Introduction

Our objectives were to characterized 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, and vein wall protein and gene expression for P-selectin, interleukin-1β, 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-1β, 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.

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism. The 2009 JUPITER trial showed a significant decrease in DVT in non-hyperlipidemic patients, with elevated C-reactive protein (CRP) levels, treated with rosuvastatin. The effects of statins on thrombosis are unclear, prompting this literature review. A literature search was performed (1950 to February 2011) with MEDLINE, EMBASE, and PUBMED databases including the following keywords: “statins”, “hydroxymethylglutaryl-CoA reductase inhibitors”, “VTE”, “PE”, “DVT”, and either “anti-coagulation” or “inflammation”. Editorials, reviews, case reports, meta-analysis and duplicates were excluded. Inflammatory biomarkers of DVT, include interleukin (IL)-6, CRP, IL-8, and monocyte chemotactic protein 1 (MCP-1). Statin therapy reduces IL-6 expression of CRP and MCP-1, usually elevated in VTE. Reduction of IL-6 induced MCP-1 has been linked to vein wall fibrosis, promoting post thrombotic syndrome (PTS) and recurrent DVT in patients. Also, our review suggests that the anti-thrombotic effects are likely exhibited through the anti-inflammatory properties of statins. This work supports that statin therapy has the ability to decrease the incidence and recurrence of VTE and the potential to decrease PTS. This is mainly due to the anti-inflammatory effects of statins and may explain why normolipidemic patients, with elevated CRP, appear to have the greatest reduction in VTE. Given their low risk of bleeding, statins have the potential to serve as a safe adjunctive pharmacological therapy to current treatments in select patients with VTE, however further investigations into this concept are needed and essential.

Objective

The combination of D-dimer and Wells score can exclude, but not confirm, the diagnosis of deep venous thrombosis (DVT). Since thrombosis and inflammation are interrelated, we evaluated the combination of soluble P-selectin (sPsel) with other inflammatory biomarkers for the diagnosis of DVT.

Methods

Sixty-two positive and 116 negative DVT patients, by duplex scan, were prospectively evaluated for sPsel, D-dimer, C-reactive protein (CRP), microparticles (total, leukocyte and platelet-derived and tissue factor positive microparticles - MP) and clinical Wells score.

Results

Biomarkers and clinical scores that differentiated DVT positives from negatives were sPsel (87.3 versus 53.4 ng/ml, p<0.0001), D-dimer (5.8 versus 2.1 mg/L, p<0.0001), CRP (2.1 versus 0.8 μg/ml, p<0.0005) and Wells score (3.2 versus 2.0, p<0.0001). For MP analysis, platelet-derived MP were found to differentiate DVT from negatives. Using multivariable logistic regression, a combination of sPsel and Wells score could establish the diagnosis of DVT (cut-point ≥90 ng/ml + Wells ≥2), with a specificity of 96% and positive predictive value (PPV) of 100%, and could exclude DVT diagnosis (cut-point ≤60 ng/ml and Wells <2) with a sensitivity of 99%, a specificity of 33% and a negative predictive value (NPV) of 96%.

Conclusion

This study establishes a biomarker and clinical profile combination that can both confirm and exclude the diagnosis of DVT.

Deep vein thrombosis and pulmonary embolism are a significant health care concern, representing a major source of mortality and morbidity. In order to understand the pathophysiology of thrombogenesis and thrombus resolution, animal models are necessary. Mouse models of venous thrombosis contribute to our understanding of the initiation, propagation, and resolution of venous thrombus, as well as allow for the evaluation of new pharmaceutical approaches to prophylaxis and treatment of deep vein thrombosis. In this work we review the ferric chloride model, the inferior vena cava ligation model, the inferior vena cava stenosis models, and the electrolytic inferior vena cava model and compare their advantages and disadvantages.

Purpose

This study evaluated histotripsy as a noninvasive, image-guided method of thrombolysis in a porcine model of deep vein thrombosis. Histotripsy therapy uses short, high-intensity, focused ultrasound pulses to cause mechanical breakdown of targeted soft tissue by acoustic cavitation, which is guided by real-time ultrasound imaging. This paper is an in-vivo feasibility study of histotripsy thrombolysis.

