Supplementary Figures 1-2 from Inactivation of Apc in the Mouse Prostate Causes Prostate Carcinoma

Oxidized low density lipoprotein (Ox-LDL) is a known biomarker of inflammation and atherosclerosis, a leading cause of death worldwide. As a new class of nanomaterials, carbon nanodots (CNDs) are widely used in bioimaging, diagnostics, and drug delivery. However, there is no current report on how these CNDs affect the cardiovascular system, particularly their potential in mediating endothelial inflammatory dysfunction. This study examined effects of CNDs on Ox-LDL-mediated endothelial dysfunction. CNDs significantly inhibited Ox-LDL-mediated adhesion of monocytes to human microvascular endothelial cells (HMEC-1), in human microvascular endothelial cells (HMEC-1). CNDs significantly inhibited Ox-LDL-mediated adhesion of monocytes to endothelial cells, which is an essential step in the development of atherosclerosis. Further, CNDs significantly inhibited OxLDL-induced expression of interleukin-8 (IL-8), a vital cytokine on monocyte adhesion to the endothelial cells. These results demonstrate CNDs possess anti-inflammatory properties. CNDs also protect cells against Ox-LDL-induced cytotoxicity. Electron paramagnetic resonance (EPR) spectroscopy studies demonstrated direct reactive oxygen species-scavenging by CNDs. This result indicates that the anti-inflammatory properties of CNDs are most likely due to their direct scavenging of reactive oxygen species. Animal studies involving mice did not show any morphological or physical changes between the CNDs and control groups. Our study provides evidence of potential of CNDs in reducing Ox-LDL-mediated inflammation and cytotoxicity in HMEC-1.

BACKGROUND:This study evaluated E-selectin inhibition with GMI-1271 (Uproleselan [GMI]) alone and in combination with the standard of care low-molecular-weight heparin (LMWH) to improve vein recanalization, decrease vein wall inflammation and protect against adverse bleeding in a primate model. We sought to examine this novel treatment of venous thrombosis. METHODS:Using a well-documented primate animal model, iliac vein thrombosis was induced by balloon occlusion of the iliac vein for 6 hours. Starting on day 2 after thrombosis, animals began treatment in two phases. In phase one, nontreated controls received no treatment (n = 5) vs animals treated with the E-selectin inhibitor GMI, 25 mg/kg, subcutaneous (SC), once daily (n = 4) for 21 days (previously published data). In phase two, animals were treated with GMI plus a combination of LMWH 1.5 mg/kg or 40 mg (GMI + LMWHc) SC once daily (n = 8) for 19 days; and animals treated with LMWH 1.5 mg/kg or 40 mg (LMWHc) SC once daily (n = 6) for 19 days. Animals were evaluated by magnetic resonance venography for vein recanalization and inflammation by gadolinium extravasation, duplex ultrasound, coagulation tests (thromboelastography, bleeding time, prothrombin time, activated partial thromboplastin time, fibrinogen) and complete blood count at baseline, days 2, 7, 14, and 21 at euthanasia. Statistical analysis included using unpaired t test with Welch's correction for direct comparisons and one-way analysis of variance for comparison between the groups. RESULTS:Percent vein recanalization by magnetic resonance venography was highest in the GMI alone group followed by GMI + LMWHc, both significantly different from control. On ultrasound examination, animals treated with GMI alone had no decrease in open vein lumen by day 21, whereas decreases were observed in groups GMI + LMWHc (-26%), LMWHc (-27%), and controls (-80%). Vein wall inflammation decreased significantly in all treated groups. Intimal fibrosis and intimal thickness was best preserved in the GMI alone group. An analysis of total vein wall collagen revealed a trend in all treatment groups of decreasing vein wall collagen. No clinically significant bleeding events were noted in any group. The LMWH groups trended to have prolonged coagulation test values, whereas E-selectin inhibition with GMI did not cause clinically significant changes in coagulation measures. CONCLUSIONS:Treatment with E-selectin inhibition results in improved vein recanalization, a decrease in vein wall inflammation and vein wall intimal thickness and fibrosis, with no changes in markers of coagulation. E-selectin inhibition with GMI alone is superior to E-selectin inhibition combined with LMWH, LMWH alone, and no treatment in this deep vein thrombosis model of iliac vein thrombosis.

