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Objective— Deep venous thrombosis is a major health problem. Thrombolytic therapies are effective in recanalizing the veins and preventing post-thrombotic complications, but there is no consensus on selection criteria. The aim of this study was to investigate a fibrin-specific MRI contrast agent (EP-2104R) for the accurate quantification of thrombus’ fibrin content in vivo and for the identification of thrombus suitable for thrombolysis. Approach and Results— Venous thrombosis was induced in the inferior vena cava of 8- to 10-week-old male BALB/C mice and MRI performed 2, 4, 7, 10, 14, and 21 days later. Eighteen mice were scanned at each time point pre and 2 hours post injection of EP-2104R (8.0 μmol/kg) with 12 mice at each time point used to correlate fibrin contrast uptake with thrombus’ histological stage and fibrin content. Six mice at each time point were immediately subjected to intravascular thrombolytic therapy (10 mg/kg of tissue-type plasminogen activator). Mice were imaged to assess response to lytic therapy 24 hours after thrombolytic treatment. Two mice at each time point were scanned post injection of 0.2 mmol/kg of Gd-DTPA (gadolinium with diethylenetriaminepentacetate, Magnevist, Schering AG, Berlin, Germany) for control purpose. Contrast uptake was correlated positively with the fibrin content of the thrombus measured by Western blotting ( R 2 =0.889; P R 1 ) post contrast and the change in visualized thrombus size on late gadolinium enhancement inversion recovery MRI pre–EP-2104R and post–EP-2104R injection were the best predictors for successful thrombolysis (area under the curve, 0.989 [95% confidence interval, 0.97–1.00] and 0.994 [95% confidence interval, 0.98–1.00] respectively). Conclusions— MRI with a fibrin-specific contrast agent accurately estimates thrombus fibrin content in vivo and identifies thrombi that are amenable for thrombolysis.

Purpose: Organization, recanalization, and contraction are common to wound healing and thrombus resolution. Monocytes are essential to wound healing and are also found in venous thrombi. We measured endogenous levels of the monocyte chemotactic protein-1 (MCP-1) in naturally resolving venous thrombi and determined the effect of injecting MCP-1 into newly formed thrombus. Methods: Endogenous MCP-1 levels were estimated in rat blood, thrombi, and the adjacent vessel wall after thrombus formation, in cohorts of eight animals at 1, 7, and 14 days. In another group (n = 10), 1 μg of MCP-1 was injected into newly formed thrombi. Carrier was injected into the thrombi of control animals (n = 10). Thrombi and adjacent vein walls were obtained for histology at 7 days. Thrombi were given an arbitrary organization score based on erythrocyte and extracellular matrix content, which was assessed by means of computerized and observer analysis. Specimen weight, thrombus area, and cellular and monocyte content were measured. Results: Endogenous MCP-1 increased between days 1 and 7 in the thrombus (1-day median, 1.1 ng/g wet wt; 1-day range, 0.8 to 1.4 ng/g wet wt; 7-day median, 5.4 ng/g wet wt; 7-day range, 1.5 to 7.4 ng/g wet wt; P < .0001) and vein wall (1-day median, 1.5 ng/g wet wt; 1-day range, 0.8 to 4.3 ng/g wet wt; 7-day median, 3.3 ng/g wet wt; 7-day range, 2.7 to 8.3 ng/g wet wt; P < .05). At 14 days, thrombus was incorporated in the vein wall, and total MCP-1 levels remained high (median, 3.9 ng/g wet wt; range, 1.1 to 7.4 ng/g wet wt). Less MCP-1 was found in the thrombus than the adjacent vessel wall at day 1 (P < .05), but there was no difference at day 7. MCP-1 could not be detected in the blood. MCP-1 injection into thrombus increased the computer (P = .016) and observer (P = .004) organization scores, reduced the thrombus area (from median, 3.4 mm2, and range, 1.5 to 5.7 mm2, to median, 0.2 mm2, and range, 0.02 to 2.6 mm2; P = .048), and increased the surrounding vessel wall monocyte content (P = .008). Specimen weights of treated animals were lower than those of control animals (P < .02). Conclusion: Venous thrombus MCP-1 levels increase during natural resolution. MCP-1 treatment increased the organization and resolution of thrombi. MCP-1 may therefore be of therapeutic use. (J Vasc Surg 1999;30:894-900.)

Purpose: The mechanisms of venous thrombogenesis have been studied by using animal models and cells in culture. The results from these systems may not, however, be relevant to the human condition. The aim of this study was to develop a method by which thrombus could be safely produced in a human vein in vivo. The model that was developed was used as a means of studying the changes in soluble adhesion molecule expression in human venous thrombogenesis. Methods: An autologous thrombin extract was used to generate experimental thrombi in the disconnected portion of the long saphenous veins of 30 patients who were undergoing routine bilateral varicose vein surgery. The contralateral vein was perfused with thrombin extract diluent buffer to act as the control. The concentration of soluble P-, E- and L-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule-1 were measured by means of specific enzyme-linked immunosorbent assays in samples of blood taken from veins in which thrombus had formed and in contralateral control veins. Results: Thrombosis invariably formed when at least 100 IU of thrombin activity was administered. Thrombus formation was independent of the time that the thrombin extract was allowed to remain within the emptied vessel. Thrombosis never developed in control vessels that were similarly treated with the buffer used to dilute the thrombin extract. Experimental thrombi were composed mainly of red cells, with layers of fibrin next to platelet and leukocyte packages. These findings are similar to those observed in samples of established human venous thrombi. There were small but significantly higher levels of the adhesion molecules, soluble P-selectin, and vascular cell adhesion molecule-1 in blood taken from veins in which experimental thrombi had formed, compared with controls ( P = .015 and .007, respectively; Wilcoxon signed rank test). Serum levels of soluble L-selectin, E-selectin, and ICAM-1 were not affected by thrombosis. Conclusion: This model is safe and reproducible. It produces thrombi with a morphology similar to that described for established human deep venous thrombi. The model may be appropriate for the study of the early changes that occur during human venous thrombogenesis and may also be of value in testing the efficacy of novel antithrombotic agents. (J Vasc Surg 1999;30:139-47.)

Background It is still unclear what initiates aneurysmal dilatation and what determines whether or not an aneurysm will continue to expand and rupture. Early detection and operative repair of an abdominal aortic aneurysm (AAA) still remains the only effective means of reducing the high mortality rate associated with the condition. Endovascular techniques are being developed in an attempt to reduce the mortality rate associated with elective repair. A variety of animal models and experimental techniques have been described in the investigation of the pathophysiology of AAA and in the development of improved endovascular surgical and pharmacological therapies. This article discusses these models and techniques, their advantages and some of the problems encountered in extrapolating experimental findings to the human condition. Methods This review is based on a search of the Medline database from 1966 to March 1998 using recognized key words and text words. A further search was then conducted on references quoted within selected relevant publications. Results and conclusion Treatment of rodent aortas with intraluminal elastase or periaortic calcium chloride creates reproducible aneurysms that have certain similarities to the human pathology; such aneurysms have been favoured in the investigation of the pathophysiology of aneurysm expansion. However, these models lack several of the prominent features of the human lesion, such as atherosclerosis and intraluminal thrombosis. The development of gene knockout mice may lead to a more analogous aneurysm formation, with associated atherosclerosis. Many large animal models have been used in the development of endovascular techniques but, in general, these do not mimic the human pathophysiology and fail to predict medium- and long-term complications.

