Rivaroxaban attenuates leukocyte adhesion in the microvasculature and thrombus formation in an experimental mouse model of type 2 diabetes mellitus Toshiaki Iba, Koichiro Aihara, Atushi Yamada, Masataka Nagayama, Yoko Tabe, Akimichi Ohsaka PII: DOI: Reference:
S0049-3848(13)00549-5 doi: 10.1016/j.thromres.2013.11.013 TR 5290
To appear in:
Thrombosis Research
Received date: Revised date: Accepted date:
26 June 2013 10 November 2013 18 November 2013
Please cite this article as: Iba Toshiaki, Aihara Koichiro, Yamada Atushi, Nagayama Masataka, Tabe Yoko, Ohsaka Akimichi, Rivaroxaban attenuates leukocyte adhesion in the microvasculature and thrombus formation in an experimental mouse model of type 2 diabetes mellitus, Thrombosis Research (2013), doi: 10.1016/j.thromres.2013.11.013
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Revised on Nov. 10, 2013
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Original article
Rivaroxaban attenuates leukocyte adhesion in the microvasculature and
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thrombus formation in an experimental mouse model of type 2 diabetes
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mellitus
1)
*, Koichiro Aihara, M.D.
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Toshiaki Iba, M.D.
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Running Head: rivaroxaban attenuates vascular inflammation
1)
, Atushi Yamada, M.D.
1)
, Masataka
Department of Emergency and Disaster Medicine
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1)
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Nagayama, M.D. 1), Yoko Tabe, M.D. 2) and Akimichi Ohsaka, M.D. 3)
2)
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Juntendo University Graduate School of Medicine Department of Clinical Laboratory Medicine Juntendo University Graduate School of Medicine 3)
Department of Transfusion Medicine and Stem Cell Regulation Juntendo University Graduate School of Medicine
* : Professor of Emergency and Disaster Medicine
Correspondence to: Toshiaki Iba, MD. 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
E-mail: [email protected] tel: 81-3-3813-3111 (X: 5818) fax: 81-3-3813-5431
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ACCEPTED MANUSCRIPT Abstract Introduction: Thrombosis is a major complication in diabetes mellitus. Since Factor Xa
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inhibitors are not only inhibit the coagulation system but also attenuate the
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leukocyte-endothelial interaction in acute inflammation models, the purpose of this study is
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to confirm the similar effects of rivaroxaban in a mouse model of type 2 diabetes mellitus.
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Materials and Methods: In the treatment groups, either 5 or 10 mg/kg of rivaroxaban dissolved in DMSO was orally given to KK-Ay mice for 7 weeks (n= 6 in each group).
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KK-Ay mice fed by chow containing DMSO without rivaroxaban for 7 weeks were served
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for the control group (n= 6). Following clamping of the mesenteric vein for 20 minutes,
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intravital microscopic observation of the intestinal microcirculation and the measurement of
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bleeding time after the needle puncture were carried-out. In another series, the calculation for blood cell counts and the measurement of blood fluidity using micro channel array flow analyzer (MC-FAN) were performed. Results: The initial event in the microvasculature is the leukocyte adhesion on endothelium. Then, the leukocytes make clusters and the platelets are involved in. These leukocyte-platelet conjugates aggregate and form thrombus. The leukocyte adherence and the microthrombus formation was significantly suppressed with the treatment of 10 mg/kg of rivaroxaban compared to the control group (P< 0.05). While, the bleeding time was significantly extended with the treatment with 10 mg/kg of rivaroxaban (P< 0.01). The blood fluidity was maintained best with the treatment of 10 mg/kg rivaroxaban.
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ACCEPTED MANUSCRIPT Conclusions: Rivaroxaban attenuates the leukocyte-platelet-endothelial interaction, which
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leads to the attenuation of microthrombus formation in a mouse model of diabetes mellitus.
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Key Words
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rivaroxaban, Factor Xa inhibitor, type 2 diabetes mellitus, leukocyte-endothelial interaction,
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venous thrombosis.
