Report on the Spring Meeting of The British Atherosclerosis Society, Magdalen College, Oxford

Atherosclerosis 156 (2001) 245– 248 www.elsevier.com/locate/atherosclerosis

Abstracts

Report on the Spring Meeting of The British Atherosclerosis Society, Magdalen College, Oxford March 29–30, 2001

Symposium on ‘Peripheral Arterial Disease and Critical Limb Ischaemia’. Organised by: Professor Janet Powell and Professor Gerry Fowkes

Aetiology of peripheral arterial disease

Gordon Lowe

Measurement of peripheral arterial disease

John Woodcock

Regulation of muscle gene expression by physical activity

Carl Sundberg

Pathogenesis of critical limb ischaemia

Jill Belch

Development and stimulation of collateral circulation in the ischaemic limb

Wolfgang Schaper

Therapeutic angiogenesis for critical limb ischaemia

Peter Vale

The John French Memorial Lecture was delivered by Professor Paul Greenhaff, and entitled ‘Mitochondrial enzymes: a promising metabolic target for pharmacological treatment of peripheral arterial disease’.

Abstracts of the ten free communications presented are given below.

The next meeting of the Society will take place on 13th and 14th September 2001 in Cambridge. The meeting will be on ‘Recent advances in high density lipoproteins and atherosclerosis’ and will be organised by Professor Paul Durrington. Further details about this meeting are available from the BAS Secretary, Professor Jeremy D. Pearson, Research Centre for Cardiovascular Biology and Medicine, School of Biomedical Sciences, King’s College, London W8 7AH (telephone/fax: + 44-20 7333 4083, e-mail: [email protected], website: www.britathsoc.ac.uk

PII: S 0 0 2 1 - 9 1 5 0 ( 0 1 ) 0 0 4 9 2 - 0

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Interleukin-6 (IL6) promoter polymorphisms and the effect on interleukin-6 levels following coronary artery bypass surgery D.J. Brull 1, H.E. Montgomery 1, L. Luong 1, J. Sanders 1, S. Dhamrait 1, A. Rumley 2, G.D.O. Lowe 2, S.E. Humphries 1 1 Cardiovascular Genetics Division, UCL, 2 Department of Medicine, Glasgow Royal Infirmary Interleukin-6 (IL6) may play an important role in bridging the inflammatory and atherosclerotic processes. Coronary artery bypass graft surgery (CABG) induces an acute phase response, releasing cytokines such as IL-6. We have examined the effect of two polymorphisms in the IL6 gene promoter − 174G\ C and − 572G\ C on IL6 levels following elective CABG. One hundred and twenty seven patients (mean age 64 9 9 years) completed the study. Serum IL6 (pg/ml) was measured preoperatively, and in serial samples 6, 24, 48 and 72 h post CABG. Rare allele frequencies ( 9 95% confidence intervals) were similar to those reported previously: −174C 0.37 (0.31–0.43)– 572C 0.07 (0.04–0.10). There was no effect of either polymorphism on baseline IL6. In contrast, peak IL6 levels 6 h post CABG, were significantly higher in those with genotype − 174 CC, compared to − 174 G allele carriers (287 9 31 vs. 227 9 15, P= 0.04); and in the presence of one or more − 572 C allele compared to genotype −572 GG (355 9 67 vs. 216 9 13, P= 0.03). These effects remained statistically significant after adjusting for possible confounders, including age, sex, smoking, duration of cardiopulmonary bypass, aortic cross clamp time and total duration of surgery. In conclusion, we demonstrate IL6 gene promoter −572G\ C and − 174G \C polymorphisms influence peak IL6 production post CABG by a magnitude that is likely to be clinically relevant. Our results suggest that these polymorphisms are functional in vivo, suggesting a genetic influence on IL6 levels after acute severe injury. This work is funded by BHF grant FS99025