Objective: There is an inter-relationship between thrombosis and inflammation. Previously, we have shown the importance of P-selectin in thrombogenesis and thrombus resolution in many preclinical animal models. The role of E-selectin has been explored in rodent models and in a small pilot study of clinical calf vein deep venous thrombosis. The purpose of this study was to determine the role of E-selectin in thrombosis in a primate model of proximal iliac vein thrombosis, a model close to the human condition. Methods: Iliac vein thrombosis was induced with a well-characterized primate model. Through a transplant incision, the hypogastric vein and iliac vein branches were ligated. Thrombus was induced by balloon occlusion of the proximal and distal iliac vein for 6 hours. The balloons were then deflated, and the primates recovered. Starting on postocclusion day 2, animals were treated with the E-selectin inhibitor GMI-1271, 25 mg/kg subcutaneously, once daily until day 21 (n = 4). Nontreated control animals received no treatment (n = 5). All animals were evaluated by magnetic resonance venography (MRV); evaluation of vessel area by ultrasound, protein analysis, hematology (complete blood count), and coagulation tests (bleeding time, prothrombin time, activated partial thromboplastin time, fibrinogen, and thromboelastography) were performed at baseline, day 2, day 7, day 14, and day 21 with euthanasia. In addition, platelet function and CD44 expression on leukocytes were determined. Results: E-selectin inhibition by GMI-1271 significantly increased vein recanalization by MRV vs control animals on day 14 (P<.05) and day 21 (P<.0001). GMI-1271 significantly decreased vein wall inflammation by MRV with gadolinium vein wall enhancement vs control also on day 14 (P<.0001) and day 21 (P<.0001). The thromboelastographic measure of clot strength (maximum amplitude) showed significant decreases in animals treated with GMI-1271 vs controls at day 2 (P<.05) and day 7 (P<.05). Animals treated with GMI-1271 had significant vessel area increase by day 21 vs controls (P<.05) by ultrasound. Vein wall intimal thickening (P<.001) and intimal fibrosis (P<.05) scores were significantly decreased in GMI-1271-treated animals vs controls. Importantly, no significant differences in hematology or coagulation test results were noted between all groups, suggesting that E-selectin inhibition carries no bleeding potential. GMI-1271 did not affect platelet function or aggregation or CD44 expression on leukocytes. In addition, no episodes of bleeding were noted in either group. Conclusions: This study suggests that E-selectin modulates venous thrombus progression and that its inhibition will increase thrombus recanalization and decrease vein wall inflammation, without affecting coagulation. The use of an E-selectin inhibitor such as GMI-1271 could potentially change how we treat deep venous thrombosis.

Luteinizing hormone (LH), produced in the anterior pituitary, has been detected in cadaver eyes and LH receptors (LHRs) have been identified in the retina, with the highest density in cone photoreceptors. Our aim was to confirm the presence of LH in the living, human eye as well as to examine the potential impact of a reduction in LHR signaling on visual processing. Vitreous samples were collected from 40 patients (23 diabetics, 17 non-diabetics) who were undergoing vitrectomies for various indications. LH concentration was quantified in each sample via an electro-chemiluminescence immunoassay and Meso Scale Discovery platform and normalized to total protein. In addition, full-field electroretinography (ERG) was performed on 11 adult LHR knockout heterozygous mice (B6;129X1-Lhcgrtm1Zmlei/J) and 11 wild types using the Celeris-Diagnosys system. The median LH values (pg/mg total protein) for non-diabetics, diabetics without proliferative diabetic retinopathy (PDR) and diabetics with PDR were 40.7, 41.9 and 167.8 respectively. LH levels were significantly higher in diabetics with PDR. In our ERG investigation, heterozygous LHRKOs were found to have significantly reduced amplitudes of a-wave and b-waves at high stimulus intensities with no significant change in a-wave or b-wave amplitudes at lower intensities; this is consistent with a selective impairment of cone-mediated responses. Our findings confirm LH is present in the adult human eye. Our findings also suggest that a reduction in LH receptor signaling negatively impacts visual processing of the cone photoreceptors. Overall, our study results support the theory that LH likely plays a physiologic role in the eye.

