Absence of IFN-γ accelerates thrombus resolution through enhanced MMP-9 and VEGF expression in mice

Deep vein thrombosis (DVT) is a major cause of pulmonary thromboembolism, a leading cause of death in individuals with DVT. Several lines of evidence indicate proinflammatory cytokines such as TNF-α are involved in thrombus formation and resolution, but the roles of IFN-γ remain unclear. To address this issue, we performed ligation of the inferior vena cava to induce DVT in WT and IFN-γ–deficient (Ifng^–/–) mice. In WT mice, intrathrombotic IFN-γ levels were elevated progressively as the postligation interval was extended. Thrombus size was substantially smaller at 10 and 14 days in Ifng^–/– mice than in WT mice. Intrathrombotic collagen content was remarkably reduced at more than 10 days after the ligation in Ifng^–/– mice compared with WT mice. The expression and activity of MMP-9, but not MMP-2, was higher at the late phase in Ifng^–/– mice than in WT mice. Moreover, intrathrombotic recanalization was increased in Ifng^–/– mice, with enhanced Vegf gene expression, compared with that in WT mice. Activation of the IFN-γ/Stat1 signal pathway suppressed PMA-induced Mmp9 and Vegf gene expression in peritoneal macrophages. Furthermore, administration of anti–IFN-γ mAbs accelerated thrombus resolution in WT mice. Collectively, these findings indicate that IFN-γ can have detrimental roles in thrombus resolution and may be a good molecular target for the acceleration of thrombus resolution in individuals with DVT.

Deep vein thrombosis (DVT) is multifactorial and often results from a combination of risk factors such as genetic conditions, obesity, drugs, pregnancy, aging, trauma, and malignancy (1–3). DVT is frequently complicated with severe morbidity, leading sometimes to mortality (4–6). Indeed, the American Heart Association reported that approximately 2,000,000 people annually suffered from DVT and that 200,000 of these died from pulmonary thromboembolism, one of the severe complications arising from DVT, in the United States alone. Thus, in order to minimize the mortality of DVT-related pulmonary thromboembolism, accurate diagnosis and prompt therapy for DVT are necessary (5). Standard treatments for DVT aim to prevent death from pulmonary embolism, propagation of the thrombus, and recurrence of venous thromboembolism. At present, anticoagulation with either unfractionated or low–molecular weight heparin, or warfarin is used to inhibit thrombus extension, to allow natural resolution, and to slowly reduce and organize the existing thrombus (4). However, anticoagulation cannot accelerate natural thrombus resolution and reciprocally increases the risk of bleeding. Moreover, any treatment modalities have not been developed to directly lyse thrombus until the present.

The pathogenesis of venous thrombosis involves 3 factors, referred to as Virchow’s triad; endothelial damages, stasis or turbulence of blood flow, and blood hypercoagulability. Damage to the vessel wall prevents the endothelium from inhibiting coagulation and initiating local fibrinolysis. Venous stasis inhibits the clearance and dilution of activated coagulation factors. Finally, congenital or acquired thrombophilia promotes coagulation. Several lines of evidence indicate the involvement of proinflammatory cytokines and chemokines in the thrombus formation and resolution (7–10). Indeed, TNF-α and IL-1β can potently activate endothelial cells, increase the expression of adhesion molecules by endothelial cells, and promote thrombosis (11–15). Moreover, TNF-α and IL-1β can induce the expression of chemokines, which can activate leukocytes to accelerate thrombus resolution and intrathrombotic neovascularization (16–18).

IFN-γ exerts pleiotropic effects on NK cells and macrophages, including enhancement of their antiviral and bactericidal activities and upregulation of MHC class II expression on macrophages (19). Moreover, accumulating evidence implicates IFN-γ as a major mediator in various types of disease models (20–25). Furthermore, we previously determined that the IFN-γ/Stat1 signal pathway played an important role in skin wound healing by regulating VEGF expression and the TGF-β signaling pathway (23). The thrombus resolution process resembles wound healing, with the injury nidus being the clot, followed by leukocyte recruitment and eventually matrix accumulation. Indeed, we immunohistochemically detected IFN-γ protein in deep vein thrombi taken from autopsy cases (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI40782DS1). These observations prompted us to explore the pathophysiological roles of IFN-γ in the process of thrombus formation and resolution in a stasis-induced thrombosis model by the use of IFN-γ–deficient (Ifng^–/–) mice.