NF-kB is essential for metalloproteinase secretion by smooth muscle and foam cells Alex J. Chase, Andrew C. Newby Bristol Heart Institute, University of Bristol, Bristol, UK Identifying the biochemical pathways that underlie atherosclerotic plaque instability is needed to define new therapies. We employed adenoviral (rAd) gene delivery of the inhibitory subunit I-kBa to investigate the importance of transcription factor NF-kB in regulating MMP 1, 3 and 9 secretion in human smooth muscle cells (VSMC), macrophages (MFs) and rabbit foam cells. Optimal adenoviral gene delivery into human VSMCs with rAd: I-kBa but not rAd:b-galactosidase or rAd:null led to dramatically reduced MMP induction; MMP-1 by 979 4%, MMP-3 by 949 7% and MMP-9 by 77 9 12% (all P B0.0001, n= 5–8). Human MF constitutively expressed MMP-9 but not MMP-1 or -3. Rad: I-kBa infected MFs overexpressed I-kBa but there was no significant difference in MMP9 secretion compared with uninfected or rAd:null infection, (mean difference 11 916%, n =10), showing constitutive MMP-9 secretion is regulated by an NF-kB independent mechanism. We next investigated the role of NF-kB in the pathological overexpression of MMP1 and 3 in MF-derived foam cells isolated from 1% cholesterol fed rabbits. Overexpression of I-kBa with rAd:I-kBa resulted in no difference in MMP-9 secretion but an 83 9 12 and 69 911% reduction in MMP-1 and MMP-3, respectively (PB0.01, n= 6), as compared with uninfected controls. We demonstrate an essential role for NF-kB in regulating cytokine induced MMP expression by VSMCs and the pathological overexpression of MMP-1 and 3 characteristic of foam cells.

A novel pecam-1 gene variant influences atherosclerosis and the response of the PECAM-1 gene to shear stress M.A. Elrayess 1, K.E. Webb 1, D. Fla6el 1, M. Sy6anne 2, M.R. Taskinen 2, J.W. Jukema 3, J.J.P. Kastelein 3, S. Humphries 1 1 Cardiovascular Genetics, RFUCLMS, UK, 2LOCAT Study Group, Department of Medicine, Helsinki University Central Hospital, Finland and 3Department of Cardiology, Leiden University Medical Centre, The Netherlands The platelet endothelial cell adliesion molecule-1 (PECAM-1) is a key participant in the adhesion cascade leading to extravasation of leukocytes during the inflarnmatory process. A novel G to A polymorphism located at nucleotide 53 in the 5%UTR of PECAM-1 gene was identified. The frequency of the rare allele is 0.01 in Finnish post coronary bypass men (LOCAT) as well as Dutch men with angiographically documented coronary atherosclerosis (REGRESS). Carriers of the 53A allele show significantly less focal progression in the LOCAT study (P =0.01). A similar trend was seen in the average mean segment diameter in the REGRESS study but this did not reach statistical significance (P =0.08). The 53G \ A change alters a putative shear stress responsive element in the 5%UTR (GGTCTC“ AGTCTC). Shear stressed cells show 1.8 total induction of PECAM-1 mRNA level compared to static cells indicating, that PECAM-1 is responsive to shear stress. In order to investigate if the 53G \A polymorphism might affect this response a luciferase reporter assay was performed. While the G allele responds to shear stress exhibiting 1.25-fold increase in the promoter activity (P= 0.007) compared to static condition, the A allele exhibits no response to shear stress. These data demonstrate that the PECAM-1 gene is shear stress inducible and that decreased PECAM-1 gene expression in 53A carriers may explain progression of atherosclerosis in LOCAT and REGRESS studies. This work was generously supported by the British Heart Foundation.

