Journal of Thrombosis and Haemostasis, 8: 808–816
DOI: 10.1111/j.1538-7836.2010.03751.x
ORIGINAL ARTICLE
Pulmonary hypertension is ameliorated in mice deficient in thrombin-activatable fibrinolysis inhibitor L. QIN,* C. N. D’ALESSANDRO-GABAZZA, S. AOKI,* P. GIL-BERNABE,à Y. YANO,§ T. TAKAGI, D. BOVEDA-RUIZ,* A. Y. RAMIREZ MARMOL,* V. T. SAN MARTIN MONTENEGRO,* M. TODA,* Y . M I Y A K E , * O . T A G U C H I , Y . T A K E I , à J . M O R S E R * , – and E . C . G A B A Z Z A * Departments of *Immunology, Pulmonary and Critical Care Medicine, àGastroenterology and Hepatology, §Metabolism and Diabetes, Mie University School of Medicine, Edobashi, Tsu city, Japan; and –Division of Hematology, School of Medicine, Stanford University, Stanford, CA, USA
To cite this article: Qin L, D’Alessandro-Gabazza CN, Aoki S, Gil-Bernabe P, Yano Y, Takagi T, Boveda-Ruiz D, Ramirez Marmol AY, San Martin Montenegro VT, Toda M, Miyake Y, Taguchi O, Takei Y, Morser J, Gabazza EC. Pulmonary hypertension is ameliorated in mice deficient in thrombin-activatable fibrinolysis inhibitor. J Thromb Haemost 2010; 8: 808–16.
Summary. Background: The fibrinolytic system has been implicated in the pathogenesis of pulmonary hypertension (PH). Thrombin-activatable fibrinolysis inhibitor (TAFI) inhibits fibrinolysis and therefore its absence would be expected to increase fibrinolysis and ameliorate PH. Objective: The objective of the present study was to evaluate the effect of TAFI deficiency on pulmonary hypertension in the mouse. Methods and results: PH was induced in C57/Bl6 wild-type (WT) or TAFI-deficient (KO) mice by weekly subcutaneous treatment with 600 mg kg)1 monocrotaline (MCT) for 8 weeks. PH was inferred from right heart hypertrophy measured using the ratio of right ventricle-to-left ventricle-plus-septum weight [RV/ (LV+S)]. Pulmonary vascular remodeling was analyzed by morphometry. TAFI-deficient MCT-treated and wild-type MCT-treated mice suffered similar weight loss. TAFI-deficient MCT-treated mice had reduced levels of total protein and tumor necrosis factor-alpha (TNF-a), interleukin-6 (IL-6), transforming growth factor-b (TGF-b) and monocyte chemoattractant protein-1 (MCP-1) in bronchial alveolar lavage compared with wild-type MCT-treated mice. The ratio of RV to (LV+S) weight was significantly higher in WT/MCT than in KO/MCT mice. The pulmonary artery wall area and vascular stenosis were both greater in MCT-treated WT mice compared with MCT-treated TAFI-deficient mice. Conclusions: TAFIdeficient MCT-treated mice had less pulmonary hypertension, vascular remodeling and reduced levels of cytokines compared with MCT-treated WT animals, possibly as a result of reduced coagulation activation. Correspondence: Esteban Cesar Gabazza, Department of Immunology, Mie University School of Medicine, Edobashi 2-174, Tsu city, Mie 514-8507, Japan. Tel.: +81 59 232 1111; fax: +81 59 331 5225. E-mail:
[email protected] Received 15 October 2009, accepted 7 January 2010
Keywords: coagulation, fibrinolysis, monocrotaline, vascular remodeling.