Using the CRISPR/Cas9 gene-editing technology, we recently produced a number of rabbits with mutations in immune function genes, including FOXN1, PRKDC, RAG1, RAG2, and IL2RG. Seven founder knockout rabbits (F0) and three male IL2RG null (−/y) F1 animals demonstrated severe combined immunodeficiency (SCID), characterized by absence or pronounced hypoplasia of the thymus and splenic white pulp, and absence of immature and mature T and B-lymphocytes in peripheral blood. Complete blood count analysis showed severe leukopenia and lymphocytopenia accompanied by severe neutrophilia. Without prophylactic antibiotics, the SCID rabbits universally succumbed to lung infections following weaning. Pathology examination revealed severe heterophilic bronchopneumonia caused by Bordetella bronchiseptica in several animals, but a consistent feature of lung lesions in all animals was a severe interstitial pneumonia caused by Pneumocystis oryctolagi, as confirmed by histological examination and PCR analysis of Pneumocystis genes. The results of this study suggest that these SCID rabbits could serve as a useful model for human SCID to investigate the disease pathogenesis and the development of gene and drug therapies.

Purpose/Aim: Luteinizing hormone (LH) is known to function as a key regulator of vascular endothelial growth factor (VEGF) expression in reproductive organs. In recent years, LH has also been detected in human vitreous and LH receptors have been identified in human retina. This study was aimed to investigate a potential correlation between LH and VEGF levels in healthy mammalian eyes to provide supporting evidence of LH's potential involvement in intraocular VEGF regulation. METHODS:18 bovine and 30 porcine eyes were procured from an abattoir and VEGF and LH levels were measured in the vitreous extracted from these eyes by commercially available bovine & porcine ELISA assay kits. Total protein of the vitreous was measured by using Micro BSA protein assay kit. RESULTS:After total protein normalization, the Pearson Correlation Coefficients (PCC) showed a strong and significant correlation between LH and VEGF levels. (Bovine LH/VEGF PCC: 0.89, p < 0.001; Porcine LH/VEGF PCC: 0.80, p < 0.001). Linear regression analyses, adjusted for gender, showed significant linear relationships between LH and VEGF levels in both bovine and porcine vitreous. (Bovine: t-value = 7.69, p < 0.0001, adjusted r2 = .79; Porcine: t-value = 6.71, p < 0.001, adjusted r2 = .62) Conclusions: We show that VEGF and LH are strongly correlated in healthy, adult mammalian eyes. The robustness of the correlation is shown both by its strength of association and reproducibility in two species. Given that LH is well known to regulate VEGF levels in several tissue types, the LH/VEGF linear relationship in vitreous potentially implicates LH in homeostatic VEGF regulation of the eye. Because we also found that the correlation between LH and VEGF only became manifest when our targeted analytes were normalized by total amount of protein, preclinical and clinical investigators should consider normalizing analytes in vitreous by total protein when assessing potential correlations among them.

