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Background and Purpose: Hematopoietic progenitor cells (HPCs) may attenuate the response to vascular injury by maintaining endothelial integrity and function. Our aim was to determine whether circulating HPC number and function correlate with restenosis after carotid endarterectomy., Methods: HPC number (CD34(+)/CD133(+) cells), early colony-forming units, migratory capacity, and senescence were analyzed in blood collected preoperatively, 1 day, and 6 weeks postoperatively. Mobilizing cytokine levels were also measured. Stenosis was assessed by duplex scanning., Results: HPC numbers (P<0.001) and early colony-forming unit count (P=0.001) fell rapidly 24 hours postoperatively. Restenosis at 6 months correlated negatively with the magnitude of postoperative falls in HPC numbers (R=-0.38, P=0.013) and early colony-forming unit counts (R=-0.42, P=0.008). The migratory capacity of preoperative HPCs correlated negatively with restenosis (R=-0.48, P=0.007). Preoperative SDF1 levels correlated with falls in HPC number (R=0.42, P=0.044) and early colony-forming unit counts (R=0.56, P=0.004)., Conclusions: HPC function appears to be linked to the development of carotid artery restenosis after endarterectomy. These data support the concept that HPCs have a role in regulating remodeling of the injured arterial wall.

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.

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)., Conclusions: Treatment with ad.VEGF enhanced thrombus recanalization and resolution, probably as a consequence of an increase in macrophage recruitment.

There is increasing evidence that modified phospholipid products of low density lipoprotein (LDL) oxidation mediate inflammatory processes within vulnerable atherosclerotic lesions. Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is present in vulnerable plaque regions where it acts on phospholipid oxidation products to generate the pro-inflammatory lysophsopholipids and oxidized non-esterified fatty acids. This association together with identification of circulating Lp-PLA(2) levels as an independent predictor of cardiovascular disease provides a rationale for development of Lp-PLA(2) inhibitors as therapy for atherosclerosis. Here we report a systematic analysis of the effects of in vitro oxidation in the absence and presence of an Lp-PLA(2) inhibitor on the phosphatidylcholine (PC) composition of human LDL. Mass spectrometry identifies three classes of PC whose concentration is significantly enhanced during LDL oxidation. Of these, a series of molecules, represented by peaks in the m/z range 594-666 and identified as truncated PC oxidation products by accurate mass measurements using an LTQ Orbitrap mass spectrometer, are the predominant substrates for Lp-PLA(2). A second series of oxidation products, represented by peaks in the m/z range 746-830 and identified by LTQ Orbitrap analysis as non-truncated oxidized PCs, are quantitatively more abundant but are less efficient Lp-PLA(2) substrates. The major PC products of Lp-PLA(2), saturated and mono-unsaturated lyso-PC, constitute the third class. Mass spectrometric analysis confirms the presence of many of these PCs within human atherosclerotic lesions, suggesting that they could potentially be used as in vivo markers of atherosclerotic disease progression and response to Lp-PLA(2) inhibitor therapy.

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, P<0.05) at the mRNA (n=12) and (2.3-fold, P<0.01) at the protein level (n=9). Analysis of aortic tissue from ApoE-/- 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 (P<0.001) and 12.2-fold (P<0.01) and that Gal-3 staining colocalizes with macrophages. In vitro, conditioned media from Gal-3-treated human macrophages induced an up to 6-fold increase in human monocyte chemotaxis (P<0.01, ANOVA), an effect that was reduced by 66 and 60% by Pertussis Toxin (PTX) and the Vaccinia virus protein 35K, respectively. Microarray analysis of human macrophages and subsequent qPCR validation confirmed the upregulation of CC chemokines in response to Gal-3 treatment., Conclusions: 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.

