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This paper describes the synthesis, solution-phase biophysical studies, and X-ray crystallographic structures of hexamers formed by macrocyclic β-hairpin peptides derived from the central and C-terminal regions of Aβ, which bear “tails” derived from the N-terminus of Aβ. Soluble oligomers of the β-amyloid peptide, Aβ, are thought to be the synaptotoxic species responsible for neurodegeneration in Alzheimer’s disease. Over the last 20 years, evidence has accumulated that implicates the N-terminus of Aβ as a region that may initiate the formation of damaging oligomeric species. Our laboratory has previously studied macrocyclic β-hairpin peptides derived from Aβ(16–22) and Aβ(30–36,) capable of forming hexamers that can be observed by X-ray crystallography and SDS-PAGE. To better mimic oligomers of full length Aβ, we use an orthogonal protecting group strategy during the synthesis to append residues from Aβ(1–14) to the parent macrocyclic β-hairpin peptide 1, which comprises Aβ(16–22) and Aβ(30–36). The N-terminally extended peptides N+1, N+2, N+4, N+6, N+8, N+10, N+12, and N+14 assemble to form dimers, trimers, and hexamers in solution-phase studies. X-ray crystallography reveals that peptide N+1 assembles to form a hexamer that is composed of dimers and trimers. These observations are consistent with a model in which the assembly of Aβ oligomers is driven by hydrogen bonding and hydrophobic packing of the residues from the central and C-terminal regions, with the N-terminus of Aβ accommodated by the oligomers as an unstructured tail.

Introduction: Endovenous stenting is being increasingly used for the management of iliofemoral venous outflow obstruction due to thrombotic or non-thrombotic iliac vein lesions (NIVL). Dedicated ve...

In chronic venous ulcers (CVUs), which account for up to 75% of leg ulcers, the inflammatory stage of wound healing fails to down-regulate, preventing progression to proliferation, remodeling and eventual epithelialisation. The roles of reactive oxygen species (ROS) in the oxidative burst and pathogen killing are well known, but ROS also have important functions in extra-cellular and intra-cellular signalling. Iron deposition, resulting from venous reflux, primes macrophages towards a persistent inflammatory response, with ongoing stimulation by bacteria potentially playing a role. Generation of excessive ROS by activated inflammatory cells causes tissue destruction and disintegration of the dermis, and then at later stages, a failure to heal. Here, we review the evidence for ROS in CVU formation and in normal and delayed healing. We also discuss how ROS modulation might be used to influence the healing of these complex wounds, which cause long-term morbidity and are associated with a significant financial burden to healthcare systems.

Vascular smooth muscle cells (VSMCs) contribute to the deposition of extracellular matrix proteins (ECMs), including Type IV collagen, in the vessel wall. ECMs coordinate communication among different cell types, but mechanisms underlying this communication remain unclear. Our previous studies have demonstrated that X-box binding protein 1 (XBP1) is activated and contributes to VSMC phenotypic transition in response to vascular injury. In this study, we investigated the participation of XBP1 in the communication between VSMCs and vascular progenitor cells (VPCs). Immunofluorescence and immunohistology staining revealed that Xbp1 gene was essential for type IV collagen alpha 1 (COL4A1) expression during mouse embryonic development and vessel wall ECM deposition and stem cell antigen 1-positive (Sca1+)-VPC recruitment in response to vascular injury. The Western blot analysis elucidated an Xbp1 gene dose-dependent effect on COL4A1 expression and that the spliced XBP1 protein (XBP1s) increased protease-mediated COL4A1 degradation as revealed by Zymography. RT-PCR analysis revealed that XBP1s in VSMCs not only upregulated COL4A1/2 transcription but also induced the occurrence of a novel transcript variant, soluble type IV collagen alpha 1 (COL4A1s), in which the front part of exon 4 is joined with the rear part of exon 42. Chromatin-immunoprecipitation, DNA/protein pulldown and in vitro transcription demonstrated that XBP1s binds to exon 4 and exon 42, directing the transcription from exon 4 to exon 42. This leads to transcription complex bypassing the internal sequences, producing a shortened COL4A1s protein that increased Sca1+-VPC migration. Taken together, these results suggest that activated VSMCs may recruit Sca1+-VPCs via XBP1s-mediated COL4A1s secretion, leading to vascular injury repair or neointima formation.

A number of dedicated self expanding nitinol stents have been developed for use in the venous system, with both open cell (OC) and closed cell (CC) designs available. Data comparing these different designs are lacking. The objective of this study was to evaluate outcomes in patients treated with open and closed cells for unilateral chronic iliac vein obstruction.A single centre retrospective cohort study was conducted, including all patients treated with a dedicated nitinol venous stent between 2014 and 2019. Stent patency and details of re-interventions (including lysis, venoplasty, reinforcement, extension, arteriovenous fistula formation) were examined in the first post-operative year. Subgroup analysis described outcomes for patients treated with OC and CC stents ending above the inguinal ligament and those who required extension into the common femoral vein. Cox regression analysis was used to identify factors associated with loss of primary patency.A total of 207 patients were included (OC 100 patients, CC 107 patients). There was no significant difference between the groups for age (OC 42 years, CC 44 years); gender (OC and CC 67% female); presence of post-thrombotic lesions (OC 71%, CC 73%); stenting across the inguinal ligament (OC 58%, CC 56%), or presence of inflow disease (OC 49%, CC 47%). Primary and cumulative patency at 12 months were similar between groups (primary: OC 63%, CC 65%; cumulative: OC 93%, CC 90%). Patients with a CC stent across the inguinal ligament had a greater risk of needing multiple re-interventions at one year compared with those with an OC stent (odds ratio 2.84, 95% confidence interval [CI] 1.16 - 6.9) but overall, the only factor significantly associated with loss of primary patency was inflow vessel disease (hazard ratio 3.39, 95% CI 1.73 - 6.62, p.001).OC and CC dedicated nitinol venous stents were observed to perform similarly in terms of patency and symptom improvement at one year. Disease of the inflow vessels was the most important factor associated with a loss of stent patency irrespective of stent design.

