Endogenous tissue factor pathway inhibitor in vascular smooth muscle cells inhibits arterial thrombosis

Abstract

Tissue factor pathway inhibitor (TFPI) is the main inhibitor of tissue factor-mediated coagulation. TFPI is expressed by endothelial and smooth muscle cells in the vasculature. Endothelium-derived TFPI has been reported to play a regulatory role in arterial thrombosis. However, the role of endogenous TFPI in vascular smooth muscle cells (VSMCs) in thrombosis and vascular disease development has yet to be elucidated. In this TFPI^Flox mice crossbred with Sma–Cre mice were utilized to establish TFPI conditional knockout mice and to examine the effects of VSMC-directed TFPI deletion on development, hemostasis, and thrombosis. The mice with deleted TFPI in VSMCs (TFPI^Sma) reproduced viable offspring. Plasma TFPI concentration was reduced 7.2% in the TFPI^Sma mice compared with TFPI^Flox littermate controls. Plasma TFPI concentration was also detected in the TFPI^Tie2 (mice deleted TFPI in endothelial cells and cells of hematopoietic origin) mice. Plasma TFPI concentration of the TFPI^Tie2 mice was 80.4% lower (P<0.001) than that of the TFPI^Flox mice. No difference in hemostatic measures (PT, APTT, and tail bleeding) was observed between TFPI^Sma and TFPI^Flox mice. However, TFPI^Sma mice had increased ferric chloride–induced arterial thrombosis compared with TFPI^Flox littermate controls. Taken together, these data indicated that endogenous TFPI from VSMCs inhibited ferric chloride–induced arterial thrombosis without causing hemostatic effects.

Keywords

arterial thrombosis / conditional knockout mice / tissue factor pathway inhibitor / vascular smooth muscle cells

Introduction

Tissue factor pathway inhibitor (TFPI), originally known as lipoprotein-associated coagulation inhibitor, has been isolated from a hepatoma cell line [1]. Under normal physiological conditions, tissue factor (TF) is located in extravascular cells, and hemostasis occurs after TF is exposed to flowing blood following a traumatic vascular injury [2]. The potential adverse effects of intravascular TF are inhibited by TFPI, a Kunitz-type serine protease inhibitor that blocks the extrinsic coagulation pathway and regulates coagulation initiation by inhibiting the TF/factor VIIa activation of factor X [3–7]. A human completely lacking TFPI has yet to be described, and TFPI knockout mice homozygous for the null allele (Tfpi^−/−) die in utero [8], highlighting the importance of TFPI anticoagulant activity during development [9]. TFPI is thought to be predominantly expressed from endothelium. However, endothelial-derived TFPI is not necessary for murine development or fertility [10]. TFPI is also expressed in vascular smooth muscle cells (VSMCs). Nevertheless, whether VSMC-derived TFPI is essential for murine development or fertility remains unclear.

TFPI is constitutively synthesized by microvascular endothelial cells and VSMCs, although the expression of TFPI has been reported in many other cell types including platelets, megakaryoctyes, T-lymphocytes, macrophages/monocytes and cardiac myocytes [10–15]. The endothelium has been thought to be the main source of circulating TFPI [16]. Tie2-directed TFPI-K1 deletion results in a 71% reduction in circulating TFPI activity [10]. However, VSMCs-derived TFPI contributing to circulating TFPI remains unkown.

Local TFPI can regulate arterial thrombosis [17,18]. For example, VSMC-directed TFPI overexpression alleviates ferric chloride (FeCl₃)-induced arterial thrombosis [19]. Endothelial-derived TFPI plays a regulatory role in arterial thrombosis [10]. Hematopoietic TFPI restricts the growth of thrombi following a vascular injury [20]. However, TFPI overexpression in VSMCs fails to indicate the role of endogenous TFPI from VSMCs under physiological conditions. The role of endogenous TFPI from VSMCs in hemostasis regulation and arterial thrombosis remains unknown. The endothelium provides an antithrombotic interface with circulating blood which is generated in part by the coordinated expression of endothelial-derived anticoagulants [10]. However, whether endothelial-derived TFPI is sufficient for anti-arterial thrombosis in the absence of TFPI in VSMCs remains unclear.

To address these concerns and define VSMC-derived TFPI and its role in development, hemostasis, and thrombosis, we established TFPI conditional knockout mice. Our results demonstrated that mice with deleted TFPI in VSMCs underwent normal reproduction. We also defined the contribution of VSMCs and endothelial and hematopoietic progenitor cells to mouse plasma TFPI concentration. Our results further revealed that endogenous TFPI from VSMCs participated in anti-arterial thrombosis.