Methods and Materials

Acute thrombi were formed in the femoral vein of juvenile pigs weighing 30–40 kg by balloon occlusion with two catheters and thrombin infusion. A 10-cm diameter 1-MHz focused transducer was used for therapy. An 8 MHz ultrasound imager was used to align the clot with the therapy focus. Therapy consisted of 5 cycle pulses delivered at a rate of 1 kHz and peak negative pressure between 14–19 MPa. The focus was scanned along the long axis of the vessel to treat the entire visible clot during ultrasound exposure. The targeted region identified by a hyperechoic cavitation bubble cloud was visualized via ultrasound during treatment.

Results

Thrombus breakdown was apparent as a decrease in echogenicity within the vessel in 10 of 12 cases, and in 7 cases, improved flow through the vein as measured by color Doppler. Vessel histology showed denudation of vascular endothelium and small pockets of hemorrhage in the vessel adventitia and underlying muscle and fatty tissue, but perforation of the vessel wall was never observed.

Conclusions

The results indicate histotripsy has potential for development as a noninvasive treatment for deep vein thrombosis.

Background

Microparticles (MP) are submicron size membrane vesicles released from activated cells that are associated with thrombosis and inflammation. MP present diverse biological expressions that may be linked to a unique subset of proteins derived from their origin cells.

Methods

To identify these proteins, plasma samples were taken from 9 patients with deep venous thrombosis (DVT) documented by duplex ultrasound, 9 with leg pain but negative for DVT by duplex, and 6 healthy controls without a history of thrombosis, for fold variation. MP were extracted from platelet-poor plasma, digested separately with trypsin and tagged using iTRAQ reagents. The digests were subjected to 2-D LC separation followed by MALDI tandem mass spectrometry. Peak lists were generated and searched against all human sequences. For protein identification, a minimum of two peptides at 95% confidence was required. Later, iTRAQ ratios were generated comparing relative protein levels of DVT patients to baseline. The proteomic analysis was performed twice for each blood sample. Proteins were considered elevated or depressed if the iTRAQ ratio (R) deviated by 20% change from normal and a p-value less than 0.05.

Results

Two proteins (Galectin-3 Binding Protein, [Gal3BP], R=1.76 and Alpha-2 macroglobulin [A2M] R=1.57) were differentially expressed on DVT patients. Nine proteins were depleted including fibrinogen beta and gamma chain precursors (R=0.65).

Conclusions

These proteins influence thrombosis through inflammation, cell shedding, inhibition of fibrinolysis and hemostatic plug formation. Further studies are needed to confirm the mechanistic role of these proteins in the pathogenesis of venous thrombosis in humans.

Background

P-selectin antagonism has been shown to decrease thrombogenesis and inflammation in animal models of deep venous thrombosis (DVT).

Objective

To determine the effectiveness of P-selectin inhibitors versus saline and enoxaparin in venous resolution in nonhuman primate models of venous thrombosis.

Methods

Studies reporting vein re-opening, inflammation expressed as Gadolinium enhancement and coagulation parameters were searched in the literature and pooled into a meta-analysis using an inverse variance with random effects.

Results

Five studies were identified comparing P-selectin/ PSGL-1 inhibitors versus saline or enoxaparin regarding venous thrombosis resolution. Vein re-opening was significantly higher on P-selectin/ PSGL-1 compounds, when compared to saline (Inverse Variance [IV] 95% CI; 44.37 [17.77–70.96], p=0.001, I2 =97%) and similar to enoxaparin (IV 95% CI; 5.03 [−8.88–18.95], p=0.48, I2 =41%). Inflammation, reflected as Gadolinium enhancement at magnetic resonance venography (MRV), was significantly decreased in the P-selectin treated group when compared to saline (IV 95% CI; −17.84 [−14.98 – −8.30], p<0.00001, I2 =80%). No significant differences on vein wall inflammation were observed between P-selectin/ PSGL-1 inhibitors and enoxaparin treated animals (IV95% CI; −3.59 [−10.67–3.48], p=0.32, I2 =66%). In addition, there was no differences in the coagulation parameters (aPTT, TCT, BT, D-Dimer, fibrinogen, platelets) between P-selectin/ PSGL-1 inhibitors and enoxaparin (IV 95% CI; −1.12[−2.36–0.11], p=0.07, I2 =92%), although there was a trend showing less prolongation in TCT with P-selectin /PSGL-1 inhibitors over enoxaparin (p<0.0001).

Conclusion

P-selectin antagonism successfully paralleled the low-molecular-weight-heparin enoxaparin, for the treatment of DVT in nonhuman primate models, by decreasing both thrombus burden and inflammation without causing any bleeding complications and increasing coagulation times.

