Crucial Involvement of IL-6 in Thrombus Resolution in Mice via Macrophage Recruitment and the Induction of Proteolytic Enzymes

After the ligation of the inferior vena cava (IVC) of wild-type (WT) mice, venous thrombi formed and grew progressively until 5 days and resolved thereafter. Concomitantly, intrathrombotic gene expression of Il6 was enhanced later than 5 days after IVC ligation. IL-6 protein expression was detected mainly in F4/80-positive macrophages in thrombus. When Il6-deficient (Il6−/−) mice were treated in the same manner, thrombus mass was significantly larger than in WT mice. Moreover, the recovery of thrombosed IVC blood flow was markedly delayed in Il6−/− compared with WT mice. F4/80-positive macrophages in thrombus expressed proteolytic enzymes such as matrix metalloproteinase (Mmp) 2, Mmp9, and urokinase-type plasminogen activator (Plau); and their mRNA expression was significantly reduced in Il6−/− mice. Consistently, the administration of anti-IL-6 antibody delayed the thrombus resolution in WT mice, whereas IL-6 administration accelerated thrombus resolution in WT and Il6−/− mice. Moreover, IL-6 in vitro enhanced Mmp2, Mmp9, and Plau mRNA expression in WT-derived peritoneal macrophages in a dose-dependent manner; and the enhancement was abrogated by a specific Stat3 inhibitor, Stattic. Thus, IL-6/Stat3 signaling pathway can promote thrombus resolution by enhancing Mmp2, Mmp9, and Plau expression in macrophages.


INTRODUCTION
Deep vein thrombosis (DVT) is a common vascular disease, often causing post-thrombotic symptoms including pain, heaviness, itching, swelling, and brownish or reddish skin discoloration, with ulcer as its long-term complication (1,2). Moreover, DVT is frequently associated with pulmonary embolism (PE), one of the major causes of sudden unexpected natural deaths (1,2). DVT formation has traditionally been thought to be caused by blood stagnancy, endothelial injury of the vein, and hypercoagulability (3). However, evidence is accumulating to indicate the involvement of proinflammatory cytokines and chemokines in the thrombus formation (4)(5)(6)(7). Moreover, venous thrombosis resolution and vein wall healing are mediated by leukocytes, particularly macrophages, and their associated chemokines, tissue-type or urokinase-type plasminogen activator (PLAT or PLAU), matrix metalloproteinases (MMPs), and proinflammatory cytokines (8)(9)(10)(11). Proinflammatory cytokines can potently activate endothelial cells, can increase the expression of adhesion molecules by endothelial cells, and can promote thrombosis (12,13). On the contrary, some chemokines can activate leukocytes to accelerate thrombus resolution and intrathrombotic neovascularization (14)(15)(16). We previously demonstrated that a proinflammatory cytokine, IFN-γ, can decelerate thrombus resolution by suppressing collagenolysis (17). Moreover, the TNF-α-TNF-Rp55 axis can promote thrombus resolution by inducing macrophages to express Plau, Mmp2, and Mmp9, the proteolytic enzymes that are crucial to collagenolysis and neovascularization (18). However, the roles of other proinflammatory cytokines in DVT formation and/or resolution remain elusive.
IL-6 is a pleiotropic cytokine involved in inflammation, autoantibody production, vascular permeability, tissue regeneration, metabolism, and hematopoiesis. IL-6 is produced by myriads of cells including T cells, monocytes, fibroblasts, endothelial cells, and keratinocytes (19). IL-6 signaling blockade has recently been proven to be therapeutically effective for a number of autoimmune and inflammatory disorders including rheumatoid arthritis, polymyalgia rheumatica, vasculitis, and Castleman disease, the diseases that exhibit aberrant IL-6 signaling pathway (20). Thrombus forms and resolves with distinct phases consisting of neutrophil and macrophage infiltration, fibrosis, and neovascularizationinduced recanalization (14,21); and the whole process resembles skin wound healing process. We previously revealed that Il6 −/− mice exhibited delayed skin wound healing with attenuated leukocyte infiltration, re-epithelialization, angiogenesis, and collagen accumulation (22). Thrombus formation and resolution processes share with skin wound healing processes various pathophysiological aspects such as leukocyte accumulation, collagen production, and angiogenesis. This promoted us to conduct our previous study, which revealed markedly enhanced IL-6 protein in thrombi of wild-type (WT) mice (23). However, the pathophysiological roles of IL-6 in venous thrombosis are still unknown.
In this study, we demonstrated that the absence of IL-6 did retard thrombus resolution, together with suppressed expression of MMP-2, MMP-9, and PLAT, compared with that in WT mice. This mirrors the observation that IL-6 could enhance the gene expression of Plau, Mmp2, and Mmp9 in macrophages in an IL-6dependent manner. These observations implied that the IL-6 can be a target molecule to design the therapeutic strategy for DVT.

