Ablation of Angiotensin IV Receptor Attenuates Hypofibrinolysis via PAI-1 Downregulation and Reduces Occlusive Arterial Thrombosis
Objectives— Reduced fibrinolytic activity is associated with adverse cardiovascular events. Although insulin-regulated aminopeptidase (IRAP) was recently identified as the angiotensin (Ang) IV receptor (AT4R), the impact of AngIV-AT4R signaling distal to AngII on the activation of type-1 plasminogen activator inhibitor (PAI-1) in the fibrinolytic process and subsequent formation of thrombosis remains unclarified.
Methods and Results— To determine whether AngIV would inhibit fibrinolysis via PAI-1 activation and promote thrombosis, we evaluated the degree of fibrinolysis in thrombosis models and investigated the roles of AT4R after vascular injury using IRAP knockout mice (IRAP−/−). In endothelial cells from control mice (WT; C57Bl6/J), both AngII and AngIV treatments increased PAI-1 mRNA expression in a dose-dependent manner, whereas the response was blunted in endothelial cells from IRAP−/− mice. FeCl3-induced thrombosis was suppressed in the carotid arteries of IRAP−/− mice when compared with WT mice. Similarly, in a model of carotid artery ligation and cuff placement, IRAP−/− mice demonstrated accelerated fibrinolysis 7 days after surgery and reduced occlusive thrombosis with negative remodeling at 28 days.
Conclusions— AngIV-AT4R signaling has a key role in fibrinolysis and the subsequent formation of arterial thrombosis after vascular injury. AT4R may be a novel therapeutic target against cardiovascular disease.
The renin–angiotensin system (RAS) is a key regulator of blood pressure and fluid homeostasis. The main effector peptide of the RAS, angiotensin II (AngII; an octapeptide hormone), causes vasoconstriction, platelet aggregation, and increased sodium uptake and water retention in the kidneys, thereby leading to hypertension, atherosclerosis, and subsequent cardiovascular disease.1 AngII exerts these actions through the activation of downstream signals by binding to its receptors, namely, angiotensin type 1 and type 2 receptors (AT1R and AT2R, respectively). Most of the well-known functions of AngII in the cardiovascular system are mediated through AT1R.2–5 In addition to AngII and these receptors, AngIV, a hexapeptide that is derived by the cleavage of 2 N-terminal amino acids from AngII by aminopeptidases, is sufficiently bioactive to have a prothrombotic potential through the production of plasminogen activator inhibitors (PAIs) by binding to the AT4 receptor (AT4R).1,6,7 However, its role in the pathogenesis of thrombosis remains unknown.
Insulin-regulated aminopeptidase (IRAP), a zinc-metallopeptidase, was identified as AT4R.8 IRAP was first identified as a major protein in intracellular vesicles isolated from low-density microsomes of rat fat and muscle cells that also colocalized with the insulin-responsive glucose transporter isotype GLUT4.9,10 In 1995, we purified placental leucine aminopeptidase (P-LAP, EC:188.8.131.52, 1024 amino acids, 170 kDa) from retroplacental serum and cloned P-LAP from a human placental cDNA library.11 It was identified as a human homologue of IRAP and confirmed to be a key enzyme regulating the serum levels of hormones such as oxytocin and vasopressin, thus maintaining homeostasis during pregnancy and tumorigenesis.12,13
Impaired fibrinolysis has been linked to thrombosis in a number of experimental and clinical studies.14,15 During fibrinolysis, type-1 PAI (PAI-1; 379 aa and 48 000 MW) binds to tissue-type and urinary-type plasminogen activators (t-PA and u-PA, respectively), protecting a blood clot from premature lysis.16–18 Under atherosclerotic conditions, PAI-1 is overexpressed in vessel walls and plaques, negatively regulating thrombolysis, and this may facilitate thrombotic events after the rupture of plaques.19 Although AngIV and AT4R are involved in the regulation of thrombus formation and inflammation under thrombogenic conditions such as atherosclerosis, acute myocardial infarction, and pregnancy, little is known as to how the AngIV/AT4R pathway could affect thrombosis, inflammation, and subsequent vascular lesion formation through induction of PAI-1. In this study, we evaluated both acute fibrinolysis and chronic thrombosis using 3 different mouse models: the acute disseminated intravascular coagulation (DIC) model induced by endotoxinemia,20 the ferric chloride (FeCl3)-induced thrombosis model,21,22 and the ligation and cuff placement model in the carotid artery.23 We aimed to determine whether the genetic ablation of AT4R could attenuate thrombosis and inflammation using IRAP-deficient mice (IRAP−/−). Our data provide an important insight into the novel association between RAS and thrombosis and heightens awareness to a pivotal role of IRAP/AT4R in the pathogenesis of cardiovascular disease.
