In Vivo Monitoring of Venous Thrombosis In Mice Using Ultrasonography

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4214-4214
Author(s):  
Meghedi Aghourian ◽  
Catherine Lemarie ◽  
Mark Blostein
Keyword(s):  
Venous Thrombosis ◽  
Murine Model ◽  
Conflicts Of Interest ◽  
Genetically Modify ◽  
Vena Cava ◽  
Venous Stasis ◽  
Clinical Aspects ◽  
Reliable Technique ◽  
Over Time

Abstract Abstract 4214 Deep venous thrombosis is an important cause of morbidity and mortality in clinical medicine. There has been extensive research dedicated to the clinical aspects of venous thrombosis, especially with regards to its diagnosis and treatment. However, animal models studying this phenomenon are scarce and, in most cases, very crude, relying on sacrificing animals to excise the formed thrombi. Developing an in vivo murine model of venous thrombosis, detecting and monitoring thrombi non-invasively as is done in humans can be a powerful tool given our ability to genetically modify the murine genome. Therefore, we developed such a murine model using the Vevo770®, a microimaging ultrasound system previously developed to study the arterial circulation of mice. Two different thrombosis models were employed to generate clots in the inferior vena cava (IVC) of wild type C57Bl6 mice: 1) ligation of the IVC to generate venous stasis and 2) application of Ferric Chloride (FeCl3) to the outer layer of the IVC to injure the endothelium. Using both of these techniques, adequate thromboses were generated in the IVCs of mice as determined pathologically. Other mice were allowed to recover after surgery, and the development of venous thrombosis was assessed by ultrasonography using the Vevo 770®. In order to assess the precision of clot measurements using this novel technique, we then sacrificed the mice and excised the clots. In both models, the measurement of the clot pathologically correlates favorably (R2= 0, 9116 for the ligation model, and R2 = 0,905 for the FeCl3 model) with measurements done by ultrasonography (n=20 for the ligation model, and n=5 for the FeCl3 injury model). In the ligation model, a thrombus develops less than an hour after ligation of the IVC, and the size of the clot increases over time. For example, five hours after the ligation of the IVC, a clot develops and has a cross sectional area of 4,5 mm2. The clot size increases significantly (p=0.001) over time to 6.2 mm2 at 24 hours post ligation (n=20). Treatment of these mice with an anticoagulant (dalteparin at a dose of 200 u/kg) prior to the procedure prevented the development of IVC thrombosis as determined by ultrasonagraphy. These data suggest that the Vevo770® can be used as a reliable technique for the non-invasive assessment of venous thrombosis in mice. Developing a murine model for thrombosis using more accurate, and clinically more relevant techniques such as ultrasonography, is a step towards better understanding the pathophysiology of venous thromboembolism. Figure 1. Clot length correlation using histology and ultrasonography, 24 hrs post ligation of the IVC in 20 mice. R2= 0,9116. Figure 1. Clot length correlation using histology and ultrasonography, 24 hrs post ligation of the IVC in 20 mice. R2= 0,9116. Disclosures: No relevant conflicts of interest to declare.

In Vivo Monitoring of Venous Thrombosis in Mice.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5061-5061
Author(s):  
Meghedi Aghourian ◽  
Mark Blostein
Keyword(s):  
Venous Thromboembolism ◽  
Venous Thrombosis ◽  
Murine Model ◽  
Conflicts Of Interest ◽  
Vena Cava ◽  
Reliable Technique ◽  
Non Invasive ◽  
Post Surgery ◽  
Ultrasound Technology