SummaryCurrent biochemical markers of thrombosis, such as d-dimer, are of little value in demonstrating the presence of thrombus postoperatively, as their levels are elevated by surgery. Thrombosis involves adhesive interactions between the endothelium, platelets and leukocytes. The aim of the study was to determine which of a panel of haemostatic and adhesion factors are altered by the presence of thrombus, but not by surgery. These factors were measured in 20 patients with established spontaneous DVTs, 13 patients having hip replacement surgery and 28 control patients. Circulating levels of P-selectin, VCAM-1 and tissue factor were found to be increased when thrombus was present (p

Monocyte infiltration into forming thrombus has been demonstrated in experimental models of venous thrombosis developed in our laboratories. These cells produce and release plasminogen activators as the thrombus organises and resolves. Monocytes are also capable of assembling and releasing procoagulant factors and the evidence for their importance in thrombogenesis is reviewed. The ability of monocytes to maintain this fibrinolytic balance suggests that they may have a role in both thrombosis and thrombus resolution. Control of the mechanisms which regulate these activities may therefore be important in preventing thrombus formation or stimulating its resolution.

Objective— Venous thromboembolism is a common complication in patients with cancer, resulting in significant morbidity and mortality. Clinical studies suggest that the incidence of venous thromboembolic events increased after treatment of these patients with antiangiogenic agents. Thrombi resolve through a process of remodeling, involving the formation of microvascular channels within the thrombus. Our aim was to determine whether inhibiting angiogenesis affects venous thrombus resolution. Approach and Results— Thrombus was induced in the inferior vena cava of mice. These mice were treated with axitinib (50 mg/kg per day), 2-methoxyestradiol (2ME, 150 mg/kg per day), or vehicle control. Thrombus size, recanalization, neovascularization, inflammatory cell content, and collagen content were assessed after axitinib (days 3, 10, 17) and 2ME (day 10 only) treatment (n=6/group). Axitinib treatment resulted in reduced thrombus resolution ( P P P P P Conclusions— Antiangiogenic therapy (using axitinib and 2ME) inhibits the resolution of venous thrombi, which could lead to persistent venous obstruction and the possibility of thrombus extension. This potential prolongation of venous occlusion by antiangiogenic agents should therefore be taken into consideration in trials of these agents and when managing the complications of venous thromboembolic events in patients with cancer.

Background— Deep vein thrombosis remains a major health problem necessitating accurate diagnosis. Thrombolysis is associated with significant morbidity and is effective only for the treatment of unorganized thrombus. We tested the feasibility of in vivo magnetization transfer (MT) and diffusion-weighted magnetic resonance imaging to detect thrombus organization in a murine model of deep vein thrombosis. Methods and Results— Deep vein thrombosis was induced in the inferior vena cava of male BALB/C mice. Magnetic resonance imaging was performed at days 1, 7, 14, 21, and 28 after thrombus induction using MT, diffusion-weighted, inversion-recovery, and T1-mapping protocols. Delayed enhancement and T1 mapping were repeated 2 hours after injection of a fibrin contrast agent. Finally, excised thrombi were used for histology. We found that MT and diffusion-weighted imaging can detect histological changes associated with thrombus aging. MT rate (MTR) maps and percentage of MT rate (%MTR) allowed visualization and quantification of the thrombus protein content, respectively. The %MTR increased with thrombus organization and was significantly higher at days 14, 21, and 28 after thrombus induction (days 1, 7, 14, 21, 28: %MTR=2483±451, 2079±1210, 7029±2490, 10 295±4356, 32 994±25 449; P ANOVA r =0.70; P −3 mm 2 /s]). Thrombus at days 7 and 14 had the highest apparent diffusion coefficient values (0.95±0.09 and 1.10±0.18 [×10 −3 mm 2 /s]). Conclusions— MT and diffusion-weighted magnetic resonance imaging sequences are promising for the staging of thrombus composition and could be useful in guiding medical intervention.

Background— Abdominal aortic aneurysm (AAA) is a common cardiovascular disease among older people and demonstrates significant heritability. In contrast to similar complex diseases, relatively few genetic associations with AAA have been confirmed. We reanalyzed our genome-wide study and carried through to replication suggestive discovery associations at a lower level of significance. Methods and Results— A genome-wide association study was conducted using 1830 cases from the United Kingdom, New Zealand, and Australia with infrarenal aorta diameter ≥30 mm or ruptured AAA and 5435 unscreened controls from the 1958 Birth Cohort and National Blood Service cohort from the Wellcome Trust Case Control Consortium. Eight suggestive associations with P −4 were carried through to in silico replication in 1292 AAA cases and 30 503 controls. One single-nucleotide polymorphism associated with P LDLR ) rs6511720 A was significantly associated overall and in 3 of 5 individual replication studies. The full study showed an association that reached genome-wide significance (odds ratio, 0.76; 95% confidence interval, 0.70–0.83; P =2.08×10 −10 ). Conclusions— LDLR rs6511720 is associated with AAA. This finding is consistent with established effects of this variant on coronary artery disease. Shared causal pathways with other cardiovascular diseases may present novel opportunities for preventative and therapeutic strategies for AAA.

Therapeutic neovascularisation using angiogenic cells has been hampered by the loss of cells from the target tissue. Encapsulation of these cells within a semi-permeable membrane could improve their retention within the ischaemic tissue without affecting the excretion of the angiogenic growth factors produced. Bio-spraying is a novel cell-handling technique that does not adversely affect cell viability. We used this technique to encapsulate human peripheral blood monocytes and found that cell viability, cell phenotype and functional downstream angiogenic signalling were preserved. Encapsulation of monocytes with macrophage-colony stimulating factor resulted in increased vascular-endothelial growth factor production and enhanced angiogenic function. Bio-spraying/encapsulation has the potential to enhance the efficacy of current angiogenic cell therapy strategies and merits further investigation.