Introduction Pulmonary hypertension (PH) is diagnosed when the mean pulmonary arterial pressure is greater than 25 mmHg at rest or greater than 30 mmHg on exertion [1]. The cause of PH may be idiopathic (primary PH) or secondary to pulmonary disorders such as pulmonary thrombo-embolic disease, left heart dysfunction, chronic obstructive pulmonary diseases and collagen vascular-associated lung diseases. PH is generally characterized by a progressive increase in pulmonary artery pressure and pulmonary vascular resistance that may result in right heart failure and death [1]. The initial lesion in all PH-associated lung disorders is endothelial injury within the pulmonary vasculature [2]. Several factors including procoagulant and anticoagulant proteins, inflammatory cytokines, growth factors and adhesion molecules have been implicated in the pathogenesis of lung vascular injury [3,4]. A consequence of endothelial injury is activation of the coagulation system, which if excessive, may lead to abnormal deposition of fibrin and extracellular matrix in lung tissue, intravascular thrombosis and remodeling of pulmonary vessels [3]. Decreased anticoagulant plus decreased profibrinolytic activity of the injured lung vascular endothelium is believed to promote hypercoagulability and fibrin deposition in the lungs of individuals with PH [4]. The tissue deposition of fibrin is regulated by the fibrinolytic system [5]. Fibrin degradation is catalyzed by plasmin which is generated from plasminogen by tissue plasminogen activator or urokinase cleavage. The rate at which plasmin is generated and thus the rate of fibrinolysis is determined by the level of C-terminal lysines on fibrin [6]. Activated thrombin-activatable fibrinolysis inhibitor (TAFIa), a circulating plasma glycoprotein synthesized by the liver, 2010 International Society on Thrombosis and Haemostasis
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removes those C-terminal lysines, thereby reducing the rate of fibrinolysis [6,7]. In the present study, we hypothesized that TAFIa exacerbates PH by reducing the rate of fibrin clearance. We tested this hypothesis in TAFI-deficient mice by inducing PH with monocrotaline (MCT) and comparing the outcome to that in control mice also given MCT. We found that mice deficient in TAFI are protected against the development of PH, pulmonary vascular remodeling and the concomitant increase in inflammatory cytokines. Materials and methods Animals
TAFI knockout mice were generated and characterized as previously described [8]. Genotyping of mice was confirmed by polymerase chain reaction using the same conditions and primers previously described [8]. C57Bl/6 wild-type littermates with the same genetic background as the TAFI knockout mice were used as controls. All animals were housed in a pathogenfree environment with air, food and water containing no foreign organisms. Mice were kept on a constant 12:12-h light/ dark cycle in a temperature- and humidity-controlled room and were given water and standard mouse food ad libitum. The Mie University Committee on animal investigation approved the experimental protocol, and the experiments were performed according to the guidelines for animal experiments of the National Institute of Health. Mouse model of PH
PH was induced by MCT treatment of male 10-week-old wild-type (WT/MCT, n = 6) or TAFI knockout (KO/ MCT, n = 9) mice by 8-weekly subcutaneous (s.c.) MCT injections as described previously [9]. WT (WT/SAL, n = 4) and TAFI knockout (KO/SAL, n = 6) control mice were given sterile saline alone following the same schedule and route of administration as MCT. This animal experiment was done three times with the same design to assure reproducibility. The mice were weighed weekly. At the end of the 8-week dosing period, sampling of bronchoalveolar lavage fluid (BALF) was performed under deep anesthesia induced by intraperitoneal pentobarbital, blood was obtained by cardiac puncture into citrate for preparation of plasma and the heart and lungs were harvested for histological analyses [10]. Determination of cardiac hypertrophy
PH was assessed by the presence of right heart hypertrophy as described [9]. To determine right heart hypertrophy, the ratio of right ventricle to left ventricle plus septum [RV/(LV+S)] weight was calculated after dissection apart, and separate weighing of, the right ventricle and the left ventricle plus septum. 2010 International Society on Thrombosis and Haemostasis
Morphometric analysis of pulmonary arteries