We demonstrated that thrombus resolution was accelerated in Ifng^–/– mice compared with WT mice without any differences in thrombosis formation. Moreover, the expression of an enzyme with collagenolytic activity, MMP-9, was enhanced at mRNA and enzymatic activity levels in Ifng^–/– mice compared with WT mice. Furthermore, intrathrombotic vascular channels were increased with enhanced VEGF expression in Ifng^–/– mice, compared with WT mice. These observations imply that IFN-γ plays a detrimental role in thrombus resolution by suppressing MMP-9 and VEGF expression.

Intrathrombotic IFN-γ expression after the inferior vena cava ligation (IVC). We examined intrathrombotic Ifng gene expression in WT mice at 3, 5, 10, and 14 days after the IVC ligation. IFN-γ mRNA was consistently detected in thrombi and its expression was elevated progressively as thrombi aged (Figure 1A). Likewise, IFN-γ proteins were detected in thrombi and increased progressively after the IVC ligation (Figure 1B). Moreover, double-color immunofluorescence analyses detected IFN-γ mainly in F4/80-positive macrophages (Figure 1C). These observations implied that IFN-γ was produced in thrombi mainly by infiltrating macrophages.

Figure 1

Intrathrombotic IFN-γ expression in WT mice after IVC ligation. (A) Ifnγ gene expression was examined by real-time RT-PCR as described in Methods. All values represent mean ± SEM (n = 6). **P < 0.01, versus day 3; ^##P < 0.01, versus day 10. (B) Intrathrombotic IFN-γ protein contents were determined at the indicated time intervals after IVC ligation as described in Methods. All values represent mean ± SEM (n = 6). **P < 0.01, versus day 5; *P < 0.05, versus day 5; ^#P < 0.05, versus day 10. (C) A double-color immunofluorescence analysis of IFN-γ–expressing cells in the thrombus. The samples were immunostained with the combination of anti-F4/80 mAbs and anti–IFN-γ pAbs as described in Methods. The fluorescent images were digitally merged in the right panel. Representative results from 6 independent experiments are shown here. Original magnification, ×400. Blue, nuclear staining by DAPI.

Enhanced thrombus resolution in the absence of IFN-γ. In order to explore the pathophysiological roles of IFN-γ in the development of stasis-induced venous thrombus, we compared thrombus formation between WT and Ifng^–/– mice. In WT mice, venous thrombi developed progressively until 5 days but remained similar in size until 10 days after the IVC ligation, decreasing thereafter (Figure 2A). In Ifng^–/– mice, thrombi started to reduce in size 10 days after the IVC ligation (Figure 2, A and B) and were smaller than those in WT mice 14 days after the IVC ligation. Because intrathrombotic collagen contents can reflect thrombus mass (18), we determined intrathrombotic collagen contents by the use of histopathological and biochemical methods. In Ifng^–/– mice, intrathrombotic collagen area was remarkably reduced compared with that in WT mice (Figure 2, C and D, and Supplemental Figure 2). Similarly, intrathrombotic contents of both collagen and hydroxyproline, a major component of collagen, were significantly diminished in Ifng^–/– mice later than 10 days after the IVC ligation, compared with WT mice (Figure 2, E and F). These observations implied that the absence of IFN-γ could accelerate the resolution of stasis-induced venous thrombi.

Figure 2

Stasis-induced deep vein thrombus in WT and Ifng^–/– mice. (A) Macroscopic appearance of intravenous thrombi in WT and Ifng^–/– mice 10 days after the IVC ligation. Representative results from 6 independent animals are shown here. (B) Thrombus weights of WT and Ifng^–/– mice at the indicated time intervals after IVC ligation. All values represent the mean ± SEM (n = 6). **P < 0.01, Ifng^–/– versus WT. (C and D) Histopathological analyses of venous thrombi obtained from WT and Ifng^–/– mice 10 days after IVC ligation. Thrombus samples were stained with H&E (C) or Masson trichrome solution (D). Representative results from 6 independent experiments are shown here. Original magnification, ×100. (E and F) Intrathrombotic contents of collagen and HP, a major component of collagen, of WT and Ifng^–/– mice at the indicated time intervals after IVC ligation. All values represent the mean ± SEM (n = 6). **P < 0.01, Ifng^–/– versus WT.