Venous valve (VV) failure causes chronic venous insufficiency, but the molecular regulation of valve development is poorly understood. A primary lymphatic anomaly, caused by mutations in the receptor tyrosine kinase EPHB4, was recently described, with these patients also presenting with venous insufficiency. Whether the venous anomalies are the result of an effect on VVs is not known. VV formation requires complex 'organization' of valve-forming endothelial cells, including their reorientation perpendicular to the direction of blood flow. Using quantitative ultrasound we identified substantial VV aplasia and deep venous reflux in patients with mutations in EPHB4. We used a GFP reporter, in mice, to study expression of its ligand, ephrinB2, and analysed developmental phenotypes following conditional deletion of floxed Ephb4 and Efnb2 alleles. EphB4 and ephrinB2 expression patterns were dynamically regulated around organizing valve-forming cells. Efnb2 deletion disrupted the normal endothelial expression patterns of the gap junction proteins connexin37 and connexin43 (both required for normal valve development) around reorientating valve-forming cells, and produced deficient valve-forming cell elongation, reorientation, polarity, and proliferation. Ephb4 was also required for valve-forming cell organization, and subsequent growth of the valve leaflets. These results uncover a potentially novel cause of primary human VV aplasia.

Objective Thrombophilia is a prothrombotic condition that increases the risk of venous thromboembolism. It is unclear whether the presence of thrombophilia alters the clinical outcomes after deep venous stenting. The aim of the present study was to examine the relationship between thrombophilia and outcomes after stenting for post-thrombotic syndrome. Methods Consecutive patients (2012-2017) receiving a nitinol venous stent for chronic post-thrombotic venous occlusive disease with a minimum of 18 months of follow-up in one center using the same anticoagulation protocol were included. The clinical history and thrombophilia testing results were reviewed. The outcomes were stent patency, which was assessed using duplex ultrasonography at 24 hours, 2 and 6 weeks, 3 months, 6 months, and annually thereafter; and reinterventions, which were performed when the stent diameter was Results Of the 136 patients who had undergone intervention, 55 (40%) had had a provoked deep vein thrombosis (DVT) and 81 (60%) had had an unprovoked DVT and had therefore undergone thrombophilia testing. Of the 81 patients, 38 (47%) had had either inherited (n = 19; 50%) or acquired (n = 19; 50%) thrombophilia. Of the 136 patients who had undergone stenting, 68 had required reintervention (50%) during follow-up to maintain stent patency. Of the 55 patients with a provoked DVT, 29 (53%) had required reintervention. Of the 81 patients with an unprovoked DVT, 39 (48%) had required reintervention (P = .420). Of the 38 patients with unprovoked DVT and thrombophilia, 17 (45%) had required reintervention. Of the 43 patients with unprovoked DVT and no thrombophilia, 22 (51%) had required reintervention (P = .766). The cumulative patency rate was 80% for patients with provoked DVT and 88% for those with unprovoked DVT (P = .193). The presence of thrombophilia was not associated with patency loss (92% cumulative patency for patients with thrombophilia and 84% for patients without thrombophilia; P = .307). Conclusions Using our anticoagulation protocol, patients with and without thrombophilia had similar clinical outcomes after deep venous stenting and should not be excluded from iliofemoral venous stenting. We found no significant differences in outcomes in conjunction with appropriate postoperative anticoagulation therapy.

Reactive oxygen species (ROS) have physiological roles as second messengers, but can also exert detrimental modifications on DNA, proteins and lipids if resulting from enhanced generation or reduced antioxidant defense (oxidative stress). Venous thrombus (DVT) formation and resolution are influenced by ROS through modulation of the coagulation, fibrinolysis, proteolysis and the complement system, as well as the regulation of effector cells such as platelets, endothelial cells, erythrocytes, neutrophils, mast cells, monocytes and fibroblasts. Many conditions that carry an elevated risk of venous thrombosis, such as the Antiphospholipid Syndrome, have alterations in their redox homeostasis. Dietary and pharmacological antioxidants can modulate several important processes involved in DVT formation, but their overall effect is unknown and there are no recommendations regarding their use. The development of novel antioxidant treatments that aim to abrogate the formation of DVT or promote its resolution will depend on the identification of targets that enable ROS modulation confined to their site of interest in order to prevent off-target effects on physiological redox mechanisms. Subgroups of patients with increased systemic oxidative stress might benefit from unspecific antioxidant treatment, but more clinical studies are needed to bring clarity to this issue.

OBJECTIVE/BACKGROUND The aim was to assess two year outcomes with placement of the Vici Venous Stent® in patients with chronic iliofemoral venous occlusions (complete blockage). METHODS This was a retrospective single centre study comprising patients treated with the Vici Venous Stent for venographically verified iliofemoral venous occlusion and post-thrombotic syndrome (Villalta score ≥ 5 points) at least 12 months after acute deep vein thrombosis. Venography and intravascular ultrasound were used peri-operatively; duplex ultrasound was used to assess stent patency during follow up. RESULTS Eighty-eight patients (101 limbs) had stent placement between March 2014 and October 2016. Median pre-treatment Villalta score was 14 (range 5-33). Stenting extended across the inguinal ligament in 63 limbs (62%) in order to land in a healthy venous segment. Six patients (7%) required endophlebectomy and fistula creation. Median imaging follow up was 21 months (range 0-41 months). Primary, assisted primary and secondary patency rates at one year were 59%, 78%, and 87%, respectively, and two years 51%, 73%, and 82%, respectively. Forty-three limbs (43%) had re-intervention (lysis, venoplasty, and/or placement of stent) during follow up; median time to re-intervention was 32 days (range 0-520 days). At 24 months, 37 of 53 limbs (70%) with available Villalta assessment showed clinically significant improvement (>30% reduction of baseline score). Villalta scores at the 6, 12, and 24 month clinical follow up were significantly lower than before stenting (p