Under deep anesthesia mice were thoracotomized and the lungs were removed after flushing the pulmonary circulation with heparinized saline. The lung was then perfused with 10% neutral buffered formalin and fixed in formalin for 24 h before embedding in paraffin. Lung sections (5-lm thick) were stained with Masson’s trichrome (Sigma-Aldrich, St Louis, MO, USA) and hematoxylin & eosin. Morphological analysis of the pulmonary arteries was performed using an Olympus BX50 microscope with a plan objective, combined with an Olympus DP70 digital camera (Tokyo, Japan) and the WinROOF image processing software (Mitani Corp., Fukui, Japan) for Windows as previously described [9]. Images of pulmonary arterial vessels (an average of 10 arteries per lung) with a diameter of < 100 lm were taken of mice from each group by an investigator blinded to the experimental group. The total vascular wall and lumen areas were then measured by a second blinded investigator. The total vessel area was considered to be the lumen area plus the wall area bounded by the outer smooth muscle cell layer of the vascular media, and the lumen area was defined as the area delineated by the inner boundary of the vessel intimal layer. The total vessel area minus the lumen area was taken as the vascular wall thickness. The percentage of vascular narrowing was calculated using the following formula: (total vessel area – lumen area)/total vessel area · 100. Biochemical analysis
The concentration of total protein in BALF was measured using a dye-binding assay (BCATM protein assay kit; Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. The total number of cells in BALF was measured using a nucleocounter from ChemoMetec (Allerød, Denmark). For differential cell counting BALF was centrifuged using a cytospin and the cells were stained with May–Grunwald– Giemsa (Merck, Darmstadt, Germany). The concentrations of cytokines in BALF were measured using commercial immunoassay kits specific for mouse cytokines. The immunoassay kits for measuring interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-a (TNF-a) and transforming growth factor-b1 (TGF-b1) were purchased from BD Biosciences Pharmingen (San Diego, CA, USA). All cytokines were measured according to the manufacturer’s instructions. Platelet-derived growth factor (PDGF) was measured using anti-PDGF (Genzyme, Boston, MA, USA) and biotin-labeled anti-PDGF antibodies. Thrombin–antithrombin complexes (TAT; Cedarlane Laboratories, Burlington, ON, Canada) and TAFI (Affinity Biologicals, Ancaster, ON, Canada) were measured using commercially available enzyme immunoassay kits according to the manufacturer’s instructions. The level of plasmin activity was measured spectrophotometrically using the synthetic chromogenic substrate S-2251 (Chromogenix, Molndal, Sweden). The level of D-dimer was measured using antibodies from Fitzgerald
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Industries International (Concord, MA, USA). The concentration of fibrin(ogen) was measured using rabbit anti-fibrinogen antibody and biotin-labeled anti-fibrinogen. The antibodies used to determine fibrin(ogen) do not discriminate between fibrin and fibrinogen. Alanine transaminase (ALT) and asparatate transaminase (AST) in plasma were measured by enzyme assay.
increased in WT/MCT compared with WT/SAL mice, and also in KO/MCT compared with KO/SAL mice. No significant difference was found between WT/SAL and KO/SAL mice. The ratio of RV to (LV+S) weight was significantly higher in WT/MCT than in KO/MCT mice (Fig. 2) showing that right ventricular hypertrophy had occurred in WT animals with PH but that the TAFI-deficient mice were protected from PH.
Statistical analysis
Data were expressed as the mean ± standard error of the means (SEM). The statistical difference between variables was calculated by analysis of variance with post hoc analysis using Fisher’s predicted least significant difference test. Statistical analyses were performed using the STATVIEW 4.5 package for Macintosh (Abacus Concepts, Berkeley, CA, USA). P < 0.05 was considered as significant.
Vascular wall remodeling
During PH pulmonary vessel remodeling occurs. Vascular wall remodeling was investigated in WT and TAFI KO mice with PH induced by MCT treatment. Total wall area and vascular narrowing in vessels of < 100 lm were both significantly increased in WT/MCT as compared with the other three groups, which did not differ significantly from each other (Fig. 3A–C).
Results Vascular permeability and lung injury Weight loss in PH
Mice treated with MCT lost weight during the experiment, irrespective of their genotype (Fig. 1). There was no significant difference in weight loss between the WT/MCT and KO/MCT mice. Control mice of both genotypes had similar weight gains that were normal over the same time period (Fig. 1). To investigate if there was general liver toxicity in mice treated with MCT, plasma ALT and AST were measured but no significant differences were found between control animals and those treated with MCT (data not shown).