Background: Cysteine proteases are potent elastolytic enzymes and together with their inhibitor, cystatin C, have been linked with the growth of abdominal aortic aneurysms (AAAs). These enzymes and their inhibitors have previously been studied in AAAs, but comparisons have always been made with wall from normal aorta. Atherosclerosis is a feature of aneurysmal disease and may therefore confound comparisons with normal wall. This study compared the expression and activity of cysteine proteases and their inhibitors in aneurysm wall with their expression in the aortic wall of patients with aortic occlusive disease (AOD)., Methods: Aortic wall was obtained from 82 patients with AAA and 13 with AOD. Protein expression and activity of cathepsin B, H, K, L and S, and cystatins A, B, and C were measured by enzyme-linked immunosorbent assay and specific fluorogenic substrate assays. Matrix metalloproteinase 9 (MMP-9) activity was measured by quantitative bioimmunoassay in the same extracts., Results: AAA wall had 330% more cathepsin H protein (P = .007) and >30% less cystatin C (P = .03) than the aortic wall from patients with AOD. The activity of cathepsins B, H, L, and S was significantly greater in AAA than AOD (376%, [P < .0001], 191%, [P = 0.019], 223%, P = 0.002, and approximately 20% [P = 0.045] respectively). MMP-9 activity was also increased in AAA compared with AOD (P<0.0001) and levels in the wall of AAA correlated positively with cathepsin L activity (r = 0.42, P<.0001) and negatively with cystatin C (r = -0.75, P<.0001)., Conclusions: The activity of four cathepsins B, H, L, and S was higher in the aneurysm wall than in aortic wall of patients with occlusive disease. This was associated with a reduced level of cystatin C in the aneurysmal wall. Cathepsin H was the only protein in which there was a correlation between protein level and activity, which suggests that post-translational modifications were responsible for activation of the other cathepsins. Increased cathepsin activity may influence the activity of MMP-9, which is thought to have an important role in aneurysm development.

Objective: The aim of this study was to assess the relationship between urinary and tissue haemosiderin in chronic leg ulcers, and its value as a diagnostic test for venous ulceration., Methods: 45 patients with chronic leg ulcers were recruited to the study (24 venous, 6 ischaemic, 6 lymphoedematous, 5 rheumatoid and 4 sickle cell). Punch biopsy of the ulcer edge was taken and early morning urine samples were collected. Positive Prussian-blue urinary haemosiderin granules were measured with a haemocytometer following Perls' staining. The percentage area of histological section staining positively with Perls' was measured using image analysis., Results: 84 urine samples and 46 ulcer biopsies were collected. Urinary haemosiderin was present in 92% of venous ulcer patients, but was absent in the ischaemic ulcer patients (p<0.0001). Significantly more urinary haemosiderin granules were detected in venous ulcer patients compared with patients who had lymphoedema (p<0.05). Tissue haemosiderin was detected in all ulcer types investigated. No correlation was found between the amounts of haemosiderin deposited in the tissue and the amount found in urine (r(2)=0.06)., Conclusions: Haemosiderin is present in the urine of most patients with venous ulcers but not in ischaemia ulcers.

Objectives: Devitalized tissue in a recalcitrant leg ulcer is common and may impede healing. The aim of this study was to evaluate the use of a non-invasive low frequency ultrasound device to debride chronic leg ulcers as an adjunct to compression bandages therapy., Methods: 19 patients with leg ulceration of at least 6 months were recruited. Low frequency ultrasound at 25kHz was delivered by a portable Sonaca--180 via a handheld probe, using normal saline as the irrigation/coupling medium. The ultrasound was applied for 10-20 seconds per probe head area onto the ulcer. Each leg underwent treatment at an interval of 2-3 weeks with compression bandages reapplied at the end of the treatment. Serial colour photographs were taken to evaluate the response at each visit., Results: Each patient received on average 5.7 treatments each ranged from 5-20 minutes depending on the ulcer size. Symptomatic relief (pain and odour reduction) was achieved in 6 patients. 7 patients achieved complete ulcer healing (mean ulcer size=4.72+/-SD 1.872cm(2)) but no response was observed in 8 patients. There were no major complications of the treatment which was relatively painless., Conclusions: The application of low frequency ultrasound debridement may heal some recalcitrant ulcers when standard compression regimens have failed. It is cheap and does not require admission. The role of simple wound cleansing requires further investigation.