Summary

Several rodent models have been used to study deep venous thrombosis (DVT). However, a model that generates consistent venous thrombi in the presence of continuous blood flow, to evaluate therapeutic agents for DVT, is not available. Mice used in the present study were wild-type C57BL/6 (WT), plasminogen activator inhibitor-1 (PAI-1) knock out (KO) and Delta Cytoplasmic Tail (ΔCT). An electrolytic inferior vena cava (IVC) model (EIM) was used. A 25G stainless-steel needle, attached to a silver coated copper wire electrode (anode), was inserted into the exposed caudal IVC. Another electrode (cathode) was placed subcutaneously. A current of 250 μAmps over 15 minutes was applied. Ultrasound imaging was used to demonstrate the persence of IVC blood flow. Analyses included measurement of plasma soluble P-selectin (sP-Sel), thrombus weight (TW), vein wall morphometrics, P-selectin and Von Willebrand factor (vWF) staining, transmission electron microscopy (TEM), scanning electron microscopy (SEM); and the effect of enoxaparin on TW was evaluated. A current of 250 μAmps over 15 minutes consistently promoted thrombus formation in the IVC. Plasma sP-Sel was decreased in PAI-1 KO and increased in ΔCT vs. WT (WT/PAI-1: p=0.003, WT/ΔCT: p=0.0002). Endothelial activation was demonstrated by SEM, TEM, P-selection and vWF immunohistochemistry and confirmed by inflammatory cell counts. Ultrasound imaging demonstrated thrombus formation in the presence of blood flow. Enoxaparin significantly reduced the thrombus size by 61% in this model. This EIM closely mimics clinical venous disease and can be used to study endothelial cell activation, leukocyte migration, thrombogenesis and therapeutic applications in the presence of blood flow.

Summary

Microparticles (MP) are lipid vesicles from platelets, leukocytes and endothelial cells that are involved in early thrombogenesis. We evaluated a detailed time-course analysis of MPs on thrombogenesis and the associated tissue factor (TF) activity in wild-type, in gene-deleted for E- and P-selectins and with high levels of P-selectin expression after the initiation of venous thrombosis in mice. Inferior vena cava (IVC) ligation was performed on C57BL/6 mice (n =191, 59 = wild-type [WT], 55 = gene-deleted for E- and P – selectins [knock-outs, EPKO] and 77 = elevated levels of soluble P-selectin, named Delta Cytoplasmic Tail (ΔCT). Animals were euthanised at various time points to assess MP production, origin and thrombus weight. MPs were re-injected into separate mice at concentrations of 80,000 and 160,000 units, as well as from different ages. In addition, MPs from thrombosed animals were pooled and TF activity quantitated using a chromogenic assay. Thrombus weight correlated negatively with MPs derived from leukocytes, and positively with MPs derived from platelets for WT animals (p<0.05), while MPs from platelets presented a positive correlation to thrombus weight in the WT and EPKO groups (p<0.01). Total MPs correlated negatively with thrombus weight in the ΔCT group (p<0.05). MP re-injections led to greater thrombus weight, while older MP reinjections tended to form larger thrombus than younger. Finally, TF bearing MPs showed a significant correlation to MP concentrations (R=0.99). In conclusion, MPs appear to be an important element in venous thrombogenesis.

SUMMARY

Microparticles (MPs) are small membrane vesicles released from activated cells and are associated with thrombosis and inflammation. Microparticles contain a unique subset of surface proteins derived from the parent cell and may be responsible for their diverse biological functions. To identify these proteins juvenile baboons (Papio anubis, n=4) underwent iliac vein thrombosis with six-hour balloon occlusion. Plasma samples were taken at baseline and at 2 days post thrombosis for MP analysis. Microparticles were extracted from platelet-poor plasma, digested separately with trypsin and tagged using iTRAQ reagents. The digests were subjected to 2-D LC separation followed by MALDI tandem mass spectrometry. Peak lists were generated and searched against all primate sequences. For protein identity, a minimum of two peptides at 95% confidence was required. Later, iTRAQ ratios were generated comparing relative protein level of day-2 to baseline. The proteomic analysis was performed twice for each blood sample, totaling 8 experiments. Proteins were considered elevated or depressed if the iTRAQ ratio deviated by 20% change from normal and a p-value less than 0.05. Significantly, 7 proteins were differentially expressed on day-2 compared to baseline, and appeared in at least two animals and regulated in at least 4 experiments, and appeared in at least three animals and regulated in at least four experiments. Among these 7 proteins, up-regulated proteins include various forms of fibrinogen and alpha-1-antichymotrypsin, and down-regulated proteins include immunoglobulins. These proteins influence thrombosis and inflammation through hemostatic plug formation (fibrinogen), inhibiting neutrophil adhesion (alpha-1-antichymoptrypsin), and immunoregulation (immunoglobulins). Further studies are needed to confirm the mechanistic role of these proteins in the pathogenesis of venous thrombosis.