The derivation of IRAP−/− mice has been described in detail elsewhere.24 The mice (a kind gift from Bayer AG, Leverkusen, Germany) were derived from a 129/C57BL/6 background, and backcrossed with a C57BL/6 strain more than 7 times. C57BL/6 mice were used as wild-type (WT) controls. Animal experiments were performed with the authorization of the institutional review board of the Animal Care and Use Committee of Nagoya University Graduate School of Medicine. This animal study conformed to the “Position of the American Heart Association on Research Animal Use” (Circulation 1985; 71:849A). All experiments were performed using 8- to 10-week-old male mice, unless otherwise indicated.
Cell Culture and Drug Treatment
Murine aortic endothelial cells (ECs) were explanted from the mouse aorta as previously described.25 ECs of passages 2 and 3, which were confirmed by CD31-positive staining, were used in the experiments (please see supplemental Figure I, available online at http://atvb.ahajournals.org).
ECs cultured to subconfluency in a 6-well plate were treated with 10 μg/mL lipopolysaccharide (LPS; Escherichia coli serotype O111:B4; Sigma Chemical Co Ltd) to assess the induction levels of PAI-1 mRNA after LPS treatment. Cells were then lysed with lysis buffer containing 1% β-mercaptoethanol after washing twice in PBS.20 Cell lysates were stored at −80°C before mRNA measurement. We evaluated the active antigen levels of PAI-1 in the culture medium using the murine PAI-1 activity assay kit (Innovative Research Inc).
Similarly, ECs were pretreated with 1 μmol/L captopril (Sigma Chemical) overnight and then exposed to AngII and AngIV (Peptide Institute) at 10−9 to 10−6 mol/L for 6 hours.6,7
Measurement of mRNA Expression Level
Total RNA was extracted from cell lysates using TRIzol Reagent (Invitrogen), and the amount was quantified by a densitometer. The first cDNA strand was synthesized using the SuperScript First-Strand Synthesis System (Invitrogen). Quantitative real-time PCR was performed using the LightCyclerT System (Roche Diagnostics) and QuantiTect SYBR Green PCR kit (Qiagen; please see supplemental materials).
Hemodynamic Analyses and Tail Bleeding Times
Systolic blood pressure and heart rate of mice (n=12, each) were determined by the tail-cuff detection system Softron BP-98A (Softron).25,26 Tail bleeding times were assessed by standard procedures at 8 weeks of age (n=6, each; please see supplemental materials).
The mice (n=8 to 10, each) were anesthetized by sodium pentobarbital (Dainippon Pharmaceutical, Tokyo, Japan, 33 mg/kg IP), and blood was drawn with 21G needles by the ventricle puncture method via the subxyphoid approach. Differential blood counts were obtained using an automated hematology analyzer. Samples of mouse plasma after LPS injection were stored at −80°C before measurement. To detect plasma proteins such as D-dimer and thrombin-antithrombin III complex, ELISA kits were used (please see supplemental materials).
To assess the fibrinolytic balance between PAI-1 and t-PA, we evaluated the active antigen levels of PAI-1 and t-PA using murine PAI-1 and t-PA activity assay kits, respectively (Innovative Research Inc).
LPS Treatment for the DIC Model
Eighteen to 20-week-old mice (n=12, each) were treated intraperitoneally with 100 μL of LPS (50 mg/kg) and euthanized at 2, 4, 8, and 24 hours after injection. We assessed the levels of active PAI-1 in platelet-poor plasma (PPP) and the degree of fibrin deposition in the kidney.20
FeCl3-Induced Arterial Injury and Thrombosis
Mice were subjected to carotid artery injury with 10% FeCl3 (n=15 to 20 in each group).21,22 Briefly, mice were anesthetized with sodium pentobarbital, the left carotid artery was dissected, and a small strip of filter paper soaked in 10% FeCl3 was applied to the surface of the adventitia for 3 minutes. Carotid flow was monitored with a flow probe (0.5VB, Transonic Systems) interfaced with a flowmeter (Power Laboratory, AD Instruments) and analytic program (Laboratory Chart 7, AD Instruments). The time to occlusion was measured.
