Colin E. Evans

Proteomics identifies thymidine phosphorylase as a key regulator of the angiogenic potential of colony-forming units and endothelial progenitor cell cultures.

Endothelial progenitor cell (EPC) cultures and colony-forming units (CFUs) have been extensively studied for their therapeutic and diagnostic potential. Recent data suggest a role for EPCs in the release of proangiogenic factors. To identify factors secreted by EPCs, conditioned medium from EPC cultures and CFUs was analyzed using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer combined with offline peptide separation by nanoflow liquid chromatography. Results were verified by RT-PCR and multiplex cytokine assays and complemented by a cellular proteomic analysis of cultured EPCs and CFUs using difference in-gel electrophoresis. This extensive proteomic analysis revealed the presence of the proangiogenic factor thymidine phosphorylase (TP). Functional experiments demonstrated that inhibition of TP by 5-bromo-6-amino-uracil or gene silencing resulted in a significant increase in basal and oxidative stress-induced apoptosis, whereas supplementation with 2-deoxy-d-ribose-1-phosphate (dRP), the enzymatic product of TP, abrogated this effect. Moreover, dRP produced in EPC cultures stimulated endothelial cell migration in a paracrine manner, as demonstrated by gene-silencing experiments in transmigration and wound repair assays. RGD peptides and inhibitory antibodies to integrin &agr;v&bgr;3 attenuated the effect of conditioned medium from EPC cultures on endothelial migration. Finally, the effect of TP on angiogenesis was investigated by implantation of Matrigel plugs in mice. In these in vivo experiments, dRP strongly promoted neovascularization. Our data support the concept that EPCs exert their proangiogenic activity in a paracrine manner and demonstrate a key role of TP activity in their survival and proangiogenic potential.

Leukocytes and the natural history of deep vein thrombosis: current concepts and future directions

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

Magnetic Resonance T1 Relaxation Time of Venous Thrombus Is Determined by Iron Processing and Predicts Susceptibility to Lysis

Background— The magnetic resonance longitudinal relaxation time (T1) changes with thrombus age in humans. In this study, we investigate the possible mechanisms that give rise to the T1 signal in venous thrombi and whether changes in T1 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 T1 relaxation time correlated with thrombus composition. The mean T1 relaxation time of thrombus was shortest at 7days following thrombus induction and returned to that of blood as the thrombus resolved. T1 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 Fe3+, which causes thrombus T1 relaxation time shortening. Studies using chemokine receptor-2–deficient mice (Ccr2−/−) revealed that the return of the T1 signal to that of blood is regulated by removal of Fe3+ by macrophages that accumulate in the thrombus during its resolution. Quantification of T1 relaxation time was a good predictor of successful thrombolysis with a cutoff point of <747 ms having a sensitivity and specificity to predict successful lysis of 83% and 94%, respectively. Conclusions— The source of the T1 signal in the thrombus results from the oxidation of iron (released from the lysis of trapped erythrocytes in the thrombus) to its paramagnetic Fe3+ form. Quantification of T1 relaxation time appears to be a good predictor of the success of thrombolysis.

Hypoxia and Upregulation of Hypoxia-Inducible Factor 1α Stimulate Venous Thrombus Recanalization

Objective—Angiogenic factors are expressed within thrombus during resolution, but the primary stimulus for neovascularization is unknown. Our aims were to determine whether (1) hypoxia and hypoxia-inducible factor 1&agr; (HIF1&agr;) are induced in resolving thrombus, (2) this stimulates angiogenic factor production, and (3) upregulating HIF1&agr; enhances thrombus resolution and vein recanalization. Methods and Results—Oxygen tension in the thrombus was negatively correlated with HIF1&agr; levels (Spearman correlation [RS]=−0.77, P<0.0001), whereas HIF1&agr; levels positively correlated with vascular endothelial growth factor (VEGF) expression (Pearson correlation [R]=0.85, P<0.0005), during resolution in a murine model. HIF1&agr; (P<0.005), VEGF (P<0.005), and VEGF receptor 1 (VEGFR1) (P<0.05) expression was 2-fold greater in the thrombus of mice treated with the prolyl hydroxylase domain inhibitor l-mimosine compared with controls. The levels of 13 other HIF1-mediated angiogenic factors were also increased. Thrombus weight (P<0.001) and volume (P<0.05) were reduced by a third in l-mimosine–treated mice compared with controls, whereas vein recanalization (P<0.005) and thrombus neovascularization (P<0.001) were 2-fold greater, and this was associated with increased inflammatory cell content. Conclusion—Hypoxia and HIF1&agr; are induced in the naturally resolving thrombus and correlate with increased angiogenic factor expression. Upregulation of HIF1&agr; enhances thrombus resolution and vein recanalization. HIF1&agr; may represent a novel target for treatments that promote resolution and recanalization and reduce the incidence of post-thrombotic syndrome.