Materials and methods

Animals

TFPI^Flox mice, which were generated using the Cre−LoxP system and a gene inactivation strategy, were previously established by our laboratory [21] and were bred normally. TFPI^Tie2 mice, which were obtained by crossbreeding TFPI^Flox mice with Tek–Cre mice, were also previously engineered by our laboratory. Sma–Cre mice was a generous gift from Xiao Yang (Academy of Military Medical Sciences, Beijing, China). All animal protocols were approved by the Institutional Committee for the Use and Care of Laboratory Animals of Fudan University.

Generation of TFPI^Sma mice and genotyping

TFPI^Flox mice were crossbred with Sma–Cre mice to generate the TFPI conditional knockout mice. TFPI^Flox mice were crossbred with Sma-Cre mice. The offsprings were then backcrossed to floxed homozygosity while maintaining the Sma-Cre transgene (TFPI^Sma). Genotyping for the Sma-Cre mice were done with genomic PCR using Sma specific primers: forward primer, 5′-CTGTGACACTCCCGCTCTTTGTGCTG-3′; reverse primer, 5′-CGAACATCTTCAGGTTCTGCGGGAAA-3′. The TFPI^Flox mice primers are as follows: forward primer, 5′-TCTTCCTGTTGTCTGGGACATCC-3′; reverse primer, 5′-AGAGCGGCCATCATAACTTCG-3′.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Aortic SMCs were isolated through collagenase digestion as previously described [22]. To measure the TFPI expression levels, we extracted total RNA from aortic SMCs by utilizing TRIzol reagent (Invitrogen, Japan) according to the manufacturer’s instructions. cDNA was synthesized using a reverse transcription kit (Takara, Japan). qRT-PCR was performed using SYBR Green (Applied Biosystems, Germany). The Tfpi and Gapdh primers are as follows: Tfpi-F, 5′-TATACGGGGGATGTGAAGGGAACG-3′ and Tfpi-R, 5′-TGCTCCAGATGCTGCCTTCACAG-3′ and Gapdh-F, 5′-AGATTGTTGCCATCAACGACCCC-3′ and Gapdh-R, 5′-CCATTCTCGGCCTTGACTGTGC-3′.

Immunohistochemistry

Dissected aortic vessel were fixed in 4% paraformaldehyde overnight at 4 °C and then eluted using gradient ethanol. Next, the vessel was embedded in paraffin, and 5 µm-thick sections were cut to produce paraffin sections, which were processed for immunohistochemical analysis. First, paraffin sections were subjected to gradient dewaxing hydration. Then, antigen retrieval was performed on paraffin-embedded sections by heating in 0.01 mol/L citrate buffer (pH 6.0) for 20 min. Immunohistochemical labeling was performed using a polyclonal rabbit anti-mouse TFPI antibody (Santa Cruz Biotechnology, USA). The secondary antibody and the DAB staining kit were utilized as described in the product manual (DakoCytomation, USA). The images of three to six representative fields from different mouse groups were captured using optical microscope (Olympus, Japan).

Mouse TFPI enzyme-linked immunosorbent assay (ELISA) assay

A 96-well microplate was coated with diluted capture antibody overnight at room temperature. The microplates were blocked with reagent diluent (1% BSA in phosphate-buffered saline (PBS)) at room temperature for 1 h after these plates were washed thrice with PBS containing 0.05% Tween-20, which was also used in subsequent washes. The plasma samples were diluted with reagent diluent at 1:2 ratio and incubated at room temperature for 2 h. The plates were washed, and detection antibody was incubated for 2 h at room temperature. The plates were washed again, and then the working dilution composed of streptavidin–horseradish peroxidase was added to each well. The plates were covered and incubated for 20 min at room temperature. The aspiration/wash process was repeated, the substrate solution was incubated for 20 min at room temperature, and then the stop solution was added to each well (R&D Systems, USA). Finally, the optical density of each well was immediately determined using a microplate reader set to 450 nm.

Hemostasis analysis

The prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured in citrated plasma using automated techniques (Sysmex CA-1500). The bleeding times were measured as described in previously published research [23]. The mice (approximately 6 weeks old) were anesthetized, and their tails were transected 5 mm from the tip with a razor blade and immediately immersed into a 5 ml test tube with 5 ml of PBS (37 °C). The time to bleeding cessation was recorded. A quantitative estimate of the bleeding amount was determined by measuring the hemoglobin content of the blood collected in PBS. After centrifugation, the red blood cells were lysed with 5 ml cell lysis buffer (1.0 g/L KHCO₃, 8.3 g/L NH₄Cl, and 0.037 g/L EDTA), and the absorbance of the sample was measured at 575 nm.