Purpose/Aim: Vascular endothelial growth factor (VEGF) dysregulation is implicated in the pathogenesis of retinopathy of prematurity (ROP). Identifying the factors that contribute to VEGF regulation during normal retinal vascularization is the key to ROP prevention. Currently, physiologic hypoxia is thought to be responsible for retinal VEGF regulation in utero. However, a potential hormonal contribution to VEGF regulation during eye development has not been fully investigated. The placental hormone, human chorionic gonadotropin and the pituitary hormone, and luteinizing hormone (LH) induce VEGF expression in several tissue types. Both of these gonadotropins activate the same LH receptor (LHR) in the human body; LHRs are expressed in the retina. In this study, we aimed to show that LHR signaling participates in VEGF regulation in the developing eye.Methods: When offspring from breeding pairs of LHR knockout mice (lhrkos) reached 21days old, eyes and serum were extracted from homozygote lhrkos and wildtype (WT) siblings. VEGF levels were measured using Mouse VEGF Quantikine immunoassay kit. Retinas were incubated with isolectin for endothelial cell staining, flat mounted and imaged by confocal microscopy. Retinal vascular density was quantified using Imaris software. Some eyes were sectioned and stained for histopathologic review.Results: Ocular VEGF and retinal vascular volumes were significantly reduced by 15% in lhrko eyes. Serum VEGF was not changed. The lhrko retinas did not display any anomalies.Conclusions: We provide evidence that LHR signaling plays a role in VEGF regulation and vascularization in the developing eye. Given that human preterm infants may have altered LHR-activity, the effect of gonadotropins on eye development should be further studied to identify novel strategies for ROP prevention.

Selectins, such as E-selectin (CD62E), function in venous thrombosis by binding and activating immune cells to initiate the coagulation cascade. GMI-1271 is a small molecule antagonist that inhibits E-selectin activity. Here we determine whether inhibition of E-selectin is sufficient to decrease acute venous thrombosis and associated inflammatory events in both prophylactic and treatment protocols without significantly affecting haemostasis. Male C57BL/6 mice underwent surgery for experimental thrombosis induction and were harvested at peak thrombus formation in our animal model, two days post induction. Groups included non-thrombosed true controls, shams, controls, and prophylactic or treatment groups of GMI-1271 (10 mg/kg intraperitoneal BID (twice a day) and low-molecular-weight heparin (LMWH, Lovenox 6 mg/kg subcutaneously (SC), once a day (SID). Compared with control animals, prophylaxis or treatment with LMWH and GMI-1271 in a dose-dependent manner significantly decreased thrombosis. GMI-1271 significantly lowered tail bleeding times when compared to LMWH. GMI-1271 and LMWH prophylactically administered significantly decreased vein wall neutrophil cell extravasation. However, all, treatment and prophylactic therapies significantly decreased vein wall monocyte extravasation versus controls. GMI-1271 prophylactic therapy significantly decreased intra-thrombus cell counts versus control animals and other treatment groups. Immunohistochemistry confirmed that both treatments with GMI-1271 and LMWH significantly decreased activated leukocyte migration. GMI-1271 therapy significantly decreased thrombus weight and resulted in significantly lower bleeding times than LMWH. GMI-1271 treated mice showed decreased local and systemic inflammatory effects while modulating neutrophil activation, suggesting that GMI-1271 is a viable therapeutic candidate for venous thrombosis prophylaxis and treatment.

Background: Specialized pro-resolving lipid mediators (SPM) derived from polyunsaturated fatty acids, such as Resolvin D2 (RvD2), play a critical role in the resolution of acute inflammation. In recent years, total organic synthesis of SPM have enabled investigation of their bioactivity in-vivo. It is known that the effects of RvD2 include limited neutrophil influx in-vivo, reduced neutrophil transmigration and counter-regulation of cytokines such as tumor necrosis factor-α TNF-α in mice. However, SPMs have not been studied in the context of experimental venous thrombosis (VT). Methods: Mice C57Bl/6, 10 week-old, 25grams. Groups: Control non-VT mice; VT-control-vehicle; VT-RvD2; Flow model: Electrolytic IVC model. Intra-peritoneal injection was used for RvD2 and vehicle administration 4 hours after surgery. Harvest was performed 2 days after surgery, acute VT. Thrombus weight, qRT-PCR for inflammatory cytokines panel and subsets of white blood cell (WBC) counts were performed at the site of thrombosis histologically. Results (Figure 1): We observed that RvD2 significantly reduced thrombus size when compared to vehicle controls (17.9mg±2.1mg vs. 4.5mg±0.8mg). The vein wall cytokine profile demonstrated a significant gene down-regulation of IL-6, CCL2, and TNF-a. In addition, the inflammatory cell counts showed reduction in all white cells subsets. No bleeding complications were observed. Conclusions: For the first time we have demonstrated that RvD2 reduces VT formation. The decreased thrombogenesis was accompanied by a decrease in WBC and proinflammatory cytokines. We demonstrated that RvD2 is a potent anti-inflammatory agent that reduced WBC and attenuated cytokines in the context of experimental VT. Further investigation of RvD2 in VT treatment and prophylaxis appears warranted.