The absence of IFN-γ had no effects on intrathrombotic leukocyte recruitment and the expression of chemokines, tPA, uPA, and PAI. The potential contribution of intrathrombotic leukocytes to the resolution of venous thrombi (12–18) prompted us to investigate the effects of IFN-γ deficiency on intrathrombotic leukocyte recruitment. Myeloperoxidase (MPO)-positive neutrophils infiltrated thrombi in WT and Ifng^–/– mice to similar extents, reaching a maximal level at 1 day after the IVC ligation and decreasing gradually thereafter (Figure 3, A and C). Likewise, F4/80-positive macrophages infiltrated thrombi in WT and Ifng^–/– mice to similar extents, reaching a maximal level at 5 days after the IVC ligation and decreasing gradually thereafter (Figure 3, B and D). Similar tendencies were also observed on the infiltration of CD11b-positive cells (data not shown). In line with this, we further detected the gene expression of several chemokines including CXCL1/KC, CXCL2/MIP-2, CCL2/MCP-1, and CCL3/MIP-1α to similar extents in WT and Ifng^–/– mice (Supplemental Figure 3). Because the plasmin system is also essential for cell migration and subsequent thrombus resolution (26–28), we examined the intrathrombotic gene expression of tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1). We failed to detect any significant differences in the expression of these genes between WT and Ifng^–/– mice at any time point after IVC ligation (Figure 4). Collectively, the absence of IFN-γ had little, if any, effects on intrathrombotic expression of chemokines, tPA, uPA, and PAI-1, and subsequent leukocyte recruitment.

Figure 3

Enumeration of intrathrombotic leukocytes in WT and Ifng^–/– mice. (A and B) Immuno­histo­chemical analysis was performed using anti-MPO pAbs at day 1 (A) or anti-F4/80 mAbs at day 5 (B) in venous thrombus samples from WT and Ifng^–/– mice. Original magnification, ×400. Representative results from 6 independent experiments are shown here. The numbers of neutrophils (C) and macrophages (D) were determined as described in Methods. All values represent the mean ± SEM (n = 6 animals).

Figure 4

Intrathrombotic gene expression of tPA, uPA, and PAI-1 determined by real-time RT-PCR. All values represent the mean ± SEM (n = 6 animals).

Enhanced enzymatic activity of MMP-9 but not MMP-2 in Ifng^–/– mice. MMP-2 and MMP-9 have crucial roles in collagenolysis during thrombus resolution (18). Thus, we examined the intrathrombotic gene expression and the enzymatic activity of MMP-2 and MMP-9. MMP-2 mRNA and enzymatic activities were detected consistently in thrombi at similar levels in WT and Ifng^–/– mice (Figure 5, A, C, and D). Mmp9 gene expression was detected in thrombi of WT mice, reaching the maximal level at 5 days after the IVC ligation and declining thereafter (Figure 5B). In Ifng^–/– mice, MMP-9 mRNA was detected, to a similar extent, at 5 days, but its levels remained similar until 14 days after the IVC ligation (Figure 5B). Likewise, the enzymatic activities of MMP-9 remained at similar levels in Ifng^–/– mice until 14 days after the IVC ligation, whereas its activities decreased in WT mice at 10 days after the ligation (Figure 5, C and E). Consistently, later than 10 days after the IVC ligation, intrathrombotic MMP-9–positive cell numbers were higher in Ifng^–/– mice than in WT mice (Figure 6, A and B). Moreover, a double-color immunofluorescence analysis demonstrated that F4/80-positive macrophages were a main cellular source of MMP-9 (Figure 6C). Collectively, these observations would imply that IFN-γ might play a detrimental role in thrombus resolution by suppressing the expression of MMP-9 but not MMP-2 in macrophages present in the thrombus.

Figure 5

Intrathrombotic MMP-2 and MMP-9 expression. (A and B) Intrathrombotic gene expression for MMP-2 (A) and MMP-9 (B) in WT and Ifng^–/– mice was determined by real-time RT-PCR as described in Methods. All values represent the mean ± SEM (n = 6). **P < 0.01, Ifng^–/– versus WT. (C–E) Intrathrombotic MMP-2 and MMP-9 activities were measured by gelatin gel zymography as described in Methods. Representative gel images from 6 independent experiments are shown in C. Gel images were semiquantitatively analyzed; analysis is shown in D and E. All values represent the mean ± SEM (n = 6). **P < 0.01, Ifng^–/– versus WT.