Abstract Abstract 5061 Venous thromboembolism afflicts 117 people per 100,000 each year and is an important cause of morbidity and mortality. There has been extensive research dedicated to the clinical aspect of venous thrombosis, especially with regards to its diagnosis and treatment. However, animal models studying this phenomenon are scarce and, in most cases, very crude. Developing a murine model of venous thrombosis using techniques similar to the ones used to detect thrombosis in humans can be a constructive step in studying this phenomenon in more detail. The model developed in our lab uses ultrasound imaging to visualize venous clots in the Inferior Vena Cava (IVC) of mice, allowing for precise measurements of the formed clot. Ligation of the IVC is one of the well established models for studying thrombosis in mice. We ligated the IVC of wild type C57B6 mice, and allowed them to recover. We then followed clot formation at several time points after the operation using micro-ultrasonography, the Vevo 770®, a novel imaging ultrasound technology designed to monitor murine vasculature. To assess the precision of the clot measurements, we then sacrificed the mice, and dissected out the thrombi in order to precisely measure and weigh them. A thrombosis develops only after 5 hours of ligation post surgery when a clot is visualized in the IVC. The clot increases slightly over the next 24 hours. The measurements of the clot after dissection correlates favourably with the measurements done by ultrasonagraphy using the Vevo770®. These data suggest that the Vevo770® can be used as a reliable technique for non-invasive assessment of venous thromboembolism in mice. Developing a murine model for thrombosis using more accurate, and clinically more relevant techniques such as ultrasonography, is a step towards better understanding and treatment of venous thromboembolism. Disclosures No relevant conflicts of interest to declare.

Leukocyte XII Regulates Venous Thrombosis Risk

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 238-238
Author(s):  
Evi X. Stavrou ◽  
Chao Fang ◽  
Alona A. Merkulova ◽  
Lalitha V. Nayak ◽  
Howard Meyerson ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Venous Thrombosis ◽  
Conflicts Of Interest ◽  
Human Peripheral Blood ◽  
Vena Cava ◽  
Leukocyte Migration ◽  
Venous Stasis ◽  
Factor Xii ◽  
Fibrin Network

Abstract Introduction: Previous studies show that Factor XII (XII) participates in the inflammatory response. XII regulates the expression of monocyte FcγII receptor and stimulates monocytes and macrophages to release interleukin (IL)-1 and IL-6. XII deficient patients have reduced leukocyte migration into skin windows. In vitro, purified XIIa corrects neutrophil aggregation and degranulation defects in XII-deficient plasma. Recent studies show that leukocytes initiate and propagate venous thrombosis in vivo. We examined the contribution of XII in the inflammatory response and venous thrombosis. Methods & Results: Sterile punch biopsy wounds were created on wild type (WT) and F12-/- mice. On Days 2 and 5, there was a ~3-fold decrease in CD11b-stained cells in F12-/- woundsvs. WT. On the thioglycolate (TG)-induced sterile peritonitis assay, lavage fluid from F12-/- mice contained significantly less peritoneal exudative cells (PEC)] on days 1 and 7, (p<0.008). To determine the contribution of XII in WBC function, we used XII siRNA (Alnylam Pharmaceuticals) to create plasma XII deficiency in WT mice. After tail vein injection, plasma XII was reduced to < 5% within 24 h (T1/2 plasma XII: 6.7 h). In the TG assay, even though plasma XII is decreased to less than 5%, PEC migration is the same as in WT mice. These data suggest that the reduced leukocyte migration observed in F12-/- mice is related to altered leukocyte function. On adoptive bone marrow (BM) transplantation (BMT) experiments, WT BM transplanted into KO hosts corrects the leukocyte migration defect on the TG assay. These data suggest that there is a pool of XII associated with BM cells that is functionally distinct than plasma-derived, hepatic XII. F12 cDNA is found in leukocytes and shares sequence homology to hepatic XII. Immunofluorescence confirms XII antigen on murine BM-derived and human peripheral blood WBCs. No XII antigen is observed in BM-derived leukocytes from F12-/- mice. When WBC are activated with fMLF, XII antigen translocates to the external membrane. F12-/- PMNs have reduced chemotaxis to fMLF and adherence to several integrin-binding glycoproteins. pAktS473 mediates neutrophil cell migration, integrin activation, and cytoskeletal assembly. Normal and F12-/- PMNs exhibit pAktS473 in response to fMLF and XII. Histologically, F12-/- wounds show a smaller wound gap and a greater percentage of wound re-epithelialization than WT controls. Inferior vena cava (IVC) thrombosis induced by 90% restriction to flow at 24h contains a smaller thrombus in F12-/- than WT mice (p<0.04). Histologically, IVC thrombi from WT mice contain abundant neutrophils that are adherent to the wall and trapped within a dense fibrin network (Fig 1). siRNA treatment results in less-occlusive thrombi (n.s) with an adequate neutrophil content but a finer fibrin network (Fig 1). F12-/- thrombi are non-occlusive and contain significantly less adherent neutrophils (Fig 1). XII itself is integrally a part of neutrophil extracellular traps (NETs) in the forming thrombus and F12-/- mice have reduced NETs at sites of occlusion. WT BM transplanted into F12-/- hosts corrects the thrombus weight and degree of inflammation in F12-/- mice to normal. Likewise, F12-/- BM into WT hosts, reduces thrombus weight and degree of inflammation. Conclusions: Leukocyte XII has a dual role in neutrophil function. We hypothesize that signaling by leukocyte XII contributes to neutrophil trafficking in sites of inflammation and venous stasis. At these sites, neutrophils become indispensable for activation of both the extrinsic and intrinsic pathways of coagulation during the early formation of intraluminal fibrin and for subsequent thrombus propagation by NETs and the activation of circulating XII. Defining the signaling pathway of XII in leukocytes will further our understanding as to the mechanism(s) by which these cells cooperate to initiate and propagate venous thrombosis in vivo. Disclosures No relevant conflicts of interest to declare.