Poloxamer 188 NF (national formulary (NF) grade of P-188) improves cardiac muscle function in the mdx mouse and golden retriever muscular dystrophy models. However in vivo effects on skeletal muscle have not been reported. We postulated that P-188 NF might protect diaphragm muscle membranes from contraction-induced injury in mdx and mdx/utrophin-/- (dko) muscular dystrophy models. In the first study 7-month old mdx mice were treated for 22 weeks with subcutaneous (s.c.) injections of saline or P-188 NF at 3 mg/Kg. In the second, dkos were treated with saline or P-188 NF (1 mg/Kg) for 8 weeks beginning at age 3 weeks. Prednisone was the positive control in both studies. Respiratory function was monitored using unrestrained whole body plethysmography. P-188 NF treatment affected several respiratory parameters including tidal volume/BW and minute volume/BW in mdx mice. In the more severe dko model, P-188 NF (1 mg/Kg) significantly slowed the decline in multiple respiratory parameters compared with saline-treated dko mice. Prednisone’s effects were similar to those seen with P-188 NF. Diaphragms from P-188 NF or prednisone treated mdx and dko mice showed signs of muscle fiber protection including less centralized nuclei, less variation in fiber size, greater fiber density, and exhibited a decreased amount of collagen deposition. P-188 NF at 3 mg/Kg s.c. also improved parameters of systolic and diastolic function in mdx mouse hearts. These results suggest that P-188 NF may be useful in treating respiratory and cardiac dysfunction, the leading causes of death in Duchenne muscular dystrophy patients.

The use of thrombolytic agents has greatly improved patient outcomes, but the prothrombotic response to these drugs in vivo is unknown. Approximately 24 h after we induced thrombosis in male Sprague-Dawley rats, we placed an infusion line in the inferior vena cava and administered either saline or a thrombolytic agent (tissue plasminogen activator [tPA] or plasmin) for 30 min. Blood was drawn immediately after infusion; rats were euthanized 24 h after infusion for collection of blood and tissue (inferior vena cava and thrombus). Thrombus size was decreased in the tPA-treated rats but not in those that received saline or plasmin; this change correlated with the significant rise in D-dimer levels noted immediately after infusion in the tPA-treated rats. Plasma soluble P-selectin, a prothrombotic marker, was elevated at 24 h in the plasmin group compared with the other treatment groups. There were no significant differences in plasma C3a, C5a, or C5b9 levels or in thrombus C3 levels between groups. According to ultrastructural analysis, thrombus structure and vein wall effects did not differ between groups. Local tPA did not induce a prothrombotic state during acute DVT or after thrombolytic therapy in a rodent model of venous thrombolysis. Conversely, levels of the prothrombotic marker plasma soluble P-selectin increased when plasmin was administered.