Figure 6

Detection of intrathrombotic MMP-9–positive cells (A) Immunohistochemical analysis was performed using anti–MMP-9 pAbs in venous thrombus samples from WT and Ifng^–/– mice. Representative results from 6 independent experiments are shown here. Original magnification, ×400. (B) The numbers of MMP-9–positive cells were determined as described in Methods and are shown in B. All values represent the mean ± SEM (n = 6 animals). *P < 0.05; **P < 0.01, Ifng^–/– versus WT. (C) A double-color immunofluorescence analysis of MMP-9–positive cells in the thrombi. The samples were immunostained with the combination of anti-F4/80 mAbs and anti-MMP-9 pAbs, as described in Methods. Representative results from 6 independent experiments are shown here (day 10). Original magnification, ×400. Blue, nuclear staining by DAPI.

Enhanced intrathrombotic recanalization in Ifng^–/– mice. Intrathrombotic recanalization was presumed to be essential for thrombus resolution (29–31). The number of intrathrombotic vWF-positive channels was significantly increased at 10 and 14 days in Ifng^–/– mice compared with WT mice (Figure 7, A and B). Moreover, we examined blood flow within and around the thrombosed IVC by the use of laser Doppler imaging (18). At 5 days after IVC ligation, there were no significant differences in the blood flow of thrombosed IVC between WT and Ifng^–/– mice. However, at 10 and 14 days after IVC ligation, the blood flow was recovered in Ifng^–/– to larger extents than in WT mice (Figure 7C). Furthermore, intrathrombotic gene expression of VEGF, a potent angiogenic factor, was significantly enhanced in Ifng^–/– mice compared with WT mice (Figure 7D). A double-color immunofluorescence analysis demonstrated that F4/80-positive macrophages were a main cellular source of VEGF (Figure 7E). These observations implied that the lack of IFN-γ might increase intrathrombotic recanalization at least partly through enhanced Vegf gene expression in macrophages.

Figure 7

Evaluation of intrathrombotic recanalization. (A) Immunohistochemical analysis was performed using anti-vWF pAbs on venous thrombus samples from WT and Ifng^–/– mice. Original magnification, ×400. Representative results from 6 independent experiments are shown (day 14). Arrows indicate intrathrombotic neovessels. (B) The vascular channels were determined as described in Methods. All values represent the mean ± SEM (n = 6 animals). **P < 0.01; *P < 0.05, Ifng^–/– versus WT. (C) Laser Doppler analysis of thrombosed blood flow. All values represent the mean ± SEM (n = 6 animals). **P < 0.01, Ifng^–/– versus WT. (D) Intrathrombotic gene expression of VEGF in WT and Ifng^–/– mice. Real-time RT-PCR was performed as described in Methods. All values represent the mean ± SEM (n = 6). **P < 0.01, Ifng^–/– versus WT. (E) A double-color immunofluorescence analysis of VEGF-expressing cells in the thrombus. The samples were immunostained with the combination of anti-F4/80 mAbs and anti-VEGF pAbs as described in Methods. The fluorescent images were digitally merged in the right column. Representative results from 6 independent experiments are shown here. Original magnification, ×400. Blue, nuclear staining by DAPI.

Effects of an MMP inhibitor on thrombus resolution. There are controversies on the roles of MMP-9 in thrombus formation and resolution (18, 32). Hence, in order to define precisely the roles of enhanced MMP expression in this process, we examined the effects of an MMP inhibitor on thrombus resolution. The administration of an MMP-2/9 inhibitor impaired thrombus resolution, as evidenced by thrombus mass and intrathrombotic collagen contents, and thrombosed blood flow (Figure 8, A to C), whereas it had no effects on intrathrombotic Vegf gene expression (Figure 8D). These observations implied that MMPs have important roles in collagenolysis and angiogenesis during thrombus resolution, with few effects on VEGF expression.

Figure 8

The effects of an MMP inhibitor on thrombus resolution. WT mice were i.p. administered with an MMP inhibitor as described in Methods. At 14 days after the IVC ligation, thrombus weight (A) and intrathrombotic collagen contents (B), thrombosed blood flow (C), and Vegf gene expression (D) were measured and shown here. All values represent the mean ± SEM (n = 6 animals). *P < 0.05, versus control.