During the course of PH, vascular permeability is increased allowing leakage of proteins and cells. Vascular permeability, measured as protein in BALF, was found to be significantly higher in the WT/MCT group than the WT/SAL group. The KO/SAL mice were not different from the WT/SAL mice, but the level of total protein in BALF was significantly reduced in KO/MCT mice compared with WT/MCT mice showing that TAFI deficiency prevents much of the increase in vascular permeability (Fig. 4A). The total cell count in BALF was significantly increased in KO/MCT mice compared with the WT/MCT and TAFI/SAL groups but no significant difference
PH and cardiac hypertrophy P = 0.5
Right ventricular hypertrophy is characteristic of PH so we determined it in WT and TAFI KO mice that had PH induced by MCT. The ratio of RV to (LV+S) weight was significantly
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Fig. 1. Changes in body weight. Mice in the WT/SAL and KO/SAL groups significantly gained weight with no difference between these two groups. However, both WT/MCT and KO/MCT markedly lost weight, also without a significant difference between the groups. The representative results from one of three separate experiments are shown. WT/SAL: wild type/saline; WT/MCT: wild type/monocrotaline; KO/SAL: TAFI knockout/saline; KO/MCT: TAFI knockout/monocrotaline.
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Fig. 2. Right ventricular hypertrophy. The RV/(LV+S) ratio was significantly different between WT/MCT and WT/SAL mice but not between WT/SAL and KO/SAL mice. There was also a significant difference between KO/SAL and KO/MCT and between WT/MCT and KO/MCT mice. The representative results from one of three separate experiments are shown. Bars represent the means ± SEM. Group designation as for Fig. 1. 2010 International Society on Thrombosis and Haemostasis
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Fig. 3. Pulmonary artery wall area and stenosis. (A) Masson’s trichrome staining showing stenosis of pulmonary vascular arteries. (B) Vascular wall area was increased in WT/MCT mice compared with WT/SAL and KO/MCT. There was no significant difference between KO/SAL and KO/MCT or between WT/SAL and KO/SAL mice. (C) Stenosis in WT/MCT was also greater than in WT/SAL and KO/MCT mice. No significant difference was found between KO/SAL and KO/MCT or between WT/SAL and KO/SAL mice. The representative results from one of three separate experiments are shown. Bars represent the means ± SEM. Group designation as for Fig. 1.
was found between the WT/SAL and WT/MCT groups (Fig. 4B). The number of macrophages was significantly decreased in KO/MCT mice compared with KO/SAL and WT/MCT mice but no difference was found between WT/ MCT and WT/SAL mice. The number of lymphocytes and neutrophils was significantly increased in WT/MCT mice compared with WT/SAL mice but there was no difference between KO/MCT and KO/SAL mice. The number of neutrophils was significantly enhanced in WT/MCT mice compared with KO/MCT mice (Fig. 4B). The total number of cells, macrophages, lymphocytes and neutrophils tended to be increased in KO/SAL compared with WT/SAL mice but was not significantly different. 2010 International Society on Thrombosis and Haemostasis
Alterations in coagulation and fibrinolysis
The concentration of thrombin–antithrombin III complex (TAT) can be used as a surrogate for activity of the coagulation cascade. The BALF concentration of TAT was significantly elevated in WT/MCT compared with WT/SAL and KO/MCT mice; there was no difference between the KO/SAL and KO/ MCT groups (Fig. 5A). The concentration of TAFI antigen was significantly increased in WT/MCT mice compared with WT/SAL mice, and as expected no TAFI antigen was detected in either the KO/SAL or KO/MCT groups (Fig. 5B). The concentrations of fibrin(ogen) (Fig. 5C) and D-dimer (Fig. 5D) in BALF were significantly higher in WT/MCT when
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Fig. 4. Lung inflammation. (A) Bronchoalveolar lavage fluid (BALF) total protein was significantly different between WT/SAL and WT/MCT and between WT/MCT and KO/MCT mice but no difference was found between KO/SAL and KO/MCT mice. (B) The total cell count was significantly different between the KO/SAL and KO/MCT groups and between the WT/MCT and KO/MCT groups. The number of macrophages in BALF was significantly different between KO/SAL and KO/MCT but not between WT/SAL and WT/MCT; the number of lymphocytes and neutrophils in BALF was significantly different between WT/SAL and WT/MCT mice but not between KO/SAL and KO/MCT mice. The number of neutrophils was significantly different between WT/MCT and KO/MCT. The representative results from one of three separate experiments are shown. Bars represent mean ± SEM. Group designation as for Fig. 1.