Magnetic resonance imaging of experimental atherosclerosis L. Hegyi 1, P.D. Hockings 4, J.N. Skepper 2, G.M. Benson 4, T.A. Carpenter 3, G.A. Whelan 4, A.L. Busza 4, D.C. Grimsditch 4, K.E. Suckling 4 and P.L. Weissberg 1 Department of Medicine1, Multi-Imaging Centre2, Wolfson Brain Imaging Centre3, Cambridge University, Glaxo SmithKline, Harlow4, UK Our objective is to develop magnetic resonance imaging (MRI) methods to image the detailed structure of atherosclerotic lesions using a rabbit model of atherosclerosis. In the present study, 18 New Zealand White (NZW) rabbits were fed a high-fat diet including 0.2% cholesterol for up to 30 weeks. The abdominal aortas of the rabbits were injured on the 4th week using a 4F Fogarty embolectomy catheter at a balloon pressure of 1 atm. MRIs were acquired with a fast spin-echo technique with or without fat suppression on a 2T Bruker Medspec at 24 – 26 weeks after injury. Image resolution was 0.25 ×0.25 × 2 mm. After imaging the rabbits were culled and their aortas were examined by histology. MR images were compared with histological sections. We found that all 18 rabbits used in the study developed atherosclerosis visible by MRI. The thickness of the lesions produced by balloon injury in NZW rabbits fed high-fat diet was variable (mean 731 9 58 mm). The maximal thickness was more than 1 mm, well within the resolution of MRI. MR images showed marked thickening of the aorta wall. Histology confirmed the presence of extensive intimal remodelling and enlargement in the same regions. We found that lesions as small as 300 mm are visible by MRI. The size of the lesions imaged by MR and the corresponding lesion area determined by histology correlated with each other when a comparison was made by simple linear regression analysis (r = 0.84; P B

Abstracts 0.01). We conclude that MRI is a suitable method to image not only large but relatively small atherosclerotic lesions in NZW rabbits.

Chemokine expression by macrophages in human atherosclerotic lesions Andrew Lucas, Dawn O’Reilly, Kate Liddiard, Christina Bursill 1, Keith Channon 1, Da6id R. Grea6es Sir William Dunn School of Pathology and 1Cardiovascular Medicine, University of Oxford, Oxford OX1 3RE, UK Complex atherosclerotic lesions in human arteries are characterised by chronic recruitment of mononuclear cells. Chemokines mediate leukocyte recruitment in a number of inflammatory pathologies and chemokines present in atherosclerotic lesions may play an important role in T cell and monocyte recruitment and differentiation. We have analysed the expression of the linked chromosome 16q13 genes that encode MDC, fractalkine and TARC in primary macrophages and human atherosclerotic lesions by RT-PCR and immunohistochemistry. We show that macrophage expression of the chemokines MDC, fractalkine and TARC is upregulated by treatment with the Th2-type cytokines Interleukin-4 (IL-4) and IL-13 but not by the Th1-type cytokine interferon-g. High levels of MDC, fractalkine and TARC mRNA expression are seen in some, but not all, human arteries with advanced atherosclerotic lesions. Immunohistochemistry shows that MDC, fractalkine and TARC are expressed by a subset of macrophages within atherosclerotic lesions that are associated with plaque microvessels. These IL-4 and IL-13 induced chemokines may be surrogate markers for the presence of Th2-type immune responses in human atherosclerotic lesions. We are currently studying the regulation of MDC expression in human and murine macrophages to better understand the role of this CC chemokine in atherogenesis. http://dunn1.path.ox.ac.uk/ greaves/

Continued benefits of photodynamic therapy 2 years after treatment of restenosis R. Mansfield, M. Jenkins, C. Bishop, J. McEwan, S. Bown Royal Free & UCL Medical School, London, UK Arterial photodynamic therapy (PDT) shows exciting potential in the prevention of restenosis. PDT involves the activation of a systemically administered photosensitiser by the trans-catheter delivery of non-thermal laser light. A pilot study of adjuvant PDT, using the sensitiser 5-aminoalevulinic acid (ALA) (60 mg/kg oral), in seven patients (eight lesions of which three were occluded) undergoing repeat femoral angioplasty for restenosis showed the technique to be safe and effective. Without PDT, such patients may be anticipated to have a re-occlusion/restenosis rate of \40%. All patients were asymptomatic at 6 months with no evidence of restenosis or local arterial complications on Duplex scanning, including peak systolic velocity ratios (PSVR), or iv DSA. These patients have now been followed up in the longer term. Methods: Patients were reviewed for symptoms of claudication and/or ischaemic rest pain. Duplex scanning of the treatment site, with calculation of PSVR as an index of restenosis, was offered. Results: All seven patients were reviewed 26 9 3 months after angioplasty and adjuvant PDT for femoral restenosis. Six patients remained asymptomatic (one had undergone repeat angioplasty to the original treatment sites at one year). Four subjects underwent Duplex scanning. In the three asymptomatic patients there was no restenosis (PSVRB 2.0). In the 4th patient, the only one developing symptoms of mild claudication after 18 months, there was Doppler evidence of restenosis (PSVR =3.7) with diffuse plaque. Importantly there was

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no re-occlusion or aneurysm formation at the treatment site in any patient examined. Conclusion: Arterial PDT with the sensitiser ALA is convenient, safe and effective in the longer term and warrants further evaluation in the treatment and prevention of restenosis.