Contains fulltext : 97601.pdf (Publisher’s version ) (Closed access) Abdominal aortic aneurysm (AAA) is a common cause of morbidity and mortality and has a significant heritability. We carried out a genome-wide association discovery study of 1866 patients with AAA and 5435 controls and replication of promising signals (lead SNP with a p value < 1 x 10(-5)) in 2871 additional cases and 32,687 controls and performed further follow-up in 1491 AAA and 11,060 controls. In the discovery study, nine loci demonstrated association with AAA (p < 1 x 10(-5)). In the replication sample, the lead SNP at one of these loci, rs1466535, located within intron 1 of low-density-lipoprotein receptor-related protein 1 (LRP1) demonstrated significant association (p = 0.0042). We confirmed the association of rs1466535 and AAA in our follow-up study (p = 0.035). In a combined analysis (6228 AAA and 49182 controls), rs1466535 had a consistent effect size and direction in all sample sets (combined p = 4.52 x 10(-10), odds ratio 1.15 [1.10-1.21]). No associations were seen for either rs1466535 or the 12q13.3 locus in independent association studies of coronary artery disease, blood pressure, diabetes, or hyperlipidaemia, suggesting that this locus is specific to AAA. Gene-expression studies demonstrated a trend toward increased LRP1 expression for the rs1466535 CC genotype in arterial tissues; there was a significant (p = 0.029) 1.19-fold (1.04-1.36) increase in LRP1 expression in CC homozygotes compared to TT homozygotes in aortic adventitia. Functional studies demonstrated that rs1466535 might alter a SREBP-1 binding site and influence enhancer activity at the locus. In conclusion, this study has identified a biologically plausible genetic variant associated specifically with AAA, and we suggest that this variant has a possible functional role in LRP1 expression. 9 p.

Background— We sought to perform a systematic lipid analysis of atherosclerotic plaques using emerging mass spectrometry techniques. Methods and Results— A chip-based robotic nanoelectrospray platform interfaced to a triple quadrupole mass spectrometer was adapted to analyze lipids in tissue sections and extracts from human endarterectomy specimens by shotgun lipidomics. Eighteen scans for different lipid classes plus additional scans for fatty acids resulted in the detection of 150 lipid species from 9 different classes of which 24 were detected in endarterectomies only. Further analyses focused on plaques from symptomatic and asymptomatic patients and stable versus unstable regions within the same lesion. Polyunsaturated cholesteryl esters with long-chain fatty acids and certain sphingomyelin species showed the greatest relative enrichment in plaques compared to plasma and formed part of a lipid signature for vulnerable and stable plaque areas in a systems-wide network analysis. In principal component analyses, the combination of lipid species across different classes provided a better separation of stable and unstable areas than individual lipid classes. Conclusions— This comprehensive analysis of plaque lipids demonstrates the potential of lipidomics for unraveling the lipid heterogeneity within atherosclerotic lesions.

Abdominal aortic aneurysms (AAA) are characterized by pathological remodeling of the aortic extracellular matrix (ECM). However, besides the well-characterized elastolysis and collagenolysis little is known about changes in other ECM proteins. Previous proteomics studies on AAA focused on cellular changes without emphasis on the ECM. In the present study, ECM proteins and their degradation products were selectively extracted from aneurysmal and control aortas using a solubility-based subfractionation methodology and analyzed by gel-liquid chromatography-tandem MS and label-free quantitation. The proteomics analysis revealed novel changes in the ECM of AAA, including increased expression as well as degradation of collagen XII, thrombospondin 2, aortic carboxypeptidase-like protein, periostin, fibronectin and tenascin. Proteomics also confirmed the accumulation of macrophage metalloelastase (MMP-12). Incubation of control aortic tissue with recombinant MMP-12 resulted in the extensive fragmentation of these glycoproteins, most of which are novel substrates of MMP-12. In conclusion, our proteomics methodology allowed the first detailed analysis of the ECM in AAA and identified markers of pathological ECM remodeling related to MMP-12 activity.

Background— Macrophages have been identified as a major contributor to plaque development and destabilization in atherosclerosis. The aim of this study was to noninvasively assess uptake of citrate coated very small iron oxide particles at different stages of plaque development in the brachiocephalic artery of apoE −/− mice. Susceptibility gradient mapping (SGM) was applied to generate positive contrast images and to quantify iron oxide uptake. Methods and Results— ApoE −/− mice were fed a high-fat diet for 4, 8, or 12 weeks; 300 μmol Fe/kg was injected 24 and 48 hours before final MRI. Increasing very small iron oxide particle uptake was observed over the course of atherosclerotic plaque development. Simultaneous administration of pravastatin led to a significant decrease in very small iron oxide particle uptake, assessed by mass spectroscopy and histology. SGM-MRI allowed the generation of positive contrast images, and magnitudes (mT/m) of contrast enhancement in SG parameter maps significantly correlated with the absolute iron oxide content ( R 2 =0.70, P R 2 =0.71, P Conclusions— This study shows an increase in iron oxide uptake (measured by in vivo SGM-MRI, histology, and mass spectroscopy) with the progression of plaque development in an apoE −/− mouse model of accelerated atherosclerosis. Positive contrast provided by SGM-MRI allowed for a clear visualization of intraplaque iron oxide depositions, and magnitudes (mT/m) of contrast enhancement in SG parameter maps allowed for the quantification of intraplaque iron oxide particles.

Observational studies have shown that inflammatory cells accumulate within the thrombus and surrounding vein wall during the natural history of venous thrombosis. More recent studies have begun to unravel the mechanisms that regulate this interaction and have confirmed that thrombosis and inflammation are intimately linked. This review outlines our current knowledge of the complex relationship between inflammatory cell activity and venous thrombosis and highlights new areas of research in this field. A better understanding of this relationship could lead to the development of novel therapeutic targets that inhibit thrombus formation or promote its resolution.

Human lymphoedema distichiasis syndrome (LDS) results from germline mutations in transcription factor FOXC2. In a mouse model, lack of lymphatic and venous valves is observed plus abnormal smooth muscle cell recruitment to initial lymphatics. We investigated the mechanism of lymphoedema in humans with FOXC2 mutations, specifically the effect of gravitational forces on dermal lymphatic function.We performed (1) quantitative fluorescence microlymphangiography (FML) on the skin of the forearm (non-swollen region) at heart level, and the foot (swollen region) below heart level (dependent) and then at heart level, and (2) immunohistochemical staining of microlymphatics in forearm and foot skin biopsies, using antibodies to podoplanin, LYVE-1 and smooth muscle actin.FML revealed a marked reduction in fluid uptake by initial lymphatics in the LDS foot during dependency, yet normal uptake (similar to controls) in the same foot at heart level and in LDS forearms. In control subjects, dependency did not impair initial lymphatic filling. Immunohistochemical microlymphatic density in forearm and foot did not differ between LDS and controls.FOXC2 mutations cause a functional failure of dermal initial lymphatics during gravitational stress (dependency), but not hypoplasia. The results reveal a pathophysiological mechanism contributing to swelling in LDS.