Supplemental Digital Content is available in the text., Background: Radiation exposure during fluoroscopically guided interventions such as endovascular aortic repair (EVAR) is a growing concern for operators. This study aimed to measure DNA damage/repair markers in operators perfoming EVAR. Methods: Expression of the DNA damage/repair marker, γ-H2AX and DNA damage response marker, phosphorylated ataxia telangiectasia mutated (pATM), were quantified in circulating lymphocytes in operators during the peri-operative period of endovascular (infrarenal, branched, and fenestrated) and open aortic repair using flow cytometry. These markers were separately measured in the same operators but this time wearing leg lead shielding in addition to upper body protection and compared with those operating with unprotected legs. Susceptibility to radiation damage was determined by irradiating operators’ blood in vitro. Results: γ-H2AX and pATM levels increased significantly in operators immediately after branched endovascular aortic repair/fenestrated endovascular aortic repair (P

Aims Dysfunctional matrix turnover is present at sites of abdominal aortic aneurysm (AAA) and leads to the accumulation of monomeric tropoelastin rather than cross-linked elastin. We used a gadolinium-based tropoelastin-specific magnetic resonance contrast agent (Gd-TESMA) to test whether quantifying regional tropoelastin turnover correlates with aortic expansion in a murine model. The binding of Gd-TESMA to excised human AAA was also assessed. Methods and results We utilized the angiotensin II (Ang II)-infused apolipoprotein E gene knockout (ApoE-/-) murine model of aortic dilation and performed in vivo imaging of tropoelastin by administering Gd-TESMA followed by late gadolinium enhancement (LGE) magnetic resonance imaging (MRI) and T1 mapping at 3 T, with subsequent ex vivo validation. In a cross-sectional study (n = 66; control = 11, infused = 55) we found that Gd-TESMA enhanced MRI was elevated and confined to dilated aortic segments (control: LGE=0.13 ± 0.04 mm2, control R1= 1.1 ± 0.05 s-1 vs. dilated LGE=1.0 ± 0.4 mm2, dilated R1 =2.4 ± 0.9 s-1) and was greater in segments with medium (8.0 ± 3.8 mm3) and large (10.4 ± 4.1 mm3) compared to small (3.6 ± 2.1 mm3) vessel volume. Furthermore, a proof-of-principle longitudinal study (n = 19) using Gd-TESMA enhanced MRI demonstrated a greater proportion of tropoelastin: elastin expression in dilating compared to non-dilating aortas, which correlated with the rate of aortic expansion. Treatment with pravastatin and aspirin (n = 10) did not reduce tropoelastin turnover (0.87 ± 0.3 mm2 vs. 1.0 ± 0.44 mm2) or aortic dilation (4.86 ± 2.44 mm3 vs. 4.0 ± 3.6 mm3). Importantly, Gd-TESMA-enhanced MRI identified accumulation of tropoelastin in excised human aneurysmal tissue (n = 4), which was confirmed histologically. Conclusion Tropoelastin MRI identifies dysfunctional matrix remodelling that is specifically expressed in regions of aortic aneurysm or dissection and correlates with the development and rate of aortic expansion. Thus, it may provide an additive imaging marker to the serial assessment of luminal diameter for surveillance of patients at risk of or with established aortopathy.

Introduction This review aims to provide an update on the management of deep vein thrombosis (DVT). Sources of data A systematic search of PubMed, Google Scholar and Cochrane databases was carried out. Areas of agreement Direct oral anticoagulants (DOACs) are as effective and easier to use than vitamin K antagonists for the treatment of DVT. Catheter-directed thrombolysis can reduce post thrombotic syndrome in patients with iliofemoral DVT. Compression bandaging can help heal a venous ulcer. Areas of controversy Compression hosiery to prevent post thrombotic syndrome. Long-term evidence to show clinical benefit of using endovenous therapies to restore deep vein patency. Growing points Developing imaging methods to identify patients who would benefit from venous thrombolysis. The evolution of dedicated venous stents. Areas timely for developing research Understanding the mechanisms that lead to stent occlusion and investigation into the appropriate treatments that could prevent in-stent thrombosis is required.

Successful management of acute deep vein thrombosis and post-thrombotic syndrome depends on careful patient selection and detailed investigation of thrombus extent, composition, and anatomy. This article reviews the use of computerized tomography and magnetic resonance imaging in the assessment of central deep veins of the pelvis and addresses new developments within the field. Despite drawbacks of each imaging modality, when contemplating deep venous reconstruction, cross-sectional imaging should be considered for preoperative planning and to compliment intraoperative imaging tools, including intravascular ultrasound and contrast venography.

We note with disappointment Doss’s suggestion that occupational radiation exposure should be considered as potentially beneficial. This comes at a time when the entire interventionist community is trying to impress on their young trainees, many of whom will be exposed to radiation several times a week, performing in some cases lengthy fluoroscopically guided procedures over a 40-year career, the importance of protecting against this exposure. We are aware that Doss and his group extol the virtues of not sparing irradiation to ensure proper screening, diagnosis, and treatment of patients. Although we would not advocate avoiding the use of radiation …

Leukocyte-released antimicrobial peptides contribute to pathogen elimination and activation of the immune system. Their role in thrombosis is incompletely understood. Here we show that the cathelicidin LL-37 is abundant in thrombi from patients with acute myocardial infarction. Its mouse homologue, CRAMP, is present in mouse arterial thrombi following vascular injury, and derives mainly from circulating neutrophils. Absence of hematopoietic CRAMP in bone marrow chimeric mice reduces platelet recruitment and thrombus formation. Both LL-37 and CRAMP induce platelet activation in vitro by involving glycoprotein VI receptor with downstream signaling through protein tyrosine kinases Src/Syk and phospholipase C. In addition to acute thrombosis, LL-37/CRAMP-dependent platelet activation fosters platelet–neutrophil interactions in other inflammatory conditions by modulating the recruitment and extravasation of neutrophils into tissues. Absence of CRAMP abrogates acid-induced lung injury, a mouse pneumonia model that is dependent on platelet–neutrophil interactions. We suggest that LL-37/CRAMP represents an important mediator of platelet activation and thrombo-inflammation., Cathelicidins are antimicrobial peptides that eliminate pathogens and contribute to the innate immune response. Here the authors show that neutrophil-derived LL-37/CRAMP induces platelet activation and promotes arterial thrombosis and thrombo-inflammation.