FeCl₃-induced thrombosis of the carotid artery

A FeCl₃-induced carotid artery injury murine thrombosis model was developed as described in a previous study [24]. The mice used in these experiments were approximately 8 weeks old. A filter paper strip saturated with 10% FeCl₃ solution was applied to the adventitial surface of the surgically exposed carotid artery for 3 min. After the filter paper was removed, blood flow was immediately recorded, and the first occlusion was defined as blood flow of less than 0.05 ml/min.

Statistics

Results were presented as mean±SEM, and statistical significance was assessed through unpaired two-tailed Student’s t-test in GraphPad Prism (*P<0.05; **P<0.01; ***P<0.001).

Results

TFPI conditional knockout genotyping and efficiency of the Sma–Cre-directed TFPI deletion

VSMC TFPI conditional knockout mice were obtained by crossing the mice expressing Cre recombinase under the control of the Sma promoter with TFPI^Flox mice. PCR was used to determine the genotyping of the offspring. A duplex PCR determined knockout Tfpi and Sma–Cre alleles as bands of 582 and 500 bp, respectively. The two PCR products (582 and 500 bp) in TFPI^Sma mice demonstrated that Cre recombinase driven by the Sma promoter is expressed in VSMCs (Fig. 1A). Next, we examined the efficiency of the Sma–Cre-directed TFPI deletion. The RNA isolated from the VSMCs of TFPI^Flox or TFPI^Sma mice were used for qRT-PCR analysis. The relative TFPI mRNA levels of TFPI^Sma mice was 20%±0.9%. The efficiency of the Sma–Cre-directed TFPI deletion was approximately 80% (Fig. 1B). Consistently, we confirmed the TFPI expression via immunohistochemistry. Our figure demonstrated that the positive staining in TFPI^Sma mice was weaker than that in the control mice (Fig. 1C).

Plasma TFPI concentration in TFPI^Flox, TFPI^Sma, and TFPI^Tie2 mice

An ELISA assay was used to detect the plasma TFPI concentration and to determine the contribution of VSMCs to mouse plasma TFPI concentration. The plasma TFPI concentration of TFPI^Sma mice was 7.2% lower than that of TFPI^Flox mice. The loss of VSMC TFPI does not significantly change the plasma TFPI concentration. We also detected the plasma TFPI concentration of TFPI^Tie2 mice for comparison. The plasma TFPI concentration of TFPI^Tie2 mice was 80.4% lower (P<0.001) than that of TFPI^Flox mice (Fig. 2). These data indicate that Sma- and Tie2-directed TFPI deletion results in a 7.2% and 80.4% reduction in plasma TFPI concentration, respectively.

Role of VSMC-derived TFPI in hemostasis

Hemostasis was assessed through tail bleeding times and quantitation of hemoglobin loss in TFPI^Flox and TFPI^Sma mice. No significant differences in tail bleeding times (Fig. 3A) or hemoglobin loss (Fig. 3B) were observed in TFPI^Sma mice compared with TFPI^Flox mice. Meanwhile, we did not observed difference in the PT (Fig. 3C) and APTT (Fig. 3D) between TFPI^Sma and TFPI^Flox mice. These data indicated that VSMC-directed TFPI deletion does not lead to bleeding or hemostatic abnormality.

VSMC-derived TFPI regulates arterial thrombosis

The function of VSMC-derived TFPI in vivowas investigated in a model of FeCl₃-induced arterial thrombosis. The blood flow was monitored by using a transit-time perivascular flowmeter in the carotid artery after a 3 min vascular injury with 10% FeCl₃. The average time to the first occlusion of the TFPI^Flox mice was 11.86±1.42 min, which was different from 7.90±0.48 min (P<0.01) of the TFPI^Sma mice. The times to occlusion of TFPI^Sma mice were 33% shorter than those of the TFPI^Flox littermate controls (Fig. 4A and 4B).

Discussion

Sma expression is highly restricted to SMCs or smooth muscle-like cells [25]. Sma–Cre-directed deletion has been previously reported as a smooth muscle-specific gene targeting approach [26]. Given that TFPI is also expressed in VSMCs, we used Sma–Cre mice crossbred with TFPI^Flox mice to establish TFPI conditional knockout mice to examine the effects of VSMC-derived TFPI on development, hemostasis, and thrombosis.

