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Report of the Spring Meeting of the British Atherosclerosis Society

Magdalen College, Oxford, 10th and 11th April, 2003
        The John French Memorial Lecture was delivered by Professor Keith Channon, Oxford, and entitled ‘Mechanisms of endothelial dysfunction in vascular disease’Further details about this meeting are available from the BAS secretary: Dr. Chris Newman, Cardiovascular Research Group, Division of Clinical Sciences (North), University of Sheffield, Sheffield, S5 7AU, UKE-mail address: [email protected], Website: www.britathsoc.ac.ukAbstracts of the 10 free communications presented are given belowAcknowledgements:Generously sponsored by Astra-ZenecaThe Young Investigator's Award is sponsored by the British Heart FoundationA POLYMORPHISM IN THE HUMAN CRP GENE INFLUENCES BASAL AND STIMULATED CRP LEVELS: IMPLICATIONS FOR THE PREDICTION AND PATHOGENESIS OF CORONARY HEART DISEASEDJ Brull; N Serranob; F Zitoa; L Jonesa; HE Montgomerya; A Rumleyc; Pankaj Sharmaa; GDO Lowec; MJ Worldd; SE Humphriesa; AD HingoraniaaCentres for Cardiovascular Genetics and Clinical Pharmacology, BHF Laboratories at UCL; bUniversidad Autónoma de Bucaramanga, Colombia; cDepartment of Medicine, University of Glasgow, UK; dRoyal Centre for Defence Medicine, Selly Oak Hospital, Birmingham, UKObjective: There is a strong association between C-reactive protein (CRP) and cardiovascular disease, and levels of CRP are heritable. We identified two novel polymorphisms in the CRP gene and tested the hypothesis that CRP genotype influences basal and stimulated CRP level. Methods and Results: CRP was measured in 250 male Army Recruits before and after strenuous exercise, and peri-operatively in 193 coronary artery bypass graft (CABG) patients. Two novel polymorphisms were identified in the CRP gene: -732G>A in the promoter and +1334C>T in the 3′UTR. The 1334C>T and -732G>A genotypes were in allelic association (P<0.001 for both cohorts). In the Army recruits, mean CRP (mg/l) increased from 0.54±0.04 at baseline to 1.14±0.09, 0.92±0.06 and 0.74±0.06 at 2-h, 48-h and 96-h after exercise. CRP was higher in +1334TT homozygotes than +1334 C-allele carriers at baseline (1.04±0.38 vs. 0.55±0.06, P=0.014) and at all time points following exercise (2.35±0.68 vs. 1.07±0.12; 2.11±0.53 vs. 0.88±0.09, and 1.77±0.44 vs. 0.71±0.09, P=0.034, P=0.007 and P=0.013, at 2-h, 48-h and 96-h post exercise). In the CABG patients, mean CRP rose by 83 fold from 1.97±0.36 at baseline to a peak 72 h post-operatively of 167.2±5.0. There was no significant difference in CRP between genotypes at baseline, however peak post-CABG CRP levels were significantly higher in +1334TT homozygotes compared to +1334C-allele carriers (198±17 vs. 164±5, P=0.032). Conclusions: The CRP gene contains a polymorphism that is associated with individual differences in basal and stimulated CRP level. The identification of a genetic factor that influences CRP concentration has implications both for the prediction and pathogenesis of coronary heart disease.PRAVASTATIN INHIBITS DE-STABILIZATION, AND PROMOTES RESTABILIZATION, OF UNSTABLE PLAQUES IN A MOUSE MODEL OF PLAQUE RUPTUREKGS Carson, H Williams, JL Johnson, CL JacksonBristol Heart Institute, University of Bristol, UKClinical therapy with statins reduces the incidence of adverse cardiovascular events to an extent that is greater than predicted from their lipid-lowering effects alone, leading to the hypothesis that they have a direct plaque-stabilizing action. We have administered pravastatin to fat-fed apolipoprotein E knockout mice in order to assess its effects on plaque stability. This is a particularly useful model because statins do not affect plasma cholesterol concentration in these animals. High-fat diet was given from 6 weeks of age (time zero), and pravastatin was administered in the drinking water. Administration from time zero to 8 weeks resulted in significantly smaller and less lipidic plaques with significantly fewer ruptures. Administration from time zero to 40 weeks also resulted in significantly smaller and less lipidic plaques with significantly fewer ruptures, and in addition mortality was significantly reduced. Administration from 16 to 40 weeks—that is, from a time when unstable plaques are already present in all mice—also resulted in significantly less mortality and fewer ruptures. These data indicate that pravastatin can prevent destabilization of plaques, and can also re-stabilize plaques that have already entered the rupture-healing cycle. They establish the fat-fed apoE knockout mouse as a pathophysiologically and pharmacodynamically validated model of plaque rupture, and strongly support the contention that statins stabilize human plaques.THE ROLE OF NITRIC OXIDE IN MEDIATING THE EFFECTS OF EXOGENOUS VEGFTJA Chico, JM Greve, HG Steinmetz, N van Bruggen, S BuntingGenentech inc, South San Francisco USVEGF may represent a