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ACCEPTED MANUSCRIPT Introduction
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The connection between acute inflammation and thrombosis formation has widely been perceived. We have previously studied the anti-thrombotic effect of Factor Xa inhibitors in
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acute infection models and reported that they not only inhibit the coagulation system but
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also exert anti-inflammatory effects expressed by decreased levels of proinflammatory
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cytokines and inhibition of leukocyte-endothelial interactions [1-4]. Furthermore, we also
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reported that the anti-inflammatory and anti-thrombotic effects of Factor Xa inhibitor are observed in the non-infectious model [5]. However, since no study has evaluated similar
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effects in a model of chronic vascular inflammation, we planned to evaluate the effects of
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oral administration of Factor Xa inhibitor on vascular inflammation and thrombosis
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formation in a diabetes mellitus model. In diabetes mellitus, advanced glycation end products (AGEs) are thought to play major roles in vascular inflammation and thrombus formation [6]. The inflammatory reaction is provoked through the binding between AGEs and their receptor for advanced glycation end products (RAGE) on the vascular endothelium [7]. The binding of AGEs-RAGE transduces signals and up-regulates the inflammatory responses [8], which leads to the thrombus formation [9, 10]. Indeed, many experimental and clinical studies demonstrated a correlation between hyperglycemia, vascular inflammation and increased risk of thrombosis [11, 12]. Thus, the objective of this study is to examine the anti-inflammatory and antithrombotic the effects of rivaroxaban, a Factor Xa inhibitor, in a diabetes mellitus model of mouse.
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ACCEPTED MANUSCRIPT Methods
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Animal preparation 7 week-old female KK-Ay mice were purchased (Japan Clea Co., Ltd., Tokyo Japan)
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and were utilized for the experiment. All experimental procedures were conducted after
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obtaining the approval of the Ethical Committee for Animal Experiments of Juntendo
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University. Total of 36 mice were randomized to 3 groups (12 mice per group): the first
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group received chow diet supplemented with 5 mg rivaroxaban (Bayer Health Care, Wuppertal, Germany) dissolved with dimethyl sulfoxide (DMSO, Sigma Chemical Co.,
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St.Louis, USA)/kg bodyweight/day for 7 weeks (low-dose group). The second group
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received rat chow supplemented with 10 mg rivaroxaban for 7 weeks (high-dose group) and
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the third group received standard chow diet with DMSO only for 7 weeks (control group). For multiple oral administration, micronized powder rivaroxaban was dissolve in 100% DMSO (20 mg/56 ml), which was then diluted with demineralized water to the final DMSO concentration ≤ 1.0 %.
Intravital microscopic observation In the first series, a total 18 mice (6 mice in each group) were utilized for the intravital microscopic examination [13]. Mice were anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneally), the abdomen was opened by a median incision, an approximately 5 cm segment of jejunum was exteriorized, then the immobilized mesentery was secured on a special stand. Next, 20 minutes of total occlusion of the superior mesenteric vein was
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ACCEPTED MANUSCRIPT applied by a special clip. After a clip was released, approximately 20 µm diameter venules
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were examined by intravital microscopy (Nikon Microphot-FX Microscope, Nikon Co., LTD., Tokyo, Japan). In each animal, six fields in succession were selected and each field
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was recorded for 5 minutes at the speed of 30 frames/second by a high-vision recording
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system (EOS 5D Mark III, Nikon Co., Ltd., Tokyo, Japan). The images obtained were used
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to document and analyze the number of adherent leukocytes. Red blood cell (RBC) velocity
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was calculated by using an off-line video analysis program (Well system Co., Ltd., Tokyo, Japan) for high-speed camera imaging. For the calculation of adherent leukocyte, two
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individuals examined the off-line video and counted the number of static leukocyte in each
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field for 5 minutes, and the average number was calculated. A leukocyte was defined as
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adherent if it remained stationary for more than 15 seconds. Finally, the venule was punctured with a 3 µm glass microneedle and the bleeding time was counted. Blood tests and Micro channel array flow analysis In the second series, a total of 18 KK-Ay mice (6 mice in each group) were utilized. The anesthesia and venous clump was applied similarly as the first series, and then the blood samples were obtained from the inferior vena cava three hours after de-clamp. White blood cell (WBC), platelet and RBC were counted using an automated device for animals (Celltac, MEK-5128; Nihon Kohden Co.,LTD., Tokyo, Japan). The blood glucose level was also measured in the same samples.
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ACCEPTED MANUSCRIPT Using another whole blood sample, blood passage time, the time taken for the passage
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of 50 μL of the mixture (0.9 ml of blood and 0.1 ml of 3.8% sodium citrate solution) of each group was measured by a micro channel array flow analyzer (MC-FAN, Hitachi,
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Tokyo, Japan) with a micro channel array (the array has parallel 8736 slits: size of each slit;
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4.5 μm wide, 30 μm long, 4.5 μm deep). The whole blood passage time required for
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passage of 50 μL of the mixture through the micro channel array was determined under a
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negative pressure of 20 cm H2O following the maker’s instruction [14]. The calibration was performed following the standard method [15]; the standard saline passage time required
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for passage of 100 μL of saline through the micro channel array was 12.0 sec. In order to
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avoid a deviation of the micro channel array, the saline passage time required for 100 μL
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was determined just before each blood sample measurement. The sample blood passage time was calculated from the following equation; whole blood passage time (sec)= observed whole blood passage time (sec) x 12.0 (sec)/observed saline passage time (sec) Statistical analysis
All data are expressed as mean ± standard error. Statistical analysis was performed using one-way analysis of variance. Fisher’s post hoc analysis was performed for the comparison of means between the groups with the statistical software package (StatView 5.0, Abacus Corporation, Berkeley, CA, USA). Statistical differences were deemed significant at a level of P< 0.05.
