E-selectin inhibitor is superior to low-molecular-weight heparin for the treatment of experimental venous thrombosis.

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.

Carbon Nanodots Inhibit Oxidized Low Density Lipoprotein-Induced Injury and Monocyte Adhesion to Endothelial Cells Through Scavenging Reactive Oxygen Species.

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.

A new way to treat proximal deep venous thrombosis using E-selectin inhibition.

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.

Elimination of Signaling by the Luteinizing Hormone Receptor Reduces Ocular VEGF and Retinal Vascularization during Mouse Eye Development.

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 21 days 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.

Confirmation of Luteinizing Hormone (LH) in Living Human Vitreous and the Effect of LH Receptor Reduction on Murine Electroretinogram.

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.

Vitreous Levels of Luteinizing Hormone and VEGF are Strongly Correlated in Healthy Mammalian Eyes.

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.

Bacterial and Pneumocystis Infections in the Lungs of Gene-Knockout Rabbits with Severe Combined Immunodeficiency.

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.

E-selectin inhibition with GMI-1271 decreases venous thrombosis without profoundly affecting tail vein bleeding in a mouse model.

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.

Chronic Dosing with Membrane Sealant Poloxamer 188 NF Improves Respiratory Dysfunction in Dystrophic Mdx and Mdx/Utrophin-/- Mice.

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.

A multi-site feasibility study for personalized medicine in canines with osteosarcoma.

BACKGROUND: A successful therapeutic strategy, specifically tailored to the molecular constitution of an individual and their disease, is an ambitious objective of modern medicine. In this report, we highlight a feasibility study in canine osteosarcoma focused on refining the infrastructure and processes required for prospective clinical trials using a series of gene expression-based Personalized Medicine (PMed) algorithms to predict suitable therapies within 5 days of sample receipt. METHODS: Tumor tissue samples were collected immediately following limb amputation and shipped overnight from veterinary practices. Upon receipt (day 1), RNA was extracted from snap-frozen tissue, with an adjacent H&E section for pathological diagnosis. Samples passing RNA and pathology QC were shipped to a CLIA-certified laboratory for genomic profiling. After mapping of canine probe sets to human genes and normalization against a (normal) reference set, gene level Z-scores were submitted to the PMed algorithms. The resulting PMed report was immediately forwarded to the veterinarians. Upon receipt and review of the PMed report, feedback from the practicing veterinarians was captured. RESULTS: 20 subjects were enrolled over a 5 month period. Tissue from 13 subjects passed both histological and RNA QC and were submitted for genomic analysis and subsequent PMed analysis and report generation. 11 of the 13 samples for which PMed reports were produced were communicated to the veterinarian within the target 5 business days. Of the 7 samples that failed QC, 4 were due to poor RNA quality, whereas 2 were failed following pathological review. Comments from the practicing veterinarians were generally positive and constructive, highlighting a number of areas for improvement, including enhanced education regarding PMed report interpretation, drug availability, affordable pricing and suitable canine dosing. CONCLUSIONS: This feasibility trial demonstrated that with the appropriate infrastructure and processes it is possible to perform an in-depth molecular analysis of a patient's tumor in support of real time therapeutic decision making within 5 days of sample receipt. A number of areas for improvement have been identified that should reduce the level of sample attrition and support clinical decision making.

Prothrombotic effects of thrombolytic therapy in a rat (Rattus norvegicus) model of venous thrombolysis.

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.

Circulating and vein wall P-selectin promote venous thrombogenesis during aging in a rodent model.