IFN-γ/Stat1 signal pathway negatively regulated the gene expression of MMP-9 and VEGF in macrophages. Our in vivo observations suggest that IFN-γ could suppress the gene expression of MMP-9 and VEGF in intrathrombotic macrophages. To validate this hypothesis, we examined the effects of IFN-γ on the gene expression of MMP-9 and VEGF in WT mouse–derived peritoneal macrophages. PMA significantly enhanced Mmp9 and Vegf gene expression in macrophages (Figure 9, A and B). IFN-γ significantly suppressed PMA-induced gene expression of MMP-9 and VEGF and increase in Stat1 phosphorylation (Figure 9, A–D). Reciprocally, these IFN-γ effects were abrogated by AG490, a JAK2 inhibitor (Figure 9, A to D). These observations strongly implied that the IFN-γ/Stat1 signal pathway negatively regulated Mmp9 and Vegf gene expression in macrophages.

Figure 9

The effects of IFN-γ/Stat1 signal pathways on the gene expression of MMP-9 and VEGF on peritoneal macrophages. Peritoneal macrophages were collected and stimulated as described in Methods. The gene expression of MMP-9 (A) and VEGF (B) was analyzed by real-time RT-PCR. All values represent the mean ± SEM (n = 6 independent experiments). **P < 0.01, versus no stimulation; ^##P < 0.01; ^#P < 0.05 PMA plus IFN-γ versus PMA alone or PMA plus IFN-γ plus AG490. (C and D) Western blotting analysis of Stat1 phosphorylation. Representative results from 6 independent experiments are shown in C. (D) The ratios of phosphorylated Stat1 to β-actin were densitometrically calculated. All values represent the mean ± SEM (n = 6 independent experiments). **P < 0.01, versus no stimulation; ^##P < 0.01, PMA plus IFN-γ plus AG490 versus PMA plus IFN-γ.

Effects of anti–IFN-γ mAbs on thrombus resolution. We finally examined the therapeutic effects of anti–IFN-γ mAbs on the resolution of stasis-induced venous thrombi. When WT mice received anti–IFN-γ mAbs or control IgG at 4, 8, and 12 days after the IVC ligation, thrombus mass and intrathrombotic collagen contents were smaller in anti–IFN-γ mAb–treated WT mice than control IgG-treated WT ones (Figure 10, A and B). Consistently, blood flow was larger in anti–IFN-γ mAb–treated WT mice than in control IgG-treated WT ones (Figure 10C). Concomitantly, the intrathrombotic gene expression of MMP-9 and VEGF was significantly higher in the anti–IFN-γ mAb–treated group than in the control IgG-treated group (Figure 10, D and E). Moreover, the administration of anti–IFN-γ mAbs did not cause any abnormalities in coagulation function as revealed by prothrombin time (PT) (control vs. anti–IFN-γ mAbs: 10.8 ± 0.53 s vs. 11.1 ± 0.25 s) and activated partial thromboplastin time (APTT) (control vs. anti–IFN-γ mAbs: 31.3 ± 1.2 s vs. 30.1 ± 0.8 s). These observations demonstrated that IFN-γ blockade was therapeutically effective in accelerating the thrombus resolution without any adverse effects on coagulation function.

Figure 10

The effects of anti–IFN-γ mAb administration on thrombus resolution. WT mice were i.p. administered anti–IFN-γ mAbs or isotype-matched IgG as described in Methods. At 14 days after the IVC ligation, thrombus weight (A) and intrathrombotic collagen contents (B), thrombosed blood flow (C), and the gene expression of MMP-9 (D) and VEGF (E) were measured and shown here. All values represent the mean ± SEM (n = 6 animals). *P < 0.05; **P < 0.01, anti–IFN-γ mAbs versus control IgG.