Shear-activated potassium channels in saphenous vein endothelium S. Qureshi, M. Gosling, N. Carey, J.T. Powell Vascular Surgery, Imperial College School of Medicine, London, UK Background. The major stimuli for saphenous vein graft remodelling are haemodynamic forces, where ion channels are implicated strongly in early mechanotransduction events. Aims. To pharmacologically dissect the identity of the cation channels involved in the upregulation of ICAM-1 on saphenous vein endothelium in response to haemodynamic forces. Methods. Human saphenous vein endothelial cells (HSVEC) were cultured in parallel plate flow chambers. ICAM-1 and DNA were measured by ELISA and picogreen assays respectively, to report ICAM-1concentration/(g DNA. Intact saphenous vein was subject to altered haemodynamic forces in an in vitro flow circuit and endothelial cell proteins assessed by immunohistochemistry. Results. In HSVEC, arterial levels of shear stress (0.8 N/m2), for periods up to 4 h, were without effect on the concentration of ICAM-1, but a 2-fold increase in ICAM-1 protein was evident after 6 h (ANOVA P =0.025). This increase at 6 h was abrogated by the non-selective K+ channel blocker tetraethylammonium, partially inhibited by charybdotoxin (an inhibitor of Ca2 + -activated K+ channels) and abolished by apamin (specific small-conductance Ca2 + -activated K+ channel inhibitor). Western blotting revealed small-conductance Ca2 + -activated K+ channel in HSVEC lysates of the SK2 subtype. The effect of apamin on the upregulation of ICAM-1 in intact vein also will be reported. Conclusions. Small conductance Ca2 + -activated K+ channels appear to play an important role in mechanotransduction in saphenous vein endothelium.

Migration of adventitial myofibroblasts during vascular remodelling following balloon injury of rat carotid arteries Richard C.M. Siow, Chandike M. Mallawaarachchi, Nichola Figg, Peter L. Weissberg Division of Cardiovascular Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, CB2 2QQ, UK Renarrowing of the vessel lumen is commonly observed after balloon angioplasty of occluded arteries. Vascular smooth muscle hyperplasia results in neointima formation, however, there is increasing evidence that balloon injury also results in proliferation of adventitial myofibroblasts, which can migrate towards the lumen, thus contributing to remodelling of the vessel wall (Shi et al., Circulation 1996;94:1655 – 1664). In the present study, we have directly labelled and tracked the migration of adventitial cells in vivo following balloon injury of rat carotid arteries using adenoviral mediated transfer of the reporter gene b-galactosidase (b-gal). Male 3 month old Sprague – Dawley rats (350 – 400 g) were anesthetized with Ketamine (75 mg/kg) and Xylazine (5 mg/kg) via intraperitoneal injection and left common carotid arteries exposed following midline neck incisions. An adenoviral vector containing the b-gal construct (AdLacZ) was suspended in ice-cold 25% pluronic gel (109 pfu/ml) and 200 ml delivered to the adventital surface of the arteries. Animals were recovered following closure of the wound and after 4 days the left common carotid arteries were re-exposed for endoluminal injury, performed by passing an inflated 2F Fogarty

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embolectomy catheter along the length of the artery 3 times with constant rotation. Animals were recovered and sacrificed by perfusion fixation at 3, 7 and 14 days following injury. b-Gal and a-actin expression in carotid arteries were determined by x-gal staining and immunohistochemistry respectively in paraffin embedded sections. In the absence of balloon injury, left carotid artery sections did not remodel and cells positive for b-gal were restricted to the adventitial layer only for up to 14 days. The medial and neointimal layer of vessels exhibited b-gal positive cells at 7 –14 days following balloon injury. Untransfected and uninjured control right carotid arteries did not show any b-gal activity. Positive a-actin staining in the adventitia was only observed in balloon injured arteries and not those treated with gel containing adenoviral vector without injury. By using in vivo gene transfer of the b-gal reporter to the adventitia prior to injury, these findings provide further direct evidence that adventital myofibroblasts exhibiting smooth muscle cell markers migrate towards the lumen following balloon injury and may thus be associated with angioplasty-induced remodelling of the vessel wall. This study was supported by the British Heart Foundation.