Mice
Pathogen-free male BALB/c mice that are 8-10 weeks old were obtained from Japan SLC (Shizuoka, Japan) and were designated as WT mice in this study. Il6 −/− mice were a kind gift of Dr. Blüthmann (24) and were back-crossed to BALB/c mice for more than 10 generations. Subsequently, age-and sexmatched Il6 −/− mice were used in these experiments (25). All mice were housed individually in cages under specific pathogenfree conditions during the experiments. All animal experiments were approved by the Committee on Animal Care and Use in Wakayama Medical University and complied with the Guidelines for the Care and Use of Laboratory Animals of Wakayama Medical University.

Inferior Vena Cava Ligation-Induced Deep Vein Thrombus Model
Intravenous thrombus formation was induced as previously described (14,17,26). In brief, after deep anesthesia with intraperitoneal injection of pentobarbital (50 mg/kg of body weight), a 2-cm incision was made along the abdominal midline. Then, inferior vena cava (IVC) was exposed carefully, and a 21gauge needle was placed along the exposed IVC. Subsequently, IVC was ligated with the needle using 3-0 silk suture, followed by pulling out the needle. This procedure can induce thrombus formation in almost all the mice. In some experiments, WT mice were intraperitoneally given anti-mouse IL-6 mAb [5 µg/mouse in 200 µl of phosphate-buffered saline (PBS)] at 1, 3, 6, and 8 days after IVC ligation. WT and Il6 −/− mice were intraperitoneally given rIL-6 (0.3 µg/mouse in 200 µl of PBS) at 1, 4, and 8 days after IVC ligation. At the indicated time intervals after the IVC ligation, mice were euthanized by an overdose of diethyl ether, and intravenous thrombi were harvested for the determination of the weights.

Histopathological Analyses
At the indicated time intervals after IVC ligation, thrombi were harvested and fixed in 4% formaldehyde buffered with PBS (pH 7.2), and paraffin-embedded sections (4 µm thick) were made. The sections were stained with hematoxylin and eosin (H&E) or Masson trichrome solution.

Immunohistochemical Analyses
Deparaffinized sections were immersed in 0.3% H 2 O 2 in methanol for 30 min to eliminate endogenous peroxidase activities. The sections were further incubated with PBS containing 1% normal serum derived from the same species as the origin of the secondary Abs and 1% bovine serum albumin (BSA) to reduce non-specific reactions. The sections were incubated with anti-mouse F4/80 mAb, anti-mouse CCL2 pAbs, anti-mouse Col1A2 mAb, anti-mouse MPO pAbs, antimouse CD3 mAb, or anti-mouse IL-6 pAbs at a concentration of 1 µg/ml at 4 • C overnight. After the incubation of biotinylated secondary Abs, immune complexes were visualized using Catalyzed Signal Amplification System (Dako) according to the manufacturer's instructions.