INTRODUCTION: The objective of this study was to identify the direct relationship between aging and selectin activation during acute venous thrombosis in mice of varying ages. We hypothesized that older animals would have increased venous thrombus formation as a result of age associated-increases of pro-inflammatory molecules within the vein wall when compared to younger animals. MATERIALS AND METHODS: Deep venous thrombosis (DVT) was induced in 4 and 18month old C57BL/6 mice using the electrolytic inferior vena cava model (EIM) of DVT. Blood and tissue samples were collected at baseline (TC), 6hours, and 2days post-thrombosis induction. RESULTS: Older mice had significantly larger thrombi versus younger mice at 6H (18.4±6.21 vs. 13.0±4.29×10(-3) grams, p=0.0033) and 2D (18.4±4.27 vs. 13.0±5.01×10(-3) grams, p=0.0005), higher soluble P-selectin levels at 6H (13±2.5 vs. 8.4±2.7ng/mg p=0.0010) and 2D (12.7±5.0 vs. 5.9±1.3ng/mg p=0.0020), and higher vein wall P-selectin levels at 6H (1.94×10(5)±3.56×10(4) vs. 4.81±2.29×10(4) pg/mg p=0.0001) and 2D (1.38×10(5)±5.65×10(4) vs. 3.73±1.66×10(4) pg/mg p=0.0177). Older animals also had significantly higher platelet numbers at 6H (841±203.8 vs. 564±164.8K/µL p=0.0001), and 2D (1002±342.9 vs. 690±186.1K/µL p=0.0003), with corresponding increases in mean platelet volume versus younger mice post thrombosis (p≤0.01). CONCLUSIONS: Older animals had significantly larger venous thrombi versus younger animals post-thombosis, as a result of high levels of P-selectin both in the circulation and locally at the level of the vein wall. Expression of local and soluble P-selectin increased with age, resulting in a pro-thrombotic environment not represented in younger mice.

Genomic characterization of explant tumorgraft models derived from fresh patient tumor tissue.

BACKGROUND: There is resurgence within drug and biomarker development communities for the use of primary tumorgraft models as improved predictors of patient tumor response to novel therapeutic strategies. Despite perceived advantages over cell line derived xenograft models, there is limited data comparing the genotype and phenotype of tumorgrafts to the donor patient tumor, limiting the determination of molecular relevance of the tumorgraft model. This report directly compares the genomic characteristics of patient tumors and the derived tumorgraft models, including gene expression, and oncogenic mutation status. METHODS: Fresh tumor tissues from 182 cancer patients were implanted subcutaneously into immune-compromised mice for the development of primary patient tumorgraft models. Histological assessment was performed on both patient tumors and the resulting tumorgraft models. Somatic mutations in key oncogenes and gene expression levels of resulting tumorgrafts were compared to the matched patient tumors using the OncoCarta (Sequenom, San Diego, CA) and human gene microarray (Affymetrix, Santa Clara, CA) platforms respectively. The genomic stability of the established tumorgrafts was assessed across serial in vivo generations in a representative subset of models. The genomes of patient tumors that formed tumorgrafts were compared to those that did not to identify the possible molecular basis to successful engraftment or rejection. RESULTS: Fresh tumor tissues from 182 cancer patients were implanted into immune-compromised mice with forty-nine tumorgraft models that have been successfully established, exhibiting strong histological and genomic fidelity to the originating patient tumors. Comparison of the transcriptomes and oncogenic mutations between the tumorgrafts and the matched patient tumors were found to be stable across four tumorgraft generations. Not only did the various tumors retain the differentiation pattern, but supporting stromal elements were preserved. Those genes down-regulated specifically in tumorgrafts were enriched in biological pathways involved in host immune response, consistent with the immune deficiency status of the host. Patient tumors that successfully formed tumorgrafts were enriched for cell signaling, cell cycle, and cytoskeleton pathways and exhibited evidence of reduced immunogenicity. CONCLUSIONS: The preservation of the patient's tumor genomic profile and tumor microenvironment supports the view that primary patient tumorgrafts provide a relevant model to support the translation of new therapeutic strategies and personalized medicine approaches in oncology.

Increased fall risk is associated with elevated co-contraction about the ankle during static balance challenges in older adults.

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.

Myeloid cell tissue factor does not contribute to venous thrombogenesis in an electrolytic injury model.

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.

Male mice have increased thrombotic potential: sex differences in a mouse model of venous thrombosis.

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 naïve true controls and surgical shams. Analyses included thrombus weight, vein wall morphometrics, vein wall protein and gene expression for P-selectin, interleukin-1ß, and tumor necrosis factor-α; 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 ± 0.5 × 10(-3) grams, p < 0.01), 6 (10.4 ± 0.8 vs. 5.4 ± 0.5 × 10(-3) grams, p < 0.01) and 14 (6.3 ± 0.5 vs. 4.1 ± 0.3 × 10(-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-1ß, 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-α expression (-66 %) at 6 hours. Females had increased tumor necrosis factor-α 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.

