooth muscle cells can reduce the occlusion time in FeCl₃ model.⁴⁷ However, in this study, we found that TFPI expression was not significantly changed in the KLF11-deficient VSMCs. Therefore, the increased TF can at least partially account for the pro-thrombotic phenotype in the KLF11-deficient VSMCs.

It is well known that thrombin causes positive feedback effects on the coagulation cascade, including promoting the contact activation pathway.⁴⁸ However, numerous studies indicated that thrombin can also induce TF expression in VSMCs.^49–52 Thrombin can bind to protease-activated receptors (PARs) on human aortic SMCs and activate protein kinase B (PKB), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) pathways, which could induce TF expression.^53–55 Our data demonstrated that KLF11 directly binds to the F3 promoter and inhibits its activity in VSMCs, which account for the KLF11-dependent decreased F3 transcription under thrombin stimulation. The effect of KLF11 on thrombin-activated signaling pathways (e.g. PARs, PKB, PKC and MAPK) warrants future investigation.

In the present study, we demonstrated that KLF11 binds to F3 promoter to inhibit its transcription (Figure 6). In basal conditions (serum starvation), TF is expressed at very low level in VSMCs, and endogenous KLF11 is abundant enough to maintain the low level of TF by direct binding to the F3 promoter. Thus, overexpression of KLF11 did not further inhibit F3 transcription (Figure 5). The ChIP (Figure 6C) and luciferase (Figure 6D-E) experiments were performed in normal growth conditions (10% FBS). Under these conditions, we observed the inhibitory effect of KLF11 on F3 transcription (Figure 5). Upon thrombin or serum stimulation, overexpressed KLF11 may compete with other transcription factors such as activator protein-1 (AP-1),⁵⁶ whose binding region (−172–160bp) at the F3 promoter overlaps with the KLF11 binding region (−177–161bp). On the other hand, KLF11 deficiency may vacate the KLF11 binding site and abolish the repressive effect of KLF11 on F3 promoter activity. Therefore, KLF11 deficiency can increase F3 transcription even under basal conditions (Figure 4).

The FeCl₃ model is a commonly used experimental model to study arterial thrombosis.⁵⁷ However, whether the endothelium is denudated and internal elastic lamina is intact in this model are still controversial.^{58, 59} The mechanisms mediating the VSMC derived effect on arterial thrombosis in the FeCl₃ model remain to be investigated. Noteworthy, despite an intact internal elastic lamina upon FeCl₃ infiltration,^{58, 59} VSMC-specific TF KO inhibited the thrombus formation in the FeCl₃ model.¹⁹ In the present study, we found that smooth muscle cell specific Klf11 KO mice had a pro-thrombotic phenotype in association with increased TF, indicating that VSMC KLF11 can maintain anti-thrombotic state through transcriptional control of F3. We also found that KLF11 overexpression potently inhibited tumor necrosis factor alpha (TNF-α)-induced TF expression in HUVECs at both the mRNA and protein levels (Supplemental Figure VII), although it should be highlighted that endogenous levels of KLF11 in ECs are low.⁶ Since the contribution of endothelium is unclear in the FeCl₃ model,^{58, 59} the function of EC KLF11 in thrombosis warrants further investigation independently in other appropriate thrombosis models.

Interestingly, although there was no significant difference in TAT complexes between WT and Klf11 KO mice at basal conditions, there was a higher TAT complexes in Klf11 KO mice after LPS treatment (Figure 1G and Supplemental Figure IV). At basal conditions, TF is constitutively expressed in vascular wall and expressed much less in circulation. ^{60, 61} After LPS stimulation, TF expression is significantly induced not only in vascular wall, but also monocytes. ^{62, 63} We have demonstrated that KLF11 inhibits TF expression in VSMCs and ECs (Figure 3–5 and Supplemental Figure VII). Whether KLF11 also has inhibitory effects on TF expression in monocytes and thereby inhibits the pro-thrombotic status under LPS conditions warrants future investigations. Moreover, the conversion of prothrombin to thrombin is the common pathway in coagulation cascade. ⁶¹ Under LPS-treated conditions, apart from the increased TF expression, the abnormality of other coagulation factors may also contribute to the elevated TAT complexes in Klf11 KO mice.

In summary, utilizing gain- and loss-of-function strategies, we demonstrated an important homeostatic role of KLF11 as an anti-thrombotic factor in the vascular wall. We uncovered KLF11-dependent transcriptional inhibition of F3 in VSMCs as the potential mechanism underlying this anti-thrombotic effect. Our findings extend the current understanding of the roles of KLF11 in the vascular system. Manipulation of this novel molecular target could contribute to therapeutic strategies aimed at controlling thrombosis under pathological conditions and diabetic vascular pathologies at large.

Highlights.

• VSMC-specific KLF11 protects against arterial thrombosis.

• Tissue factor is increased in the KLF11-deficient VSMCs.

• KLF11 inhibits F3 at the transcriptional level.

Nonstandard Abbreviations and Acronyms

KLF

krüppel-like factor

VSMC

vascular smooth muscle cell

HASMC

human aortic smooth muscle cell

MASMC

mouse aortic smooth muscle cell

Myh11

smooth muscle cell myosin heavy chain 11

WT

wild type

KO

knockout

TF

tissue factor

TFPI

tissue factor pathway inhibitor

TIEG

transforming growth factor-β-inducible gene

aPTT

activated partial thromboplastin time

PT

prothrombin time

BMT

bone marrow transplantation

siRNA

small interfering RNA

Ad

adenovirus

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

FeCl₃

ferric chloride