therapy for occlusive arterial disease. Nitric oxide (NO) has been proposed to mediate its effects. To test this hypothesis, C57bl6 mice treated with or without chronic oral 1.5g/l L-NAME (a non-specific NO synthase inhibitor) were injected with 50μg slow-release murine VEGF165 or vehicle into the left calf. Results: L-NAME treatment inhibited the hypotensive effect of intravenous VEGF, proving NO synthase was inhibited. Seven days after injection, VEGF treatment induced angiogenesis in the injected region, increasing capillary density and inducing a 262% increase in PECAM1 (CD31) density compared to control (n=6 per group, P<0.01). This effect was unaltered by chronic L-NAME treatment, suggesting that NO is not required for angiogenesis in response to exogenous VEGF. Serial magnetic resonance angiography demonstrated a time-dependent expansion in vessels proximal to the injected region in response to VEGF which was similarly unaffected by either chronic oral L-NAME from one week before injection or intravenous L-NAME seven days after injection (n=5–8 per group), suggesting that VEGF induces enlargement of conduit vessels and that this process similarly does not require NO for either its development or maintenance. VEGF treatment greatly increased resting perfusion of the injected left lower leg (129±20 versus 13±3 ml/min/100 g, n=6–7 per group, P<0.01) seven days after injection, an effect almost completely abolished by chronic L-NAME treatment (20±3 ml/min/100 g), suggesting that the effect of VEGF on tissue perfusion is dependent upon NO synthase. We conclude that whilst NO mediates the haemodynamic effects of VEGF, it is not absolutely required for the effects of VEGF on vascular expansion.CIRCULATING PROGENITOR CELLS REGENERATE ENDOTHELIUM OF VEIN GRAFTSYanhua Hu, Zhongyi Zhang, Evelyn Torsney, Fergus Davison, Qingbo XuDepartment of Cardiovascular Sciences, St George's Hospital Medical School, LondonPreviously we showed that a large number of endothelial cells in vein grafts undergo apoptosis or necrosis during the first several days followed by endothelial regeneration. In the present study we investigated the kinetics of endothelial cell death and regeneration in vein grafts using transgenic mice carrying LacZ genes driven by endothelial TIE2 promoter. We demonstrated that the endothelium of donor vessels denuded completely 72 h, and neoendothelium appeared 24 h after grafting. When vein isografts were performed between TIE2-LacZ and wild-type mice, β-gal+ cells were observed on the surface of vein segments at 48 h. These β-gal+ cells were only seen on the endothelial surface of vein segments grafted into TIE2-LacZ mice 1, 2 and 4 weeks after surgery, suggesting recipient origins. Interestingly, β-gal+ cells were evenly distributed on the surface of the whole vein segment grafted into TIE2-LacZ mice, indicating a contribution of circulating progenitor cells. When wild-type veins were grafted into a chimeric mouse carrying TIE2-LacZ genes in bone marrow cells, about 30% of total endothelial cells displayed a β-gal+ staining. Thus, we provide the first evidence that endothelial cells of vein grafts are derived from circulating progenitor cells, of which 30% are derived from bone marrow progenitor cells.MATRIX METALLOPROTEINASES-9 AND -12 HAVE OPPOSITE EFFECTS ON ATHEROSCLEROTIC PLAQUE STABILITYJason Johnson, Sarah George, Andrew Newby, Christopher JacksonBristol Heart Institute, University of Bristol, UKMatrix metalloproteinases (MMPs) are thought to be involved in the destabilization and rupture of atherosclerotic lesions. We are testing this hypothesis in apolipoprotein E (apoE) knockout mice that have been crossed with various MMP knockouts and fed a high-fat diet for 8 weeks. The present study focuses on apoE/MMP-9 (gelatinase B) or apoE/MMP-12 (macrophage metalloelastase) double knockouts. Plaques in the proximal 150 m of the brachiocephalic artery were 57% smaller in apoE/MMP-12 double knockouts than in age-matched, strain-matched apoE knockout controls (P<0.0001). In marked contrast, plaques were 110% larger in apoE/MMP-9 double knockouts than in controls (P=0.005). The frequency of silent plaque rupture (seen as buried fibrous layers within the plaque) was 64% lower in apoE/MMP-12 double knockouts than in controls (P=0.01), but very surprisingly was 93% higher in apoE/MMP-9 double knockouts than in controls (P<0.05). These data indicate that MMP-12 plays an essential role in plaque growth and destabilization, but MMP-9 appears to have a protective role: it limits plaque growth and promotes plaque stability. This challenges the concept that MMPs simply degrade matrix and thus destabilise plaques, and suggest instead that the MMP family has diverse effects on plaque stability. Notwithstanding this, the present data suggest that inhibition of MMP-12 may be an attractive target for prevention of atherosclerotic plaque rupture.EPIDERMAL GROWTH FACTOR: A NOVEL MONOCYTE