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.

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.

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.

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.

scholarly journals Current Challenges in Diagnosis of Venous Thromboembolism

2020 ◽  
Vol 9 (11) ◽  
pp. 3509
Author(s):  
Zachary Liederman ◽  
Noel Chan ◽  
Vinai Bhagirath
Keyword(s):  
Venous Thromboembolism ◽  
Venous Thrombosis ◽  
Cost Effective ◽  
Diagnostic Approach ◽  
Medical Patients ◽  
Vein Thrombosis ◽  
Non Invasive ◽  
Risk Patients ◽  
Diagnostic Framework ◽  
Deep Vein

In patients with suspected venous thromboembolism, the goal is to accurately and rapidly identify those with and without thrombosis. Failure to diagnose venous thromboembolism (VTE) can lead to fatal pulmonary embolism (PE), and unnecessary anticoagulation can cause avoidable bleeding. The adoption of a structured approach to VTE diagnosis, that includes clinical prediction rules, D-dimer testing and non-invasive imaging modalities, has enabled rapid, cost-effective and accurate VTE diagnosis, but problems still persist. First, with increased reliance on imaging and widespread use of sensitive multidetector computed tomography (CT) scanners, there is a potential for overdiagnosis of VTE. Second, the optimal strategy for diagnosing recurrent leg deep venous thrombosis remains unclear as is that for venous thrombosis at unusual sites. Third, the conventional diagnostic approach is inefficient in that it is unable to exclude VTE in high-risk patients. In this review, we outline pragmatic approaches for the clinician faced with difficult VTE diagnostic cases. In addition to discussing the principles of the current diagnostic framework, we explore the diagnostic approach to recurrent VTE, isolated distal deep-vein thrombosis (DVT), pregnancy associated VTE, subsegmental PE, and VTE diagnosis in complex medical patients (including those with impaired renal function).

Prostaglandin E Synthase Is Upregulated By Gas6 during Cancer-Induced Venous Thromboembolism

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 111-111
Author(s):  
Meghedi N Aghourian ◽  
Catherine A Lemarie ◽  
Francois-Rene Bertin ◽  
Mark D Blostein
Keyword(s):  
Endothelial Cells ◽  
Venous Thromboembolism ◽  
Microarray Analysis ◽  
Conflicts Of Interest ◽  
Prostaglandin E ◽  
Wild Type ◽  
Prostaglandin E Synthase ◽  
Murine Lung

Abstract Venous thromboembolism (VTE) is the most common morbid complication related to cancer and its treatments. Although malignancies are characterized by a hypercoagulable state leading to VTE, the pathophysiology of this state has not been well studied. Growth arrest specific 6 (Gas6) is a protein that has pro-coagulant properties. Gas6 deficient mice develop smaller venous thrombi as compared to wild type mice, and express less tissue factor in the endothelium when challenged with thrombotic stimuli. We hypothesize that Gas6 may be involved in cancer-induced venous thrombosis. In order to test this hypothesis, venous thrombi were induced in wild type (WT) and Gas6 null (-/-) mice injected with M27 murine lung cancer cell lines. Thrombus size was measured using ultrasonography, thrombus weight and histology. We observed that WT mice with cancer developed larger thrombi than their healthy counterparts (p<0.05). However, these larger thrombi induced by cancer were not seen in Gas6-/- mice, suggesting that Gas6 has a pathophysiologic role in promoting malignancy associated VTE. Whole genome microarray analysis was then used to identify differential gene expression in WT and Gas6-/- endothelial cells co-cultured with M27 murine lung carcinoma cells. Microarray analysis revealed that prostaglandin E synthase (PTGES) was increased in WT endothelial cells but not in Gas6-/- cells co-cultured with M27. These results were confirmed using real-time PCR and immunofluorescence staining (p<0.05). In WT endothelial cells, PTGES expression was regulated through ERK1/2 phosphorylation. We also show that co-culture of WT endothelial cells with M27 augments the secretion of PGE2, the enzymatic product of PTGES. PGE2 activates platelets in vitro after binding to its receptor, EP3. In vivo, EP3 receptor antagonism reversed the effect of cancer-induced thrombosis in WT mice. These results show that Gas6, through upregulation of PGE2, contributes to cancer-induced VTE. Disclosures No relevant conflicts of interest to declare.

Therapeutic efficacy of an oncolytic adenovirus monitored by in vivo imaging of an ovarian cancer model

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 10061-10061
Author(s):  
Y. Tsuruta ◽  
L. Pereboeva ◽  
D. T. Rein ◽  
M. Breidenbach ◽  
D. T. Curiel
Keyword(s):  
Ovarian Cancer ◽  
Tumor Growth ◽  
Cancer Cells ◽  
Murine Model ◽  
Ovarian Cancer Cell ◽  
Imaging System ◽  
Wild Type ◽  
Non Invasive ◽  
Ovarian Cancer Cell Lines