Antiangiogenic Therapy Inhibits Venous Thrombus Resolution

Objective— Venous thromboembolism is a common complication in patients with cancer, resulting in significant morbidity and mortality. Clinical studies suggest that the incidence of venous thromboembolic events increased after treatment of these patients with antiangiogenic agents. Thrombi resolve through a process of remodeling, involving the formation of microvascular channels within the thrombus. Our aim was to determine whether inhibiting angiogenesis affects venous thrombus resolution. Approach and Results— Thrombus was induced in the inferior vena cava of mice. These mice were treated with axitinib (50 mg/kg per day), 2-methoxyestradiol (2ME, 150 mg/kg per day), or vehicle control. Thrombus size, recanalization, neovascularization, inflammatory cell content, and collagen content were assessed after axitinib (days 3, 10, 17) and 2ME (day 10 only) treatment (n=6/group). Axitinib treatment resulted in reduced thrombus resolution (P<0.002) and vein recanalization (P<0.001) compared with vehicle-treated controls. This was associated with inhibition of organization as seen through reduced thrombus neovascularization (P<0.0001) and collagen (P<0.0001) content, as well as reduced macrophage accumulation in the thrombus (P<0.001). Treatment with a second antiangiogenic agent, 2ME, mirrored these findings, with a similar order of magnitude of effect of treatment over vehicle control in all of the parameters measured, with the exception of neutrophil content, which was significantly reduced after 2ME treatment but not affected by axitinib. Conclusions— Antiangiogenic therapy (using axitinib and 2ME) inhibits the resolution of venous thrombi, which could lead to persistent venous obstruction and the possibility of thrombus extension. This potential prolongation of venous occlusion by antiangiogenic agents should therefore be taken into consideration in trials of these agents and when managing the complications of venous thromboembolic events in patients with cancer.

The effect of colorectal cancer upon host peripheral immune cell function.

Objective  Colorectal cancer is immunogenic. However, it is also associated with suppression of host immunity. Identifying the mechanisms involved in immune suppression is necessary to develop future immunotherapeutic strategies. The aim of this study was to assess immune cell function in colorectal cancer patients.

Assessment of Venous Thrombosis in Animal Models

Deep vein thrombosis and common complications, including pulmonary embolism and post-thrombotic syndrome, represent a major source of morbidity and mortality worldwide. Experimental models of venous thrombosis have provided considerable insight into the cellular and molecular mechanisms that regulate thrombus formation and subsequent resolution. Here, we critically appraise the ex vivo and in vivo techniques used to assess venous thrombosis in these models. Particular attention is paid to imaging modalities, including magnetic resonance imaging, micro–computed tomography, and high-frequency ultrasound that facilitate longitudinal assessment of thrombus size and composition.

Techniques of assessing hypoxia at the bench and bedside.

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.

The soluble urokinase plasminogen activator receptor and its fragments in venous ulcers.

OBJECTIVE 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Protein fragments from the VEGF binding domain of fibronectin are expressed in distinct spatial and temporal patterns during venous thrombus resolution

Deep vein thrombosis (DVT) affects 1-2% of the general population [1,2] and can lead to chronic and debilitating complications including leg pain, swelling, lipodermatosclerosis, and ulceration [3,4]. Clinical outcome is improved in patients whose thrombi resolve rapidly by a process of vein recanalisation and organisation that is similar to wound healing [5,6]. This process is enhanced by infiltration of inflammatory and endothelial cells, which mediate resolution by releasing a catalogue of angiogenic chemokines, proteases, and growth factors [5]. These factors include the potent angiogenic chemokine vascular endothelial growth factor (VEGF), which is highly expressed in resolving thrombus and accelerates this process [7,8]. Although spatial and temporal patterns of VEGF expression have been identified during venous thrombus resolution [8], the mechanisms that control natural resolution remain unclear. Fibronectin is found in blood plasma, extracellular matrix, and platelets, and this angiogenic glyocoprotein has roles in cell adhesion, migration, differentiation, and proliferation [9]. Fibronectin specifically binds with VEGF or VEGF receptor 1 (VEGFR1) and in doing so enhances processes that contribute to venous thrombus resolution including macrophage and endothelial cell migration [10,11]. VEGF binds with fibronectin at the N-terminal collagen binding domain and the C-terminal Hep II binding domain, the latter of which enhances endothelial cellmigration andmitogen-activated protein kinase activity [10,11]. The role of the N-terminal VEGF binding domain in fibronectin, however, is unclear. Proteolytic fragments of fibronectin, for example, can enhance monocyte migration in response to VEGF and placental growth factor (PLGF) via VEGFR1 [12], suggesting that the N-terminal VEGF/VEGFR1 binding region of fibronectin could mediate VEGFdependent tissue remodelling responses during thrombus resolution. It follows that fibronectin-VEGF binding could amplify VEGF activity and accelerate venous thrombus resolution. It was therefore our aim to identify spatial and temporal expression patterns of fibronectin and its proteolytic fragments during natural thrombus resolution in an established mouse model of venous thrombosis. We show that fibronectin and several of its proteolytic fragments are expressed in distinct temporal patterns in the thrombus and surrounding vein throughout natural resolution, suggesting that fibronectin could amplify the angiogenic and chemotactic effects of VEGF during thrombus resolution and in doing so accelerate this process.