compared with WT/SAL and KO/MCT mice, but no significant differences were observed between KO/MCT and KO/ SAL mice. Plasmin activity in BALF tended to be higher in both TAFI-deficient groups (KO/SAL, KO/MCT) but did not reach significance (Fig. 5E). Because the level of fibrin degradation products (D-dimer level) depends on both the amount of coagulation as well as the plasmin activity, the BALF level of D-dimer was corrected for that of TAT; the D-dimer/TAT ratio was 6-fold higher in the KO/MCT mice than in the WT/ MCT mice (Fig. 5F). The level of plasmin activity in relation to coagulation was also compared between WT/MCT and KO/ MCT mice; the plasmin activity/TAT ratio in BALF was significantly increased in KO/MCT compared with WT/MCT mice (Fig. 5F). Taken together these data show that the TAFI concentration is increased in WT mice with MCT-induced PH and that activation of coagulation is higher and fibrinolysis is
Growth factors in the lung
As there was vascular wall remodeling in WT/MCT mice, we investigated whether there were increased levels of growth factors that could promote vascular smooth muscle cell growth. PDGF in BALF was significantly elevated in WT/MCT mice compared with the three other groups, which did not differ significantly from each other (Fig. 7A). Similarly, TGF1-b in BALF was significantly elevated in WT/MCT mice as compared with the other three groups, which again did not differ significantly from each other (Fig. 7B). No significant differences in the plasma levels of PDGF or TGF-b1 were detected among the different groups of animals (data not shown). Thus there were increased levels of growth factors in the lungs of WT/MCT mice but not in the lungs of KO/MCT mice. Discussion The present study shows that mice deficient in TAFI have significantly less PH induced by MCT treatment, vascular remodeling in the lung, lung injury and significantly decreased expression of inflammatory cytokines and growth factors as compared with WT mice, implicating TAFI in the pathogenesis of PH. The coagulation system in PH
The role of the coagulation system in the pathological process that causes PH has been previously documented in human studies as well as in animal models of the disease [1,4]. The high frequency of microvessel thrombosis in the lung, the elevated circulating level of markers of clotting activation in PH patients and the improvement in survival of PH patients treated with anticoagulants show the importance of hypercoagulability in the pathogenesis of PH [5,11,12]. Rats with PH have increased fibrin deposition and microthrombi in the lungs and increased plasma levels of TAT have been reported 2010 International Society on Thrombosis and Haemostasis
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Fig. 5. Alteration of coagulation and fibrinolytic systems. (A) Thrombin–antithrombin III complex (TAT) levels in bronchoalveolar lavage fluid (BALF) were significantly different between WT/SAL and WT/MCT and between WT/MCT and KO/MCT but not between KO/SAL and KO/MCT mice. (B) The BALF levels of thrombin-activatable fibrinolysis inhibitor (TAFI) antigen were significantly different between WT/SAL and WT/MCT. (C) Fibrin levels in BALF were significantly different between WT/SAL and WT/MCT and between WT/MCT and KO/MCT but not between KO/SAL and KO/ MCT. (D) The BALF level of D-dimer (D-D) in WT/MCT was significantly different from WT/SAL and KO/MCT. (E) Plasmin activity (PA) was not significantly different among the groups. (F) The DD/TAT ratio was 6-fold higher in KO/MCT mice than in WT/MCT mice, and the plasmin activity/ TAT ratio in BALF was significantly different between WT/MCT and KO/MCT mice. The representative results from one of three separate experiments are shown. Bars represent the means ± SEM. Group designation as for Fig. 1.