Milroy disease is an inherited autosomal dominant lymphoedema caused by mutations in the gene for vascular endothelial growth factor receptor-3 (VEGFR-3, also known as FLT4). The phenotype has to date been ascribed to lymphatic aplasia. We further investigated the structural and functional defects underlying the phenotype in humans.The skin of the swollen foot and the non-swollen forearm was examined by (i) fluorescence microlymphangiography, to quantify functional initial lymphatic density in vivo; and (ii) podoplanin and LYVE-1 immunohistochemistry of biopsies, to quantify structural lymphatic density. Leg vein function was assessed by colour Doppler duplex ultrasound.Milroy patients exhibited profound (86-91%) functional failure of the initial lymphatics in the foot; the forearm was unimpaired. Dermal lymphatics were present in biopsies but density was reduced by 51-61% (foot) and 26-33% (forearm). Saphenous venous reflux was present in 9/10 individuals with VEGFR3 mutations, including two carriers.We propose that VEGFR3 mutations in humans cause lymphoedema through a failure of tissue protein and fluid absorption. This is due to a profound functional failure of initial lymphatics and is not explained by microlymphatic hypoplasia alone. The superficial venous valve reflux indicates the dual role of VEGFR-3 in lymphatic and venous development.

Intimal hyperplasia leading to restenosis is the major process that limits the success of cardiovascular intervention. The emergence of vascular progenitor cells and, in particular, endothelial progenitor cells has led to great interest in their potential therapeutic value in preventing intimal hyperplasia. We review the mechanism of intimal hyperplasia and highlight the important attenuating role played by a functional endothelium. The role of endothelial progenitor cells in maintaining endothelial function is reviewed and we describe how reduced progenitor cell number and function and reduced endothelial function lead to an increased risk of intimal hyperplasia. We review other potential sources of endothelial cells, including monocytes, mesenchymal stem cells and tissue resident stem cells. Endothelial progenitor cells have been used in clinical trials to reduce the risk of restenosis with varied success. Progenitor cells have huge therapeutic potential to prevent intimal hyperplasia but a more detailed understanding of vascular progenitor cell biology is necessary before further clinical trials are commenced.

Atherosclerosis and its consequences remain the main cause of mortality in industrialized and developing nations. Plaque burden and progression have been shown to be independent predictors for future cardiac events by intravascular ultrasound. Routine prospective imaging is hampered by the invasive nature of intravascular ultrasound. A noninvasive technique would therefore be more suitable for screening of atherosclerosis in large populations. Here we introduce an elastin-specific magnetic resonance contrast agent (ESMA) for noninvasive quantification of plaque burden in a mouse model of atherosclerosis. The strong signal provided by ESMA allows for imaging with high spatial resolution, resulting in accurate assessment of plaque burden. Additionally, plaque characterization by quantifying intraplaque elastin content using signal intensity measurements is possible. Changes in elastin content and the high abundance of elastin during plaque development, in combination with the imaging properties of ESMA, provide potential for noninvasive assessment of plaque burden by molecular magnetic resonance imaging (MRI).

To determine the changes in the endocrine mechanisms of fluid balance after cardiopulmonary bypass in children.Prospective study; analysis of numbered plasma samples performed blind with respect to clinical data.Regional paediatric cardiothoracic unit.Nine patients, median age 4, range 2 to 9 years, five males. Patients under the age of 1 year were excluded because of the frequent blood sampling involved.Plasma concentrations of atrial natriuretic peptide (ANP), arginine vasopressin, plasma renin activity, aldosterone, noradrenaline and adrenaline, and urinary concentrations of cyclic guanosine monophosphate (cGMP) as measured by radioimmunoassay.After 30 minutes of cardiopulmonary bypass plasma atrial natriuretic peptide (ANP) decreased from (mean (SEM)) 151 (71) pg/ml to 52 (44) pg/ml (NS), and urinary production of its second messenger cyclic guanosine monophosphate (cGMP) decreased from 1286 (600) pmol/ml to 151 (414) pmol (p0.05). Other plasma concentrations of hormones studied did not change significantly although arginine vasopressin, adrenaline, and noradrenaline increased whereas aldosterone and plasma renin activity decreased. After cardiopulmonary bypass stopped there was an immediate and significant rise in plasma ANP, but within the next 24 hours plasma ANP declined significantly (p0.05), decreasing from 294 (49) pg/ml to 64 (29) pg/ml at 22 hours. In the postoperative period there was a significant correlation between plasma ANP and both mean fluid balance (r = 0.96, p0.001) and mean urine output (r = 0.97, p0.001). Plasma aldosterone peaked (p0.05) at 22 hours after operation, and argine vasopressin peaked (p0.05) at two hours and then declined (p0.05) to a trough at 24 hours. Plasma renin activity, adrenaline, noradrenaline, and urinary cGMP concentrations, and mean central venous pressure did not change significantly in the postoperative period.The changes documented show the differing pattern of release of water balance hormones invoked by cardiopulmonary bypass. The central role of ANP is shown by its strong correlation with urinary output and its similarly strong relation to fluid balance.

The cysteine protease, legumain, is thought to have a role in the processing and activation of proteases such as cathepsin-L, which have been implicated in plaque rupture. This study aimed to determine: if legumain activity is up-regulated in unstable areas of plaque; the effect of legumain over-expression on the activity of cathepsin-L and the effect of mutation of the legumain RGD sequence on its cellular location.Legumain was measured in human carotid plaque extracts (n=17) using a novel ELISA and modified activity assay. Unstable regions of plaque contained more than twice the amount of legumain protein (P0.001) and activity (P0.03) compared with stable regions of the same plaque. Over-expression of legumain in THP-1 macrophages using an adenoviral construct resulted in the processing of cathepsin-L from its 30kDa to its 25kDa form compared with controls.Unstable regions of plaque contain increased levels of active legumain. Over-expression of legumain in macrophages alters intracellular processing of cathepsin-L to its mature 25kDa form. This may be a means by which legumain could contribute to plaque instability.