Abstract 32: Ly6C Lo Monocyte/Macrophages Are Essential for Thrombus Resolution in a Murine Model of Venous Thrombosis

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Andrew S Kimball ◽  
Cathy Luke ◽  
Qing Cai ◽  
Andrea Obi ◽  
Farouc Jaffer ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Murine Model ◽  
Inferior Vena ◽  
Tissue Injury ◽  
Vena Cava ◽  
Venous Reflux ◽  
Western Immunoblotting ◽  
Vein Wall ◽  
Wall Injury

Venous thrombosis (VT) results in vein wall injury by promoting inflammation and fibrosis leading to venous reflux, swelling, pain, and potentially, recurrent thrombosis. While prior work has shown that infiltrating leukocytes are important for VT resolution, as of yet, the precise roles of different leukocyte subsets are not well understood. Monocyte/macrophages (Mo/MΦs) are essential for the repair and resolution of tissue injury in other models, and come in inflammatory (Ly6C Hi ) or pro-resolution (Ly6C Lo ) subtypes. We hypothesized that infiltrating Mo/MΦs would be critical to VT resolution. In order to study this in vivo , we utilized a conditional macrophage depletion technique, using CD11b-DTR mice, to examine the effects of Mo/MΦs in a murine model of stasis VT by inferior vena cava ligation. Administration of 10ng/g diphtheria toxoid (DTx) every 48 hours by intra-peritoneal injection in CD11b-DTR mice resulted in an 89% and 55% decrease in circulating monocytes at 24hrs and 48hrs, respectively. When compared to saline controls, DTx injection had no effects on thrombogenic response or IVC thrombus cell populations in C57BL/6 control mice. At 8 days’ post-ligation, DTx treated CD11b-DTR mice had preferentially decreased vein wall-thrombus Ly6C Lo Mo/MΦs as compared with controls. DTx treated mice had significantly larger thrombi (1.7-fold) and less TGF-β, FSP-1, and plasminogen by western immunoblotting (all P-values ≤ 0.01). Consistent with a reduction in Ly6C Lo Mo/MΦs was a significant decrease in cellular TGF-β by intra-cellular flow cytometry. These findings suggest that Ly6C Lo Mo/MΦs are essential for normal VT resolution and may promote thrombus resolution via a plasminogen-mediated mechanism.