Measurements of Intrathrombotic Leukocytes
Intrathrombotic macrophage and CCL2-positive cell numbers were determined as previously described (17,18). Briefly, after F4/80-positive macrophages, MPO-positive neutrophils, or CCL2-positive cells were counted in five high power fields (×1,000) within the thrombus, the total numbers in the five fields were combined. All measurements were performed by an examiner without a prior knowledge of the experimental procedures.

Double-Color Immunofluorescence Analyses
Deparaffinized sections were incubated with PBS containing 1% normal donkey serum and 1% BSA to reduce nonspecific reactions as previously described (17). Thereafter, the sections were further incubated with the combinations of anti-F4/80 and anti-IL-6, anti-MMP-2, anti-MMP-9, or anti-PLAU (uPA); anti-MPO and anti-IL-6; or anti-CD3 and anti-IL-6. All Abs were used at a concentration of 1 µg/ml. After the incubation with fluorochrome-conjugated secondary Abs (15 µg/ml) at room temperature for 30 min, the sections were observed under a fluorescence microscopy. In some experiments, nuclei were stained using 4 ′ ,6-diamidino-2-phenylindole (DAPI; Roche Diagnostics, Indianapolis, IN) according to the manufacturer's instructions.

Inferior Vena Cava Blood Flow Measurement by Laser Doppler Flowmeter
At 5, 10, and 14 days after IVC ligation, microvascular IVC blood flow was evaluated by laser Doppler imaging (OMEGAFLO FLO-C1 BV, OMEGAWAVE) as described previously (17). Blood flow through the exposed IVC region of the interest was assessed at three time points; immediately after laparotomy, at the indicated time points after the ligation, and at the harvest. The intensities were reported as the percentage of the baseline blood flow of each animal, in order to ensure consistency.

Extraction of Total RNAs and Real-Time Reverse Transcription-PCR
Real-time reverse transcription (RT)-PCR was performed as described previously (17). Briefly, total RNAs were extracted from tissue samples (100 µg) using ISOGENE (Nippon Gene, Toyama, Japan) according to the manufacturer's instructions, and 5 µg of total RNAs were reverse-transcribed into cDNA at 42 • C for 1 h in 20 µl of reaction mixture containing mouse Moloney leukemia virus reverse transcriptase (PrimeScript, Takara Bio, Kusatsu, Japan) with random 6 primers (Takara Bio). Thereafter, generated cDNA was subjected to a real-time PCR analysis using SYBR R Premix Ex Taq TM II kit (Takara Bio) with the sets of specific primers ( Table 1). Relative quantity of the target gene expression to Actb gene was measured by comparative Ct method.

ELISA for IL-6
At the indicated time intervals, thrombus samples were obtained and homogenized with 0.3 ml of PBS (pH 7.2) containing complete Protease Inhibitor Cocktail (Roche Diagnostics). The homogenates were centrifuged at 12,000 g for 15 min. IL-6 levels in the supernatant were measured using a specific ELISA kit (Murine IL-6 ELISA kit, Diaclone, Besancon Cedex, France), according to the manufacturer's instructions. The detection limit was 10 pg/ml. Total protein in the supernatant was measured with a commercial kit (BCA Protein Assay Kit; Pierce) using BSA as a standard. The data were expressed as IL-6 (ng/ml)/total protein (mg/ml) for each sample.

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Cell Culture
WT mice were i.p. injected with 2 ml of 3% thioglycollate (Sigma-Aldrich), to obtain intraperitoneal macrophages 3 days later as described previously (17). The obtained cells were judged to consist of more than 95% macrophages as determined by flowcytometry (FCM) using anti-F4/80 Ab. The resultant cells were suspended in antibiotic-free Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (FBS) and incubated at 37 • C in three 6-well cell culture plates. 2 h later, non-adherent cells were removed, and the medium was replaced. After the cells were incubated for 24 h in the presence of the indicated concentrations of rIL-6 (1,000 U/ml), together with or without anti-mouse IL-6 mAb (5 µg/ml), or Stattic (20 µM), the cells were subjected to subsequent analyses.