10061 Background: Although a number of advances in ovarian cancer treatment have occurred in the last decade, most patients will experience a recurrence after standard therapies. Recently, virotherapy has been proposed as a new therapeutic approach for ovarian cancer. Conditionally replicative adenovirus (CRAd) contains tumor-specific promoters that restrict virus replication to cancer cells and has shown particular promise as oncolytic viral agents. However, the lack of a tumor-volume monitoring system hinders the evaluation of CRAd impact on cancer treatment. Therefore, methods for analyzing CRAd efficacy and tumor response are required. Mesothelin, a cell surface glycoprotein, is overexpressed in ovarian cancer but not in normal ovarian tissues. The purpose of this study was to explore the therapeutic utility of a mesothelin promoter-based CRAd in a murine model of ovarian cancer, using a non-invasive biological imaging system. Methods: We constructed a mesothelin promoter based CRAd which also contains a modified fiber (Ad5/3 fiber) previously shown to improve infectivity of many ovarian cancer cells (Ad5/3MSLN). Viral replication and oncolysis were assessed in a panel of ovarian cancer cell lines. To test the oncolytic efficacy of Ad5/3MSLN in murine model, firefly luciferase-expressing SK-OV-3-luc cells were injected intraperitoneally (i.p.), followed by an i.p. injection of viruses. Then, bioluminescence imaging of tumor luciferase activity was carried out. Results: Ad5/3MSLN achieved up to 10,000-fold higher cell killing effect and up to 120-fold higher levels of viral replication in all ovarian cancer cell lines tested, compared to wild type Ad5. In vivo tumor imaging confirmed that Ad5/3MSLN significantly inhibited tumor growth, while the untreated mice had rapid tumor growth (p<0.05). Survival with Ad5/3MSLN was significantly enhanced when compared with no virus, or wild type Ad5-treated group (p<0.05). Conclusions: The robust replication, oncolysis, and in vivo therapeutic efficacy of Ad5/3MSLN demonstrated that this CRAd is a promising candidate for treating ovarian cancer. Importantly, we have established an in vivo non-invasive imaging system, which has allowed repeated and longitudinal measurements of tumor growth after CRAd treatment. No significant financial relationships to disclose.

Human Vascular Progenitor Cells Can Guide Mesodermal Lineage Choice of Mesenchymal Stem and Progenitor Cells After Co-Transplantation In Vivo.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 939-939
Author(s):  
Andreas Reinisch ◽  
Nathalie Etchart ◽  
Nicole A Hofmann ◽  
Anna Ortner ◽  
Eva Rohde ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Time Course ◽  
Conflicts Of Interest ◽  
Differentiation Potential ◽  
Environmental Conditioning ◽  
Non Invasive ◽  
Developmental Fate ◽  
Stem And Progenitor Cells ◽  
Proliferation Potential

Abstract Abstract 939 Background: Multilineage differentiation potential of mesenchymal stem and progenitor cells (MSPCs) make them attractive candidates for tissue regeneration purposes. Guiding the differentiation of MSPCs towards single lineages would facilitate their application for targeted therapies in vivo. We have previously shown that MSPCs are essential for endothelial colony-forming progenitor cell (ECFC)-derived patent vessel formation in vivo*[Blood 2009; 113 (26):6716-25]. Preliminary data indicate that the ratio of co-applied cells can change mesenchymal lineage differentiation from vascular support towards either osteogenesis with subsequent bone marrow (BM) ingrowth or chondrogenesis. We hypothesized that environmental conditioning by ECFCs plays an instructive role during the developmental fate decision of MSPCs in vivo. Methods: MSPCs as well as ECFCs were isolated from adult BM, white adipose tissue (WAT), umbilical cord blood (UCB) and perivascular cord tissue**[J Vis Exp. 2009;(32) pii: 1525]. Proliferation potential and clonogenicity were monitored. Phenotype was analyzed by flow cytometry and immune cytochemistry. Cell function was studied in differentiation assays and during vascular network assembly in vitro. Models for in vivo human vessel as compared to bone, BM or cartilage formation were established in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ). Non-invasive imaging was performed using computed tomography (CT), magnetic resonance (MRI) and near-infrared fluorescence imaging to elucidate the time course of heterotopic tissue development. Immune histochemistry was applied for morphologic studies of organogenesis. Results: Baseline analysis confirmed MSPC and ECFC purity, immune phenotype and sustained proliferation potential. We could show that human BM-derived MSPCs are capable of forming bone in vivo. Osteogenic differentiation and heterotopic ossicle formation was followed by attraction of mouse hematopoiesis and the establishment of entire murine BM including red and white blood cells and megakaryocytes within a human endosteal niche. Co-transplanted human ECFCs could instruct the MSPCs to differentiate also into pericytes or chondrocytes in vivo, depending on the applied MSPC/ECFC cell ratio. Non-invasive imaging and histological staining revealed that ectopic organogenesis had already started after 2–4 weeks and was stable during the observation period of 20 weeks. Non-BM-derived populations, although phenotypically identical, invariably lacked the capacity to build bone and marrow environment in this model in vivo. Conclusion: These data indicate that human ECFCs can instruct MSPCs and induce developmental fate decisions early in the time course of organ regeneration after transplantation. We suppose that effective regenerative stem cell therapy in vivo requires more than the injection of one single cell population. For vascular repair as compared to bone and marrow environment reconstitution our model is a promising tool to study the therapeutic applicability and risk profile of such ECFC/MSPC-based transplantation strategies. 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.

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