Germline met mutations in mice reveal mutation- and background-associated differences in tumor profiles.

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.

A multilevel analysis of individual, household, and neighborhood correlates of intimate partner violence among low-income pregnant women in Jefferson county, Alabama.

OBJECTIVES: We examined individual, household, and neighborhood correlates of intimate partner violence (IPV) before and during pregnancy. METHODS: We used multilevel modeling to investigate IPV among 2887 pregnant women in 112 census tracts who sought prenatal care in 8 public clinics in Jefferson County, Alabama, from 1997 through 2001. Data were collected from the Perinatal Emphasis Research Center project, the 2000 Census, and the local Sheriff and Police Departments Uniform Crime Reports for 1997 through 2001. RESULTS: Participants were predominantly young, African American, on Medicaid, and residents of low-income neighborhoods. The prevalence of past-year male partner-perpetrated physical or sexual violence was 7.4%. Neighborhood residential stability, women performing most of the housework (lack of involvement among partners), being unmarried (being in an uncommitted relationship), and alcohol use were positively associated with elevated IPV risk. Significant protective factors for IPV included older age at first vaginal intercourse and a greater sense of mastery (e.g., the perception of oneself as an effective person). CONCLUSIONS: Both neighborhood contextual and individual and household compositional effects are associated with IPV among low-income pregnant women. The results imply that combined interventions to improve neighborhood conditions and strengthen families may effectively reduce IPV.

Met induces diverse mammary carcinomas in mice and is associated with human basal breast cancer.

Understanding the signaling pathways that drive aggressive breast cancers is critical to the development of effective therapeutics. The oncogene MET is associated with decreased survival in breast cancer, yet the role that MET plays in the various breast cancer subtypes is unclear. We describe a knockin mouse with mutationally activated Met (Met(mut)) that develops a high incidence of diverse mammary tumors with basal characteristics, including metaplasia, absence of progesterone receptor and ERBB2 expression, and expression of cytokeratin 5. With gene expression and tissue microarray analysis, we show that high MET expression in human breast cancers significantly correlated with estrogen receptor negative/ERBB2 negative tumors and with basal breast cancers. Few treatment options exist for breast cancers of the basal or trastuzumab-resistant ERBB2 subtypes. We conclude from these studies that MET may play a critical role in the development of the most aggressive breast cancers and may be a rational therapeutic target.

Tissue inhibitor of metalloproteinase-1 is an early marker of acute endothelial dysfunction in a rodent model of venous oxidative injury.

Although there are extensive research data regarding arterial endothelial dysfunction, the effects of venous endothelial dysfunction are not well characterized. Matrix metalloproteinases (MMPs) have a defined role in vascular remodeling. MMPs are endopeptidases that are capable of degrading extracellular matrix proteins. We hypothesize that tissue inhibitor of metalloproteinase-1 (TIMP-1) can serve as an indicator of acute venous endothelial dysfunction in a rat model of oxidative injury. The experimental groups evaluated were as follows: rats not undergoing oxidative injury (controls), rats that received rose bengal but no laser (shams), and rats that received both rose bengal and laser illumination, resulting in an oxidative injury. Animals were evaluated at baseline (control, shams) and at 1 hr and 1 day post-oxidative injury. mRNA expression was determined by gene array technology and real-time polymerase chain reaction, plasma and vein wall TIMP-1 protein concentrations were determined by enzyme-linked immunosorbent assay, and vein wall morphometrics (cells/five high-power fields) were performed. B-cell lymphoma 2-like gene expression was upregulated at both 1 hr and 1 day post-injury. TIMP-1 protein and mRNA expression were significantly increased post-oxidative injury. One hour postinjury, vein wall polymorphonuclear leukocytes were present in significant numbers. Our results support the hypothesis that increased expression of TIMP-1 in venous endothelium and plasma may serve as an early indicator of endothelial dysfunction.