Western Blotting
The obtained macrophages were homogenized with a lysis buffer [20 mM of Tris-HCl (pH 7.6), 150 mM of NaCl, 1% Triton X-100, and 1 mM of EDTA] containing complete Protease Inhibitor Cocktail (Roche Diagnostics) and were centrifuged to obtain lysates. The lysates (equivalent to 30 µg of protein) were electrophoresed in a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and were transferred onto a nylon membrane. After the membrane was sequentially reacted with optimally diluted primary Abs and horseradish peroxidase (HRP)-conjugated secondary Abs, the immune complexes were visualized using ECL system (Amersham Biosciences, Pittsburgh, PA). The band intensities were measured using NIH Image Analysis Software version 1.48 (National Institutes of Health).

Measurement of Prothrombin Time and Activated Partial Thromboplastin Time
Blood samples were taken with 3.8% citrate solution and centrifuged to obtain plasma samples. Prothrombin time (PT) and activated partial thromboplastin time (APTT) of citrated plasma samples were measured by using COAGSEARCH (A&T) according to the manufacturer's instructions.

Statistical Analyses
Data were expressed as the mean ± SEM. For the comparison between WT and Il6 −/− mice at multiple time points, a twoway ANOVA followed by Dunnett's post-hoc test was used. To compare the values between two groups, unpaired Student's ttest was performed. In the series of IL-6 stimulation on peritoneal macrophages in vitro, a one-way ANOVA followed by Dunnett's post-hoc test was used. p < 0.05 was considered statistically significant. All statistical analyses were performed using Statcel3 software under the supervision of a medical statistician.

Intrathrombotic IL-6 Expression After the Inferior Vena Cava Ligation
The detection of IL-6 in venous thrombi in autopsy cases (our unpublished data) prompted us to examine intrathrombotic gene expression of Il6 in WT mice after IVC ligation. Il6 mRNA was detected in the thrombus 5 days after IVC ligation, and its expression was decreased later than 10 days ( Figure 1A). Consistently, IL-6 protein could be detected at day 5 and later ( Figure 1B). IL-6 protein was immunohistochemically found in macrophage-like cells inside thrombus ( Figure 1C). Consistently, double-color immunofluorescence analyses identified F4/80 + macrophages but not MPO + neutrophils and CD3 + T cells as a main cellular source of IL-6 ( Figure 1D and Figure S1). Thus, these observations would imply the involvement of macrophage-derived IL-6 in the formation and/or resolution of deep vein thrombi.

Impaired Thrombus Resolution in the Absence of IL-6
In order to explore the pathophysiological roles of IL-6 in IVC ligation-induced venous thrombus, we compared thrombus formation between WT and Il6 −/− mice. At 5 days after IVC ligation, Il6 −/− mice developed a larger thrombus than did WT mice (Figures 2A,B) (Figures 2C,D). Consistently, at 10 and 14 days after IVC ligation, intrathrombotic Col1 mRNA expression was significantly higher in Il6 −/− mice than WT mice (Figure 2E), indicating that the thrombus in Il6 −/− mice was replaced with collagen to a larger extent than that in WT mice. Mirroring collagen contents in thrombus, blood flow recovery was delayed in Il6 −/− mice compared with WT mice (Figure 2F). Collectively, these observations implied that the lack of IL-6 could retard thrombus resolution with excessive collagen deposition.