Introduction: There is an increase in the natural level of urokinase-type plasminogen activator (uPA) activity within the thrombus during venous thrombus resolution. The use of uPA as a thrombolytic agent in the treatment of acute iliofemoral deep vein thrombosis is not suitable for all patients. This study aimed to determine whether thrombus resolution could be enhanced by upregulating uPA expression using adenoviral gene transfer as an alternative method of delivery., Methods: The production of functional uPA by an adenoviral gene construct (ad.uPA) was confirmed by a colorimetric substrate assay and fibrin plate lysis. Thrombus was formed in the inferior vena cava of wild-type mice and injected, 48-hours after induction, with either a control virus at 10(8) plaque-forming units (pfu) or ad.uPA at 10(7) or 10(8) pfu. Thrombi were removed and weighed 7 days after treatment. Activity of metalloproteinase (MMP) 2 and 9 was measured by zymography and the release of vascular endothelial growth factor (VEGF) and D-dimer levels by enzyme-linked immunoabsorbent assay. The results were expressed as a mean +/- SEM. Values were standardized for wet weight or for soluble protein content (mg/sol protein)., Results: Treatment with ad.uPA reduced thrombus weight by twofold compared with thrombi treated by control virus (15.1 +/- 1.1 mg vs 7.4 +/- 1.3 mg, P = .004). Urokinase activity (17 +/- 3 pg/mg wet weight) was detected in all treated thrombi, but there was no dose-dependent effect. D-dimer activity was increased twofold after treatment with ad.uPA (1.7 +/- 0.15 ng/mg of sol protein vs 0.8 +/- 0.1 ng/mg of sol protein, P = .0015) and was associated with a reduction in thrombus size (P = .03). Urokinase overexpression did not affect the activity of MMP2, MMP9, or VEGF in the thrombus., Conclusion: Increasing urokinase activity within the thrombus significantly enhanced natural thrombus resolution by a fibrinolytic action. Therapeutic delivery of ad.uPA in patients may provide a novel method of treating deep vein thrombosis., Clinical Relevance: The use of urokinase as a thrombolytic agent in the treatment of acute iliofemoral deep vein thrombosis is not suitable for all patients. This study aimed to determine whether thrombus resolution could be enhanced by upregulating urokinase expression using adenoviral gene transfer as an alternative method of therapeutic delivery. The study shows that by increasing urokinase activity within the thrombus, natural thrombus resolution can be significantly enhanced. The delivery of ad.uPA in patients may provide a novel method of treating deep vein thrombosis.

Objective: Comparison of gene expression in stable versus unstable atherosclerotic plaque may be confounded by interpatient variability. The aim of this study was to identify differences in gene expression between stable and unstable segments of plaque obtained from the same patient., Methods and Results: Human carotid endarterectomy specimens were segmented and macroscopically classified using a morphological classification system. Two analytical methods, an intraplaque and an interplaque analysis, revealed 170 and 1916 differentially expressed genes, respectively using Affymetrix gene chip analysis. A total of 115 genes were identified from both analyses. The differential expression of 27 genes was also confirmed using quantitative-polymerase chain reaction on a larger panel of samples. Eighteen of these genes have not been associated previously with plaque instability, including the metalloproteinase, ADAMDEC1 (approximately 37-fold), retinoic acid receptor responder-1 (approximately 5-fold), and cysteine protease legumain (approximately 3-fold). Matrix metalloproteinase-9 (MMP-9), cathepsin B, and a novel gene, legumain, a potential activator of MMPs and cathepsins, were also confirmed at the protein level., Conclusions: The differential expression of 18 genes not previously associated with plaque rupture has been confirmed in stable and unstable regions of the same atherosclerotic plaque. These genes may represent novel targets for the treatment of unstable plaque or useful diagnostic markers of plaque instability.

Objective: Our 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 Methods: The 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 VEGF(165) 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 VEGF(121), VEGF(189), and VEGF(165); 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 chi(2) test., Results: There was no difference in VEGF(165) 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 VEGF(165) 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 VEGF(165) protein concentration in the ulcer biopsy specimen and wound fluid from the same ulcer (R(2) = 0.2; P =.019; n = 27). Expression of mRNA for VEGF receptors and Tie-2 was poor. VEGF(121) was expressed in all samples, and VEGF(165) in 43 of 48 samples. mRNA expression of VEGF(189) (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 VEGF(165) (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 VEGF(165) 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)., Conclusion: In 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.