Superoxide-nitric oxide interactions in experimental venous bypass graft biology Nick E.J. West, Keith M. Channon Department of Cardiovascular Medicine, University of Oxford, UK Background: Vascular remodelling in vein grafts (VG) results in smooth muscle cell (SMC) intimal hyperplasia and provides the substrate for VG failure. Nitric oxide (NO) bioactivity is reduced in vascular disease states, in association with increased vascular superoxide (SO) production. We aimed to investigate sources of SO production in VG and the relationship between SO/NO levels and VG emodelling. Methods: Normocholesterolaemic New Zealand White rabbits underwent jugular-carotid interposition bypass grafting with or without intraoperative adenoviral gene transfer of NOS. Vascular SO production was assessed by lucigenin-enhanced chemiluminescence and dihydroethidium staining. Immunohistochemistry and Westem blotting techniques were used to characterise sources of SO production and SMC differentiation. Results: SO production was trebled in VG compared with JV from the same animal; the source of this enhanced SO generation was NAD(P)H oxidase in dedifferentiated intimal SMC. NOS gene transfer reduced inflammation in early (3 day) VG and decreased intimal hyperplasia in mature (28 day) VG, where intimal SMC were directed

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towards a more differentiated phenotype. SO generation was reduced by NOS gene transfer in early VG, and NAD(P)H oxidase-derived SO production reduced in mature VG. Conclusions: These data suggest a role for NAD(P)H oxidase-mediated SO production in regulating SMC proliferation and differentiation following vascular injury in VG. In addition, early modulation of NO bioactivity has sustained favourable effects on VG remodelling and SMC differentiation.

From gene to function: discovery of vasodilator properties of novel endogenous ligands to orphan receptors ligands to orphan receptors in human arteries Katherine E. Wiley, Anthony P. Da6enport Clinical Pharmacology Unit, University of Cambridge, Centre for Clinical Investigation, Addenbrooke’s Hospital, Cambridge, CB2 2QQ, UK The endogenous peptides apelin-13 and ghrelin have recently been paired to the ‘orphan’ G protein-coupled receptors (GPCR) APJ and GSHR. These ‘orphan’ receptors were previously predicted to exist from the human genome, but their function was unknown. The potent and long-acting vascoconstrictor endothelin-1 (ET-1) is upregulated in coronary heart disease. Our objective was to determine the vascodilator potential of the known dilators nitric oxide (NO) and the natriuretic peptides against constrictions to ET-1 in human internal mammary (IMA) and coronary (CA) arteries, and to compare them with these novel ligands. Resistance CA were mounted in a wire myrograph, and conductance CA and IMA in 5 ml ogan baths for the measurement of isometric tension. Constrictions were induced with 10 nM ET-1 and concentration– response curves to the dilator molecules were constructed. The NO-donor DEA/NO completely reversed ET-1 constrictions in conductance CA and IMA, and caused a 78 9 8% reversal in resistance CA. Atrial natriuretic peptide (ANP; 0.1 – 300 nM) reversed constrictions to ET-1 with nanomolar potency in all three vessel types, however the maximum response (EMAX) was only approximately 50% relaxation in CA compared to 100% in IMA. In IMA, Apelin-13 (0.1 – 300 nM) caused a modest reversal of the ET-1 response (EMAX of 28 9 5%). Ghrelin caused sustained relaxations that were slow in onset. The EMAX was comparable to the effects of ANP (77 9 1%) and achieved with nanomolar potency (EC50 4.16 (95% CI; 0.1 –182) nM). In conclusion we have shown that two novel endogenous ligands recently paired to orphan GPCR have vascodilator activity in human arteries.