CHEMOTACTIC FACTOR AND MACROPHAGE MITOGENDavid J Lamb, Helmout Modjtahedi, Gordon AA FernsCentre for Clinical Science and Measurement, School of Biomedical and Life Sciences, University of Surrey, Guildford, GU2 7XH, UKThe recruitment of peripheral monocytes to the sub-endothelial space, their metamorphosis to macrophages and subsequent proliferation is critical during atherosclerosis. We show here that epidermal growth factor receptor (EGFR) is expressed on the surface of rabbit peripheral monocytes and epidermal growth factor (EGF) promotes chemotaxis of peripheral rabbit mononuclear cells (P<0.005 vs. saline). Furthermore, antibodies that blockade the EGF receptor (EGFR) inhibit mononuclear cell chemotaxis (P<0.05 vs. IgG) and antibodies that increase EGF binding to EGFR promote chemotaxis (P<0.001 vs. IgG). EGF induces proliferating cell nuclear antigen (PCNA), changes in nuclear morphology and EGFR mRNA in monocyte-derived macrophages. We have also found that EGFR co-localises with macrophages in atherosclerotic lesions from cholesterol-fed rabbits. EGFR mRNA was also increased in these lesions compared to non-atherosclerotic aorta that did not contain macrophages. Furthermore, both mRNA and immunocytochemical stain intensity increased in proportion to the size of the atherosclerotic lesion. In conclusion, these data demonstrate for the first time that both monocytes and macrophages possess functional receptors for EGF. Furthermore, we show that EGF is both chemotactic for monocytes and mitogenic for macrophages. These findings increase our understanding of atherogenesis and provide a novel target for potential intervention in coronary disease.This work was sponsored by the British Heart Foundation.PROTEOMIC ANALYSIS REVEALS A KEY ROLE FOR PKC-DELTA IN SMOOTH MUSCLE CELL APOPTOSIS AND MIGRATIONManuel Mayra, Ursula Mayra, Emma McGregorb, Yanhua Hua, Michael J. Dunnc, Qingbo XuaaDepartment of Cardiological Sciences, St George′s Hospital Medical School, London, UK, bProteome Sciences, King′s College, London, UK; cInstitute of Psychiatry, King's College, London, UKWe demonstrated previously that neointima formation in vein grafts is markedly accelerated in PKC-Delta-knockout mice. To study the mechanisms of PKC-Delta-enhanced arteriosclerosis, we undertook proteomic analysis of myocytes and smooth muscle cells (SMCs) from PKC-Delta??? mice. Profound changes in protein profiles were observed for mitochondrial enzymes related to energy metabolism and free radical generation. Mass spectrometry revealed a mis-processing of the mitochondrial precursor sequence in certain mitochondrially-targeted enzymes. Functional assays confirmed a decreased production of reactive oxygen species in PKC-Delta??? SMCs, which was associated with increased resistance to apoptosis induced by cytokine treatment. In contrast, PKC-Delta??? SMCs rapidly underwent cell death after exposure to sodium nitroprusside (SNP), but not to other NO donors. SNP spontaneously releases NO and cyanide, a potent inhibitor of mitochondrial respiratory chain reactions. Moreover, changes in small heat shock proteins and the cytosolic chaperonin containing the T-complex polypeptide 1 were related to an abnormal cytoskeletal structure in PKC-Delta-/- SMCs, resulting in diminished SMC migration. Taken together, our findings demonstrate that PKC-Delta plays an essential role in SMC apoptosis and migration.MACROPHAGES AND LIPOPROTEIN CHOLESTEROL CLEARANCEO. Steina, Y. SteinbLipid Research Laboratory, Hebrew University-Hadassah Medical Schoola, Hadassah University Hospitalb, Jerusalem, IsraelRecruitment of macrophages [MP] plays a prominent role in initiation and progression of atherosclerosis, but their role in regression, via cholesterol removal, is unknown. To elucidate this aspect, a cholesterol depot was created in the rectus femoris muscle of mice by injecting cationized LDL (200 μg cholesterol). The clearance of the injected cholesterol, 70% in the form of cholesteryl ester [CE], requires CE hydrolysis, and coincides with the appearance of an inflammatory infiltrate, evoked by the cat LDL; it seems, therefore, that these cells are responsible for CE hydrolysis (Atherosclerosis 1997, 133:15 and 2002, 164:73). In the present experiments LDL cholesterol clearance from muscle of CCR2−/− mice, which lack the receptor responsible for MCP-1 mediated monocyte mobilization, was slower than in controls. This was due to a decrease in CE hydrolysis and marked delay in MP recruitment to the injected muscle. The slower rate of CE hydrolysis in the injected muscle was related to macrophage paucity and not to impaired MP CE-hydrolase, as CE hydrolysis by resident peritoneal MP from CCR2−/− mice and controls was similar. In conclusion, these results show that MP, considered to be mainly proatherogenic, display also antiatherogenic properties required for regression of atherosclerosis.
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