Katherine Mattock

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.

The monocyte/macrophage as a therapeutic target in atherosclerosis.

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.

TIE2-expressing monocytes/macrophages regulate revascularization of the ischemic limb

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.

Noninvasive Assessment of Atherosclerotic Plaque Progression in ApoE(-/-) Mice Using Susceptibility Gradient Mapping

Background— Macrophages have been identified as a major contributor to plaque development and destabilization in atherosclerosis. The aim of this study was to noninvasively assess uptake of citrate coated very small iron oxide particles at different stages of plaque development in the brachiocephalic artery of apoE−/− mice. Susceptibility gradient mapping (SGM) was applied to generate positive contrast images and to quantify iron oxide uptake. Methods and Results— ApoE−/− mice were fed a high-fat diet for 4, 8, or 12 weeks; 300 &mgr;mol Fe/kg was injected 24 and 48 hours before final MRI. Increasing very small iron oxide particle uptake was observed over the course of atherosclerotic plaque development. Simultaneous administration of pravastatin led to a significant decrease in very small iron oxide particle uptake, assessed by mass spectroscopy and histology. SGM-MRI allowed the generation of positive contrast images, and magnitudes (mT/m) of contrast enhancement in SG parameter maps significantly correlated with the absolute iron oxide content (R2=0.70, P<0.05) and the macrophage density (R2=0.71, P<0.05). Conclusions— This study shows an increase in iron oxide uptake (measured by in vivo SGM-MRI, histology, and mass spectroscopy) with the progression of plaque development in an apoE−/− mouse model of accelerated atherosclerosis. Positive contrast provided by SGM-MRI allowed for a clear visualization of intraplaque iron oxide depositions, and magnitudes (mT/m) of contrast enhancement in SG parameter maps allowed for the quantification of intraplaque iron oxide particles.

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.

Legumain and cathepsin-L expression in human unstable carotid plaque

OBJECTIVE The cysteine protease, legumain, is thought to have a role in the processing and activation of proteases such as cathepsin-L, which have been implicated in plaque rupture. This study aimed to determine: if legumain activity is up-regulated in unstable areas of plaque; the effect of legumain over-expression on the activity of cathepsin-L and the effect of mutation of the legumain RGD sequence on its cellular location. METHODS AND RESULTS Legumain was measured in human carotid plaque extracts (n=17) using a novel ELISA and modified activity assay. Unstable regions of plaque contained more than twice the amount of legumain protein (P<0.001) and activity (P<0.03) compared with stable regions of the same plaque. Over-expression of legumain in THP-1 macrophages using an adenoviral construct resulted in the processing of cathepsin-L from its 30kDa to its 25kDa form compared with controls. CONCLUSION Unstable regions of plaque contain increased levels of active legumain. Over-expression of legumain in macrophages alters intracellular processing of cathepsin-L to its mature 25kDa form. This may be a means by which legumain could contribute to plaque instability.

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.

Upregulation of hypoxia-inducible factor 1 alpha in local vein wall is associated with enhanced venous thrombus resolution

Introduction Venous thrombus resolution may be regulated by an angiogenic process that involves the surrounding vein wall. The aims of this study were to determine whether: (i) thrombosis stimulates activation of the angiogenic transcription factor, hypoxia-inducible factor (HIF) 1α, and downstream expression of growth factors in vein wall; and (ii) upregulation of HIF1α in vein wall leads to increased growth factor expression and enhanced thrombus resolution. Materials and methods HIF1α, vascular endothelial growth factor (VEGF), and placental growth factor (PLGF) were quantified in mouse inferior vena cava (IVC) at days 1, 3, 7, and 14 after thrombus formation (n = 10-13 per group). An additional group of thrombosed mice were treated with the prolyl-hydroxylase domain (PHD) inhibitor, L-mimosine (L-mim) or vehicle control. HIF1α, VEGF, and PLGF in IVC were measured at days 1 and 7; and vein recanalisation and thrombus resolution were measured at days 7 and 10 (n = 6-7 per group). Results HIF1α was expressed in thrombosed IVC and its levels remained relatively constant throughout natural resolution. The levels of VEGF in thrombosed IVC were elevated at days 1 (P < 0.0001) and 3 (P < 0.05); and PLGF at days 1 (P < 0.0001), 3 (P < 0.0001), and 7 (P < 0.0001). Treatment with L-mim led to: increased HIF1α (P < 0.05), VEGF (P < 0.005), and PLGF (P < 0.001) levels in the IVC; decreased thrombus size (P < 0.01); and increased vein recanalisation (P < 0.001). Conclusions HIF1α levels in vein wall are not affected by thrombosis and it appears that the angiogenic drive in the vein surrounding resolving thrombus is regulated independently of HIF1α. Stimulating HIF1α levels in the vein wall leads to an increased angiogenic drive and promotes vein recanalisation and thrombus resolution.

Encapsulation of angiogenic monocytes using bio-spraying technology

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

HIF1 signalling regulates venous thrombus resolution.

The number of deaths that are attributable to pulmonary embolism resulting from deep vein thrombosis (DVT) in the UK (>25 000/year) is greater than the number caused by breast cancer, HIV, and road traffic accidents combined [1]. Approximately one third of patients with DVT develop post-thrombotic syndrome, which is characterised by long-term and debilitating symptoms including leg pain, swelling, and ulceration [2]. Post-thrombotic complications are not, however, a corollary of DVT, as clinical studies show that rapid resolution improves patient outcome and reduces post-thrombotic complications [3–5]. Current treatments for DVT include anticoagulation and thrombolysis. Anticoagulants prevent thrombus extension, but do little to remove the thrombus, which naturally resolves through a slow process of organisation similar to wound healing [6]. Thrombolysis removes the thrombus rapidly, but its use is restricted to fresh thrombus, and furthermore, both anticoagulation and thrombolysis predispose to an increased risk of pathological bleeding. Novel therapies that accelerate thrombus resolution without the risk of haemorrhage may arise from a better understanding of the molecular and cellular mechanisms that control this remodelling process. Natural thrombus resolution involves infiltration of inflammatory cells and the formation of new vascular channels both within and around the thrombus [6]. Hypoxia is a major stimulus for the production of many molecular mediators of tissue remodelling and neovascularisation (angiogenesis) that we know regulate thrombus remodelling [6,7]. These include monocyte chemotactic protein (MCP) 1, matrix metalloproteinases (MMPs), placental growth factor (PLGF), stromal cell-derived factor (SDF) 1, urokinase-type plasminogen activator (uPA), and vascular endothelial growth factor (VEGF) [6–12], which are directly and transcriptionally upregulated by the nuclear transcription factor, hypoxia-inducible factor 1 (HIF1) [13,14]. We hypothesised that cells within venous thrombus become hypoxic during thrombogenesis, which results in HIFmediated increases in the production of angiogenic targets that enhance neovascularisation within the thrombus and subsequently accelerate vein recanalization and thrombus resolution.