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ACCEPTED MANUSCRIPT Results
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Intravital microscopic observation The initial intravascular event after de-clamp was the tethering, rolling and adhesion of
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the leukocytes on the venular endothelium. After the release of 20 min venous occlusion,
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the rolling leukocytes lost their transformability and the shape changed from tear-drop to
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round-stiff, then began to stick to the endothelial surface (Fig 1, right column). In the
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control group, the adherent leukocytes assembled and made clusters with platelets (Fig 1, left column), which gradually formed larger conjugates. The expanded leukocyte-platelet
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conjugates sometimes grew to obstructive thrombi, however, since the conjugates were not
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stiff enough, most of them were wiped away. These serial changes were significantly
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suppressed in the high-dose group, while the changes were not remarkable in the low-dose group. The bleeding after the venous puncture was immediately stopped by the platelet aggregation in the control group (Fig. 2, left). In the rivaroxaban groups, the platelet aggregations were smaller and easy to detach, and the bleeding continued longer in the high-dose group (Fig. 2, right). With regard to the adherent cell-count, it was significantly reduced in high-dose group compared to the control (P< 0.05) (Fig 3, left). The RBC velocity was maintained better in the high-dose group (3.55 ± 0.18 /sec) compared to the control (2.57 ± 0.18 /min)(P< 0.01), while the similar effect was not recognized in the low-dose group (Fig 3, middle). The bleeding time after the puncture was significantly elongated in the high-dose group (6.95 ±
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ACCEPTED MANUSCRIPT 0.87 sec) compared to the control and the low-dose group (2.53 ± 0.28 sec, 3.92 ± 0.52 sec)
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(P< 0.01, respectively). Blood cell counts, glucose level and blood fluidity
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The high-dose group demonstrated significantly higher number of the WBC and platelet
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counts compared to the control group (P< 0.01, respectively); in contrast, the difference did
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not reach statistical significance in the low-dose group. In contrast, the count of RBC did
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not change significantly between the groups. The blood glucose levels were approximately 500 mg/dL in each group and there was no difference between the groups (Table 1).
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A summary of the measurement results for each groups’ blood transit time measurements
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is shown in Fig. 4. Compared with the values in the control group, the blood transit times for
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the high-dose group was shortened significantly (52.8±4.9 sec vs. 47.6±4.2 sec, P< 0.01).
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ACCEPTED MANUSCRIPT Discussion
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KK-Ay mouse exhibits obesity and insulin resistant hyperglycemia, including high levels of HbA1c and albuminuria [16], therefore, utilized widely as a type 2 diabetes mellitus
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model [17]. Previously, we examined the pathogenesis of venous thrombosis in KK-Ay
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mouse and reported that thrombus formation was initiated by the activation in
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leukocyte-endothelial interaction in the venule and leukocyte-platelet conjugate formation
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[18]. As for the mechanisms of thrombosis in hyperglycemia, Joshi et al. [19] reported that hyperglycemia mimics a state of constitutively active pro-inflammatory condition. They also
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reported that glucose level modulate IL-6 mediated inflammatory responses. In the current
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study, the blood glucose level reached approximately 500 mg/dL in each group, and the
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increase in adhesion of the leukocytes onto the vascular endothelium and the increase in formation of leukocyte-platelet conjugate were observed after ischemia/reperfusion and these sequences were effectively attenuated by high-dose rivaroxaban. Rivaroxaban is an orally active direct coagulation factor Xa inhibitor. In phase III clinical trials, rivaroxaban regimens reduced the rates of venous thromboembolism in patients after total hip or knee arthroplasty compared with enoxaparin regimens without significant differences in rates of major bleeding, showing that rivaroxaban has a favorable benefit-to-risk profile [20, 21]. As for the dose setting, Zhou et al. [22] reported that 5 mg/kg/day of rivaroxaban could stabilize the atherosclerotic plaque in a mouse model. In addition, Perzborn et al. [23]
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ACCEPTED MANUSCRIPT demonstrated the dose-dependent antithrombotic activity of rivaroxaban in a mouse model
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of thrombosis. Therefore, we set 5 mg/kg (low-dose) and 10 mg/kg (high-dose) in this study.