Background: 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., Objectives: 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., Patients/methods: 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., Results: 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., Conclusion: 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.

Background: Monocytes may have an important role in the resolution of venous thrombosis. Increased expression of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) is associated with an ingress of monocytes into the thrombus. This study was designed to evaluate the importance of these activators in thrombus resolution., Methods and Results: Inferior caval vein thrombosis was induced in cohorts of adult wild-type, uPA gene-knockout (uPA-/-), and tPA gene-knockout (tPA-/-) mice in a flow model. Thrombi were harvested from wild-type and uPA-/- mice (n=60 per group) between 1 and 60 days. Thrombi were also obtained from groups of wild-type and tPA-/- mice (n=24 per group) between 1 and 28 days. Thrombus size and macrophage content were measured by computer-assisted image analysis. Thrombus resolution was significantly impaired in the uPA-/- mice compared with wild-type controls (P<0.0001) but was unaffected in tPA-/- mice. Monocyte content in wild-type mice was highest at 14 days after thrombus induction and was approximately 4 times greater than in uPA-/- mice (P=0.0043). Thrombus size in uPA-/- mice transplanted with wild-type marrow (0.29+/-0.06 mm2) was significantly smaller than in uPA-/- mice given uPA-/- bone marrow (3.9+/-1.1 mm2) (P=0.0022). Donor bone marrow-derived cells expressing LacZ were present in the thrombus after transplantation., Conclusions: The resolution of experimental venous thrombus is dependent on uPA but is unaffected by the absence of tPA. Absence of uPA is also associated with delayed monocyte recruitment into the thrombus. Transplanting wild-type bone marrow restores thrombus resolution in uPA-/- animals, suggesting an important role for bone marrow-derived cells in this process.

Introduction: Lymphoedema-distichiasis syndrome (LD) (OMIM 153400) is a rare, primary lymphoedema of pubertal onset, associated with distichiasis. Causative mutations have now been described in FOXC2, a forkhead transcription factor gene. Numerous clinical associations have been reported with this condition, including congenital heart disease, ptosis, varicose veins, cleft palate, and spinal extradural cysts., Subjects: We report clinical findings in 74 affected subjects from 18 families and six isolated cases. All of them were shown to have mutations in FOXC2 with the exception of one family who had two affected subjects with lymphoedema and distichiasis and linkage consistent with the 16q24 locus., Results: The presence of lymphoedema was highly penetrant. Males had an earlier onset of lymphoedema and a significantly increased risk of complications. Lymphatic imaging confirmed the earlier suggestion that LD is associated with a normal or increased number of lymphatic vessels rather than the hypoplasia or aplasia seen in other forms of primary lymphoedema. Distichiasis was 94.2% penetrant, but not always symptomatic. Associated findings included ptosis (31%), congenital heart disease (6.8%), and cleft palate (4%). Other than distichiasis, the most commonly occurring anomaly was varicose veins of early onset (49%). This has not been previously reported and suggests a possible developmental role for FOXC2 in both venous and lymphatic systems. This is the first gene that has been implicated in the aetiology of varicose veins., Conclusion: Unlike previous publications, the thorough clinical characterisation of our patients permits more accurate prediction of various phenotypic abnormalities likely to manifest in subjects with FOXC2 mutations.

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<0.005. TIMP-3 expression was significantly higher in AAA (mean, -1.7; range, -2.9 to -1.0) than AOD (mean, -3.6; range, -5.7 to -1.8), P<0.01. Expression of 8 other MMPs (1, 2, 7, 9, 11, 12, 14, and 17) was detected and was similar in AAA and AOD. Expression of the remaining 5 MMPs (-8, -10, -13, -15, and -16) was not detected in any of the samples., 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.