To assess the effects of PAI-1 inhibition on thrombosis, we administered a specific PAI-1 inhibitor (T-686; a kind gift from Mitsubishi Tanabe Pharmaceutical) by gavage (100 mg/kg/d) for 7 days before the experiments.27
Carotid Artery Ligation and Cuff Placement Procedure and Morphometric Analysis
To assess the process of thrombolysis, we performed ligation and cuff placement in the carotid artery, which is characterized by blood flow cessation and intracuff EC injury.23 At 0, 4, 7, and 28 days after surgery, the carotid arteries were removed and stained by hematoxylin and eosin (H&E), Masson trichrome, or Elastica van Gieson staining, followed by morphometric analyses (Please see supplemental materials). T-686 was administered for 7 days before and 28 days after the surgery.
To assess NFκB activation and the inflammatory response, the sections taken 4 days after surgery were immunostained with phosphorylated IκB-α (Abcam, Cambridge) and MCP-1 (rat monoclonal antibody for CCL2, ABR/Thermo Fischer Scientific), visualized with Alexa Fluor 488 (Molecular Probe/Invitrogen), counterstained with DAPI (Molecular Probe), and observed with an epifluorescent microscope (BZ8000, Keyence).
The data are presented as mean±SEM values. Statistical analysis of multiple comparisons among the groups was conducted by 1-way ANOVA followed by the Bonferroni test. Statistical analysis of comparisons between 2 groups over time used repeated measures ANOVA. Multiple comparisons in nonparametric analysis were performed by the Kruskal–Wallis test. A probability value of P<0.05 was considered significant.
Hemodynamics and Hemostasis in IRAP−/− Mice
We initially measured the parameters of hemodynamics and hemostasis to determine the basal characteristics of IRAP−/− mice. Blood pressure, body weight, fat weight evaluated by the ratio of epididymal fat weight to body weight, and heart rate were similar between groups. With respect to glucose and lipid metabolism, IRAP−/− mice showed no differences in the basal levels of glucose, insulin, cholesterol, triglycerides, or free fatty acids, compared with WT. IRAP−/− mice showed no differences in basal hematologic characteristics (please see supplemental Table I).24
PAI-1 mRNA Expression and Active PAI-1 Levels After LPS Treatment in Cultured ECs
To assess the roles of AT4R on PAI-1 expression in ECs and subsequent fibrinolysis, we compared the magnitude of PAI-1 mRNA expression between 2 groups in response to LPS treatment, which is a potent activator that induces PAI-1 in ECs to a maximum level. There were no differences observed in PAI-1 mRNA expression between the groups before treatment. After LPS treatment, in ECs isolated from WT and IRAP−/− mice, PAI-1 mRNA levels increased by 4.2±0.5 and 3.8±0.4 fold, respectively (P<0.005 versus control). The active PAI-1 levels in the culture medium after LPS treatment were lower in IRAP−/− mice than in WT mice (57±2.4 and 86±3.5 ng/mL, P<0.01, respectively).
PAI-1 mRNA Expression After AngII and AngIV Treatment in Cultured ECs
In ECs from WT mice, PAI-1 mRNA expression increased in a dose-dependent manner and reached a 3.3±0.5 fold increase with 10−6 mol/L AngII (P<0.01 versus control) and a 2.8±0.6-fold increase by 10−6 mol/L AngIV (P<0.01 versus control, Figure 1A). In ECs from IRAP−/− mice, PAI-1 mRNA induction was suppressed after AngII and AngIV treatment (2.0±0.3 and 1.7±0.3-fold at 10−6 mol/L, respectively, P<0.05 versus WT, Figure 1B).
Active PAI-1 and t-PA Levels After LPS Treatment
To evaluate the real-time fibrinolytic balance after LPS injection, we observed the alteration of active PAI-1 and t-PA antigen levels in PPP over time. In WT mice, the curve of the active PAI-1 antigen levels was biphasic with peaks 2 and 8 hours after LPS treatment (Figure 2A). In contrast, the active PAI-1 antigen levels had only one peak 2 hours after LPS treatment in IRAP−/− mice, which then gradually decreased. The increases in the active PAI-1 antigen levels 2, 4, and 8 hours after LPS injection in IRAP−/− mice were significantly lower than those in WT mice (P<0.01, P<0.05, and P<0.01 for IRAP−/− mice versus WT mice, with respect to each time point). In general, the active PAI-1 antigen levels were suppressed after LPS injection in IRAP−/− mice compared with those in WT mice (P<0.001 each versus WT mice). In contrast to the alteration of active PAI-1 levels, active t-PA levels gradually increased with a peak at 8 hours after LPS injection and no difference was observed between the 2 groups (Figure 2B).