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It is well known that persistent chronic vascular inflammation accelerates thrombus
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formation in atherosclerotic diseases, while only a few studies have reported that chronic
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inflammation of the vein contributes to the formation of venous thrombosis [24, 25]. In the
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present study, the venous thrombi were formed after the short period ischemia, which was not occurred in the non-diabetic mouse. We speculate that thrombus was easily formed by
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additional stimulation such as hypoxia and decreased blood flow because the persistent
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vascular inflammation existed. In addition to the endothelial damage, activation in
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leukocyte, platelet and coagulation system will be involved in the development of venous thrombosis. It is reported that the activation of protease-activated receptors (PARs) by thrombin generation play roles in the regulation of adhesion molecule expression on the leukocyte [26]. Rivaroxaban could suppress the leukocyte adhesion through suppressing the thrombin formation and inhibition of Factor Xa. Other than the above mechanism, anti-inflammatory effect could be involved. Since RAGE is a multi-ligand receptor and its activation is attenuated by anticoagulants [27], Factor Xa inhibitor might affect the inflammation through this mechanism. Currently, there are several lines of evidence supporting the interplay between coagulation and inflammation in the propagation of various disease processes, including venous thromboembolism (VTE) and inflammatory diseases
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ACCEPTED MANUSCRIPT [28]. Indirect Factor Xa inhibitors such as low-molecular-weight heparin (LMWH) and
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fondaparinux represent improvements over the traditional drugs such as unfractionated heparin for acute treatment of VTE [29]. From the results of the present study, we assume
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that direct Factor Xa inhibitor rivaroxaban could be a choice for the prevention and the
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treatment of thrombosis in chronic vascular inflammatory disease including diabetes
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mellitus.
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The associated finding of a decrease of WBC and platelet counts in circulating blood may support the activation of these cells, and they were maintained best with the treatment
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of high-dose rivaroxaban. These attenuations in WBC and platelet count changes suggest
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the preventive effects of thrombosis.
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The effect of rivaroxan on blood fluidity was examined by a micro channel array flow analyzer MC-FAN. This system has a unique feature in assuming the microthrombus formation in blood flow through the minute watercourses produced on a siliconized chip [30] and utilized in numbers of studies [31-33] As a result, high-dose of rivaroxaban showed a significant reduction of the passage time, which reflects the decreased thrombus formation. The benefit from rivaroxaban is, in addition to its primary anticoagulant function, expressed
through
anti-inflammatory
effects
including
the
regulation
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leukocyte-endothelial and leukocyte-platelet interactions in the current type 2 diabetic model. However, since the bleeding risk increases with rivaroxaban, risk-benefit balance should be carefully considered.
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ACCEPTED MANUSCRIPT Finally, the limitations of this study are; first, plasma levels of rivaroxaban have not
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been assessed. Thus, it remains open if the results from the current study were applicable to the clinical issue. Second, we could not demonstrate the direct effects of rivaroxaban on
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AGEs-RAGE system. The mechanisms of leukocyte adhesion onto the endothelium are not
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questions need to be addressed in the next step.
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fully elucidated, and we still do not know how does Factor Xa inhibition affect. These
Conclusion
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In the diabetes mellitus mouse model, rivaroxaban, an oral Factor Xa inhibitor,
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attenuated thrombus formation by blocking the leukocyte-endothelial and leukocyte-platelet
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interactions. These anti-thrombotic and anti-inflammatory effects would be favorable for the prevention and the treatment for venous thromboembolism.
Competing Interests
This work was financially supported by Bayer Health Care. The authors state that they have no other conflict of interest.
Author’s contribution
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ACCEPTED MANUSCRIPT Iba T., Tabe Y. and Ohsaka A. designed the experiment and wrote the manuscript. Iba T.
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performed the animal experiment. Aihara K., Yamada A. and Nagayama M. participated in
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the data analysis.
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ACCEPTED MANUSCRIPT References
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Legends
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Fig. 1 Microscopic view of the venule after de-clamp of mesenteric vein Figures of mesenteric microcirculation in the control group (left panels) and the
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high-dose group (right panels). The leukocyte-platelet conjugate (white arrows) is formed
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in the control group (above: x20, below: x40). Rolling and adherent leukocytes (white
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arrows) were observed, but leukocyte-platelet conjugates are rarely observed in the
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high-dose group.
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Fig. 2 Microscopic view of the venule after the puncture
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Figures after the puncture in the control group (left panel) and the high-dose (right
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panel) group. The thrombus (white arrows) is formed at the puncture site and the bleeding area (black arrowheads) was limited in the control group. In contrast, bleeding spread over the scene in the high-dose group. The thrombus (white arrows) is formed at the puncture site (shite arrows), but it is not firm enough and splits apart (black arrows).