in mice with MTC-induced PH [5]. The precise mechanism by which coagulation is activated in PH is unclear but vascular endothelial injury is believed to be important [2]. Vascular injury causes not only a reduction in the endothelial expression of anticoagulants (e.g. thrombomodulin) and pro-fibrinolytic factors such as tissue plasminogen activator but also an increase in the endothelial expression of procoagulant (e.g. tissue factor) and antifibrinolytic (e.g. plasminogen activator inhibitor-1) factors, leading to a net imbalance between the coagulation and fibrinolysis systems [13,14]. In support of this theory, the present study showed that WT mice with MCTinduced PH have significantly increased lung levels of TAT compared with control animals indicating that there is activation of coagulation in these mice. On the other hand, mice deficient in TAFI have significantly decreased PH values along with a significantly lower concentration of TAT in the lungs, suggesting that TAFI plays a role in the mechanism of hypercoagulability in PH. The fact that TAFI-deficient mice treated with MCT have high ratios of plasmin activity/TAT and D-dimer/TAT in the lungs compared with their WT type 2010 International Society on Thrombosis and Haemostasis
counterparts suggests that TAFI may also promote PH development by reducing fibrinolysis. Inflammation in PH
Inflammatory mediators including cytokines and chemokines also play important roles in the pathogenesis of PH in patients and in animal models [15,16]. Here, induction of pulmonary hypertension with MCT was associated with more lung vascular inflammation in WT mice than in TAFI-deficient mice, as shown by the significant difference in the levels of total protein and the number of lymphocytes and neutrophils in BALF between WT/SAL and WT/MCT mice and by the lack of difference between KO/SAL and KO/MCT mice. The exaggerated inflammatory response in WT mice compared with TAFI-deficient mice may be explained by the high BALF concentrations of the proinflammatory mediators TNF-a, IL-6 and MCP-1 in WT mice. TNF-a promotes inflammation by stimulating leukocyte recruitment, oxidant stress and the expression of several adhesion molecules, IL-6 initiates the
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secretion of liver-derived acute-phase proteins and the activation of several types of immune cells and MCP-1 triggers inflammation by stimulating the activation and migration of monocytes, macrophages and lymphocytes at sites of tissue injury [17]. The beneficial effect of anti-cytokine therapy in PH induced in experimental animal models supports the biological relevance of inflammatory cytokines in the pathogenesis of PH [11]. Importantly, the present study shows that in TAFIdeficient mice, the MCT-induced increase in inflammatory cytokines (TNF-a, IL-6 and MCP-1) and growth factors
(PDGF and TGF-b1) in WT mice is completely prevented, and along with it the development of pulmonary vascular remodeling and much of the associated RV hypertrophy. It should be noted that, compared with control animals, the number of macrophages in BALF was significantly decreased in TAFIdeficient mice treated with MCT but not in MCT-treated WT mice; the mechanism is unclear but it may be that macrophages from TAFI knockout mice are more susceptible than those from WT of mice to the toxic effects of MCT [18,19]. Reduced recruitment of monocytes from peripheral blood is unlikely 2010 International Society on Thrombosis and Haemostasis
TAFI in pulmonary hypertension 815
because both lymphocyte and neutrophil numbers were increased. Interaction between the coagulation system and inflammation in PH
Fibrin and its derivatives have been previously reported to exacerbate inflammation by stimulating the migration of neutrophils, the expression of leukocyte adhesion molecules and chemokines from endothelial cells, and proinflammatory cytokines from monocytic cells, by increasing vascular permeability and by stimulating angiogenesis [20–25]. Proinflammatory cytokines (e.g. TNF-a) may, in turn, further promote coagulation activation and subsequent fibrin deposition by increasing the expression of tissue factor from surrounding cells. Thus, reduced fibrin formation and faster clearance could be the explanation for the reduced inflammatory reaction and coagulation activation in lungs from TAFI-deficient mice. TAFIa, besides its fibrin-mediated proinflammatory activity, may also have anti-inflammatory effects as a result of its ability to regulate inflammatory mediators including C5a, C3a and bradykinin [26]. For example, TAFI-deficient mice with obstructive nephropathy have enhanced renal interstitial fibrosis and significantly increased plasma concentration of proinflammatory cytokines (IL-1b, IL-6) and those with sepsis have increased peritoneal neutrophil recruitment and high plasma concentration of TNF-a and IL-6 compared with their wild-type counterparts [27,28]. These previous observations suggest that the inflammatory response in the TAFI knockout mice depends on the experimental disease model. Cardio-pulmonary structural responses to MCT in WT and TAFI mice