BACKGROUND: Hypoxia within acute venous thrombi is thought to drive resolution through stabilisation of hypoxia inducible factor 1 alpha (HIF1α). Prolyl hydroxylase domain (PHD) isoforms are critical regulators of HIF1α stability. Non-selective inhibition of PHD isoforms with l-mimosine has been shown to increase HIF1α stabilisation and promote thrombus resolution. OBJECTIVE: The aim of this study was to investigate the therapeutic potential of PHD inhibition in venous thrombus resolution. METHODS: Thrombosis was induced in the inferior vena cava of mice using a combination of flow restriction and endothelial activation. Gene and protein expression of PHD isoforms in the resolving thrombus was measured by RT-PCR and immunohistochemistry. Thrombus resolution was quantified in mice treated with pan PHD inhibitors AKB-4924 and JNJ-42041935 or inducible all-cell Phd2 knockouts by micro-computed tomography, 3D high frequency ultrasound or endpoint histology. RESULTS: Resolving venous thrombi demonstrated significant temporal gene expression profiles for PHD2 and PHD3 (P 0.05). CONCLUSIONS: This data suggests that PHD-mediated thrombus neovascularisation has a limited role in the resolution of venous thrombi. Directly targeting angiogenesis alone may not be a viable therapeutic strategy to enhance venous thrombus resolution. ispartof: THROMBOSIS RESEARCH vol:169 pages:105-112 ispartof: location:United States status: published

Surgery is associated with an increased risk of venous thromboembolic events (VTE) including deep vein thrombosis and pulmonary embolism. Although the current treatment regiments such as mechanical manipulation and administration of pharmacological prophylaxis significantly reduced the incidence of postsurgical VTE, they remain a major cause of postoperative morbidity and mortality worldwide. The pathophysiology of venous thrombosis traditionally emphasizes the series of factors that constitute Virchow triad of factors. However, inflammation can also be a part of this by giving rise to a hypercoagulable state and endothelial damage. The inflammatory response after surgery, which is initiated by a cytokine "storm" and occurs within hours of surgery, creates a prothrombotic environment that is further accentuated by several cellular processes including neutrophil extracellular traps formation, platelet activation, and the generation of tissue factor-bearing microparticles. Although such inflammatory markers are elevated in undergoing surgery, the precise mechanism by which they give rise to venous thrombosis is poorly understood. Here, we discuss the potential mechanisms linking inflammation to thrombosis, and highlight strategies that may minimize surgical inflammation and reduce the incidence of postoperative VTE.

Activation of proteolytic mechanisms at the cell surface through the activity of urokinase-type plasminogen activator (uPA) bound to its receptor, uPAR, is an important process in wound healing. The soluble forms of uPAR (suPAR and its fragments I, II, and III) have nonproteolytic functions that include chemotaxis, adhesion, and proliferation, which also have a role in wound healing. The aim of this study was to determine whether suPAR and its cleaved fragments are present in venous ulcers and whether their levels are associated with healing.Ulcer exudates were collected from patients with venous leg ulcers (n = 30). Healing was defined as complete re-epithelialization within 6 months of compression therapy. Time-resolved fluorescence immunoassays were validated for quantification of suPAR and its fragments in ulcer exudates. The effect of exudates on keratinocyte migration was analyzed by an in vitro scratch assay.Ulcer exudates from patients who healed (n = 9) had approximately threefold higher levels of intact suPAR (P = .005), twofold higher levels of suPARI (P = .03), and approximately threefold higher levels suPARII-III (P.0001) compared with nonhealers (n = 21). Exudate from healing ulcers stimulated keratinocyte migration (P = .02), whereas depletion of suPAR from exudates resulted in cell apoptosis.We conclude that suPAR and its fragments are present in the environs of venous ulcers and may act as indicators of the propensity of venous ulcers to heal, with suPARII-III being the best discriminator. We speculate that suPAR and its fragments may have a role in the maintenance of an optimal ulcer-healing environment.

Rationale : Abdominal aortic aneurysms constitute a degenerative process in the aortic wall. Both the miR-29 and miR-15 families have been implicated in regulating the vascular extracellular matrix. Objective : Our aim was to assess the effect of the miR-15 family on aortic aneurysm development. Methods and Results : Among the miR-15 family members, miR-195 was differentially expressed in aortas of apolipoprotein E–deficient mice on angiotensin II infusion. Proteomics analysis of the secretome of murine aortic smooth muscle cells, after miR-195 manipulation, revealed that miR-195 targets a cadre of extracellular matrix proteins, including collagens, proteoglycans, elastin, and proteins associated with elastic microfibrils, albeit miR-29b showed a stronger effect, particularly in regulating collagens. Systemic and local administration of cholesterol-conjugated antagomiRs revealed better inhibition of miR-195 compared with miR-29b in the uninjured aorta. However, in apolipoprotein E–deficient mice receiving angiotensin II, silencing of miR-29b, but not miR-195, led to an attenuation of aortic dilation. Higher aortic elastin expression was accompanied by an increase of matrix metalloproteinases 2 and 9 in mice treated with antagomiR-195. In human plasma, an inverse correlation of miR-195 was observed with the presence of abdominal aortic aneurysms and aortic diameter. Conclusions : We provide the first evidence that miR-195 may contribute to the pathogenesis of aortic aneurysmal disease. Although inhibition of miR-29b proved more effective in preventing aneurysm formation in a preclinical model, miR-195 represents a potent regulator of the aortic extracellular matrix. Notably, plasma levels of miR-195 were reduced in patients with abdominal aortic aneurysms suggesting that microRNAs might serve as a noninvasive biomarker of abdominal aortic aneurysms.