Fig. 3 WBC adhesion, RBC velocity and bleeding time Adhesion of the leukocyte was significantly suppressed in in the high-dose group compared to the control group (left). RBC velocity was maintained best in the control group (middle). Bleeding time after the venule puncture was significantly elongated in the
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ACCEPTED MANUSCRIPT high-dose group (right). The bar graphs express mean + standard error. *: P< 0.05, **: P
S0049-3848(13)00549-5 doi: 10.1016/j.thromres.2013.11.013 TR 5290
To appear in:
Thrombosis Research
Received date: Revised date: Accepted date:
26 June 2013 10 November 2013 18 November 2013
Please cite this article as: Iba Toshiaki, Aihara Koichiro, Yamada Atushi, Nagayama Masataka, Tabe Yoko, Ohsaka Akimichi, Rivaroxaban attenuates leukocyte adhesion in the microvasculature and thrombus formation in an experimental mouse model of type 2 diabetes mellitus, Thrombosis Research (2013), doi: 10.1016/j.thromres.2013.11.013
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Revised on Nov. 10, 2013
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Original article
Rivaroxaban attenuates leukocyte adhesion in the microvasculature and
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thrombus formation in an experimental mouse model of type 2 diabetes
SC
mellitus
1)
*, Koichiro Aihara, M.D.
MA
Toshiaki Iba, M.D.
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Running Head: rivaroxaban attenuates vascular inflammation
1)
, Atushi Yamada, M.D.
1)
, Masataka
Department of Emergency and Disaster Medicine
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1)
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Nagayama, M.D. 1), Yoko Tabe, M.D. 2) and Akimichi Ohsaka, M.D. 3)
2)
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Juntendo University Graduate School of Medicine Department of Clinical Laboratory Medicine Juntendo University Graduate School of Medicine 3)
Department of Transfusion Medicine and Stem Cell Regulation Juntendo University Graduate School of Medicine
* : Professor of Emergency and Disaster Medicine
Correspondence to: Toshiaki Iba, MD. 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
E-mail: [email protected] tel: 81-3-3813-3111 (X: 5818) fax: 81-3-3813-5431
1
ACCEPTED MANUSCRIPT Abstract Introduction: Thrombosis is a major complication in diabetes mellitus. Since Factor Xa
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inhibitors are not only inhibit the coagulation system but also attenuate the
RI
leukocyte-endothelial interaction in acute inflammation models, the purpose of this study is
SC
to confirm the similar effects of rivaroxaban in a mouse model of type 2 diabetes mellitus.
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Materials and Methods: In the treatment groups, either 5 or 10 mg/kg of rivaroxaban dissolved in DMSO was orally given to KK-Ay mice for 7 weeks (n= 6 in each group).
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KK-Ay mice fed by chow containing DMSO without rivaroxaban for 7 weeks were served
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for the control group (n= 6). Following clamping of the mesenteric vein for 20 minutes,
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intravital microscopic observation of the intestinal microcirculation and the measurement of
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bleeding time after the needle puncture were carried-out. In another series, the calculation for blood cell counts and the measurement of blood fluidity using micro channel array flow analyzer (MC-FAN) were performed. Results: The initial event in the microvasculature is the leukocyte adhesion on endothelium. Then, the leukocytes make clusters and the platelets are involved in. These leukocyte-platelet conjugates aggregate and form thrombus. The leukocyte adherence and the microthrombus formation was significantly suppressed with the treatment of 10 mg/kg of rivaroxaban compared to the control group (P< 0.05). While, the bleeding time was significantly extended with the treatment with 10 mg/kg of rivaroxaban (P< 0.01). The blood fluidity was maintained best with the treatment of 10 mg/kg rivaroxaban.
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ACCEPTED MANUSCRIPT Conclusions: Rivaroxaban attenuates the leukocyte-platelet-endothelial interaction, which
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leads to the attenuation of microthrombus formation in a mouse model of diabetes mellitus.
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Key Words
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rivaroxaban, Factor Xa inhibitor, type 2 diabetes mellitus, leukocyte-endothelial interaction,
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venous thrombosis.
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ACCEPTED MANUSCRIPT Introduction
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The connection between acute inflammation and thrombosis formation has widely been perceived. We have previously studied the anti-thrombotic effect of Factor Xa inhibitors in
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acute infection models and reported that they not only inhibit the coagulation system but
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also exert anti-inflammatory effects expressed by decreased levels of proinflammatory
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cytokines and inhibition of leukocyte-endothelial interactions [1-4]. Furthermore, we also
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reported that the anti-inflammatory and anti-thrombotic effects of Factor Xa inhibitor are observed in the non-infectious model [5]. However, since no study has evaluated similar
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effects in a model of chronic vascular inflammation, we planned to evaluate the effects of
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oral administration of Factor Xa inhibitor on vascular inflammation and thrombosis
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formation in a diabetes mellitus model. In diabetes mellitus, advanced glycation end products (AGEs) are thought to play major roles in vascular inflammation and thrombus formation [6]. The inflammatory reaction is provoked through the binding between AGEs and their receptor for advanced glycation end products (RAGE) on the vascular endothelium [7]. The binding of AGEs-RAGE transduces signals and up-regulates the inflammatory responses [8], which leads to the thrombus formation [9, 10]. Indeed, many experimental and clinical studies demonstrated a correlation between hyperglycemia, vascular inflammation and increased risk of thrombosis [11, 12]. Thus, the objective of this study is to examine the anti-inflammatory and antithrombotic the effects of rivaroxaban, a Factor Xa inhibitor, in a diabetes mellitus model of mouse.