Background Our previous studies showed that the direct injection of an adenovirus construct expressing urokinase-type plasminogen activator (uPA) into experimental venous thrombi significantly reduces thrombus weight. The systemic use of adenovirus vectors is limited by inherent hepatic tropism and inflammatory response. As macrophages are recruited into venous thrombi, it is reasonable to speculate that these cells could be used to target the adenovirus uPA (ad-uPA) gene construct to the thrombus. The aims of this study were to determine whether macrophages transduced with ad-uPA have increased fibrinolytic activity and whether systemic injection of transduced cells could be used to target uPA expression to the thrombus and reduce its size. Methods The effect of up-regulating uPA was examined in an immortalized macrophage cell line (MM6) and macrophages differentiated from human blood monocyte-derived macrophages (HBMMs). Cells were infected with ad-uPA or blank control virus (ad-blank). Fibrinolytic mediator expression, cell viability, and cytokine expression were measured by activity assays and enzyme-linked immunosorbent assays. Monocyte migration was measured using a modified Boyden chamber assay. A model of venous thrombosis was developed and characterized in mice with severe combined immunodeficiency (SCID). This model was used to study whether systemically administered macrophages over-expressing uPA reduced thrombus size. Uptake of HBMMs into the thrombus induced in these mice was confirmed by a combination of PKH2-labeled cell tracking and colocalization with human leukocyte antigen (HLA) by immunohistology. Results Compared with ad-blank, treated HBMMs transduction with ad-uPA increased uPA production by >1000-fold (P = .003), uPA activity by 150-fold (P = .0001), and soluble uPA receptor (uPAR) by almost twofold (P = .043). Expression of plasminogen activator inhibitor (PAI-1) and PAI-2 was decreased by about twofold (P = .011) and threefold (P = .005), respectively. Up-regulation of uPA had no effect on cell viability or inflammatory cytokine production compared with ad-blank or untreated cells. Ad-uPA transduction increased the migration rate of HBMMs (about 20%, P = .03) and MM6 cells (>twofold, P = .005) compared with ad-blank treated controls. Human macrophage recruitment into the mouse thrombus was confirmed by the colocalization of HLA with the PKH2-marked cells. Systemic injection of uPA-up-regulated HBMMs reduced thrombus weight by approximately 20% compared with ad-blank (P = .038) or sham-treated controls (P = .0028). Conclusion Transduction of HBBM with ad-uPA increases their fibrinolytic activity. Systemic administration of uPA up-regulated HBBMs reduced thrombus size in an experimental model of venous thrombosis. Alternative methods of delivering fibrinolytic agents are worth exploring., Clinical Relevance The use of thrombolysis in the treatment of acute iliofemoral deep vein thrombosis is not suitable for all patients. Our previous studies have shown that direct injection of an adenovirus construct expressing urokinase plasminogen activator (uPA) into experimental venous thrombi significantly reduced thrombus weight. The systemic use of adenovirus vectors is, however, limited by both their inherent hepatic tropism, which precludes targeted delivery to disease sites, and by the associated host inflammatory response. As macrophages are recruited into venous thrombi, these cells could be used to target uPA gene constructs to the thrombus after systemic administration.

An investigation of restriction fragment length polymorphisms (RFLPs) and association with blood pressure was carried out for the candidate genes coding for renin (REN) and atrial natriuretic peptide (ANP). The relationship between blood pressure, REN and ANP gene RFLPs was tested in a population of black Afro-Caribbean and white European subjects sampled randomly from family practice registers. Upper and lower quintiles for diastolic blood pressure were selected for analysis. Digests of DNA were prepared from leucocytes and RFLPs were determined using Southern blotting analysis with REN and ANP gene probes. There were highly significant ethnic differences found for BglI, Bg/lI and TaqI polymorphisms with 5'REN probe and for BglI polymorphism with an ANP probe. There was also an association between blood pressure and the BglI/REN polymorphism in Afro-Caribbeans but not with any other polymorphisms studied in either ethnic group. The findings show significant ethnic RFLP differences at the gene loci for both renin and ANP and provide evidence for a possible link between variations within or close to the renin gene and elevated blood pressure in Afro-Caribbeans.

Objective— Rapid thrombus recanalization reduces the incidence of post–thrombotic complications. This study aimed to discover whether adenovirus-mediated transfection of the vascular endothelial growth factor gene (ad.VEGF) enhanced thrombus recanalization and resolution. Methods and Results— In rats, thrombi were directly injected with either ad.VEGF (n=40) or ad.GFP (n=37). Thrombi in SCID mice (n=12) were injected with human macrophages transfected with ad.VEGF or ad.GFP. Thrombi were analyzed at 1 to 14 days. GFP was found mainly in the vein wall and adventitia by 3 days, but was predominantly found in cells within the body of thrombus by day 7. VEGF levels peaked at 4 days (376±299 pg/mg protein). Ad.VEGF treatment reduced thrombus size by >50% (47.7±5.1 mm 2 to 22.0±4.0 mm 2 , P =0.0003) and increased recanalization by >3-fold (3.9±0.69% to 13.6±4.1%, P =0.024) compared with controls. Ad.VEGF treatment increased macrophage recruitment into the thrombus by more than 50% ( P =0.002). Ad.VEGF-transfected macrophages reduced thrombus size by 30% compared with controls (12.3±0.89 mm 2 to 8.7±1.4 mm 2 , P =0.04) and enhanced vein lumen recanalization (3.39±0.34% to 5.07±0.57%, P =0.02). Conclusion— Treatment with ad.VEGF enhanced thrombus recanalization and resolution, probably as a consequence of an increase in macrophage recruitment.

Mammalian orthologues of the Drosophila tribbles protein (Trb1, Trb2 and Trb3) are a recently described family of signalling molecules that regulate gene expression by modulation of protein kinase signalling pathways. In the present study, a screen for mRNA species specifically regulated in vulnerable regions of human atherosclerotic plaque demonstrated the up-regulation of both Trb1 and Trb2, the latter by more than 8-fold. In vitro experiments in primary human monocyte-derived macrophages showed that Trb2 expression was up-regulated by treatment with oxidized LDL (low-density lipoprotein), and that expression of recombinant Trb2 specifically reduced macrophage levels of IL-10 (interleukin-10) mRNA. Our results thus identify Trb2 as a highly regulated gene in vulnerable atherosclerotic lesions, and demonstrate inhibition of macrophage IL-10 biosynthesis as a potential pro-inflammatory consequence of high Trb2 expression, which may contribute to plaque instability.

Background Deep vein thrombosis of the leg affects 1–2 per cent of the population with an annual incidence of 0·5–1 per 1000. It presents with non-specific symptoms and signs making clinical diagnosis difficult. Techniques to image and diagnose this condition are advancing rapidly. Methods and results A literature review from 1980 to 2007 was undertaken using PubMed, The Cochrane Library, Medline and Embase. The most frequently used diagnostic test is duplex ultrasonography which is accurate above the knee and has a low cost, but is limited by inaccuracy when assessing the pelvic and distal veins and in diagnosing a new thrombosis in the post-thrombotic limb. Magnetic resonance imaging (MRI) and sonographic elasticity imaging are more recent techniques that have shown promise in overcoming these limitations. However, their availability is currently restricted because they are expensive. Computed tomography (CT) is sensitive, specific and provides good imaging of the pelvis. It has the advantage that it can be performed at the same time as CT pulmonary angiography. Conclusion MRI has some specific advantages over duplex ultrasonography, but requires refinement before it can be used clinically. Venography or CT venography should be considered when duplex scanning is inadequate.

Background The presence of fibrous tissue in poorly healing venous leg ulcers suggests abnormal collagen metabolism. The aim was to determine whether there were differences in collagen turnover and matrix metalloproteinase (MMP) activity between ulcers that healed, those that did not heal and normal skin. Methods Biopsies were taken from the ulcers of 12 patients whose venous ulcers went on to heal and 15 patients whose ulcers failed to heal despite 12 months of compression bandaging. Biopsies were taken from 15 normal controls. Collagen turnover (collagen III N-terminal propeptide (PIIINP) and degraded collagen), and total MMP, MMP-1 and MMP-3 activities were measured. Results PIIINP and degraded collagen levels were higher in ulcers that healed compared with lesions that failed to heal (P = 0·005 and P < 0·001 respectively) and normal skin (P = 0·003 and P < 0·001). MMP-1 activity was also higher in healing ulcers than resistant ulcers (P < 0·001) and normal skin (P < 0·001). Significantly more total MMP activity was present in all ulcers than in normal skin (P < 0·001), but there was no difference in total MMP (and MMP-3 activity) between ulcers that healed and those that did not. Conclusion Rapidly healing venous leg ulcers had increased collagen turnover and MMP-1 activity, which appeared to differentiate them from those that failed to heal within 12 months.