Renal Glomerular Fibrin Deposition After LPS Treatment
As well as acute PAI-1 release from ECs, LPS injection causes a loss of integrity in the EC monolayer of blood vessels. Permeability increases and platelets can adhere to the extracellular matrices (ECMs) beneath the EC layer and activate aggregation, thereby forming fibrin deposits mainly in capillaries and in renal glomeruli. We counted the rate of renal fibrin deposition 4 hours after LPS injection at the point when fibrin deposition is most significant, as observed previously.20 As shown in Figure 3, the percentage of fibrin deposition observed within glomeruli per section were 18.9±2.5% in WT and 4.32±1.9% in IRAP−/−mice (P<0.01 versus WT).
Assessment of Occlusive Thrombosis in the FeCl3-Induced Model
To investigate the in vivo relationship between AT4R and PAI-1 in thrombosis, we administered the specific PAI-1 inhibitor T-686 to WT mice and compared the pathophysiological changes in the carotid arteries. Firstly, we analyzed the conventional FeCl3-induced thrombosis model among three groups. On induction of injury, complete thrombotic occlusion was observed in 100% of WT mice but in only 87% and 72% of T-686–treated mice and IRAP−/− mice, respectively (each P<0.001 versus WT mice). In occluded arteries, the mean time for occlusion was significantly longer in IRAP−/− mice (14.8±2.8 minutes, P<0.001 versus WT) and in T-686–treated mice (11.7±2.1 minute, P<0.01 versus WT) than in WT mice (8.6±1.7 minutes; Figure 4).
Assessment of Occlusive Thrombosis in the Model of Ligation and Cuff Placement
For the assessment of chronic occlusive thrombus formation and fibrinolysis in the artery, we used a newly developed model by combined treatments of murine carotid artery ligation with perivascular cuff placement (supplemental Figure II). We confirmed that the intraluminal thrombus formation was evident 7 days after surgery in WT mice (Figure 5).21 Thrombi were remarkably apparent 4 days after surgery in sections from WT mice and the percentage was 65.2±5.0% (Figure 5A). In contrast, the thrombus area was as low as 21.4±3.1% in IRAP−/− mice and 24.5±3.7% in T-686–treated mice (P<0.01 versus WT). Organized thrombus formation was confirmed in over 90% of the sections from WT mice 7 days after surgery, whereas in T-686–treated mice and IRAP−/− mice, fibrinolysis was accelerated and the total area of thrombi in the inner lumen area was as low as 12.5±3.2% and 14.2±3.5% (P<0.001 versus WT), respectively. At 28 days, in the sections from WT mice, thrombus was rarely observed and the lumen was occupied with rigid ECMs such as collagen and elastin produced by protruded myofibroblasts. In T-686–treated mice, the lumen was patent, whereas neointimal formation was observed and the adventitia was rich with myofibroblasts which was similar to the sections from WT mice. In IRAP−/− mice, the lumen was patent and thrombus and neointimal formation were rarely observed. However, negative remodeling (hypertrophic vessel narrowing) at a remote period after injury was observed. The average lumen diameter was smaller than that of WT mice (1.20±0.2 mm, 1.22±0.2 mm and 1.37±0.3 mm, respectively, P<0.05 each, Figure 5B).
Immunostaining of sections from the carotid arteries revealed that NFκB activation (confirmed with phosphorylated IκB-α) was prominent in T-686–treated and WT mice, whereas NFκB activation was suppressed in IRAP−/− mice (Figure 6). As NFκB activity was attenuated, the expression levels of inflammation markers such as MCP-1 was concomitantly suppressed in the sections from IRAP−/− mice.
We have tested the importance of the AngIV-AT4R-PAI-1 axis in fibrinolysis and thrombosis by comparing IRAP−/− mice with WT controls. In ECs from IRAP−/− mice, PAI-1 mRNA induction was blunted after AngII and AngIV treatment when compared to WT mice. We demonstrated that the genetic ablation of AT4R attenuated thrombosis in acute and chronic thrombosis mouse models.