Peripheral arterial disease (PAD) affects 27 million individuals in Europe and North America.1 This condition causes a severe restriction of blood flow that can lead to critical limb ischemia (CLI), a condition characterized by rest pain, ulceration, or gangrene, which is associated with limb loss in ≈25% of cases.2 The ischemic limb can be revascularized by endovascular techniques (angioplasty/stenting) or surgical bypass. Imaging modalities, including duplex ultrasound, MR angiography, computer tomographic angiography, or intra-arterial angiography, are used to assess the extent of disease, plan intervention, and confirm the patency of major blood vessels after intervention. These techniques do not provide information about perfusion at the tissue level. An early relatively crude method for assessing ischemic limbs, venous occlusion plethysmography3 relies on the principle that the volume of the limb is dependent on the arterial inflow when venous drainage is occluded. Under resting conditions, ≤70% of blood flow to the limb is directed to skeletal muscle, with the remainder to the skin circulation.4 Venous occlusion plethysmography provides a global indication of limb perfusion but gives no anatomic information and cannot delineate segmental perfusion deficits. It is influenced by numerous factors, such as ambient temperature and arteriovenous shunting that may lead to erroneous readings. Objective measurement of limb perfusion would particularly benefit patients with CLI but most attempts at addressing this have used patients with claudication. The latter can lead to CLI but is a less severe form of PAD that is often treated conservatively and is characterized by pain only when walking as opposed to pain at rest or gangrene. An accurate, noninvasive method of measuring limb perfusion would aid the diagnosis and treatment of PAD and could be combined with conventional angiography to provide both functional …

Background— The magnetic resonance longitudinal relaxation time (T 1 ) changes with thrombus age in humans. In this study, we investigate the possible mechanisms that give rise to the T 1 signal in venous thrombi and whether changes in T 1 relaxation time are informative of the susceptibility to lysis. Methods and Results— Venous thrombosis was induced in the vena cava of BALB/C mice, and temporal changes in T 1 relaxation time correlated with thrombus composition. The mean T 1 relaxation time of thrombus was shortest at 7days following thrombus induction and returned to that of blood as the thrombus resolved. T 1 relaxation time was related to thrombus methemoglobin formation and further processing. Studies in inducible nitric oxide synthase ( iNOS −/− )–deficient mice revealed that inducible nitric oxide synthase mediates oxidation of erythrocyte lysis–derived iron to paramagnetic Fe 3+ , which causes thrombus T 1 relaxation time shortening. Studies using chemokine receptor-2–deficient mice ( Ccr2 −/− ) revealed that the return of the T 1 signal to that of blood is regulated by removal of Fe 3+ by macrophages that accumulate in the thrombus during its resolution. Quantification of T 1 relaxation time was a good predictor of successful thrombolysis with a cutoff point of Conclusions— The source of the T 1 signal in the thrombus results from the oxidation of iron (released from the lysis of trapped erythrocytes in the thrombus) to its paramagnetic Fe 3+ form. Quantification of T 1 relaxation time appears to be a good predictor of the success of thrombolysis.

Critical limb ischaemia is a severe manifestation of peripheral arterial disease characterised by intractable pain and tissue gangrene. Conventional treatments include percutaneous angioplasty and surgical bypass but up to one third of patients are not amenable to these interventions and will ultimately require amputation. Therapeutic neovascularisation has been proposed as an alternative treatment in these 'no option' patients and both cytokines and cells have shown impressive efficacy in the laboratory. Clinical trials in man, however, have had modest results. This discrepancy has put into question the relevance of the pre-clinical assays that are used to test potential agents. One of the most widely used of these assays is the hind limb ischaemia model that is often performed in young, healthy animals. This review critiques the techniques used to induce and assess ischaemia in this model and outlines the reasons why healthy rodents cannot fully recapitulate critical limb ischaemia in aged patients. Strategies that may produce a hind limb model that better simulates the human condition are proposed.

Venous thrombosis is a common complication in cancer patients, and some cancer chemotherapies are associated with an increased risk of venous thromboembolism. The regulatory mechanisms that control thrombus formation and subsequent resolution in patients with cancer, however, are incompletely understood, and novel treatments for cancer-associated thrombosis may arise from a better understanding of such mechanisms. In this chapter, pathways that regulate cancer-associated thrombus formation are outlined, and the effects of anti-angiogenic cancer chemotherapies on venous thrombus resolution are highlighted. Potentially pro-thrombotic effects of anti-angiogenic agents are important considerations when managing the complications of venous thrombosis in cancer patients.

BACKGROUND: Lymphatic endothelial cells from primary lymphedema skin have never been cultured nor characterized. A subgroup of patients with primary lymphedema undergo surgery to bring about an improvement in their quality of life. The aim of this study was to culture and characterize LECs from the skin of these patients. METHODS AND RESULTS: Lymphatic endothelial cells were isolated and cultured from the skin of patients with primary lymphedema and from normal skin. The isolated cells were compared in their ability to form microvascular networks in a three-dimensional culture medium, and in their response to treatment with vascular endothelial growth factors A, C, and D. Whole tissue transcriptional profiling was carried out on two pools of isolated lymphatic endothelial cells--one from primary lymphedema skin and the other from normal skin. Lymphatic endothelial cells from primary lymphedema skin form tubule-like structures when cultured in three-dimensional media. They respond in a similar fashion to stimulation with the vascular endothelial growth factors A, C, and D. Comparative analysis between lymphedema tissue and normal tissue (fold change >2) showed differential expression of 2793 genes (5% of all transcripts), 2184 upregulated, and 609 downregulated. Genes involved in cellular apoptosis (vascular endothelial growth inhibitor, zinc finger protein), extracellular matrix turnover (matrix metalloproteinase inhibitor-16), and type IV collagen deposition were upregulated. Various pro-inflammatory genes (interleukin-6, interleukin-8, interleukin-32, E-selectin) were downregulated. CONCLUSION: Cellular adhesion, apoptosis, and increased extracellular matrix turnover play a more prominent role in primary lymphedema than previously thought. In addition, the acute inflammatory response is attenuated as evidenced by the downregulation of various pro-inflammatory genes.This sheds further light on the interplay of the various pathological processes taking place in primary lymphedema.

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 (≈37-fold), retinoic acid receptor responder-1 (≈5-fold), and cysteine protease legumain (≈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.

A sugar-specific transfection mechanism is introduced by the glycosylation of adenoviruses (AVs, see picture), and manipulation of the glycosylation pattern allows the selective transfection of human macrophages in favor of the usual target. This dramatic retargeting holds promise for the fine-tuning of adenoviruses for applications such as gene therapy. © 2005 Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim.