Objective: Resolution of venous thrombi is accompanied by an ingrowth of capillaries, which appears to be analogous to angiogenesis in other tissues. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are major regulators of angiogenesis. The aim of this study was to determine the temporal changes and the location of VEGF and bFGF expression in a rat model of venous thrombus resolution., Design and Methods: Thrombi were induced in the inferior venae cavae of rats by creating a stenosis to reduce blood flow by 80% to 90%. Thrombi, adjacent inferior vena cava wall, and systemic blood were collected at 1, 3, 7, 14, 21, and 28 days after thrombus generation (n = 9). Sham operations were performed (n = 5), and blood was collected at 1, 3, and 7 days. VEGF and bFGF were measured with specific immunoassays, and levels in tissues were expressed as picogram per milligram soluble protein. Tissues from two animals that were humanely killed 7 days after thrombus formation were prepared for histological examination. Immunohistochemistry was performed by the use of antibodies against VEGF, bFGF, and ED-1 (a monocyte/macrophage marker)., Results: Laminated thrombi were reliably produced with a median weight at 1 day of 39 mg (range, 23-63 mg). There was a significant increase in thrombus VEGF concentration between 1 day (median, 247; range, 0-514) and 7 days (median, 556; range, 254-1741) (P =.02). There was no difference between the seventh day and subsequent days. There was a positive linear correlation between thrombus bFGF concentration and time (R = 0.74, P <.0001), with a more than 300-fold increase in bFGF concentration over the 28 days of the study. VEGF and bFGF concentrations in the adjacent vena caval wall did not change significantly with time. The serum VEGF was significantly raised at 1 day (median, 5520 pg/mL; range, 4040-7912 pg/mL) and 3 days (median, 3880 pg/mL; range, 2564-7232 pg/mL) compared with 7 days (median, 1790 pg/mL; range, 232-3228 pg/mL) (P <.0001). Similar changes in the serum VEGF also developed in the sham-operated animals. The serum bFGF (day 1 median, 15.5 pg/mL; range, 1-42 pg/mL) did not change with time. Immunohistochemistry showed that the VEGF antigen was localized to monocytes, endothelial cells, and spindle-shaped cells within the thrombus. The bFGF antigen was localized to mononuclear cells and spindle-shaped cells and was also present in the extracellular matrix., Conclusion: VEGF and bFGF are found in organizing thrombi and have characteristic temporal expression patterns, which suggest that they have a role in thrombus resolution. Augmenting angiogenic growth factor expression may enhance thrombus recanalization, thus reducing long-term complications.

Objective: to describe an intraoperative technique using a Fogarty balloon to treat arterial spasm following vascular bypass and endarterectomy., Design: prospective case control study., Subjects and Treatment: twenty-two patients following femorodistal bypass surgery and one patient following carotid endarterectomy, with arterial spasm in the distal run-off on completion angiography, were treated with Fogarty balloon dilatation., Materials: Fogarty balloon catheter (Baxtertrade mark)., Results: twenty-three patients (100%) with arterial spasm were successfully treated by Fogarty balloon as demonstrated on completion angiography. No complications were seen., Conclusion: this simple technique removes vascular spasm rapidly and produces an excellent angiographic result., (Copyright 2000 Harcourt Publishers Ltd.)

Oxidized low-density lipoprotein (OxLDL) plays a critical role in foam cell formation and atherosclerogenesis. A cDNA encoding adipophilin was identified in cultured human macrophages stimulated with OxLDL using mRNA differential display. Adipophilin is a 50 kDa protein known to be a specific marker for adipocyte cell differentiation and lipid accumulation in a variety of cells. The time-dependent induction of adipophilin mRNA in macrophages was specific to OxLDL but not native LDL, and not to various cytokines and serum. In human atherosclerotic lesions, adipophilin mRNA expression was localized in a subset of lipid-rich macrophages. These data suggest that adipophilin-expressing macrophages may represent foam cells and this gene expression is likely to be associated with the lipid accumulation in foam cells of the atherosclerotic lesions.

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 microgram 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 mm(2), and range, 1.5 to 5.7 mm(2), to median, 0.2 mm(2), and range, 0.02 to 2.6 mm(2); 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.