Plasma atrial natriuretic peptide was measured in 13 children between the ages of 1 week and 2 years 9 months during atrioventricular re-entrant tachycardia and 15 minutes after the restoration of sinus rhythm. There was a significant decline in atrial natriuretic peptide during sinus rhythm. Plasma concentrations of the peptide were significantly higher during tachycardia in seven infants under 1 year than in the six older children. The heart rates and the duration of tachycardia were not significantly different in the two age groups. Cardiac failure was present in five of seven children under 18 weeks of age during tachycardia but in none of the older children. The plasma concentration of atrial natriuretic peptide did not significantly correlate with duration of tachycardia or heart rate. If tachycardia occurs in young infants the low functional reserve capacity of the developing heart leads to cardiac failure more frequently and it is likely that this was the cause of the significantly higher plasma concentration of atrial natriuretic peptide in the younger children.

Objective—Galectin-3 (Gal-3) is a 26-kDa lectin known to regulate many aspects of inflammatory cell behavior. We assessed the hypothesis that increased levels of Gal-3 contribute to atherosclerotic plaque progression by enhancing monocyte chemoattraction through macrophage activation.Methods and Results—Gal-3 was found to be upregulated in unstable plaque regions of carotid endarterectomy (CEA) specimens compared with stable regions from the same patient (3.2-fold,PP−/−mice on a high fat diet (n=14) and wild-type controls (n=9) showed that Gal-3 mRNA and protein levels are elevated by 16.3-fold (PPPConclusions—Our data suggest that Gal-3 is both a marker of atherosclerotic plaque progression and a central contributor to the pathology by amplification of key proinflammatory molecules.

This study examined whether intraluminal thrombus in abdominal aortic aneurysms (AAAs) is a source of fibrinolytic activity and proteolysis that could weaken the aneurysm wall. Plasmin, tissue plasminogen activator (tPA), and urokinase plasminogen activator (uPA) activity, plasminogen activator inhibitor 1 (PAI-1), and α2-antiplasmin (α2AP) antigen were measured in the AAA wall and juxtamural and luminal aspects of intraluminal thrombus in 18 patients. The aneurysm wall contained 100-fold higher tPA activity (1.06 ± 0.34 [standard error of measurement] U/mg soluble protein) compared with juxtamural thrombus (JMT) (0.011 ± 0.001 ) and luminal thrombus (LT) (0.01 ± 0.001) ( p < .00001) and over 6-fold higher uPA activity (29.3 ± 3.4 IU/mg compared with the JMT (4.3 ± 2.4, p = .00024) and LT (7.9 ± 1.76, p = .0005). The LT had significantly lower levels of PAI-1 (1.26 ± 0.34 ng/mg) than the AAA wall (2.08 ± 0.51, p = .04) and the JMT (3.94 ± 0.85, p = .007). The levels of α2AP in the wall (19.4 ± 3.1 ng/mg) were lower than in the JMT or LT (43.0 ± 7.9 ng/mg, p = .013, and 47.6 ± 6.0 ng/mg, p = .002, respectively). There was no significant difference, however, in plasmin activity among the AAA wall, JMT, and LT. There were significant amounts of latent gelatinase B (matrix metalloproteinase [MMP]-9) in the AAA, JMT, and LT. Mean levels of activated MMP-9 activity were similar in the AAA, JMT, and LT. Plasmin activation of MMPs at the interface between intraluminal thrombus and the aneurysm wall may enhance proteolysis and accelerate aneurysm expansion.

ObjectiveTo investigate the importance of monocyte recruitment in thrombus resolution and the role of cysteine-cysteine (CC) chemokines and the CC chemokine receptor, CCR2, in this process.MethodsPeritoneal macrophages, monocyte chemotactic protein 1 (MCP1), or carrier solutions were injected into thrombi induced in the vena cava of rats. Caval thrombi were also formed in CCR2−/− and MCP1−/− mice and in wild-type mice transfected with an adenoviral construct expressing a broad-spectrum CC receptor antagonist.ResultsDirect administration of peritoneal macrophages decreased thrombus size by more than fivefold and increased recanalization by more than fourfold compared with controls (P < .001). A 100-ng MCP1dose reduced thrombus size by more than sixfold (P < .01) and increased recanalization by more than sevenfold (P < .01), without affecting macrophage recruitment. Deletion of CCR2 or blockade of all CC chemokines inhibited both monocyte recruitment (P < .05) and thrombus resolution (P < .01), but knocking out MCP-1 had no effect.ConclusionIncreasing macrophage numbers in the thrombus enhances its resolution. MCP1 treatment enhances resolution by stimulating recanalization, independent of an effect on monocyte recruitment. CCR2 deficiency has the same effect as blockade of all CC chemokines. CCR2 receptor activation may therefore be an important mechanism in monocyte recruitment into venous thrombi and could be targeted to promote their resolution.Clinical RelevanceDeep vein thrombosis may lead to residual venous obstruction or reflux and result in post-thrombotic complications, which are debilitating and have a substantial socioeconomic impact. Enhancing the resolution of venous thrombi may reduce post-thrombotic complications.

Background—The purpose of this study was to determine whether endothelial cells of bone marrow origin are involved in thrombus recanalization.Methods and Results—Irradiated mice were reconstituted with bone marrow from transgenic donors expressing green fluorescent protein (GFP) linked to the Tie2 promoter. Thrombi were formed in 2 groups of 6 mice. GFP-expressing cells were located and quantified in sections of the thrombi taken after 7 and 14 days. The cell markers Mac-3, F4/80, CD68 (macrophage), and vascular endothelial growth factor receptor 2 (VEGFR2; endothelial cells) were used to determine colocalization with GFP expression in tissue sections and peritoneal macrophages. The markers CD34 and VEGFR2 were used to quantify changes in circulating endothelial cells by flow cytometry of blood from 3 cohorts of wild-type animals that had either a thrombus induced (n=18), a sham operation (n=18), or no operation (n=10). The number of GFP-expressing cells was found to increase by ≈3-fold in thrombi formed in transplanted animals between 7 and 14 days after induction (P=0.0022). No GFP-expressing cells were found lining the new vascular channels that formed at either time interval, but many of the GFP-expressing cells also expressed Mac-3, CD68, and VEGFR2. Approximately twice as many circulating CD34+/VEGFR2+cells were found by day 3 in animals with thrombus compared with sham controls (CD45−,P=0.046 and CD45+,P=0.016).Conclusions—Bone marrow–derived, Tie2-expressing cells were recruited into the thrombus during resolution but did not line the new vessels. Many of these cells expressed a macrophage phenotype and may represent a population of plastic stem cells that orchestrate thrombus recanalization.