Chronic venous disease and venous hypertension are common consequences of valve insufficiency, yet the molecular mechanisms regulating the formation and maintenance of venous valves have not been studied. Here, we provide what we believe to be the first description of venous valve morphogenesis and identify signaling pathways required for the process. The initial stages of valve development were found to involve induction of ephrin-B2, a key marker of arterial identity, by venous endothelial cells. Intriguingly, developing and mature venous valves also expressed a repertoire of proteins, including prospero-related homeobox 1 (Prox1), Vegfr3, and integrin-α9, previously characterized as specific and critical regulators of lymphangiogenesis. Using global and venous valve-selective knockout mice, we further demonstrate the requirement of ephrin-B2 and integrin-α9 signaling for the development and maintenance of venous valves. Our findings therefore identified molecular regulators of venous valve development and maintenance and highlighted the involvement of common morphogenetic processes and signaling pathways in controlling valve formation in veins and lymphatic vessels. Unexpectedly, we found that venous valve endothelial cells closely resemble lymphatic (valve) endothelia at the molecular level, suggesting plasticity in the ability of a terminally differentiated endothelial cell to take on a different phenotypic identity.

Tissues require an adequate supply of oxygen in order to maintain normal cell function. Low oxygen tension (hypoxia) is characteristic of a number of conditions, including cancer, atherosclerosis, rheumatoid arthritis, critical limb ischaemia, peripheral vascular disease, and ischaemic heart disease. Tissue hypoxia is found in tumours, atherosclerotic plaque, and ischaemic myocardium. There is a growing interest in methods to detect and assess hypoxia, given that hypoxia is important in the progression of these diseases. Hypoxia can be assessed at the level of the whole organ, tissue, or cell, using both invasive and non-invasive methods, and by a range of immunohistochemical, biochemical, or imaging techniques. This review describes and critiques current methods of assessing hypoxia that are used at the bench and in clinical practice.

Background— Despite a concerted effort by many laboratories, the critical subunits that participate in vascular smooth muscle cell (VSMC) NADPH oxidase function have yet to be elucidated. Given the potential therapeutic importance of cell-specific inhibition of NADPH oxidase, we investigated the role of Nox activator 1 (NoxA1), a homolog of p67phox, in VSMC NADPH oxidase function and atherosclerosis. Methods and Results— The presence of NoxA1 in mouse aortic VSMCs was confirmed by reverse-transcription polymerase chain reaction and sequencing. NoxA1/p47phox interaction after thrombin treatment was observed by immunoprecipitation/Western analysis of lysates from p47phox −/− VSMCs transfected with adenoviral HA-NoxA1 and Myc-p47phox. Infection with adenoviral NoxA1 significantly enhanced thrombin-induced reactive oxygen species generation in wild-type but not in p47phox −/− and Nox1 −/− VSMCs. Thrombin-induced reactive oxygen species production and VSMC proliferation were significantly reduced after downregulation of NoxA1 with shRNA. Infection with NoxA1 shRNA but not scrambled shRNA significantly decreased thrombin-induced activation of the redox-sensitive protein kinases (Janus kinase 2, Akt, and p38 mitogen-activated protein kinase) in VSMCs. Adenovirus-mediated overexpression of NoxA1 in guidewire-injured mouse carotid arteries significantly increased superoxide production in medial VSMCs and enhanced neointimal hyperplasia. NoxA1 expression was significantly increased in aortas and atherosclerotic lesions of ApoE −/− mice compared with age-matched wild-type mice. Furthermore, in contrast to p67phox, immunoreactive NoxA1 is present in intimal and medial SMCs of human early carotid atherosclerotic lesions. Conclusions— NoxA1 is the functional homolog of p67phox in VSMCs that regulates redox signaling and VSMC phenotype. These findings support the potential for modulation of NoxA1 expression as a viable approach for the treatment of vascular diseases.

The aim of this work was to investigate fast T (1)-mapping for the characterization of deep vein thrombosis (DVT).The accuracy and reproducibility of the T (1)-mapping sequence was tested in phantoms and in 8 healthy volunteers on a 1.5 T clinical scanner using a 32-channel array coil. Furthermore, the feasibility of the technique was tested in 5 patients diagnosed with DVT by measuring the volume and T (1) values of the thrombus at 5 time points over a period of 6 months.The results of the phantom and volunteer study showed a high accuracy and reproducibility for the quantification of T (1). The resolution of the T (1)-maps was high enough to identify small anatomical structures. T (1) values derived for normal blood and various other tissues were comparable to those reported in the literature. In all patients, the T (1) times of thrombi showed decreased values (T (1) = 843 +/- 91 ms) in the acute phase and recovered back to normal values of blood (T (1) = 1,317 +/- 36 ms) after 6 months.Measurement of all relevant T (1) values of acute thrombi and normal blood achieved accurate and reproducible results in vivo. Fast T (1) quantification of the thrombus can provide information about tissue characteristics such as thrombus resolution. Such a quantitative MRI technique may be valuable in studying the factors that influence natural resolution and in evaluating treatment effects that enhance this process.

It is now clear that the monocyte/macrophage has a crucial role in the development of atherosclerosis. This cell appears to be involved in all stages of atherosclerotic plaque development and is increasingly seen as a candidate for therapeutic intervention and as a potential biomarker of disease progression and response to therapy. The main mechanisms related to the activity of the monocyte/macrophage that have been targeted for therapy are those that facilitate recruitment, cholesterol metabolism, inflammatory activity and oxidative stress. There is also increasing evidence that there is heterogeneity within the monocyte/macrophage population, which may have important implications for plaque development and regression. A better insight into how specific phenotypes may influence plaque progression should facilitate the development of novel methods of imaging and more refined treatments.

The aim of this study was to investigate whether intravenous heparin administration was associated with a reduction in perioperative mortality and late distal thrombectomy in patients with ruptured abdominal aortic aneuryms (AAAs). One hundred thirty-one patients had repair of ruptured AAA between January 1999 and January 2004. Sixty-three received heparin according to the consultant's preference at the time of the operation. Data were prospectively collected, and multivariate analysis was performed for independent predictive factors. Thirty-day mortality was 29%. Patients receiving heparin had lower perioperative mortality (16% vs 42%; p = .001). Heparin administration was not associated with increased hemorrhage or transfusion. Multivariate analysis confirmed that heparin administration was independently predictive of survival ( p = .036). Other factors found to reduce survival were age ( p = .023), smoking ( p = .042), and systolic blood pressure (< 100 mmHg) at presentation ( p = .045). Fewer patients had thrombectomy after heparin (8% vs 12%), but this was not statistically significant. Perioperative complications were similar in both groups. The administration of systemic heparin before the clamp is applied to leaking aneurysms does not appear to increase hemorrhage and subsequent mortality and may reduce the need for early thrombectomy.