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ACCEPTED MANUSCRIPT Methods
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Animal preparation 7 week-old female KK-Ay mice were purchased (Japan Clea Co., Ltd., Tokyo Japan)
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and were utilized for the experiment. All experimental procedures were conducted after
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obtaining the approval of the Ethical Committee for Animal Experiments of Juntendo
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University. Total of 36 mice were randomized to 3 groups (12 mice per group): the first
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group received chow diet supplemented with 5 mg rivaroxaban (Bayer Health Care, Wuppertal, Germany) dissolved with dimethyl sulfoxide (DMSO, Sigma Chemical Co.,
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St.Louis, USA)/kg bodyweight/day for 7 weeks (low-dose group). The second group
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received rat chow supplemented with 10 mg rivaroxaban for 7 weeks (high-dose group) and
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the third group received standard chow diet with DMSO only for 7 weeks (control group). For multiple oral administration, micronized powder rivaroxaban was dissolve in 100% DMSO (20 mg/56 ml), which was then diluted with demineralized water to the final DMSO concentration ≤ 1.0 %.
Intravital microscopic observation In the first series, a total 18 mice (6 mice in each group) were utilized for the intravital microscopic examination [13]. Mice were anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneally), the abdomen was opened by a median incision, an approximately 5 cm segment of jejunum was exteriorized, then the immobilized mesentery was secured on a special stand. Next, 20 minutes of total occlusion of the superior mesenteric vein was
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ACCEPTED MANUSCRIPT applied by a special clip. After a clip was released, approximately 20 µm diameter venules
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were examined by intravital microscopy (Nikon Microphot-FX Microscope, Nikon Co., LTD., Tokyo, Japan). In each animal, six fields in succession were selected and each field
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was recorded for 5 minutes at the speed of 30 frames/second by a high-vision recording
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system (EOS 5D Mark III, Nikon Co., Ltd., Tokyo, Japan). The images obtained were used
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to document and analyze the number of adherent leukocytes. Red blood cell (RBC) velocity
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was calculated by using an off-line video analysis program (Well system Co., Ltd., Tokyo, Japan) for high-speed camera imaging. For the calculation of adherent leukocyte, two
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individuals examined the off-line video and counted the number of static leukocyte in each
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field for 5 minutes, and the average number was calculated. A leukocyte was defined as
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adherent if it remained stationary for more than 15 seconds. Finally, the venule was punctured with a 3 µm glass microneedle and the bleeding time was counted. Blood tests and Micro channel array flow analysis In the second series, a total of 18 KK-Ay mice (6 mice in each group) were utilized. The anesthesia and venous clump was applied similarly as the first series, and then the blood samples were obtained from the inferior vena cava three hours after de-clamp. White blood cell (WBC), platelet and RBC were counted using an automated device for animals (Celltac, MEK-5128; Nihon Kohden Co.,LTD., Tokyo, Japan). The blood glucose level was also measured in the same samples.
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ACCEPTED MANUSCRIPT Using another whole blood sample, blood passage time, the time taken for the passage
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of 50 μL of the mixture (0.9 ml of blood and 0.1 ml of 3.8% sodium citrate solution) of each group was measured by a micro channel array flow analyzer (MC-FAN, Hitachi,
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Tokyo, Japan) with a micro channel array (the array has parallel 8736 slits: size of each slit;
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4.5 μm wide, 30 μm long, 4.5 μm deep). The whole blood passage time required for
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passage of 50 μL of the mixture through the micro channel array was determined under a
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negative pressure of 20 cm H2O following the maker’s instruction [14]. The calibration was performed following the standard method [15]; the standard saline passage time required
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for passage of 100 μL of saline through the micro channel array was 12.0 sec. In order to
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avoid a deviation of the micro channel array, the saline passage time required for 100 μL
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was determined just before each blood sample measurement. The sample blood passage time was calculated from the following equation; whole blood passage time (sec)= observed whole blood passage time (sec) x 12.0 (sec)/observed saline passage time (sec) Statistical analysis
All data are expressed as mean ± standard error. Statistical analysis was performed using one-way analysis of variance. Fisher’s post hoc analysis was performed for the comparison of means between the groups with the statistical software package (StatView 5.0, Abacus Corporation, Berkeley, CA, USA). Statistical differences were deemed significant at a level of P< 0.05.
