Primary B cells from B cell chronic lymphocytic leukaemia (B-CLL) were resistant to the novel selective cytotoxic agent, TNF-related apoptosis-inducing ligand (TRAIL). Low levels of the death-inducing TRAIL receptors, TRAIL-R1 and TRAIL-R2 but not the putative 'decoy' receptors, TRAIL-R3 and TRAIL-R4, were expressed on the surface of B-CLL cells. Resistance to TRAIL was upstream of caspase-8 activation, as little or no caspase-8 was processed in TRAIL-treated B-CLL cells. Low levels of a TRAIL death-inducing signalling complex (DISC) were formed in these cells, accompanied by the recruitment of endogenous FADD, caspase-8 and c-FLIP L but not c-FLIP S . Both caspase-8 and c-FLIP L were cleaved to form two stable intermediates of *43 kDa, which remained associated with the DISC. Caspase-8 was not further processed to its active heterotetramer. Thus the resistance of B-CLL cells to TRAIL may be due partly to low surface expression of the death receptors resulting in low levels of DISC formation and also to the high ratio of c-FLIP L to caspase-8 within the DISC, which would prevent further activation of caspase-8. Our results highlight the possibility of sensitising B-CLL cells to TRAIL by modulation of c-FLIP levels or by upregulation of surface expression of death receptors.
Endothelial-monocyte activating polypeptide (EMAP)-II is a novel molecule with cytokine-like pro-inflammatory properties, inducing procoagulant activity on the surface of endothelial cells and monocyte/macrophages in vitro, as well as up-regulating E-and Pselectin expression. EMAP-II is chemotactic for monocytes/macrophages and neutrophils, and stimulates myeloperoxidase release from neutrophils. Injection of EMAP-II into the mouse footpad induces an acute inflammatory response, although some regression occurs in response to direct injection of EMAP-II into murine tumors. Very little is known about the expression of EMAP-II in normal tissues of mice or humans, or about its function in vivo. We developed polyclonal antibodies against EMAP-II using recombinant protein produced in Escherichia coli, and used these antibodies to carry out an immunohistochemical study of the occurrence and distribution of EMAP-II in human tissues. The distribution of EMAP-II protein is relatively restricted, occurring primarily in endocrine organs, in cells of neuroendocrine origin, but also in tissues with high turnover. EMAP-II is strongly expressed in secretory epithelial cells of the thyroid, pancreas, adrenal and salivary glands, among others, as well as in neurons and subsets of monocytes/macrophages. It is also found in the epithelium of the small and large intestines. We conclude that EMAP-II expression is usually, but not always, associated with tissues that display high turnover and high levels of protein synthesis. EMAP-II is a novel molecule with pleiotropic activities toward endothelial cells, monocytes/macrophages, and neutrophils.
Percutaneous coronary intervention (PCI) is now the dominant revascularisation therapy for coronary artery disease, with nearly 3 million PCIs undertaken per annum world-wide. One of the last remaining challenges to PCI success is in cases of chronic total occlusions (CTO), where average UK success rates are around 66% (BCIS Data). It is only in the more experienced hands that success rates of 80–90% are achieved (EuroCTO club data).There is a need to develop alternative, effective therapies for CTO failures and to this end we have explored therapeutic angiogenesis to enhance antegrade collateral circulation and provide symptom relief in difficult CTO cases. We have investigated the stimulation of endothelial cell (EC) angiogenesis via stabilisation of hypoxia inducible factor (HIF) using two prolyl-hydroxylase inhibitors (PHI). We previously reported that di-methyl oxalylglycine (DMOG) loaded onto a polymer-coated stent increased collateral neovascularisation in a porcine CTO model. We now describe the actions of the PHI FG-2216/BIQ, which unlike DMOG has already been approved for use in man being trialled in patients with renal anaemia. Human umbilical vein endothelial cells (HUVECs) were cultured on growth factor reduced Matrigel in the presence of FG-2216 at a range of doses (from 0 to 500 μM). Endothelial cell (EC) tubule formation was measured at 6 h by image analysis of photomicrographs (image J). Western blotting was used to assess expression of HIF-1α in HUVEC after treatment with 500 μM FG-2216/BIQ for up to 24 h. Quantitative real time RT-PCR (qPCR) was used to examine the expression of vascular endothelial cell growth factor (VEGF) mRNA from treated HUVEC (up to 24 h). We report that as with DMOG, FG-2216 has a dose response, pro-angiogenic effect on HUVEC, with significantly enhanced tubule formation on Matrigel, compared to control (107 vs 21, p < 0.05) (Figure 1). Western blotting analysis of HUVEC protein demonstrated stabilisation of HIF-1α protein within 1–2 h of treatment with FG-2216, and qPCR results showed an increase in mRNA for the main angiogenic target of HIF-1α, vascular endothelial cell growth factor (VEGF). Abstract 74 Figure 1 These proof of concept investigations confirm that FG-2216 has a pro-angiogenic effect in-vitro and is valid for testing in in-vivo models of CTO, with the potential for translation to man.We are currently developing novel strategies for the local, sustained delivery/release of FG-2216 such as drug eluting absorbable stenting and micro-sphere delivery as well as routine DES delivery to ensure slow release over long periods. These will be tested in the porcine CTO model developed by our group. We have a novel approach to perfusion by inducing therapeutic angiogenesis as an effective, innovative therapy for difficult CTO in man. Spin off therapies include peri-infarct ischaemia.
Background:Large registries have shown a survival benefit in patients with successful chronic total occlusion (CTO)-PCI as compared to unsuccessful and/or unattempted CTO-PCI. Sustained vessel patency may be considered as a main variable related to long-term survival. Few data exist about the angiographic outcome of patients who performed CTO-PCI, due to lack or very low angiographic follow-up rate of CTO registries. The aim of this study was to assess the incidence of reocclusion and identifications of predictors of angiographic failure after successful CTO-PCI in the drug-eluting stent (DES) era. Methods: From the large prospective Florence CTO-PCI registry, since 2004 to 2010, 1005 patients underwent a PCI for CTO (Ͼ3 months). The protocol treatment includes routine 6-to 9-month angiographic FU. Clinical, angiographic, and procedural variables were included into the model of multivariable binary logistic regression analysis for the identification of the predictors of reocclusion. Results: From January 2004 to December 2010 1,005 patients underwent a PCI attempt for at least 1 CTO (Ͼ3 months). Out of these, 770 (77%) had a successful PCI. Baseline and procedural characteristics: mean age 68 Ϯ 11, male 84%, diabetes 24%, previous myocardial infarction 51%, LVEFϽ0.40 37%, 3-vessel disease 50%, everolimus-eluting stent (EES) 36%, stent lenght Ͼ40 mm 54%, long subintimal tracking and very distal re-entry (STAR) 4%. STAR technique was used exclusively after failed intraluminal anterograde or retrograde attempt. The angiographic FU rate was 80% (578 patients). Reocclusion rate was 7.6%, while binary restenosis (Ͼ50%) or reocclusion rate was 21%. EES, was associated with a significant lower re-occlusion rate than other DES (2.4% vs. 9.9%; pϭ0.001). A successful STAR technique was associated with a 55% of rocclusion rate (pϽ0.001). At multivariable analysis STAR technique (OR 32; pϽ0.001) and EES (OR 0.19; pϭ0.002) were independently related to the risk of reocclusion. Conclusions: Successful EES supported CTO-PCI is associated with a very high patency rate (97.6%). Successful STAR technique is associated with a very low mid-term patency rate whatever the type of stent used.
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