Abstract Abstract 3422 Introduction: Selectins function in venous thrombosis presumably by binding and activating immune cells to initiate the coagulation cascade. E-selectin (CD62E) is known to bind and activate both monocytes and neutrophils. GMI-1271 is a small molecule antagonist that specifically inhibits E-selectin and is rationally designed to mimic the bioactive conformation of the sialyl-Lex carbohydrate ligand. Here we determine whether specific inhibition of E-selectin is sufficient to inhibit acute venous thrombosis and associated inflammatory events in both prophylactic and treatment protocols without causing the broader effects of increased bleeding time. Methods: Male C57BL/6J mice underwent our electrolytic inferior vena cava (IVC) model to produce a non-occlusive thrombosis via electrical stimulation (250 μAmp). Animals were divided into prophylactic or treatment groups. Both groups included the following: non-thrombosed animals (TC, no surgery or drug), 2 Day sham (needle inside the IVC and no current or drug), 2 Day CTR (current and no drug), 2 Day GMI-1271 (10mg/kg IP BID), and LMWH (Lovenox®, 6mg/kg SQ QD). Animals were divided into prophylactic or treatment groups. Mice in the prophylactic group were dosed one day pre-thrombus induction through day 1. Animals in the treatment groups received the first dose of the drug following thrombus induction on day 1. Mice were euthanized 2 days post-thrombosis for tissue harvest and blood collection for the following evaluations: thrombus weight; vein wall inflammatory cell counts per high power field; vein wall-thrombus histology; and intra-thrombus polymorphonuclear cell (PMN) counts. A separate group of mice received IV administration of compounds for tail bleeding time evaluation (seconds). Results: GMI-1271 Significantly Decreases Venous Thrombus Weight (Figure 1). Treatment with GMI-1271 decreased venous thrombus formation in a dose-dependent manner with significant inhibition at 10mg/kg (P=0.0271). Treatment with LMWH significantly decreased thrombus formation 2 day post induction at 6mg/kg (P=0.0203). All mice pre-treated prophylactically with GMI-1271 or LMWH followed the same pattern of decreasing thrombus weight 2 days post injury (P<0.05). E-selectin Inhibition with GMI-1271 Does Not Increase Bleeding Potential (Figure 2) LMWH at 6 mg/kg dose significantly elevated tail bleeding times in mice versus controls (341±27, 491±60 vs. 82±6 seconds, P<0.01). GMI-1271 (10mg/kg, IV) had significantly lower tail bleeding times compared to an IV dose of LMWH (6mg/kg, P<0.01). Vein Wall Morphometrics and Histology Treatment: Only treatment with GMI-1271 significantly decreased vein wall monocyte extravasation compared to controls (P<0.05). Prophylaxis: GMI-1271 and LMWH prophylaxis significantly decreased vein wall PMN extravasation 2 days post thrombosis (P=0.027 and P=0.007 respectively). The same pattern held true for prophylaxis with GMI-1271 and LMWH on vein wall monocyte extravasation at the same time point (P<0.01). Intra-Thrombus PMN Counts: GMI-1271 prophylactic therapy significantly decreases intra-thrombus cell counts versus control animal (14.5±3.7 vs. 37.4±4.7 PMNs/HPF, P=0.009), and these animals had decreased venous thrombus burden. Of interest, only mice receiving GMI-1271 therapy visually have more intra-thrombus vascular channels compared to control animals and mice receiving LMWH therapy. Conclusion: GMI 1271 inhibits venous thrombosis and significantly decreases thrombus weight. GMI 1271 proposes a much lower risk of patients having bleeding complications. Vascular channels exclusively present in thrombi from mice receiving GMI-1271 therapy may aide in thrombus resolution which is currently under investigation. Delayed inflammatory cell recruitment of all cell types into the vein wall post thrombus induction indicates a possible decrease in leukocyte activation. This data suggest that inhibition of E-selectin is sufficient to inhibit venous thrombosis without an increased bleeding risk and the small molecule E-selectin specific antagonist GMI-1271 is a viable therapeutic candidate for venous thrombosis treatment and prophylaxis. Disclosures: Patton: GlycoMimetics: Employment. Magnani:GlycoMimetics: Employment, Equity Ownership.