The Role of gas6 in Venous Thrombosis In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3057-3057
Author(s):  
Richard Robins ◽  
Peter Carmeliet ◽  
Mark Blostein
Keyword(s):  
Bone Marrow ◽  
Venous Thrombosis ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Recovery Period ◽  
Vena Cava ◽  
Specific Gene ◽  
Combined Genotypes

Abstract Abstract 3057 Poster Board II-1033 Gas6 is the vitamin-K dependent protein product of growth arrest specific gene 6. A genetic deficiency of this protein protects mice against experimentally induced thrombosis without causing a bleeding diathesis. Protection from thrombosis results from a deficiency in platelet aggregation and secretion. In addition to being expressed by platelets, Gas6 and its receptors are also expressed by vascular cells including the endothelium, an organ known to play a role in the hemostatic balance. While endothelial Gas6 has been shown to promote inflammation and cell survival, it remains unknown if it contributes to the pathophysiology of venous thrombosis. To answer this question, we employed a bone marrow transplantation (BMT) strategy using wild type and Gas6 null mice to create chimeric mice with combined genotypes in the vascular and platelet compartments. Mice were exposed to a dose of radiation optimized to maximize both survival and ablation of recipient marrow. Irradiated mice were then infused with bone marrow cells isolated from the femurs and tibias of donor mice and were allowed a one month recovery period for hematologic reconstitution. Success of marrow uptake was confirmed by PCR. They were then subjected to the Ferric Chloride model of venous thrombosis in the Inferior Vena Cava (IVC). Four groups of transplanted mice were studied. Results from these BMT experiment show a contributing effect by both endothelial as well as platelet Gas6 to thrombus formation (n=8, p<0.01). Mice with combined genotypes (Gas6-/- into WT and WT into Gas6 -/-) show an intermediate thrombus weight suggesting that both vascular and platelet derived Gas6 are both responsible for thrombosis pathology. Therefore, Gas6 at both sites could be potential targets in treating venous thrombosis. Disclosures No relevant conflicts of interest to declare.

Gas6 Regulates Thrombin-Induced of VCAM-1 Expression by a FoxO1 Dependent Mechanism

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5192-5192
Author(s):  
Richard Robins ◽  
Catherine A. Lemarie ◽  
Mark D. Blostein
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Venous Thrombosis ◽  
Cell Biology ◽  
Cell Activation ◽  
Conflicts Of Interest ◽  
Vascular Dysfunction ◽  
Soluble Form ◽  
Endothelial Cell Activation

Abstract Abstract 5192 Forkhead proteins play a broad role in endothelial cell biology. These factors mediate cell adhesion to extracellular matrix, regulate the expression of pro-inflammatory and pro-thrombotic genes, and participate in cell repair, proliferation and apoptosis. FoxOs are known downstream targets of the PI3K/Akt signaling pathway. Phosphorylation of FoxO transcription factors results in their translocation from the nucleus to the cytoplasm, thereby inhibiting their transcriptional activity. It has recently been shown that the deletion of the three FoxO isoforms in endothelial cells protects mice from vascular dysfunction. Gas6, a member of the vitamin K-dependent family of proteins, has been shown to protect endothelial cells from apoptosis and promote endothelial cell activation in vivo. It has been shown that the expression of ICAM-1 and VCAM-1 were blunted in the absence of gas6. Interestingly, a role for VCAM-1 in the pathogenesis of venous thrombosis has been proposed. Elevated levels of the soluble form of VCAM-1 have been detected in the serum of patients with venous thrombosis. We previously demonstrated that the anti-apoptotic effect of gas6 was mediated partially through FoxO1, but overall, the signalling mechanisms occurring downstream of gas6 remain largely unknown. We hypothesize that gas6 promotes thrombin-induced VCAM-1 expression through the regulation of FoxO1 in endothelial cells. Western blot analysis demonstrated that thrombin induced time dependent phosphorylation of FoxO1 with a maximum at 30 minutes in WT (p<0. 05) but not in gas6 deficient (−/−) cells. In addition, thrombin reduced the nuclear content of FoxO in WT (p<0. 05) but not in gas6−/− endothelial cells. Using qPCR, we found that mRNA expression of VCAM-1 was increased after 30 minutes of stimulation with thrombin in WT cells (p<0. 05). More importantly, thrombin-mediated induction of VCAM-1 was blunted in gas6−/− endothelial cells. We found that FoxO1 siRNA increased basal VCAM-1 expression in WT endothelial cells. Taken together, our data demonstrate that gas6 is a crucial mediator of FoxO1 that regulates thrombin-induced VCAM-1 expression. This pathway may explain the pro-thrombotic and pro-inflammatory role of gas6. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Association Between SERPINC1 C.883G>a and VT in the Chinese Population

