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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., (Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.)

Background: Our previous studies showed that the direct injection of an adenovirus construct expressing urokinase-type plasminogen activator (uPA) into experimental venous thrombi significantly reduces thrombus weight. The systemic use of adenovirus vectors is limited by inherent hepatic tropism and inflammatory response. As macrophages are recruited into venous thrombi, it is reasonable to speculate that these cells could be used to target the adenovirus uPA (ad-uPA) gene construct to the thrombus. The aims of this study were to determine whether macrophages transduced with ad-uPA have increased fibrinolytic activity and whether systemic injection of transduced cells could be used to target uPA expression to the thrombus and reduce its size., Methods: The effect of up-regulating uPA was examined in an immortalized macrophage cell line (MM6) and macrophages differentiated from human blood monocyte-derived macrophages (HBMMs). Cells were infected with ad-uPA or blank control virus (ad-blank). Fibrinolytic mediator expression, cell viability, and cytokine expression were measured by activity assays and enzyme-linked immunosorbent assays. Monocyte migration was measured using a modified Boyden chamber assay. A model of venous thrombosis was developed and characterized in mice with severe combined immunodeficiency (SCID). This model was used to study whether systemically administered macrophages over-expressing uPA reduced thrombus size. Uptake of HBMMs into the thrombus induced in these mice was confirmed by a combination of PKH2-labeled cell tracking and colocalization with human leukocyte antigen (HLA) by immunohistology., Results: Compared with ad-blank, treated HBMMs transduction with ad-uPA increased uPA production by >1000-fold (P = .003), uPA activity by 150-fold (P = .0001), and soluble uPA receptor (uPAR) by almost twofold (P = .043). Expression of plasminogen activator inhibitor (PAI-1) and PAI-2 was decreased by about twofold (P = .011) and threefold (P = .005), respectively. Up-regulation of uPA had no effect on cell viability or inflammatory cytokine production compared with ad-blank or untreated cells. Ad-uPA transduction increased the migration rate of HBMMs (about 20%, P = .03) and MM6 cells (>twofold, P = .005) compared with ad-blank treated controls. Human macrophage recruitment into the mouse thrombus was confirmed by the colocalization of HLA with the PKH2-marked cells. Systemic injection of uPA-up-regulated HBMMs reduced thrombus weight by approximately 20% compared with ad-blank (P = .038) or sham-treated controls (P = .0028)., Conclusion: Transduction of HBBM with ad-uPA increases their fibrinolytic activity. Systemic administration of uPA up-regulated HBBMs reduced thrombus size in an experimental model of venous thrombosis. Alternative methods of delivering fibrinolytic agents are worth exploring.

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.

Mammalian orthologues of the Drosophila tribbles protein (Trb1, Trb2 and Trb3) are a recently described family of signalling molecules that regulate gene expression by modulation of protein kinase signalling pathways. In the present study, a screen for mRNA species specifically regulated in vulnerable regions of human atherosclerotic plaque demonstrated the up-regulation of both Trb1 and Trb2, the latter by more than 8-fold. In vitro experiments in primary human monocyte-derived macrophages showed that Trb2 expression was up-regulated by treatment with oxidized LDL (low-density lipoprotein), and that expression of recombinant Trb2 specifically reduced macrophage levels of IL-10 (interleukin-10) mRNA. Our results thus identify Trb2 as a highly regulated gene in vulnerable atherosclerotic lesions, and demonstrate inhibition of macrophage IL-10 biosynthesis as a potential pro-inflammatory consequence of high Trb2 expression, which may contribute to plaque instability.

Objective: Galectin-3 (Gal-3) is a 26-kDa lectin known to regulate many aspects of inflammatory cell behavior. We assessed the hypothesis that increased levels of Gal-3 contribute to atherosclerotic plaque progression by enhancing monocyte chemoattraction through macrophage activation., Methods and Results: Gal-3 was found to be upregulated in unstable plaque regions of carotid endarterectomy (CEA) specimens compared with stable regions from the same patient (3.2-fold, P<0.05) at the mRNA (n=12) and (2.3-fold, P<0.01) at the protein level (n=9). Analysis of aortic tissue from ApoE-/- mice on a high fat diet (n=14) and wild-type controls (n=9) showed that Gal-3 mRNA and protein levels are elevated by 16.3-fold (P<0.001) and 12.2-fold (P<0.01) and that Gal-3 staining colocalizes with macrophages. In vitro, conditioned media from Gal-3-treated human macrophages induced an up to 6-fold increase in human monocyte chemotaxis (P<0.01, ANOVA), an effect that was reduced by 66 and 60% by Pertussis Toxin (PTX) and the Vaccinia virus protein 35K, respectively. Microarray analysis of human macrophages and subsequent qPCR validation confirmed the upregulation of CC chemokines in response to Gal-3 treatment., Conclusions: Our data suggest that Gal-3 is both a marker of atherosclerotic plaque progression and a central contributor to the pathology by amplification of key proinflammatory molecules.