Reduced Intrathrombotic Macrophage Infiltration With Attenuated CCL2 Expression
Accumulating evidence implicated the crucial roles of infiltrating macrophages in thrombus resolution (27,28). F4/80-positive macrophages infiltrated into thrombus in WT and Il6 −/− mice, reaching a maximal level at 7 days and decreasing thereafter, but intrathrombotic macrophage numbers were larger in WT mice than Il6 −/− mice (Figures 3A,B). CCL2 is a potent chemotactic cytokine for the intrathrombotic recruitment of monocytes/macrophages via CCR2 (14). Hence, we examined intrathrombotic CCL2 expression in WT and Il6 −/− mice. The intrathrombotic expression of Ccl2 mRNA was up-regulated in WT mice at 5 days after IVC ligation, whereas the enhancement was significantly attenuated in Il6 −/− mice ( Figure 3C). Moreover, immunohistochemical analyses demonstrated that the intrathrombotic CCL2-positive cell numbers were significantly lower at 5 and 10 days after IVC ligation in Il6 −/− mice than in WT mice (Figures 3D,E). Thus, the lack of IL-6 reduced intrathrombotic macrophage infiltration with attenuated CCL2 expression.

Reduced Expression of Macrophage-Derived Proteolytic Enzymes in the Absence of IL-6
We previously revealed that intrathrombotic macrophages were a major source of proteolytic enzymes such as MMP-2, MMP-9, and PLAU, which were essentially involved in thrombus resolution (17,18). Consistent with our previous reports (17,18), double-color immunofluorescence analyses revealed that intrathrombotic F4/80-positive macrophages were a main cellular source of MMP-2, MMP-9, and PLAU (Figures 4A-C). Furthermore, later than 10 days after IVC ligation, when thrombus resolution started, mRNA expression of Mmp2, Mmp9, and Plau was markedly enhanced in thrombus in WT mice, whereas the increments were significantly depressed in Il6 −/− mice compared with WT mice (Figures 4D-F). These observations would indicate that delayed thrombus resolution in Il6 −/− mice can be ascribed to depressed expression of Mmp2, Mmp9, and Plau, the proteolytic enzymes that were expressed mainly by intrathrombotic macrophages.

Effects of Anti-IL-6 Antibody and Recombinant IL-6 on Thrombus Resolution
We next examined the effects of anti-IL-6 mAb and rIL-6 on the resolution of IVC ligation-induced venous thrombi. Anti-IL-6 mAb significantly increased thrombus weights and decelerated blood flow recovery compared with those in control Abtreated mice, together with depressed intrathrombotic Mmp2, Mmp9, and Plau gene expression (Figures 5A-F), similarly as observed in Il6 −/− mice. On the contrary, when rIL-6 administration started 1 day after IVC ligation, it reduced significantly the thrombus weights and accelerated blood flow recovery with increased intrathrombotic Mmp2, Mmp9, and Plau gene expression in WT (Figures 5G-L), but without any significant effects on coagulation tests (PT in WT mice: PBS, 9.74 ± 0.28 s vs. rIL-6, 9.85 ± 0.31 s; APTT in WT mice: PBS, 36.6 ± 0.27 s vs. rIL-6, 37.8 ± 3.08 s). These observations would indicate that IL-6 could be therapeutically effective for thrombus formation at least at its early stage. Moreover, supplementation of IL-6 reduced thrombus weights and enhanced blood flow recovery in Il6 −/− mice ( Figure S2). These observations would indicate that IL-6 could regulate the thrombosis resolution without affecting coagulation activities.

