Anwar Ahmad

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.

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.

Hematopoietic Progenitor Cells and Restenosis After Carotid Endarterectomy

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.

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.

Renal transplantation after excision of the inferior vena cava for residual renal cell carcinoma

A rare case is reported of a 47-year-old patient who had residual tumour left in the stump of his left renal vein and inferior vena cava (IVC) after a left nephrectomy for renal cell carcinoma, having previously had a right nephrectomy for a non-malignant disease. He underwent delayed excision of the residual renal tumour after seven months with a prosthetic graft replacement of the IVC. Five years later he developed a graft infection caused by a graft-enteric duodenal fistula. The infected graft was excised and the duodenum closed. The patient has subsequently undergone a successful renal transplantation despite a lack of major venous outflow and remains alive and well eight years after initial removal of the tumour from the IVC.