Previous ultrasound elasticity imaging experiments supported a generally accepted concept that the hardness of deep venous thrombi increases with thrombus aging. Results also showed that this non-invasive imaging technique can accurately predict thrombus age through strain estimates in a well controlled animal study. In the present study, as an alternative means to characterize elastic properties of thrombi, we employed a direct mechanical measurement system to estimate Young’s modulus of ex vivo thrombi. Unlike conventional indentation tests, the device utilizes a specific compression geometry for cylindrical tissue specimens. We also proposed an approximation scheme to retrieve Young’s modulus from force-displacement measurements made using the device. Finite element simulations and calibrations on tissue-mimicking phantoms validated the system. Then, using two groups of rats with surgically induced thrombi, we further investigated the correlation between Young’s modulus measured ex vivo and elasticity images reconstructed in vivo. This comparison was accomplished by converting the intra-thrombus strains measured in the in vivo studies into Young’s modulus estimates using a model based approach. Good agreement between time-dependent Young’s modulus estimates observed in vivo and direct measurements of Young’s modulus using the mechanical device helps to confirm the ability of elasticity imaging to age DVT for efficient treatment.

Elasticity imaging is a reconstructive imaging technique where tissue motion in response to mechanical excitation is measured using modern imaging systems, and the estimated displacements are then used to reconstruct the spatial distribution of Young's modulus. Here we present an ultrasound elasticity imaging method that utilizes the model-based technique for Young's modulus reconstruction. Based on the geometry of the imaged object, only one axial component of the strain tensor is used. The numerical implementation of the method is highly efficient because the reconstruction is based on an analytic solution of the forward elastic problem. The model-based approach is illustrated using two potential clinical applications: differentiation of liver hemangioma and staging of deep venous thrombosis. Overall, these studies demonstrate that model-based reconstructive elasticity imaging can be used in applications where the geometry of the object and the surrounding tissue is somewhat known and certain assumptions about the pathology can be made.

Background

RNA editing is the process whereby an RNA sequence is modified from the sequence of the corresponding DNA template. In the mitochondria of land plants, some cytidines are converted to uridines before translation. Despite substantial study, the molecular biological mechanism by which C-to-U RNA editing proceeds remains relatively obscure, although several experimental studies have implicated a role for cis-recognition. A highly non-random distribution of nucleotides is observed in the immediate vicinity of edited sites (within 20 nucleotides 5' and 3'), but no precise consensus motif has been identified.

Results

Data for analysis were derived from the the complete mitochondrial genomes of Arabidopsis thaliana, Brassica napus, and Oryza sativa; additionally, a combined data set of observations across all three genomes was generated. We selected datasets based on the 20 nucleotides 5' and the 20 nucleotides 3' of edited sites and an equivalently sized and appropriately constructed null-set of non-edited sites. We used tree-based statistical methods and random forests to generate models of C-to-U RNA editing based on the nucleotides surrounding the edited/non-edited sites and on the estimated folding energies of those regions. Tree-based statistical methods based on primary sequence data surrounding edited/non-edited sites and estimates of free energy of folding yield models with optimistic re-substitution-based estimates of ~0.71 accuracy, ~0.64 sensitivity, and ~0.88 specificity. Random forest analysis yielded better models and more exact performance estimates with ~0.74 accuracy, ~0.72 sensitivity, and ~0.81 specificity for the combined observations.

Conclusions

Simple models do moderately well in predicting which cytidines will be edited to uridines, and provide the first quantitative predictive models for RNA edited sites in plant mitochondria. Our analysis shows that the identity of the nucleotide -1 to the edited C and the estimated free energy of folding for a 41 nt region surrounding the edited C are the most important variables that distinguish most edited from non-edited sites. However, the results suggest that primary sequence data and simple free energy of folding calculations alone are insufficient to make highly accurate predictions.

The authors discuss the relationship between atypical facial pain and psychiatric disturbance. They present contemporary viewpoints and describe four cases that illustrate underlying psychodynamic mechanisms associated with pain in patients who had undergone various dental procedures and other treatments without success. They identify factors which might lead to the early detection of underlying psychological problems and discuss the role of learning, the family system and other factors in producing a chronic pain syndrome.