DISCUSSION
Accumulating evidence implicates IL-6 as a key regulator of several inflammatory diseases (19). Consistently, we previously revealed that the lack of IL-6 decelerated skin wound healing with a concomitant reduced leukocyte accumulation and collagen deposition (22). Skin wound healing and thrombus resolution share several pathophysiological features: initial platelet aggregation, subsequent leukocyte infiltration, and collagen deposition (26), and final neovascularization. These shared features prompted us to investigate the roles of IL-6 in thrombus formation and resolution. Actually, macrophages were a major source of IL-6 in thrombus, and Il6 −/− mice exhibited retarded thrombus resolution as evidenced by larger venous thrombi similarly as observed in skin wound healing sites. However, Il6 −/− mice exhibited increased intrathrombotic collagen contents than did WT mice, suggesting that IL-6 may have distinct roles between skin wound healing and thrombus resolution, in terms of collagen accumulation in the lesions. Accumulating evidence revealed that various types of leukocytes were crucially involved in thrombus resolution. Neutrophil depletion impaired thrombus resolution (27), whereas intrathrombus injection of peritoneal macrophages accelerated thrombus resolution (28). Moreover, Luther and colleagues demonstrated that the absence of effector memory T cells accelerated thrombus resolution (29). In line with these observations, we previously revealed the potential contribution of intrathrombotic leukocyte accumulation to thrombus resolution (30). Consistently, Il6 −/− mice exhibited reduced neutrophil ( Figure S3) and macrophage accumulation after IVC ligation than did WT mice. Thus, the depressed leukocyte infiltration may contribute to delayed thrombus resolution in Il6 −/− mice.
Chemokine system, a major controller of leukocyte trafficking, can also regulate thrombus formation and resolution by manipulating the migration of leukocytes, key players in the processes (31). This notion was substantiated by the   observations that the administration of exogenous MCP-1/CCL2, a potent macrophage chemoattractant, accelerated venous thrombus resolution together with enhanced F4/80 + macrophage infiltration (16). Consistently, the genetic deletion of CCR2 (a specific receptor of CCL2) impaired thrombus resolution with the reduced recruitment of F4/80 + macrophages (14). These observations implied that the CCL2-CCR2 axis could promote thrombus resolution by inducing macrophage infiltration into thrombus. Given the observations that IL-6 could up-regulate CCL2 expression in macrophages (32,33), we determined intrathrombotic CCL2 expression. Macrophages were identified as a major source of CCL2 in thrombus, and intrathrombotic CCL2 expression was significantly attenuated in IVC-ligated Il6 −/− mice compared with IVC-ligated WT mice. Thus, IL-6 can induce macrophages to express CCL2, which can boost the infiltration of macrophages, the cell component crucially involved in thrombus resolution. No significant differences were observed in APTT and PTT between WT and Il6 −/− mice, indicating that coagulation dysfunction can account for the different phenotype of these two strains. On the contrary, Il6 −/− mice exhibited reduced intrathrombotic gene expression of Plau, a major plasminogen activator, whereas IL-6 augmented Plau mRNA expression in macrophages, a major cell type present in thrombus. Tissue-and urokinase-type plasminogen activators can promote the generation from plasminogen to plasmin, which has an important role in clotting, fibrinolysis, inflammatory angiogenesis, and tissue remodeling (34). Reflecting the crucial roles of the balance between plasminogen activators and inhibitors in proteolytic and anti-proteolytic activities, the lack of PLAU markedly impaired thrombus resolution (35). Thus, it is probable that IL-6 can promote thrombus resolution by enhancing Plau expression in macrophages.
Venous thrombus is replaced by deposited collagen as time passes (26), and therefore, thrombus resolution requires collagen  degradation. Il6 −/− mice displayed enhanced intrathrombotic collagen contents than did WT mice, indicating that IL-6 deficiency can increase collagen synthesis or decrease collagenolysis. The latter possibility was supported by our present observations that Il6 −/− mice exhibited reduced intrathrombotic expression of MMP-2 and MMP-9, the enzymes that have important roles in collagen turnover during thrombus resolution owing to their potent collagenolysis activities (14,17). Moreover, evidence is accumulating to indicate that Mmp2 and Mmp9 expression can be enhanced by inflammatory cytokines such as IL-1, TNF-α, and IL-6 (33,(36)(37)(38). Consistently, we also revealed that IL-6 can augment Mmp2 and Mmp9 gene expression in peritoneal macrophages and that these effects were canceled by anti-IL-6 antibody. Thus, IL-6 could be a potent inducer for MMP-2 and MMP-9 in intrathrombotic macrophages.