PAI-1, a member of serine-protease inhibitors (serpins), is the important inhibitor of plasma fibrinolytic activity as well as α-2 plasmin inhibitor and serves as a pseudosubstrate for PAs.15–18,28–30 Although PAI-1 induction was observed in ECs from each type of mice in response to either AngII or AngIV, the dose-dependency, which was obvious in ECs from WT mice, disappeared in ECs from IRAP−/− mice. The ablation of AT4R reduced the susceptibility to both AngII and AngIV; however, PAI-1 expression was not completely abolished by the lack of the AT4R gene. This may suggest an interaction between AT1R and AT4R in PAI-1 expression.31 In contrast, when we examined the active PAI-1 antigen levels in the PPP after LPS treatment, the PAI-1 levels of IRAP−/− mice were markedly suppressed compared with those of WT mice. As PAI-1 antigen levels increased, active t-PA levels increased concomitantly by 4.1 fold. However, the difference of increases between mice was not observed. These findings suggest that the difference in the process of clot lysis between mice may be due to the magnitude of PAI-1 induction in response to stimuli and the abundance of the active form of PAI-1. In support of this, the degree of renal glomerular fibrin deposition was also suppressed in IRAP−/− mice. In a previous report, PAI-1–deficient mice showed lower active PAI-1 antigen levels in plasma after LPS injection and diminished glomerular fibrin deposition.20,30 In IRAP−/− mice, PAI-1 induction is impaired, and these results are similar to those of PAI-1–deficient mice.32
In the present study, we used a newly developed model using combined treatments of murine carotid artery ligation and perivascular cuff placement.23 This model is characterized by chronic blood flow cessation and EC injury limited within an intracuff lesion, which allowed us to quantitate thrombus formation in arteries. Sections from the intracuff lesion from T-686–treated mice and IRAP−/− mice showed reduced thrombosis and the residual thrombus area was less than 20% in the cross-sectional area 7 days after surgery. In contrast, in sections from WT mice, the lumen was occluded as early as 7 days after surgery (Figure 5) and occupied with protruded myofibroblasts and rich ECMs at 28 days. At a remote period, the carotid arteries of T-686–treated mice and IRAP−/− mice exhibited lumen narrowing and thickening of the intima–media and adventitia without thrombus occlusion. This demonstrates a negative remodeling phenomenon (hypertrophic vessel narrowing) after arterial injury, which is another clinical issue after coronary intervention. Based on these findings over the 28-day observation period, we considered that although IRAP−/− mice showed decreased thrombosis and delayed restructuring after arterial injury, the reparative process in response to flow cessation and EC injury was similar to control mice. Also, pharmacological blockade of PAI-1 activity prolonged the mean time to occlusion induced by FeCl3 and inhibited thrombus formation in WT mice similar to those observed in IRAP−/− mice, suggesting that reduced occlusive thrombosis in IRAP−/− mice was at least, in part, attributable to suppressed induction of PAI-1.
Regarding NFκB activation and PAI-1 induction in the injured arteries, although the administration of T-686 could inhibit thrombus formation in the acute phase, T-686 failed to suppress NFκB activation and inflammatory responses. In contrast, both NFκB activation and inflammation were suppressed in the arteries of IRAP−/− mice.31 In a remote period, sections from T-686–treated mice demonstrated an intermediate phenotype between those of WT and IRAP−/− mice for luminal patency, hyperproliferation of myofibroblasts, overproduction of ECMs, and neointimal formation. These findings strongly suggest that the AngIV-AT4R pathway could regulate both thrombus formation through PAI-1 induction and inflammation through NFκB activation and leukocyte infiltration. To support this, it has been demonstrated that endosomal peptide trimming by IRAP is essential for MHC class 1 cross-presentation.33 This may suggest that the control of IRAP activity leads to suppression of leukocyte infiltration and inflammatory responses as shown in our thrombosis models. We hypothesized 3 AT4R mediated mechanisms to explain the in vivo biological effects: (1) AngIV binding causes an accumulation of different bioactive peptides by preventing their degradation by IRAP; (2) AngIV binding to IRAP activates certain intracellular signaling pathways; and (3) AngIV binding to IRAP modulates peptide trimming or proteasomal degradation, thereby regulating the physiological cellular processes.33,34 Further investigations are required to clarify the pivotal role of IRAP in immune response and inflammation.
In conclusion, we have demonstrated that the genetic ablation of IRAP/AT4R attenuated hypofibrinolysis and inhibited thrombosis after arterial injury. These findings may raise awareness of the importance of the AngIV-AT4R axis in the pathogenesis of cardiovascular diseases, and thus the concept of protection distal to AngII and its receptors may open new avenues for therapeutic intervention in patients with cardiovascular disease.
The animal study was performed mainly at the Institute for Laboratory Animal Research, Nagoya University.
Sources of Funding
This study was partially funded by a grant from the Mitsubishi Pharma Research Foundation (Y.N., M.I., and T. Murohara) and a grant for investigating clinical vascular function from the Kimura Memorial Heart Foundation (Y.N.).
Received September 2, 2008; revision accepted September 2, 2009.
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