Julia Humphries

Electrospray ionization mass spectrometry identifies substrates and products of lipoprotein-associated phospholipase A2 in oxidized human low density lipoprotein

There is increasing evidence that modified phospholipid products of low density lipoprotein (LDL) oxidation mediate inflammatory processes within vulnerable atherosclerotic lesions. Lipoprotein-associated phospholipase A2 (Lp-PLA2) 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-PLA2 levels as an independent predictor of cardiovascular disease provides a rationale for development of Lp-PLA2 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-PLA2 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-PLA2. 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-PLA2 substrates. The major PC products of Lp-PLA2, 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-PLA2 inhibitor therapy.

The role of neovascularisation in the resolution of venous thrombus.

Deep 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.

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.

Intraluminal thrombus enhances proteolysis in abdominal aortic aneurysms

This study examined whether intraluminal thrombus in abdominal aortic aneurysms (AAAs) is a source of fibrinolytic activity and proteolysis that could weaken the aneurysm wall. Plasmin, tissue plasminogen activator (tPA), and urokinase plasminogen activator (uPA) activity, plasminogen activator inhibitor 1 (PAI-1), and α2-antiplasmin (α2AP) antigen were measured in the AAA wall and juxtamural and luminal aspects of intraluminal thrombus in 18 patients. The aneurysm wall contained 100-fold higher tPA activity (1.06 ± 0.34 [standard error of measurement] U/mg soluble protein) compared with juxtamural thrombus (JMT) (0.011 ± 0.001 ) and luminal thrombus (LT) (0.01 ± 0.001) (p < .00001) and over 6-fold higher uPA activity (29.3 ± 3.4 IU/mg compared with the JMT (4.3 ± 2.4, p = .00024) and LT (7.9 ± 1.76, p = .0005). The LT had significantly lower levels of PAI-1 (1.26 ± 0.34 ng/mg) than the AAA wall (2.08 ± 0.51, p = .04) and the JMT (3.94 ± 0.85, p = .007). The levels of α2AP in the wall (19.4 ± 3.1 ng/mg) were lower than in the JMT or LT (43.0 ± 7.9 ng/mg, p = .013, and 47.6 ± 6.0 ng/mg, p = .002, respectively). There was no significant difference, however, in plasmin activity among the AAA wall, JMT, and LT. There were significant amounts of latent gelatinase B (matrix metalloproteinase [MMP]-9) in the AAA, JMT, and LT. Mean levels of activated MMP-9 activity were similar in the AAA, JMT, and LT. Plasmin activation of MMPs at the interface between intraluminal thrombus and the aneurysm wall may enhance proteolysis and accelerate aneurysm expansion.

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.

The Role of the Monocyte in the Generation and Dissolution of Arterial and Venous Thrombi

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.

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.

Adenovirus-Mediated VEGF Gene Therapy Enhances Venous Thrombus Recanalization and Resolution

Objective—Rapid thrombus recanalization reduces the incidence of post–thrombotic complications. This study aimed to discover whether adenovirus-mediated transfection of the vascular endothelial growth factor gene (ad.VEGF) enhanced thrombus recanalization and resolution. Methods and Results—In rats, thrombi were directly injected with either ad.VEGF (n=40) or ad.GFP (n=37). Thrombi in SCID mice (n=12) were injected with human macrophages transfected with ad.VEGF or ad.GFP. Thrombi were analyzed at 1 to 14 days. GFP was found mainly in the vein wall and adventitia by 3 days, but was predominantly found in cells within the body of thrombus by day 7. VEGF levels peaked at 4 days (376±299 pg/mg protein). Ad.VEGF treatment reduced thrombus size by >50% (47.7±5.1 mm2 to 22.0±4.0 mm2, P=0.0003) and increased recanalization by >3-fold (3.9±0.69% to 13.6±4.1%, P=0.024) compared with controls. Ad.VEGF treatment increased macrophage recruitment into the thrombus by more than 50% (P=0.002). Ad.VEGF-transfected macrophages reduced thrombus size by 30% compared with controls (12.3±0.89 mm2 to 8.7±1.4 mm2, P=0.04) and enhanced vein lumen recanalization (3.39±0.34% to 5.07±0.57%, P=0.02). Conclusion—Treatment with ad.VEGF enhanced thrombus recanalization and resolution, probably as a consequence of an increase in macrophage recruitment.