Several distinct signaling pathways have been presumed to be involved in MMP gene expression (39)(40)(41). IL-6 can utilize Stat3 and other MAP kinases to transduce its intracellular signals (19). However, we unraveled that IL-6 significantly enhanced the phosphorylation of Stat3 but not other MAP kinases such as ERK, JNK, and p38, thereby enhancing the mRNA expression of Mmp2, Mmp9, and Plau in macrophages. Moreover, the Stattic, a Stat3 inhibitor, significantly suppressed IL-6-induced the gene expression of these molecules. Thus, these observations provided the evidence to indicate the crucial involvement of the IL-6/Stat3 signal pathways in venous thrombus resolution through MMP-2, MMP-9, and PLAU.
There are still discrepancies in pathophysiological roles of IL-6 in DVT. Clinically, several inflammatory mediators including IL-6 are proposed to increase the risk of DVT in various types of pathological conditions such as surgery, obesity, cystic fibrosis, sepsis, systemic infection, cancer, inflammatory bowel disease, and lupus (42). Cancer patients, particularly, show a four-fold increased risk for DVT depending on multiple factors such as patient conditions, tumor characteristics, and treatment modalities (43). The presence of the tumor may affect the host coagulation system, and anticancer treatments also increase the risk of venous thromboembolism (VTE) in cancer patients (43). Accumulating evidence implied the close association of IL-6 with the incidence of DVT in cancer patients (44)(45)(46). Malaponte and colleagues demonstrated that IL-6 levels in plasma and monocyte samples were higher in cancer patients with DVT than in those without DVT (45). In line with this observation, Stone et al. showed that tumor-derived IL-6 promoted thrombocytosis through the induction of hepatic thrombopoietin, eventually increasing the incidences of DVT in a mouse model of ovarian cancer (44). These observations suggested that IL-6 could promote thrombus formation through an increase of coagulation activity. On the contrary, we observed that IL-6 administration had few effects on coagulation functions.
Thrombosis results from the imbalance caused by increases of formation rate and/or a delay of resolution. However, the previous clinical study on cancer patients did not examine the relationship between IL-6 and resolution-related molecules (44)(45)(46). From our observations, IL-6 can promote thrombus resolution by regulating macrophage recruitment via the upregulation of CCL2 and enhancing their expression of MMPs and PLAU in thrombus. In line with this, Malaponte (46) found a positive correlation between IL-6 and MMP-9 plasma concentrations in both DVT and non-DVT cancer patients. These observations may imply that IL-6 expression may be enhanced in the presence of DVT to accelerate thrombus resolution in cancer patients. This assumption has been strengthened by the observations that the administration of exogenous IL-6 accelerated thrombus resolution in WT mice after the IVC ligation.
Anti-coagulant therapy such as warfarin is mainly employed against DVT to prevent pulmonary thromboembolism (PTE), but it increases the incidence of bleeding complications. On the contrary, the administration of IL-6 had few effects on coagulation functions. It is clinically important that IL-6 administration under the presence of thrombus could reduce thrombus size. Our observations implied that IL-6 administration after thrombus formation might be effective for the reduction of thrombus size, because intrathrombotic IL-6 protein levels started to increase at 5 days after the IVC ligation. Thus, IL-6 may be a target molecule to induce DVT resolution, although more work on human clinical conditions is warranted.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

ETHICS STATEMENT
The animal study was reviewed and approved by Wakayama Medical University Animal Care and Use Committees (no. 879).

AUTHOR CONTRIBUTIONS
TK and NM formulated the hypothesis and initiated and organized the study. MN and YI performed the main experimental work and analyzed the data. AK, YK, ATar, MO, and ATan helped with some experimental procedures. TK and NM oversaw the experiments, analyzed the data, provided the main funding for the research, and prepared the final manuscript.