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3505-3505
Author(s):  
Jingdi Liu ◽  
Liang Tang ◽  
Wei Zeng ◽  
Yu Hu ◽  
Han Liu
Keyword(s):  
Venous Thrombosis ◽  
Chinese Population ◽  
Case Control Study ◽  
Conflicts Of Interest ◽  
Hot Spot ◽  
Case Control ◽  
Missense Mutations ◽  
Increased Risk ◽  
Control Study

Abstract Background: Antithrombin(AT) is a major anticoagulation molecule in vivo and is encoded by the gene SERPINC1. AT plays a key role as an inhibitor of physiological haemostasis by inhibiting the procoagulation factors, especially the factor Xa and thrombin. Objectives: To explore the variations of SERPINC1 gene associated with venous thrombosis in the Chinese population. Methods: SERPINC1 gene sequencing was carried out. A case-control study involving 1335 patients diagnosed with VT and 1315 Age- and sex-matched control individuals without a history of thrombosis were further carried out. Furthermore, plasma AT activity, AT antigen, and thrombin generation tests (TGT) were performed to evaluate the influences of the mutations. Results: Four different missense mutations were identified in an unreported hot spot region of SERPINC1. They were c.880C>T(p.Arg294Cys), c.881G>T(p.Arg294Leu), c.881G>A(p.Arg294His) and c.883G>A(p.Val295Met). All of the affected individuals were heterozygotes. In addition, c.883G>A was found to be a predominant mutation. In the case-control study, the mutation was proved to be a strong risk factor for venous thrombosis with an OR of 10.92(p<0.01, 95%CI 1.41-84.68). Functional assays showed that both the activities and antigens of plasma AT decreased mildly. Conclusion: A hot spot mutation region of SERPINC1 gene was discovered. The predominant mutation of SERPINC1 c.883G>A is the most frequent cause of AT deficiency and is associated with an increased risk of venous thrombosis in the Chinese population. Disclosures No relevant conflicts of interest to declare.

scholarly journals Preclinical Evaluation of Invariant Natural Killer T-Cells in the 5T33 Multiple Myeloma Model

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 938-938
Author(s):  
Haneen Nur ◽  
Sandrine Aspeslagh ◽  
Els Van Valckenborgh ◽  
Elke De Bruyne ◽  
Dirk Elewaut ◽  
...  
Keyword(s):  
Natural Killer ◽  
Murine Model ◽  
Conflicts Of Interest ◽  
Nkt Cells ◽  
Spleen Cells ◽  
Ifn Γ ◽  
End Stage ◽  
Natural Killer T