Research into the healing of venous leg ulcers is increasing as they are a common problem. The wound fluid bathing an ulcer is thought to reflect the wound microenvironment, and the properties of wound fluids have been studied in attempts to find ways to promote healing. After a brief summary of normal wound healing, this article reviews some of the research that has been carried out on venous ulcer wound fluid, with respect to its biochemistry, proteolytic nature, growth factor profile, and effects on cell cultures. Some of the problems and pitfalls inherent in performing and interpreting wound fluid studies are discussed. Finally, a proposal is made for standardizing research on wound fluids that would improve comparisons between different studies.

ObjectivesEnhancing thrombus resolution may reduce the long-term complications of venous thrombosis. The aim of this study was to examine whether a sustained release of vascular endothelial growth factor (VEGF) would further improve thrombus recanalization.MethodsInferior caval vein thrombosis was induced in a cohort of 21 male Wistar rats. A plasmid encoding the human VEGF gene (phVEGF) was injected directly into thrombus (30 to 50 μg) and the muscle adjacent to the inferior vena cava (300 to 400 μg). A plasmid containing the gene encoding β-galactosidase (pCMVβ) was injected into the same sites of a separate cohort of rats to act as a control. Tissues were harvested after 1 and 2 weeks, and β-galactosidase activity was measured to estimate transfection efficiency. Muscle and serum VEGF were measured by enzyme-inked immunosorbent assay. Thrombus size, recanalization, and organization were determined by computer-assisted image analysis.ResultsThe efficiency of control plasmid transfection into muscle was about 1%. No serum hVEGF was detected in phVEGF- or pCMVβ-treated animals. Significantly raised levels of hVEGF (P < .01) were detected in the muscle injected with phVEGF after 2 weeks compared with control muscle. There was a significant reduction in thrombus size of 23% (P < .05) and 48% (P < .001) in phVEGF-treated animals compared with pCMVβ-treated controls after 1 and 2 weeks, respectively. Thrombus recanalization was a significantly greater in the phVEGF-treated group after 2 weeks (mean 19% ± 2% [SEM]) compared with controls (mean 13% ± 2%, P < .01). There were no differences in the thrombus organization score.ConclusionVEGF gene therapy of venous thrombus resulted in smaller thrombi with greater recanalization. Angiogenic gene therapy may form the basis of a novel treatment that may improve the resolution of venous thrombi.Clinical RelevanceDeep vein thrombosis may lead to residual venous obstruction or reflux and result in post-thrombotic complications that are debilitating and have a substantial socioeconomic impact. Enhancing the resolution of venous thrombi may reduce post thrombotic complications.

Lymphoedema-distichiasis (LD) is a syndromic form of primary lymphoedema, where mutations in the gene for the developmental transcription factor FOXC2 have been shown to be causative. The disorder has been considered very rare, but our group has now ascertained 34 families and 11 sporadic cases in the UK. Two families with LD have no mutation in the coding region of FOXC2, although both are consistent with linkage to the FOXC2 locus. A deletion has been ruled out as a possible cause of LD in these families, leaving promoter mutations as the most likely cause. Sixteen previously unpublished mutations are reported, plus an estimate of the frequency of new mutations in this disorder.

ObjectiveOur previous work demonstrated that angiogenesis is inhibited in nonhealing venous ulcers. The object of this study was to determine whether local expression of vascular endothelial growth factor (VEGF) and other major regulators of vessel growth are related to healing of venous ulcers.Subjects and methodsThe study included 35 patients with venous ulcers (CEAP 6) and 9 patients whose ulcers had healed (CEAP 5). Control subjects were 18 patients undergoing routine operations (8 with closed suction drains, 10 standard skin biopsies). Healing ulcers were defined as having healed in less than a year from entry to the study; nonhealing ulcers failed to heal in this period. A 1-cm square biopsy specimen was taken from the edge of the ulcer or from a site of lipodermatosclerosis around a healed ulcer. Wound fluids were aspirated from beneath transparent occlusive dressings. Concentrations of VEGF165 and VEGF-R1 were measured in tissue homogenates with enzyme-linked immunosorbent assay, and results are expressed as mean ± SEM per milligram of soluble protein (SP). Expression of mRNA transcripts for the VEGF splice variants VEGF121, VEGF189, and VEGF165; the receptors VEGF-R1 and VEGF-R2; the angiopoietins Ang-1 and Ang-2; and their receptor, Tie-2, were measured in biopsy samples with multiplex polymerase chain reaction. Expression of each transcript was normalized to that of the housekeeping gene, GAPDH. Results were analyzed with analysis of variance, t test, and χ2 test.ResultsThere was no difference in VEGF165 protein concentration between biopsy specimens from healing ulcers (2.12 ± 0.34 ng/mg SP; n = 18) and nonhealing ulcers (2.36 ± 0.39 ng/mg SP; n = 12), but concentration was higher in all ulcer samples compared with healthy skin (0.57 ± 0.20 ng/mg SP; n = 10; P < .01)) and healed ulcers (0.33 ± 0.06 ng/mg SP; n = 9; P < .01). Concentration of VEGF165 protein in wound fluid was significantly higher in nonhealing venous ulcers (67.17 ± 13.87 ng/mg SP; n = 13) compared with healing venous ulcers (32.19 ± 7.90 ng/mg SP; n = 19; P < .05) or acute wounds (12.26 ± 4.50; n = 8; P < .01). Concentration of VEGF-R1 was similar in wound fluid obtained from healing ulcers (7.18 ± 1.34 ng/mg SP; n = 13) and nonhealing ulcers (7.02 ± 1.21 ng/mg SP; n = 19), and acute wounds (7.12 ± 2.35 ng/mg SP; n = 8). There was a weak but significant correlation between VEGF165 protein concentration in the ulcer biopsy specimen and wound fluid from the same ulcer (R2 = 0.2; P = .019; n = 27). Expression of mRNA for VEGF receptors and Tie-2 was poor. VEGF121 was expressed in all samples, and VEGF165 in 43 of 48 samples. mRNA expression of VEGF189 (P = .001), Ang-1 (P = .002), and Ang-2 (P = .026) was found in more samples from unhealed ulcers than from other sites. Healed ulcers had reduced mRNA expression of VEGF165 (0.181 ± 0.003) than did healing ulcers (0.307 ± 0.016; P = .007) or nonhealing ulcers (0.375 ± 0.033; P = .001). Relative expression of VEGF165 to Ang-2 was much lower in healed ulcers (0.4236 ± 0.060) than in healing ulcers (1.382 ± 0.235; P = .010) and nonhealing ulcers (1.887 ± 0.280; P = .003).ConclusionIn nonhealing venous ulcers there is a consistently high level of expression of VEGF, at both the gene transcript and protein level. As our previous data demonstrated that angiogenesis is depressed in these poorly healing ulcers, an increase in VEGF production may indicate an increased but ineffectual angiogenic drive. It is also possible that undiscovered inhibitors are released in the ulcer environment.