Falls are a leading contributor to disability in older adults. Increased muscle co-contraction in the lower extremities during static and dynamic balance challenges has been associated with aging, and also with a history of falling. Co-contraction during static balance challenges has not been previously linked with performance on clinical tests designed to ascertain fall risk. The purpose of this study was to investigate the relationship between co-contraction about the ankle during static balance challenges with fall risk on a commonly used dynamic balance assessment, the Four Square Step Test (FSST). Twenty-three volunteers (mean age 73 years) performed a series of five static balance challenges (Romberg eyes open/closed, Sharpened Romberg eyes open/closed, and Single Leg Standing) with continuous electromyography (EMG) of bilateral tibialis anterior and gastrocnemius muscles. Participants then completed the FSST and were categorized as ‘at-risk’ or ‘not-at-risk’ to fall based on a cutoff time of 12 s. Co-contraction was quantified with co-contraction index (CCI). CCI during narrow base conditions was positively correlated with time to complete FSST. High CCIs during all static balance challenges with the exception of Romberg stance with eyes closed were predictive of being at-risk to fall based on FSST time, odds ratio 19.3. The authors conclude that co-contraction about the ankle during static balance challenges can be predictive of performance on a dynamic balance test.

INTRODUCTION:Tissue factor (TF) is a potent initiator of the extrinsic coagulation cascade. The role and source of TF in venous thrombotic disease is not clearly defined. Our study objective was to identify the contribution of myeloid cell TF to venous thrombogenesis in mice.MATERIALS AND METHODS:The mouse electrolytic inferior vena cava model was used to induce thrombosis. The following groups of mice were used (1) TF(flox/flox)LysMCre(+) mice that have reduced TF expression in myeloid cells, (2) TF(flox/flox)LysMCre(-) littermate controls, (3) Wild type mice given a monoclonal anti-mouse TF antibody (1H1) to inhibit TF activity, and (4) Wild type mice given rat IgG. Evaluations at baseline, day 2, and day 6 post thrombosis included thrombus weight, vein wall inflammatory cell migration, vein wall TF mRNA, and plasma D-dimer levels.RESULTS:Inhibition of TF significantly decreased thrombus weight 2days post venous thrombosis. In contrast, TF(flox/flox)LysMCre(+) had no change in thrombus weight when compared to littermate controls. The absence of myeloid cell TF did not affect infiltration of neutrophils or monocytes into the vein wall. TF mRNA expression in the vein wall decreased at 2days but then returned to baseline levels by 6days post thrombosis. D-dimer levels peaked at 2days post thrombosis in mice with or without myeloid cell TF.CONCLUSIONS:TF is important in the formation of venous thrombi in the macrovasculature. However, TF expression by myeloid cells does not significantly contribute to venous thrombogenesis in this model.

Introduction: Our objectives were to characterize sex differences during venous thrombosis, using the electrolytic inferior vena cava model of the disease.Materials and methods: Male and female C57BL/6 mice (6-8 weeks) underwent inferior vena cava thrombosis. Time points included 6 hours, day 2, day 6, and day 14 post surgery, along with surgically nave true controls and surgical shams. Analyses included thrombus weight, vein wall morphometrics, vein wall protein and gene expression for P-selectin, interleukin-1 beta, and tumor necrosis factor-alpha; hematology, soluble P-selectin, and plasma microparticle tissue factor activity assays.Results: Male venous thrombi were significantly larger than females at days 2 (13.1+/-1.0 vs. 6.8+/-0.5x10(-3) grams, p<0.01), 6 (10.4+/-0.8 vs. 5.4+/-0.5x10(-3) grams, p<0.01) and 14 (6.3+/-0.5 vs. 4.1+/-0.3x10(-3) grams, p<0.01). Both male and female mice exhibited significantly increased vein wall P-selectin at 6 hours, vs. true controls (p<0.05). Males had increased vein wall interleukin-1 beta, versus females, at 6 hours (180.926+/-24.596 vs. 60.417+/-10.478 pg/mL, p<0.05) and day 6 (76.966+/-13.081 vs. 33.834+/-4.198 pg/mL, p<0.01). Males showed decreased tumor necrosis factor-a expression (-66%) at 6 hours. Females had increased tumor necrosis factor-a expression at 6 hours (+541%) and day 6 (+539%). Both sexes demonstrated decreased peripheral platelets at 6 hours (p<0.05), coinciding with thrombogenesis. Plasma P-selectin increased in both sexes, versus controls, through day 6 (p<0.05).Conclusions: Males had significantly larger venous thrombi than females. Sex differences in vascular anatomy and response to inflammation may influence thrombus formation in our mouse thrombosis model. (C) 2011 Elsevier Ltd. All rights reserved.