Local immune responses are thought to play an important role in the development of atherosclerosis. Histological studies have shown that human atherosclerotic lesions contain T lymphocytes throughout all stages of development, many of which are in an activated state. A number of novel CC chemokines have been described recently, which are potent chemoattractants for lymphocytes: PARC (pulmonary and activation-regulated chemokine), ELC (EBI1-ligand chemokine), LARC (liver and activation-regulated chemokine), and SLC (secondary lymphoid-tissue chemokine). Using reverse transcriptase-polymerase chain reaction and in situ hybridization, we have found gene expression for PARC and ELC but not for LARC or SLC in human atherosclerotic plaques. Immunohistochemical staining of serial plaque sections with specific cell markers revealed highly different expression patterns of PARC and ELC. PARC mRNA was restricted to CD68+ macrophages (n = 14 of 18), whereas ELC mRNA was widely expressed by macrophages and intimal smooth muscle cells (SMC) in nearly all of the lesions examined (n = 12 of 14). ELC mRNA was also found to be expressed in the medial SMC wall of highly calcified plaques (n = 4). Very low levels of ELC mRNA expression could also be detected in normal mammary arteries but no mRNA expression for PARC was detected in these vessels (n = 4). In vitro, ELC mRNA was found to be up-regulated in aortic SMC stimulated with tumor necrosis factor-a and interferon-gamma but not in SMC stimulated with serum. Both PARC and ELC mRNA were expressed by monocyte-derived macrophages but not monocytes. The expression patterns of PARC and ELC mRNA in human atherosclerotic lesions suggest a potential role for these two recently described CC chemokines in attracting T lymphocytes into atherosclerotic lesions.

Fourteen patients have required vena caval interruption in a period of 12 years at St Thomas' Hospital. Half of these patients had an underlying malignancy. None of these patients have had clinical or lung scan evidence of recurrent emboli. There were no deaths related to filter insertion and no patient died of a pulmonary embolism. Vena caval interruption is a procedure that is rarely necessary but may be valuable in carefully selected patients.

Purpose: The aim of this study was to measure the distribution of endogenous plasminogen activators during thrombolysis with an endothelial-conserving model of laminated thrombosis., Methods: Thrombi were raised in the inferior vena cava of rats with thrombin and flow reduction. The thrombi, adjacent vein wall, and distant veins (the superior vena cava) were removed at intervals from 1 hour to 21 days from formation and then cryohomogenized and assayed with specific bioimmunoassays for tissue-type (t-PA) and urokinase-type plasminogen activators (u-PA)., Results: The measured t-PA activity of the vein wall around the thrombus was reduced compared with the control inferior vena cava at 4 days. Both the u-PA and t-PA content of the thrombus increased progressively during thrombolysis. The t-PA activity increased significantly in the distant vein walls in the animals with thrombi. Immunocytochemistry and in situ hybridization localized the t-PA to a mononuclear cell infiltrate and showed up-regulation of mRNA for rat t-PA in these monocytes., Conclusions: The local plasminogen activator response was predominantly within the thrombus itself. Increased t-PA activity was additionally found in distant veins but was reduced in the vessel wall adjacent to the thrombus. This is the first report to show that u-PA activity is increased within organizing thrombus in vivo and that most of the t-PA activity is localized to a monocyte infiltrate.

A 52 year old man with chronic pancreatitis presented with recurrent upper gastrointestinal bleeding. Gastroscopy was normal, but visceral angiography suggested that there were gastric varices. Despite treatment with propranolol he had further episodes of bleeding and so underwent splenectomy to decompress the gastric varices. When the spleen was removed, however, an inflammatory mass in the head of the pancreas adherent to the posterior gastric wall was noted. Within it the splenic artery was visible and communicated with the gastric lumen through a small opening in the gastric wall. The artery was ligated and the patient has since had no further bleeding. Thus, chronic pancreatitis should be considered as a cause of recurrent upper gastro-intestinal bleeding, especially when gastroscopy is normal.

Two patients with Klinefelter's syndrome and three with 47,XYY who had venous ulcers, and one patient with 47,XYY and a post thrombotic limb are described. Sex chromosome abnormalities should be suspected in tall males with leg ulcers, especially those who have no progeny and are relatively young. Venous ulcers may be more common in males with 47,XYY.