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ACCEPTED MANUSCRIPT Results
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Intravital microscopic observation The initial intravascular event after de-clamp was the tethering, rolling and adhesion of
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the leukocytes on the venular endothelium. After the release of 20 min venous occlusion,
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the rolling leukocytes lost their transformability and the shape changed from tear-drop to
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round-stiff, then began to stick to the endothelial surface (Fig 1, right column). In the
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control group, the adherent leukocytes assembled and made clusters with platelets (Fig 1, left column), which gradually formed larger conjugates. The expanded leukocyte-platelet
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conjugates sometimes grew to obstructive thrombi, however, since the conjugates were not
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stiff enough, most of them were wiped away. These serial changes were significantly
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suppressed in the high-dose group, while the changes were not remarkable in the low-dose group. The bleeding after the venous puncture was immediately stopped by the platelet aggregation in the control group (Fig. 2, left). In the rivaroxaban groups, the platelet aggregations were smaller and easy to detach, and the bleeding continued longer in the high-dose group (Fig. 2, right). With regard to the adherent cell-count, it was significantly reduced in high-dose group compared to the control (P< 0.05) (Fig 3, left). The RBC velocity was maintained better in the high-dose group (3.55 ± 0.18 /sec) compared to the control (2.57 ± 0.18 /min)(P< 0.01), while the similar effect was not recognized in the low-dose group (Fig 3, middle). The bleeding time after the puncture was significantly elongated in the high-dose group (6.95 ±
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ACCEPTED MANUSCRIPT 0.87 sec) compared to the control and the low-dose group (2.53 ± 0.28 sec, 3.92 ± 0.52 sec)
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(P< 0.01, respectively). Blood cell counts, glucose level and blood fluidity
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The high-dose group demonstrated significantly higher number of the WBC and platelet
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counts compared to the control group (P< 0.01, respectively); in contrast, the difference did
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not reach statistical significance in the low-dose group. In contrast, the count of RBC did
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not change significantly between the groups. The blood glucose levels were approximately 500 mg/dL in each group and there was no difference between the groups (Table 1).
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A summary of the measurement results for each groups’ blood transit time measurements
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is shown in Fig. 4. Compared with the values in the control group, the blood transit times for
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the high-dose group was shortened significantly (52.8±4.9 sec vs. 47.6±4.2 sec, P< 0.01).
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ACCEPTED MANUSCRIPT Discussion
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KK-Ay mouse exhibits obesity and insulin resistant hyperglycemia, including high levels of HbA1c and albuminuria [16], therefore, utilized widely as a type 2 diabetes mellitus
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model [17]. Previously, we examined the pathogenesis of venous thrombosis in KK-Ay
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mouse and reported that thrombus formation was initiated by the activation in
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leukocyte-endothelial interaction in the venule and leukocyte-platelet conjugate formation
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[18]. As for the mechanisms of thrombosis in hyperglycemia, Joshi et al. [19] reported that hyperglycemia mimics a state of constitutively active pro-inflammatory condition. They also
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reported that glucose level modulate IL-6 mediated inflammatory responses. In the current
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study, the blood glucose level reached approximately 500 mg/dL in each group, and the
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increase in adhesion of the leukocytes onto the vascular endothelium and the increase in formation of leukocyte-platelet conjugate were observed after ischemia/reperfusion and these sequences were effectively attenuated by high-dose rivaroxaban. Rivaroxaban is an orally active direct coagulation factor Xa inhibitor. In phase III clinical trials, rivaroxaban regimens reduced the rates of venous thromboembolism in patients after total hip or knee arthroplasty compared with enoxaparin regimens without significant differences in rates of major bleeding, showing that rivaroxaban has a favorable benefit-to-risk profile [20, 21]. As for the dose setting, Zhou et al. [22] reported that 5 mg/kg/day of rivaroxaban could stabilize the atherosclerotic plaque in a mouse model. In addition, Perzborn et al. [23]
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ACCEPTED MANUSCRIPT demonstrated the dose-dependent antithrombotic activity of rivaroxaban in a mouse model
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of thrombosis. Therefore, we set 5 mg/kg (low-dose) and 10 mg/kg (high-dose) in this study.