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.

BackgroundCysteine 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).MethodsAortic 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.ResultsAAA 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

Background The aim of this study was to examine the effect of statin treatment on the activity of proteases in the wall of abdominal aortic aneurysms (AAAs). Methods The activities of matrix metalloproteinases (MMPs) 9 and 3, cathepsins B, H, K, L and S, and the cystatin C level were measured in extracts of AAA wall taken from 82 patients undergoing AAA repair; 21 patients were receiving statin treatment before surgery. All values were standardized against soluble protein (SP) concentration in the extract, and reported as median (interquartile range) or mean(s.e.m.). Results The two groups had similar demographics. Reduced activity of MMP-9 (43 (34–56) versus 80 (62–110) pg per mg SP; P < 0·001), cathepsin H (183 (117–366) versus 321 (172–644) nmol 4-methylcoumarin-7-amide released per mg SP; P = 0·016) and cathepsin L (102 (51–372) versus 287 (112–816) µmol 7-amino-4-trifluoromethylcoumarin released per mg SP; P = 0·020) was found in the statin-treated aortas compared with AAAs from patients not taking a statin. The statin-treated group had lower MMP-3 activity, but this did not reach statistical significance (P = 0·053). Cystatin C levels were higher in statin-treated aortas than in controls (41·3(3·1) versus 28·9(2·1) ng per mg SP; P = 0·003). Conclusion Statins decreased the activity of proteases that have been implicated in aneurysm disease.

Matrix metalloproteinase 3 (MMP3), is over expressed in the wall of abdominal aortic aneurysms (AAA), while inactivation of the gene expressing this enzyme is associated with reduced aneurysm formation in an experimental model. The 5A allele of the 5A/6A polymorphism in the promoter region of the MMP3 gene is associated with enhanced MMP3 expression. This study aimed to determine whether the presence of the 5A allele in the MMP3 promoter is a risk factor for AAA, and if this allele is associated with an increased expression of MMP3 in the aneurysm wall. We compared the frequencies of the 5A and 6A alleles in AAA (n = 405), aortic occlusive disease (AOD) (n = 123) and controls (n = 405). The 5A allele frequency was higher in AAA compared with controls (odds ratio - OR 1.32, P = 0.005) and AOD (OR 1.684, P = 0.0004), but was similar in AOD compared to controls (OR 0.78, P = 0.1). The ORs of the 5A/6A and the 5A/5A genotypes were 1.35 and 1.79, compared with 6A homozygotes. Although wall from 5A homozygotes contained 17% more MMP3 mRNA than homozygotes (P = 0.049) the significance of this was lost when adjusted for age and sex (P = 0.069), and size (P = 0.30). Wall from 5A homozygotes did however contain over 45% more MMP3 protein than heterozygotes (P = 0.009 when corrected for age and sex and P = 0.043 when corrected for aneurysm size). It appears that an abnormality in the MMP3 gene is part of the genetic profile that predisposes to aneurysmal disease.

Background— Mutations in the FOXC2 gene cause lymphedema distichiasis, an inherited primary lymphedema in which a significant number of patients have varicose veins. Because lymphedema distichiasis is believed to be caused by lymphatic valve failure (reflux), and FOXC2 is highly expressed on venous valves in mouse embryos, we tested the hypothesis that FOXC2 mutations may be linked to venous valve failure and reflux. Methods and Results— The venous system of the leg was investigated with Duplex ultrasound. Pathological reflux was recorded by color Duplex ultrasound in all 18 participants with a FOXC2 mutation, including 3 without lymphedema. Every participant with a mutation in FOXC2 showed reflux in the great saphenous vein (n=18), compared with only 1 of 12 referents (including 10 family members; P Conclusions— FOXC2 is the first gene in which mutations have been strongly associated with primary venous valve failure in both the superficial and deep veins in the lower limb. This gene appears to be important for the normal development and maintenance of venous and lymphatic valves.

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 25 kHz 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 1.872 cm2) 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.

Use of blood oxygenation level-dependent cardiovascular magnetic resonance (BOLD-CMR) to assess perfusion in the lower limb has been hampered by poor reproducibility and a failure to reliably detect post-revascularization improvements in patients with critical limb ischemia (CLI).This study sought to develop BOLD-CMR as an objective, reliable clinical tool for measuring calf muscle perfusion in patients with CLI.The calf was imaged at 3-T in young healthy control subjects (n = 12), age-matched control subjects (n = 10), and patients with CLI (n = 34). Signal intensity time curves were generated for each muscle group and curve parameters, including signal reduction during ischemia (SRi) and gradient during reactive hyperemia (Grad). BOLD-CMR was used to assess changes in perfusion following revascularization in 12 CLI patients. Muscle biopsies (n = 28), obtained at the level of BOLD-CMR measurement and from healthy proximal muscle of patients undergoing lower limb amputation (n = 3), were analyzed for capillary-fiber ratio.There was good interuser and interscan reproducibility for Grad and SRi (all p 0.0001). The ischemic limb had lower Grad and SRi compared with the contralateral asymptomatic limb, age-matched control subjects, and young control subjects (p 0.001 for all comparisons). Successful revascularization resulted in improvement in Grad (p 0.0001) and SRi (p 0.0005). There was a significant correlation between capillary-fiber ratio (p 0.01) in muscle biopsies from amputated limbs and Grad measured pre-operatively at the corresponding level.BOLD-CMR showed promise as a reliable tool for assessing perfusion in the lower limb musculature and merits further investigation in a clinical trial.