Longitudinal tracheal tear (of the trachealis muscle), an unusual but acknowledged complication of pharyngolaryngectomy, was encountered during a total pharyngo-oesophagolaryngectomy with gastric replacement. Due to serious ventilatory difficulties a rapid repair was required to obtain an airtight seal to allow continued mechanical ventilation. A reinforced polytetrafluoroethylene (PTFE) vascular graft was used as an intratracheal stent to seal the air leak. This technique proved effective and the tracheal defect had healed by the time the stent was removed 10 days later.

Subcutaneous mastectomy with immediate silicone prosthetic implantation has been performed on 32 patients. The main indications for this were severe fibrocystic disease and non-invasive carcinoma. Five patients with advanced invasive carcinoma were also included. An improved operative technique is described. The indications and results are discussed.

A technique for assessing the anatomy and luminal diameter of the long saphenous vein by direct injection phlebography in patients being considered for reversed femoropopliteal vein bypass surgery is described. In 25 consecutive patients, 20 single veins, four double veins and one network of veins were all correctly delineated by this technique and subsequently confirmed at operation. Five saphenous veins could not be demonstrated above the knee but at operation one vein was found to be present and double from knee to groin. The minimum mean luminal diameter of the saphenous vein estimated from the X-ray after correction for magnification was 3.5 mm +/- 1.12 which was significantly less than the mean external diameter found at operation, 4.6 mm +/- 1.33 (P less than 0.001). The vein diameter found at operation invariably proved to be larger than the luminal diameter estimated from the X-ray. This allows a confident preoperative assumption of the minimum diameter that will be obtained after dissection and distension of the vein.

Forty-one biopsy specimens, taken from the ulcer-bearing skin of 41 legs of 21 patients attending the varicose vein clinic, were selectively stained for fibrin with phosphotungstic acid haemotoxylin before being blindly assessed,. Layers of fibrin were found surrounding the dermal capillaries in all 26 legs with lipodermatosclerosis. None of the specimens from the 15 legs with clinically normal skin contained fibrin. There was also an increased number of dermal capillaries cut in cross section per high powered field in 24 of the 26 legs with lipodermatosclerosis compared with two of the 15 legs with normal skin (p less than 0.001). The mean reduction in foot vein pressure during exercise was significantly less in the 26 limbs with pericapillary fibrin than in the other 15 limbs (p less than 10(-6). Lipodermatosclerosis is synonymous with pericapillary fibrin deposition and is associated with, and probably secondary to, both a persistently raised venous pressure and an increase in the size of the dermal capillary bed. This extravascular deposition of fibrin probably stimulates tissue fibrosis and blocks the diffusion of oxygen to the overlying epidermis, producing cellular death and venous ulceration.

Graft sensitization was measured in dogs receiving autotransplants of perfused, flushed or hypothermically preserved kidneys. Five kidney treatments were studied: (1) continuous pulsatile perfusion with Ross solution for 24 h; (2) flush with Ross solution followed by 24h cold storage; (3) removal and immediate reimplantation; (4) flush with Ross solution and immediate reimplantation; (5) cool and immediate reimplantation. Continuous perfusion resulted in a lower mean creatinine (measured over the first five days after transplant) than did flushing followed by cold storage (P less than 0.001). Creatinines were lower in the groups in which kidneys were not stored (regardless of treatment) than in the stored groups (P less than 0.001). Dogs were said to be immune if their leucocytes were inhibited by nephrectomy kidney antigen in the leucocyte migration inhibition assay [LMI]. Immune dogs had a higher mean creatinine than non-immune animals (P less than 0.05). All autografts were examined for deposition of IgG, IgM and C3 at post-mortem by tissue immunofluorescence [IF]. Positive immune responses (LMI or IF) were seen more often in dogs receiving long preserved grafts than in those receiving immediate graft implantation (P less than 0.05).

A method of washing the outside of surgeons' gloves with cetrimide is described. Experiments on animals showed that the method resulted in a considerable reduction in the amount of intraperitoneal contamination by starch powder.