As many as 10% of airline passengers travelling without prophylaxis for long distances may develop a venous thrombosis. There is, however, no evidence that economy class travellers are at increased risk of thrombosis.A suitably powered prospective study, based on the incidence of deep-vein thrombosis (DVT) reported in previous studies on long-haul flights, was designed to determine the incidence of positive venous duplex scans and D-dimer elevations in low and intermediate-risk passengers, comparing passengers travelling in business and economy class.Eight hundred and ninety-nine passengers were recruited (180 travelling business class and 719 travelling economy). D-dimers were measured before and after the flight. A value greater than 500 ng/ml was accepted as abnormal. A thrombophilia screen was conducted which included the factor V Leiden mutation, the prothombin 20210A mutation, protein C and S levels, antithrombin levels, and anticardiolipin antibodies immunoglobulin G (IgG) and immunoglobulin M (IgM). On arrival, lower limb compression ultrasonography of the deep veins was performed. Logistical regression analysis was used to determine the risk factors related to abnormally high D-dimer levels.Only 434 subjects had a full venous duplex scan performed. None had ultrasonic evidence of venous thrombosis. Nine passengers tested at departure had elevated D-dimer levels and these volunteers were excluded from further study. Seventy-four of the 899 passengers had raised D-dimers on arrival. Twenty-two of 180 business class passengers (12%) developed elevated D-dimers compared with 52 of 719 economy class passengers (7%). There was no significant association between elevation of D-dimers and the class flown (odds ratio (OR) 0.61, p = 0.109). The factor V Leiden mutation, factor VIII levels and the use of aspirin were, however, associated with raised D-dimers (OR 3.36, p = 0.024; OR 1.01, p = 0.014; and OR 2.04, p = 0.038, respectively). Five hundred and five passengers were contacted within 6 months and none reported any symptoms of a clinical thrombosis or pulmonary embolus.The incidence of ultrasonically proven DVT is much lower than previously reported. However, more than 10% of all passengers developed raised D-dimers, which were unrelated to the class flown. A rise in D-dimers is associated with an inherent risk of thrombosis and/or thrombophilia, demonstrates activation of both the coagulation and fibrinolytic systems during long-haul flights, and may indicate the development of small thrombi.

SummaryVascular endothelial growth factor (VEGF) is a regulator of physiological and pathological angiogenesis and is found in naturally resolving experimental venous thrombi, where it may also regulate recanalisation. In this study VEGF protein was injected into venous thrombi to determine if this enhanced recanalisation and organisation. A rat model of inferior vena cava (IVC) thrombosis was used. Thrombi were formed in 3 groups (n = 3 per group). 10 μl 125I-VEGF was directly injected into thrombus thirty minutes after induction. Three hours, 1 day and 6 days later thrombus, IVC, and other tissues were harvested. 125I-VEGF was mostly distributed in the thrombus and the IVC, with smaller amounts in other tissues. Thrombi were formed in a further 4 groups (n = 6 per group).Thirty minutes after induction control solution or 1 ng, 10 ng or 100 ng recombinant human VEGF165 was injected directly into the thrombus. Lumen recanalisation, thrombus organisation and monocyte content were measured on digitised sections by image analysis. In animals treated with 10 ng of VEGF there was a greater area of lumen recanalisation [mean 5492 pixels, standard error of mean (sem) 922] compared to controls (mean 2974, sem 385) (P = 0.005). There was a significant increase in the organisation score in all treated animals (1 ng: mean 70, sem 1.7, P = 0.0025; 10 ng: mean 70, sem 2.0, P = 0.0042; 100 ng: mean 72, sem 1.9, P = 0.0003) compared to controls (mean 63, sem 1.7). The monocyte content was lower in the animals treated with 1 ng VEGF (mean 3.8% of thrombus area, sem 0.3%) compared to controls (mean 5.5%, sem 0.4%) (P = 0.0008).The proportion of monocytes migrating to the centre of the thrombus increased in a dose-related manner. VEGF may prove to be of use in the treatment of venous thrombosis.

The human monoclonal antibody mAb-LE2E9 partially inactivates human factor VIII (FVIII), leaving approximately 10% residual activity. The antithrombotic efficacy of the antibody was evaluated in mouse models of inferior vena cava thrombosis. Thrombi were induced in wild-type mice given either the antibody or saline. No thrombi occurred in any of 8 mice treated with mAb-LE2E9, whereas 6 of 8 control mice developed thrombi (P =.007). Treatment with mAb-LE2E9 did not result in a severe bleeding phenotype: a tail-cutting experiment that resulted in death of C57BL/6 FVIII-deficient (FVIII(-/-)) mice did not cause hemorrhagic death in mice treated with mAb-LE2E9. To evaluate the antithrombotic effect of mAb-LE2E9 in presence of human FVIII, thrombus formation was induced in FVIII(-/-) mice reconstituted intravenously with recombinant human FVIII (rhFVIII) or rhFVIII preincubated with mAb-LE2E9. Only 1 of 9 mice produced a thrombus in the rhFVIII/antibody complex-treated group, compared with 7 of 9 in the control group (P =.015). FVIII(-/-) mice were also reconstituted with rhFVIII and then injected with either mAb-LE2E9 or saline. One of 14 mice in the group treated with the antibody developed a thrombus, compared with 10 of 14 in the control group (P =.001). The thrombi occurring in antibody-treated animals were smaller than in controls (P

Background — Atherosclerosis is implicated in the pathogenesis of abdominal aortic aneurysm (AAA) but more often causes aortic occlusive disease (AOD). The matrix metalloproteinases (MMPs) degrade extracellular matrix and may play a central role in the pathogenesis of AAA. The aim of this study was to examine differences in the patterns of MMP and MMP inhibitor expression between AAA and AOD. Methods and Results — The expression of mRNA for 14 MMPs and 4 tissue inhibitors of metalloproteinases (TIMPs) was estimated in samples of aortic wall from 8 patients with AAA and 8 with AOD using the reverse-transcriptase polymerase chain reaction with a synthetic multicompetitor standard. AAA wall expressed significantly more stromelysin-1 (MMP-3) (mean log 10 ratio [copy enzyme cDNA/copy GAPDH cDNA], −1.9; range, −3.3 to −0.7) than the AOD wall (mean, 4; range, −5.7 to −2.4), P P Conclusions — Both AAA and AOD walls express similar levels of a wide range of MMPs, including cell membrane–bound MT-MMPs. Stromelysin-1 (MMP-3) and TIMP-3 were, however, over expressed in the AAA samples and may be involved aneurysm pathogenesis. Stromelysin-1 could provide a target for pharmacological inhibition.