OBJECTIVES Increased stiffening of the aortic wall could contribute to the development of abdominal aortic aneurysm (AAA). We investigated regional aortic wall pulse wave velocity (PWV) in patients with AAA. METHODS Forty-six men diagnosed with a small AAA and 42 control men were recruited from the AAA surveillance and screening programmes at Guy's and St Thomas' Hospital. Phase-contrast cardiovascular MRI was performed to determine regional PWV along the thoracic (PWVTHOR) and abdominal aorta (PWVABD). PWV over the total aorta (PWVTOTAL) was calculated from the combined regions. RESULTS PWVTOTAL was significantly higher in patients with AAA compared to controls (10.0 ± 2.1 versus 8.4 ± 1.6 m/s, respectively; P

Aims: Increased oxidative stress and vascular inflammation are implicated in increased cardiovascular disease (CVD) incidence with age. We and others demonstrated that NOX1/2 NADPH oxidase inhibition, by genetic deletion of p47phox, in Apoe−/− mice decreases vascular reactive oxygen species (ROS) generation and atherosclerosis in young age. The present study examined whether NOX1/2 NADPH oxidases are also pivotal to aging-associated CVD. Results: Both aged (16 months) Apoe−/− and Apoe−/−/p47phox−/− mice had increased atherosclerotic lesion area, aortic stiffness, and systolic dysfunction compared with young (4 months) cohorts. Cellular and mitochondrial ROS (mtROS) levels were significantly higher in aortic wall and vascular smooth muscle cells (VSMCs) from aged wild-type and p47phox−/− mice. VSMCs from aged mice had increased mitochondrial protein oxidation and dysfunction and increased vascular cell adhesion molecule 1 expression, which was abrogated with (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) treatment. NOX4 expression was increased in the vasculature and mitochondria of aged mice and its suppression with shRNA in VSMCs from aged mice decreased mtROS levels and improved function. Increased mtROS levels were associated with enhanced mitochondrial NOX4 expression in aortic VSMCs from aged subjects, and NOX4 expression levels in arterial wall correlated with age and atherosclerotic severity. Aged Apoe−/− mice treated with MitoTEMPO and 2-(2-chlorophenyl)-4-methyl-5-(pyridin-2-ylmethyl)-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione had decreased vascular ROS levels and atherosclerosis and preserved vascular and cardiac function. Innovation and Conclusion: These data suggest that NOX4, but not NOX1/2, and mitochondrial oxidative stress are mediators of CVD in aging under hyperlipidemic conditions. Regulating NOX4 activity/expression and using mitochondrial antioxidants are potential approaches to reducing aging-associated CVD. Antioxid. Redox Signal. 23, 1389–1409.

SummaryDeep vein thrombosis (DVT) can give rise to chronic debilitating complications, which are expensive to treat. Anticoagulation, the standard therapy for DVT, prevents propagation, but does not remove the existing thrombus, which undergoes slow natural resolution. Alternative forms of treatment that accelerate resolution may arise from a better understanding of the cellular and molecular pathways that regulate the natural resolution of thrombi. This review will outline our current understanding of the mechanisms of thrombus resolution and the role of neovascularisation in this process. Novel experimental treatments that may one day find clinical use are also discussed. The process of thrombus resolution resembles wound healing. The mainly monocytic inflammatory infiltrate, which develops, is associated with the appearance of vascular channels. These cells may drive resolution by encouraging angiogenesis, which contributes to restoration of the vein lumen. Significant numbers of bone marrow-derived progenitor cells have also been found in naturally resolving thrombi, but their precise phenotype and their role in thrombus recanalisation is unclear. Enhanced thrombus neovascularisation and rapid vein recanalisation have been achieved in experimental models with proangiogenic agents. Recent reports of the role of bone marrow-derived progenitor cells in the revascularisation of ischaemic tissues suggest that it may be possible to obtain the same effect by delivering pluripotent or lineage specific stem cells into thrombus. These cells could contribute to thrombus recanalisation by expressing a variety of proangiogenic cytokines or by lining the new vessels that appear within the thrombus.

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 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 Angiogenesis, the formation of new from existing capillaries, is an important mechanism in venous ulcer healing. The aim of this study was to determine whether venous leg ulcer wound exudates stimulate or inhibit angiogenesis. Methods Fluid exudate was obtained from 16 venous ulcers over a 4-h interval. Five of the ulcers had not healed after more than 1 year of compression bandaging, and five were rapidly healing ulcers. As a control, acute wound fluids were collected from subcutaneous drains in seven patients. Vascular endothelial growth factor (VEGF) at 2 ng/ml acted as a positive control. Tubules stained with an antiendothelial antibody were quantified using an image analysis system. The extent of angiogenesis was expressed as the ratio of the mean tubule length in the test wells over that in blank control wells. Results Venous ulcer exudates significantly inhibited angiogenesis (mean (95 per cent confidence interval) 0·72 (0·48 to 0·96)) compared with acute wound fluids (2·48 (0·86 to 4·10)) (P < 0·002) and VEGF (1·47 (1·32 to 1·61)) (P = 0·01). Exudates from the five non-healing venous ulcers inhibited angiogenesis (0·31 (0·15 to 0·46)) significantly more than exudates from the five rapidly healing venous ulcers (0·93 (0·21 to 1·65)) (P = 0·03). Conclusion Fluid exudate from venous ulcers, in particular those that healed slowly, inhibited experimental angiogenesis in this study.

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.)

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.

A third of patients with critical limb ischemia (CLI) will eventually require limb amputation. Therapeutic neovascularization using unselected mononuclear cells to salvage ischemic limbs has produced modest results. The TIE2-expressing monocytes/macrophages (TEMs) are a myeloid cell subset known to be highly angiogenic in tumours. This study aimed to examine the kinetics of TEMs in patients with CLI and whether these cells promote neovascularization of the ischemic limb. Here we show that there are 10-fold more circulating TEMs in CLI patients, and removal of ischemia reduces their numbers to normal levels. TEM numbers in ischemic muscle are two-fold greater than normoxic muscle from the same patient. TEMs from patients with CLI display greater proangiogenic activity than TIE2-negative monocytes in vitro. Using a mouse model of hindlimb ischemia, lentiviral-based Tie2 knockdown in TEMs impaired recovery from ischemia, whereas delivery of mouse macrophages overexpressing TIE2, or human TEMs isolated from CLI patients, rescued limb ischemia. These data suggest that enhancing TEM recruitment to the ischemic muscle may have the potential to improve limb neovascularization in CLI patients.

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.