Tfpi^−/− mice died at two stages during embryonic development. On a C57Bl/6 background, ~30% died between E9.5 and E11.5 due to yolk sac hemorrhage and circulatory collapse, whereas the remaining 70% died from an apparent consumptive coagulopathy before birth [8,9]. In a previous study, neither endothelial-derived TFPI nor myelomonocytic-derived TFPI is essential for murine development or fertility [10]. Considering that TFPI is also constitutively synthesized by the VSMCs, we aim to determine whether VSMC-derived TFPI is implicated in murine development or fertility. Our data suggested that embryonic lethality did not occur in TFPI^Sma mice. Therefore, VSMC-derived TFPI is not essential during embryonic development. Tfpi^−/− mice are rescued through TF deficiency [9]. This observation demonstrated the importance of TF–TFPI balance maintenance in embryonic development. Trophoblast cells may possess an ability to regulate hemostasis at the fetomaternal interface [27]. Therefore, TFPI from both trophoblasts and maternal circulation in the TFPI^Tie2 [10] or TFPI^Sma mice may be sufficient to maintain the TF–TFPI balance required for placental hemostasis and embryogenesis.

We demonstrated that Tie2-expressing cells, namely, endothelial cells and hematopoietic cells, produce a majority 80% of circulating TFPI, which differs from the 71% reduction in circulating TFPI activity detected in another study [10]. This difference might be attributed to the whole TFPI gene knockout we used in the present study, rather than the TFPI-K1 domain deletion [10]. After all, the TFPI-K1 domain could not take the place of TFPI function completely. TFPI protein without KD1 but with intact KD2, KD3, and C-terminus domains are normally expressed and useful. For instance, the TFPI Kunitz domain 3 residue Glu226 is essential for TFPI enhancement by protein S [28–30]. We also determined that approximately 7% of circulating TFPI concentration originates from VSMCs. VSMC TFPI deletion does not significantly change the plasma TFPI concentration. Therefore, the hemostatic parameters, including PT, APTT, and tail bleeding, did not significantly differ between TFPI^Sma and TFPI^Flox mice. Given that Sma- and Tie2-directed TFPI deletion results in approximately 7% and 80% plasma TFPI concentration reduction, respectively, non-VSMC, nonendothelial, and nonhematopoietic cell-derived source for TFPI must exist. This potential source comes from cardiomyocytes [31].

VSMCs express TF in vivo, which may contribute to vascular thrombosis [32,33]. Given that locally expressed TFPI can inhibit local TF activity, we anticipated that VSMC-derived TFPI might have a role in anti-vascular thrombosis. Our findings suggested that VSMC-derived TFPI deficiency promotes FeCl₃-induced carotid arterial occlusion. Considering that no differences were detected in the TFPI concentrations between TFPI^Sma and TFPI^Flox mice, we conclude that plasma TFPI is not responsible for the differences in FeCl₃-induced arterial thrombosis. Possibly, the increased arterial thrombosis derived from VSMCs lacking TFPI, rather than plasma lacking TFPI. Mice lacking endothelial TFPI have a shorter time to occlusion than their TFPI^Flox littermate controls in a FeCl₃-induced thrombosis model [10]. In the present study, the times to occlusion in TFPI^Sma mice were 33% shorter than those of their TFPI^Flox littermate controls, although VSMCs only contributed to 7% of plasma TFPI. This finding indicated that VSMC-derived TFPI plays an important role in anti-vascular thrombosis, and endothelial-derived TFPI is insufficient for anti-arterial thrombosis in the absence of TFPI in VSMCs.

In summary, a conditional knockout mouse was generated to delete TFPI in VSMCs. These mice provide a unique resource to investigate the TFPI function in vascular diseases, including thrombosis and atherosclerosis. In the present study, the role of VSMC-derived TFPI was determined in the regulation of murine development, hemostasis, and arterial thrombosis. VSMC-derived TFPI is not necessary during embryonic development. Approximately 7% and 80% of circulating TFPI concentrations originate from VSMCs and endothelial and hematopoietic origin cells, respectively. Hemostatic parameters, including PT, APTT, and tail bleeding times, did not differ between TFPI^Sma and TFPI^Flox mice. However, endogenous TFPI from VSMCs plays a regulatory role in arterial thrombosis.

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