Abstract Abstract 938 Natural killer T (NKT) cells are T-lymphocytes that co-express conventional T-cell (CD3) and NK cell (NK1.1) surface receptors, while invariant NKT cells (iNKTs) also express a unique semi-invariant TCR α-chain encoded by Vα14.Jα18 in mice and Vα24.Jα18 in human. These iNKTs can recognize glycolipid antigens such as α-Galactosylceramide (α-GalCer) presented by the class I-like major histocompatibility complex (MHC) molecule CD1d. Activation of iNKTs can lead to an anti-tumor Th1 (IFN-γ) response or an immunosuppressive Th2 (IL-4) response. Clinical studies in MM patients (1, 2) showed a low frequency and dysfunction of iNKTs which resulted in a low IFN-γ secretion. This defect could be overcome in vitro by stimulating the iNKTs with α-GalCer loaded DCs. Furthermore, when MM patients were injected with loaded DCs their iNKT pool expanded 100 fold. This make MM cells an interesting target for iNKT therapy. However, the data on NKT activity in MM patients is limited and the use of α-GalCer as a drug has not been preclinically evaluated yet. Therefore, in this study, we investigated the characteristics of iNKTs in the syngeneic 5T33MM murine model, which is an immunocompetent model of myeloma which mimics the human disease closely. We first investigated the frequency of iNKTs in the blood, BM, spleen and liver of both healthy and terminally diseased 5T33MM mice. The highest percentage of iNKTs was found in the liver (naive 7%) with a significant decrease in 5T33MM mice (2.6%). The percentage was also slightly decreased in spleen (from 1.5% in naive to 0.7% in 5T33MM mice) while no significant differences were observed in the other tissues. Next, we followed the frequency of iNKTs in the liver and spleen of MM mice during the development of the disease. We analyzed the number of iNKTs in the first, second, third and terminal week. We found that the percentage of iNKTs declined at the end stage of MM disease. To analyze the activity of iNKTs in vitro, liver iNKTs were cocultured with naive matured BM derived DCs in the presence or absence of 100 ng/ml α-GalCer. Naive iNKTs could secrete up to 2.3 ng/ml IFN-γ when stimulated with α-GalCer, and this level increased with the progression of MM to reach 3.3 ng/ml at week 2. However, the activity of the iNKTs dropped to undetectable levels upon further progression of the disease (week 4). In contrast, very slight IL-4 production was observed indicating that liver iNKTs are skewed to a Th1 profile and can therefore be used as an immunotherapeutic tool in MM. The activity of the NKTs was also followed in vivo. The serum level of IFN-γ peaked at 18h after α-GalCer injection in naive and non-terminal diseased mice and returned to baseline by 48h, however, the response of IFN-γ in diseased mice was twice (6 ng/ml) that measured in naive mice, confirming the possibility of inducing Th1 responses with α-GalCer in vivo in healthy and diseased mice. No response could be detected from terminally diseased mice. It has been described previously that CD1d is significantly downregulated in patients with advanced stages of MM (3). To investigate if this is similar in the 5T33MM model, we followed the expression of CD1d on spleen and BM cells during the course of the disease. Results showed a significant downregulation of CD1d expression on spleen cells from 93% CD1d (naive) to 68% at end stage. On BM cells, CD1d was less expressed compared to spleen cells, 52% in naive mice and expression declined significantly to 35%. CD1d expression on the MM cells themselves was high (79%) and did not alter during the course of the disease. We finally evaluated the effect of α-GalCer on the survival of MM mice. Survival was significantly increased when mice were injected with α-GalCer loaded DCs on the same day of 5T33MM inoculation (29 days survival) compared to mice injected with unloaded DCs (22 days survival). Taken together, our data demonstrate for the first time the possibility of using a murine model as a preclinical MM model to study the effects of α-GalCer on iNKTs and shows promising results of treating MM patients with a low tumorload. Disclosures: No relevant conflicts of interest to declare.

Abstract 141: Anti-thrombin Perfluorocarbon Nanoparticles Decrease Clot Burden in a Murine Model of Venous Thrombosis

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Chandu Vemuri ◽  
Batool Arif ◽  
Susannah A Grathwohl ◽  
John S Allen ◽  
Peter K Henke ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Murine Model ◽  
Safety Margin ◽  
Vena Cava ◽  
Clot Lysis ◽  
Control Group ◽  
Treatment Regimens ◽  
Significant Difference ◽  
Initial Work ◽  
Clot Burden

Objective: Venous thromboembolism (VTE) afflicts nearly an million Americans with significant mortality and long-term morbidity. Current medical treatment regimens pose significant bleeding risks and recurrence risks. The purpose of this work is to determine if anti-thrombin perfluorocarbon nanoparticles (NP-PPACK) can attenuate clot progression after vascular injury in a murine model of venous thrombosis. Methods: Male, C57 black-6 mice underwent inferior vena cava (IVC) ligation through an institutionally approved protocol. Following ligation, groups of ten mice were randomized to receive intravenous, weight-based (1 ml/kg) tail vein injections of saline, plain nanoparticles, NP-PPACK or heparin (80 units/kg). After 6 hours the animals were sacrificed, IVC with clot excised and weight and length recorded. Clot integrity (N=4) analysis was then performed by incubating clots with 750 units of streptokinase for 90 minutes at 37 degrees Celsius, removing liquid clot and recording the percent change in clot weight. Results: There was a significant difference in clot burden between NP-PPACK and the control group (0.59 mg/mm ± 0.063 vs. 1.26mg/mm ± 0.85, p=.0001). Immunofluorescent histology performed on a subgroup of animals verified nanoparticle tracking to venous thrombus. Additionally, using exogenous clot lysis as a surrogate for clot integrity, NP-PPACK treated animals exhibited a trend towards enhanced lysis over saline treatments (change in clot weight over 90 minutes: 57.8 ±14.4 vs. 21.5 ± 7.49, NP PPACK vs saline p=.067). Conclusions: This initial work demonstrates that NP-PPACK significantly decreases clot burden by local targeting and reduces clot strength in this model of VTE. We have shown previously that the system is locally active for hours against thrombosis yet produces no sustained systemic anticoagulant effect beyond 60 minutes, indicative of its significant safety margin for clinical application.