IFN-γ is produced by Th1 lymphocytes, NK cells, and macrophages (19). Moreover, under several pathological conditions, neutrophils also can produce IFN-γ (20, 22). We proved that macrophage-derived IFN-γ could negatively regulate skin wound healing by dampening the expression of VEGF and TGF-β (23). The resemblance between wound healing and thrombus resolution prompted us to investigate IFN-γ expression in thrombus caused by stasis. Indeed, IFN-γ protein was immunohistochemically detected in venous thrombi obtained from human autopsy cases (Supplemental Figure 1). In the present study, IFN-γ was expressed mainly by infiltrating macrophages and its expression was progressively enhanced until 14 days after the initiation of stasis, suggesting that IFN-γ might be involved in thrombus resolution.

Henke and colleagues previously demonstrated that Ifng^–/– mice showed similar rates of thrombus resolution in the early phase compared with WT mice, but they did not examine the later phase (18), when intrathrombotic IFN-γ contents were still enhanced in WT mice. Hence, we prolonged the observation period up to 14 days after the initiation of stasis. Although we failed to detect any significant differences in the thrombus mass between WT and Ifng^–/– mice until 5 days after the initiation of stasis, thrombus mass was significantly reduced in Ifng^–/– mice compared with WT mice later than 10 days after the initiation of stasis. These observations implied that locally produced IFN-γ might decelerate thrombus resolution in the later phase.

Rats developed significantly larger thrombi when they were rendered neutropenic by the treatment with antipolymorphonuclear leukocyte serum (33). Intrathrombotic neutrophil recruitment was decreased and subsequent thrombus resolution was diminished by genetic disruption of CXCR2, a receptor for CXC chemokines such as CXCL1/KC and CXCL2/MIP-2 with potent chemotactic activities for neutrophils (16). Supporting the notion that macrophage/monocyte recruitment has an important role in thrombus resolution, Ali and colleagues demonstrated that intrathrombus injection of peritoneal macrophages reduced the thrombus size (34). Moreover, the administration of exogenous MCP-1/CCL2, a potent chemoattractant for macrophage, promoted thrombus resolution (17). Conversely, thrombus resolution was impaired with a concomitant reduction in intrathrombotic macrophage recruitment in mice lacking CCR2, a single and specific receptor for CCL2 (18). Thus, these observations indicate that resolution of venous thrombus is regulated by chemokines and subsequent chemokine-mediated neutrophil and/or macrophage recruitment into the thrombi (6–10). In contrast, the absence of IFN-γ failed to affect the intrathrombotic gene expression of CXC and CC chemokines and subsequent leukocyte recruitment. Thus, accelerated thrombus resolution in Ifng^–/– mice was not directly ascribed to changes in leukocyte recruitment and/or chemokine expression.

Plasmin is formed from plasminogen by the action of tPA and uPA (26–28). This pathway is presumed mainly to regulate fibrin deposition in vascular trees and to activate pericellular fibrinolysis, which is required for cell migration in tissues. Singh and colleagues (35) demonstrated that the absence of uPA impaired thrombus resolution, indicating the essential involvement of plasmin system in thrombus resolution. Evidence is accumulating to indicate that IFN-γ regulated the gene expression of plasminogen activators and their inhibitor (36–39). However, in the present study, there was no significant difference in the intrathrombotic gene expression of uPA, tPA, and PAI-1 between WT and Ifng^–/– mice. Thus, it is unlikely that the absence of IFN-γ could accelerate thrombus resolution by affecting uPA, tPA, and PAI-1 expression.

Collagenolysis is indispensable for thrombus resolution (10). Reduced intrathrombotic collagen contents would indicate enhanced collagenolysis in Ifng^–/– mice compared with WT mice. Among several enzymes with collagenolysis activity, MMP-2 and MMP-9 are presumed to have an important role in collagen turnover during thrombus resolution (18). IFN-γ deficiency increased intrathrombotic MMP-9 but not MMP-2 mRNA expression, particularly at the later phase of thrombus resolution. Moreover, MMP-9 activities were higher in Ifng^–/– mice than WT mice. Transient overexpression of MMP-9 alone could promote intravascular thrombus formation induced by arterial balloon injury, whereas the combination of MMP-9 and tissue inhibitor of metalloproteinase 1 (TIMP-1) could prevent it (32). TIMP-1 expression was enhanced, to a similar extent, in thrombus formation sites of both strains in this model (our unpublished observations), and consequently, enhanced MMP-9 expression in the early phase may have few effects on thrombus formation in the presence of TIMP-1. Alternatively, discrepant observations may arise from the use of different thrombosis models. Nevertheless, retardation of thrombus resolution by an MMP-2/9 inhibitor suggested the potential roles of MMP-9 in thrombus resolution in the later phase when MMP-9 but not MMP-2 expression was higher in Ifng^–/– than WT mice, although the contribution of MMP-2 cannot be completely excluded.