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It is well known that persistent chronic vascular inflammation accelerates thrombus
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formation in atherosclerotic diseases, while only a few studies have reported that chronic
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inflammation of the vein contributes to the formation of venous thrombosis [24, 25]. In the
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present study, the venous thrombi were formed after the short period ischemia, which was not occurred in the non-diabetic mouse. We speculate that thrombus was easily formed by
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additional stimulation such as hypoxia and decreased blood flow because the persistent
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vascular inflammation existed. In addition to the endothelial damage, activation in
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leukocyte, platelet and coagulation system will be involved in the development of venous thrombosis. It is reported that the activation of protease-activated receptors (PARs) by thrombin generation play roles in the regulation of adhesion molecule expression on the leukocyte [26]. Rivaroxaban could suppress the leukocyte adhesion through suppressing the thrombin formation and inhibition of Factor Xa. Other than the above mechanism, anti-inflammatory effect could be involved. Since RAGE is a multi-ligand receptor and its activation is attenuated by anticoagulants [27], Factor Xa inhibitor might affect the inflammation through this mechanism. Currently, there are several lines of evidence supporting the interplay between coagulation and inflammation in the propagation of various disease processes, including venous thromboembolism (VTE) and inflammatory diseases
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ACCEPTED MANUSCRIPT [28]. Indirect Factor Xa inhibitors such as low-molecular-weight heparin (LMWH) and
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fondaparinux represent improvements over the traditional drugs such as unfractionated heparin for acute treatment of VTE [29]. From the results of the present study, we assume
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that direct Factor Xa inhibitor rivaroxaban could be a choice for the prevention and the
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treatment of thrombosis in chronic vascular inflammatory disease including diabetes
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mellitus.
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The associated finding of a decrease of WBC and platelet counts in circulating blood may support the activation of these cells, and they were maintained best with the treatment
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of high-dose rivaroxaban. These attenuations in WBC and platelet count changes suggest
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the preventive effects of thrombosis.
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The effect of rivaroxan on blood fluidity was examined by a micro channel array flow analyzer MC-FAN. This system has a unique feature in assuming the microthrombus formation in blood flow through the minute watercourses produced on a siliconized chip [30] and utilized in numbers of studies [31-33] As a result, high-dose of rivaroxaban showed a significant reduction of the passage time, which reflects the decreased thrombus formation. The benefit from rivaroxaban is, in addition to its primary anticoagulant function, expressed
through
anti-inflammatory
effects
including
the
regulation
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leukocyte-endothelial and leukocyte-platelet interactions in the current type 2 diabetic model. However, since the bleeding risk increases with rivaroxaban, risk-benefit balance should be carefully considered.
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ACCEPTED MANUSCRIPT Finally, the limitations of this study are; first, plasma levels of rivaroxaban have not
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been assessed. Thus, it remains open if the results from the current study were applicable to the clinical issue. Second, we could not demonstrate the direct effects of rivaroxaban on
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AGEs-RAGE system. The mechanisms of leukocyte adhesion onto the endothelium are not
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questions need to be addressed in the next step.
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fully elucidated, and we still do not know how does Factor Xa inhibition affect. These
Conclusion
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In the diabetes mellitus mouse model, rivaroxaban, an oral Factor Xa inhibitor,
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attenuated thrombus formation by blocking the leukocyte-endothelial and leukocyte-platelet
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interactions. These anti-thrombotic and anti-inflammatory effects would be favorable for the prevention and the treatment for venous thromboembolism.
Competing Interests
This work was financially supported by Bayer Health Care. The authors state that they have no other conflict of interest.
Author’s contribution
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ACCEPTED MANUSCRIPT Iba T., Tabe Y. and Ohsaka A. designed the experiment and wrote the manuscript. Iba T.
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performed the animal experiment. Aihara K., Yamada A. and Nagayama M. participated in
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the data analysis.
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ACCEPTED MANUSCRIPT References
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Legends
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Fig. 1 Microscopic view of the venule after de-clamp of mesenteric vein Figures of mesenteric microcirculation in the control group (left panels) and the
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high-dose group (right panels). The leukocyte-platelet conjugate (white arrows) is formed
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in the control group (above: x20, below: x40). Rolling and adherent leukocytes (white
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arrows) were observed, but leukocyte-platelet conjugates are rarely observed in the
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high-dose group.
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Fig. 2 Microscopic view of the venule after the puncture
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Figures after the puncture in the control group (left panel) and the high-dose (right
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panel) group. The thrombus (white arrows) is formed at the puncture site and the bleeding area (black arrowheads) was limited in the control group. In contrast, bleeding spread over the scene in the high-dose group. The thrombus (white arrows) is formed at the puncture site (shite arrows), but it is not firm enough and splits apart (black arrows).
Fig. 3 WBC adhesion, RBC velocity and bleeding time Adhesion of the leukocyte was significantly suppressed in in the high-dose group compared to the control group (left). RBC velocity was maintained best in the control group (middle). Bleeding time after the venule puncture was significantly elongated in the
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ACCEPTED MANUSCRIPT high-dose group (right). The bar graphs express mean + standard error. *: P< 0.05, **: P