Mutations in the gap junction protein connexin47 (Cx47) are associated with lymphedema. However, the role of Cx47 in lymphatic pathophysiology is unknown. We demonstrate that Cx47 is expressed in lymphatic endothelial cells by whole-mount immunostaining and qPCR. To determine if Cx47 plays a role in lymphatic vessel function we analysed Cx47-/- mice. Cx47-deficiency did not affect lymphatic contractility (contractile amplitude or frequency) or lymphatic morphology (vessel diameter or number of valves). Interstitial fluid drainage or dendritic cell migration through lymphatic vessels was also not affected by Cx47-deficiency. Cx47 is dispensable for long-chain fatty acid absorption from the gut but rather promotes serum lipid handling as prolonged elevated triglyceride levels were observed in Cx47-deficient mice after oral lipid tolerance tests. When crossed with Apolipoprotein E-deficient (Apoe-/-) mice, LDL-cholesterol was decreased in young Cx47-/-Apoe-/- adults as compared to Apoe-/- mice, which was inverted later in life. Finally, advanced atherosclerotic plaques in thoracic-abdominal aortas of 15 months-old mice tended to be larger in Cx47-/-Apoe-/- mice. These plaques contained fewer macrophages but similar amounts of T lymphocytes, collagen and lipids than plaques of Apoe-/- mice. In conclusion, Cx47 is expressed in lymphatic endothelium and seems modestly implicated in multiple aspects of lymphatic pathophysiology. [ABSTRACT FROM AUTHOR]

Objective: To investigate the importance of monocyte recruitment in thrombus resolution and the role of cysteine-cysteine (CC) chemokines and the CC chemokine receptor, CCR2, in this process. Methods: Peritoneal macrophages, monocyte chemotactic protein 1 (MCP1), or carrier solutions were injected into thrombi induced in the vena cava of rats. Caval thrombi were also formed in CCR2−/− and MCP1−/− mice and in wild-type mice transfected with an adenoviral construct expressing a broad-spectrum CC receptor antagonist. Results: Direct administration of peritoneal macrophages decreased thrombus size by more than fivefold and increased recanalization by more than fourfold compared with controls (P < .001). A 100-ng MCP1dose reduced thrombus size by more than sixfold (P < .01) and increased recanalization by more than sevenfold (P < .01), without affecting macrophage recruitment. Deletion of CCR2 or blockade of all CC chemokines inhibited both monocyte recruitment (P < .05) and thrombus resolution (P < .01), but knocking out MCP-1 had no effect. Conclusion: Increasing macrophage numbers in the thrombus enhances its resolution. MCP1 treatment enhances resolution by stimulating recanalization, independent of an effect on monocyte recruitment. CCR2 deficiency has the same effect as blockade of all CC chemokines. CCR2 receptor activation may therefore be an important mechanism in monocyte recruitment into venous thrombi and could be targeted to promote their resolution. [Copyright &y& Elsevier]

Cell therapies to treat critical limb ischaemia have demonstrated only modest results in clinical trials, and this has been partly attributed to poor cell retention following their delivery directly into the ischaemic limb. The aim of this study was to determine whether alginate encapsulation of therapeutic pro-angio/arteriogenic macrophages enhances their retention and ultimately improves limb perfusion. A reproducible GMP-compliant method for generating 300 µm alginate capsules was developed to encapsulate pro-angio/arteriogenic macrophages. Longitudinal analysis revealed no detrimental effect of encapsulation on cell number or viability in vitro, and macrophages retained their pro-angio/arteriogenic phenotype. Intramuscular delivery of encapsulated macrophages into the murine ischaemic hindlimb demonstrated increased cell retention compared with injection of naked cells (P = 0.0001), and that this was associated both enhanced angiogenesis (P = 0.02) and arteriogenesis (P = 0.03), and an overall improvement in limb perfusion (P = 0.0001). Alginate encapsulation of pro-angio/arteriogenic macrophages enhances cell retention and subsequent limb reperfusion in vivo. Encapsulation may therefore represent a means of improving the efficacy of cell-based therapies currently under investigation for the treatment of limb ischaemia. Limb ischemia: Encapsulation enhances therapeutic promise of immune cells in mice Blood vessel-promoting immune cells stay longer in the body and help promote blood flow to the feet and toes of mice with critical limb ischemia when the therapeutic cells are packaged inside tiny bubbles of a biocompatible seaweed derivative called alginate. A team led by Bijan Modarai from King's College London, UK, developed a reliable method for placing artery-stimulating macrophage cells inside alginate capsules measuring 300 micrometres in diameter, about the thickness of a postcard. In culture, the alginate coating had no effect on the macrophage viability; and when injected into the muscles of mice with artery blockages to their hindlimbs, the encapsulated cells were retained longer and offered greater therapeutic benefit than uncoated cells. This encapsulation strategy may improve the efficacy of comparable cell-based therapies for humans with limb ischemia. [ABSTRACT FROM AUTHOR]

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 7 days 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. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]