MMP-9 was expressed predominantly by infiltrating macrophages. Because macrophages are a major target cell of IFN-γ, genetic disruption of IFN-γ could modulate MMP-9 expression by macrophages, similarly reported on several other proinflammatory cytokines, which can regulate Mmp9 gene expression (40). Ma and colleagues revealed that the IFN-γ/Stat1 signal pathway suppressed Mmp9 gene expression in human astroglioma and HeLa cell lines (41, 42). In line with this, IFN-γ suppressed PMA-induced Mmp9 gene expression together with depression of Stat1 activation in murine peritoneal macrophages, while AG490, a JAK2 inhibitor, abrogated these effects. Thus, IFN-γ would prevent macrophages from transcribing the Mmp9 gene in an autocrine and/or paracrine manner, and IFN-γ deficiency would cancel the suppression, thereby resulting in enhanced MMP-9 expression and subsequent acceleration of collagen degradation in thrombus.

Neovascularization is another key event in the process of thrombus resolution (29–31), as evidenced by retarded thrombus resolution with impaired intrathrombotic neovascularization (16, 18). Several in vitro studies demonstrated that IFN-γ had direct antiangiogenic effects, as evidenced by the inhibition of capillary growth and development (43, 44). In support, we previously observed that Ifng^–/– mice exhibited enhanced angiogenesis during skin wound healing (23). Likewise, intrathrombotic neovascularization was augmented in Ifng^–/– mice compared with WT mice. Moreover, MMP-9 also exhibits proangiogenic activity by mediating basement membrane remodeling for endothelialization of tissue matrix (45, 46), and the inhibition of MMP-9 impaired neovascularization in several conditions such as cardiac ischemia (47). Consistently, when WT mice received an MMP-2/9 inhibitor, intrathrombotic recanalization was reduced with no effects on intrathrombotic Vegf gene expression, thereby resulting in impaired thrombus resolution. Thus, enhanced MMP-9 expression could promote intrathrombotic recanalization in Ifng^–/– mice independently of VEGF.

Accumulating evidence indicates that angiogenic growth factors such as VEGF and bFGF are crucial for intrathrombotic neovascularization (48–50). In the present study, the absence or immunoneutralization of IFN-γ augmented Vegf gene expression in the thrombi, and MMP inhibitors had no influence on intrathrombotic Vegf gene expression. Thus, IFN-γ can negatively regulate Vegf gene expression. Kommineni and colleagues demonstrated that IFN-γ suppressed Vegf gene expression induced by IL-1β and TNF-α in human corneal epithelial cells (51). Consistently, our study demonstrated that IFN-γ/Stat1 signal pathways attenuated Vegf gene expression in murine peritoneal macrophages. Collectively, the absence of IFN-γ promoted intrathrombotic recanalization directly and indirectly.

At present, warfarin is mainly employed against DVT to prevent pulmonary thromboembolism, but this anticoagulation therapy increases the risk of bleeding. Ifng^–/– mice did not show any abnormalities in coagulation functions but exhibited accelerated thrombus resolution, probably by upregulating the expression of MMP-9 and VEGF, the factors crucially involved in thrombus resolution. Thus, IFN-γ may be efficacious in preventing DVT and its complications. Moreover, IFN-γ blockade also accelerated thrombus resolution without any effects on coagulation function, even if given after thrombus formation. Thus, IFN-γ may be a novel molecular target for the development of therapies that accelerate thrombus resolution in patients with DVT.

View Supplemental data

We thank Yoichiro Iwakura (University of Tokyo, Tokyo, Japan) for providing us with Ifng^–/– mice. This study was financially supported in part by Grants-in-Aids for Scientific Research from the Ministry of Education, Culture, Science, and Technology of the Japanese government.

Address correspondence to: Toshikazu Kondo, Department of Forensic Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-8509, Japan. Phone: 81.73.441.0641; Fax: 81.73.441.0641; E-mail: kondot@wakayama-med.ac.jp.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2011;121(7):2911–2920. doi:10.1172/JCI40782.

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