TLR3 Activation Is Involved in Venous Thrombosis Development

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3309-3309
Author(s):  
Catherine A Lemarie ◽  
Angela Le ◽  
Meghedi Aghourian ◽  
Jianqiu Wu ◽  
Mark D Blostein
Keyword(s):  
Endothelial Cells ◽  
Venous Thrombosis ◽  
Mrna Expression ◽  
Conflicts Of Interest ◽  
Experimental Studies ◽  
Vena Cava ◽  
Vehicle Control ◽  
Sterile Inflammation ◽  
Polycytidylic Acid ◽  
Tlr3 Activation

Abstract Abstract 3309 Venous thromboembolism (VTE) afflicts 117 people per 100,000 each year and is an important cause of morbidity and mortality. The pathophysiology of VTE is now better understood from experimental studies. Leukocytes, chemokines, and proinflammatory cytokines are involved in VTE resolution and vein wall healing. This process resembles sterile wound healing with distinct phases of polymorphonucleic neutrophil and monocyte influx followed by fibrosis. Inflammation is emerging as a central mechanism in both the genesis and resolution of VTE. Recently, the role of toll-like receptor (TLR) signaling in modulating sterile inflammation has become better defined in experimental injury models. TLR3 senses dsRNA, a by-product of viral replication, in the endosome. In addition, TLR3 has been increasingly linked to tissue damage. Endogenous RNA released by damaged tissue or necrotic cells is able to induce TLR3 expression and signaling. Thus, we hypothesize that TLR3 might be involved in the inflammatory development of VTE. Intravenous injection of polyinosine polycytidylic acid (polyI:C), a synthetic double-stranded RNA analog, increases the size of thrombi after FeCl3-induced inferior vena cava injury (IVC) compared to mice treated with vehicle control (p<0.05). In TLR3 deficient (TLR3−/−) mice, polyI:C did not induce a further increase in thrombus size compared to vehicle control. Recently, neutrophils have been shown to initiate and propagate venous thrombosis. PolyI:C injection was associated with an increased of neutrophil infiltration in the thrombus in WT but not in TLR3−/− mice (p<0.05). We found that polyI:C injection was associated with increased neutrophil activation. In the thrombus of WT mice, immunofluorescence staining for myeloperoxidase and citrulinated H3, markers for neutrophils activation, were increased by polyI:C. Interestingly, in TLR3−/− mice, no increase of these markers was found after polyI:C injection as compared to vehicle control. In vitro incubation of endothelial cells with polyI:C induces production of pro-inflammatory cytokines IL-8 and CCL5 involved in the recruitment of neutrophils as demonstrated by a cytokine array. We found that mRNA expression of TLR3, IL-8 and CCL5 were dramatically increased with polyI:C. When endothelial cells were transfected with siRNA for TLR3, mRNA expression of TLR3, IL-8 and CCL5 was blunted in response to polyI:C. These results suggest that release of IL-8 and CCL5 from endothelial cells after TLR3 activation are involved in neutrophil recruitment. Taken together, these results strongly suggest that TLR3 stimulation after endothelial cell injury participate in thrombus formation by inducing a pro-inflammatory response leading to the recruitment and activation of neutrophils. Disclosures: No relevant conflicts of interest to declare.

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