The hallmark of chronic PH is the presence of vascular remodeling in the pulmonary vasculature. In the present study extensive vascular remodeling as judged by vascular stenosis and increased vessel area in mice treated with MTC was observed confirming earlier reports [29,30]. Both the vessel wall area and the degree of vascular stenosis were significantly greater in WT mice compared with TAFI-deficient mice. Importantly, these parameters were not significantly different between TAFI-deficient mice treated with MCT and those treated with saline. Thus, TAFI deficiency prevents pulmonary vascular remodeling in response to MTC. Tissue remodeling may result from disruption of the balance between processes of synthesis and degradation that is regulated by several growth factors such as TGF-b1 and PDGF [31,32]. PDGF favors vascular remodeling by promoting the proliferation of smooth muscle cells and fibroblasts and TGF-b1 by stimulating the secretion of extracellular matrix proteins including collagens [30–32]. Inflammatory mediators including TNF-a, MCP-1 and thrombin, one of the effector enzymes of the coagulation system, up-regulate the expression of growth factors including TGF-b1 and PDGF [17]. In our study, the 2010 International Society on Thrombosis and Haemostasis
BALF concentrations of TAT (a marker of thrombin generation), total protein (a marker of lung inflammation), the inflammatory cytokines TNF-a and IL-6, and the proinflammatory chemokine MCP-1 were all significantly lower in MCT-treated TAFI-deficient mice than in MCT-treated WT mice. Thus, the relative protection of TAFI-deficient mice from vascular remodeling and PH may be because of an attenuated inflammatory response with subsequently reduced expression of factors involved in the process of vascular remodeling. In support of this theory, the BALF concentrations of PDGF and TGF-b1 were significantly decreased in MTC-treated TAFI-deficient mice as compared with their wild-type counterparts. In summary, mice deficient in TAFI are protected against MCT-induced increases in PH, vascular remodeling and cytokines found in MCT-treated wild-type animals, possibly as a result of decreased coagulation activation, more fibrinolysis and an associated reduction in inflammatory response. Addendum L. Qin, C. N. D’Alessandro-Gabazza, P. Gil-Bernabe and D. Boveda prepared the mouse model of PH. E. C. Gabazza wrote the first draft of the manuscript and contributed intellectually for the completion of the study. S. Aoki. M. Toda, T. Takagi and V.T. San Martin measured cytokines and growth factors in BALF and plasma. J. Morser made corrections to the manuscript and important intellectual contributions. A.Y. Ramirez, Y. Yano and Y. Miyake carried out tissue staining. Y. Takei and O. Taguchi contributed to the intellectual content of the manuscript. Acknowledgements This research was supported in part by the Ministry of Education, Culture, Sports, Science, and Technology of Japan (grants-in-aid 15591053, 16590745, 17590788) and the Mie Medical Research Foundation (grants-in-aid 2002, 2004). J. Morser was supported by a scholarship from the Japanese Society for Promotion of Science. Disclosure of Conflict of Interests The authors state that they have no conflict of interest. References 1 Farber HW, Loscalzo J. Pulmonary arterial hypertension. N Engl J Med 2004; 351: 1655–65, 351/16/1655 [pii]; doi: 10.1056/NEJMra035488. 2 Budhiraja R, Tuder RM, Hassoun PM. Endothelial dysfunction in pulmonary hypertension. Circulation 2004; 109: 159–65; doi: 10.1161/ 01.CIR.0000102381.57477.50109/2/159 [pii]. 3 Welsh CH, Hassell KL, Badesch DB, Kressin DC, Marlar RA. Coagulation and fibrinolytic profiles in patients with severe pulmonary hypertension. Chest 1996; 110: 710–7. 4 Hassell KL. Altered hemostasis in pulmonary hypertension. Blood Coagul Fibrinolysis 1998; 9: 107–17.
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2010 International Society on Thrombosis and Haemostasis