Abstract Understanding the signaling pathways that drive aggressive breast cancers is critical to the development of effective therapeutics. Over 20% of breast cancers overexpress ERBB2 and are associated with an aggressive tumor phenotype and poor outcome. Patients with ERBB2-positive tumors are often treated with the humanized monoclonal antibody trastuzumab, yet the majority of patients develop resistance. Recent studies show that the receptor tyrosine kinase MET is expressed in ERBB2 positive tumors and contributes to trastuzumab resistance in ERBB2-expressing cell lines. Other studies have shown that MET correlates with poor clinical outcome independent of ERBB2. We are utilizing mouse models to examine the intrinsic signaling differences between MET and ERBB2-mediated breast cancers. We have developed a novel mouse model of mutationally activated MET (Metmut) that develops a high incidence of diverse mammary tumors with basal characteristics. Using this mouse model and human breast cancer tissue, we have examined the role of MET in aggressive breast cancers. By gene expression and tissue microarray analysis, we observe that high MET expression in human breast cancers significantly correlates with estrogen receptor negative/ERBB2 negative tumors and with basal breast cancers (Graveel et al., 2009). We performed gene expression analysis on mammary tumors from Metmut mice and activated ErbB2 (Neu-Ndl2-5) mice. Unsupervised hierarchical analysis revealed that solid Metmut tumors clustered separately from Metmut tumors with a mixed pathology and that solid Metmut tumors have expression patterns very similar to ERBB2 tumors. The similarity of solid Metmut tumors to ErbB2 tumors suggests that MET signaling may be influential in ERBB2-driven tumors. To investigate this hypothesis further, we performed a parametric gene set enrichment analysis (PGSEA) and observed that the Ras activation signature was significantly associated with the Metmut mixed tumors. Currently, we are also investigating expression patterns of MET and ERBB2 in human breast cancers by coimmunostaining. Collectively, our studies indicate that MET may play a critical role in the development of the most aggressive breast cancers and may be a rational therapeutic target for trastuzumab-resistant ERBB2 breast cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3256.

Background The receptor tyrosine kinase Met is involved in the progression and metastasis of numerous human cancers. Although overexpression and autocrine activation of the Met signaling pathway are commonly found in human cancers, mutational activation of Met has been observed in small cell and non-small cell lung cancers, lung adenocarcinomas, renal carcinomas, and mesotheliomas. Methodology/Principal Findings To investigate the influence of mutationally activated Met in tumorigenesis, we utilized a novel mouse model. Previously, we observed that various Met mutations developed unique mutation-specific tumor spectra on a C57BL/6 background. Here, we assessed the effect of genetic background on the tumorigenic potential of mutationally activated Met. For this purpose, we created congenic knock-in lines of the Met mutations D1226N, M1248T, and Y1228C on the FVB/N background. Consistent with the mutation-specific tumor spectra, several of the mutations were associated with the same tumor types as observed on C57BL/6 background. However, on the FVB/N background most developed a high incidence of mammary carcinomas with diverse histopathologies. Conclusions/Significance This study demonstrates that on two distinct mouse backgrounds, Met is able to initiate tumorigenesis in multiple cell types, including epithelial, hematopoietic, and endothelial. Furthermore, these observations emphasize that even a modest increase in Met activation can initiate tumorigenesis with both the Met mutational spectra and host background having profound influence on the type of tumor generated. Greater insight into the interaction of genetic modifiers and Met signaling will significantly enhance our ability to tailor combination therapies for Met-driven cancers.