- Email: [email protected]
We-W30:7 Fasting induces hyperlipidemia in mice overexpressing PCSK9
306
Wednesday, June 21, 2006: Workshop We-W30 Familial hyper- and hypolipidemias
provide novel strategies for gene identification in FCHL. Linkage signals obtadned in the previous low-density genome-wide scans can be fine mapped by using high-density of single nucleotide polymorphisms (SNPs) for association analyses and monitoring gene expression differences between FCHL cases and controls. The human sequence variation data provided by the HapMap Project can be utilized in fine mapping to define the regional linkage disequilibrium patterns. The goal is to select the tag SNPs, capturing most of the genetic variation in the linked regions, for the association testing in extended FCHL families. Results: I will describe our recent studies aiming to identify FCHL genes using these kinds of approaches. We recently identified the upstream transcription factor 1 for FCHL and high triglycerides in Finnish FCHL families (Pajukanta et al. 2004). USF1 is a transcription factor regulating several genes in lipid and glucose metabolism. Variants of this gene axe also implicated in Mexican FCHL families (Huertas-Vazquez et al. 2005). Although genetic evidence is supportive of a role for USF1 in the etiology of FCHL, the mechanisms of causality for the identified variants still remain largely unknown. Therefore, we axe currently systematically investigating the associated variants for functional significance. We axe also in the process to fine map other chromosomal regions linked to FCHL or its component traits in several study samples. Accordingly, the linked regions on chromosomes l l p , 16q, and 20q are being studied to identify the underlying genes. As an alternative strategy, gene expression data from fat biopsies is utilized to identify regional, differentially expressed candidate genes for sequencing. Conclusions: High-resolution approaches with dense sets of SNPs can now be utilized in the linked regions and at the genomic level to identify novel genes for FCHL and its component traits. Furthermore, as interactions between DNA sequence variants are expected to play a crucial role in complex diseases, such as FCHL, identified variants should be investigated for the possibility of genetic interactions to identify the full spectrum of DNA sequence variants that contribute to the complex FCHL phenotype References [1] Pajukanta P et al. Familial combined hyperlipidemia is assodated with upstream transcription factor 1 (USF1). Nature Genetics, 2004, 36:371376. 2. Huertas-Vazquez A e t al. Familial Combined Hyperlipidemia in Mexicans: Association with Upstream Transcription Factor 1 and Linkage on Chromosome 16q24.1. Arteriosclerosis Thrombosis and Vascular Biology, 2005, 25:1985-1991. Funding: NIH grants HL-28481 and HL-70150, and A H A grant 0430180N.
We-W30:4 ] GENOME-WIDE EXPRESSION ANALYSIS OF CELLS EXPRESSING WILD-TYPE OR GAIN OF FUNCTION M U T A N T (D374Y) P C S K 9 A. Chester, X.M. Sun, A.K. Soutar. Lipoproteb~ Group, MRC Cl#ueal
Sciences Centre, Hanwrsmith Hospital, London, United Kingdom Objective: Mutations in PCSK9 can cause familial hypercholesterolemia (FH), however, loss of function mutations result in low LDL cholesterol. PCSK9 is a proprotein convertase which undergoes self cleavage but its role in cholesterol metabolism is unclear. Microarray studies have linked PCSK9 to sterol metabolism, overexpression in rat liver causes hypercholesterolemia by reducing LDL receptor (LDLR) protein, but gain of function mutations increases secretion of apoB containing lipoproteins without changes in LDLR. Comparison of gene expression from cells expressing wild-type (WT) PCSK9 or a naturally occurring D374Y mutation will identify genes regulated by PCSK9. M e t h o d s : Rat McArdleRH7777 hepatoma liver cells stably expressing WT, D374Y PCSK9 or empty vector were generated. Total RNA extracted from these cells in triplicate was used to generate biotin-labelled cRNA which was hybridised to Affymetrix GeneChip ® Rat 230 arrays. Results: Data was normalised and analysed using Rosetta Resolver software. An error weighted, one-way ANOVA was performed on the 3 groups of combined data. 1647 genes were determined to have altered expression levels in at least one of the groups. Pair wise comparison reveals 116 genes that are differentially regulated between W T and D374Y with a 2.0 or greater fold change between the means of the two groups. This greatly exceeds the differences between W T and vector (36) or D374Y and vector (15) analysed using the same criteria. Conclusions: Gene expression analysis has identified a number of genes that axe differentially regulated in cells expressing W T or D374Y PCSK9. Funding: BHF (UK)
EFFECT OF FREQUENT IWe-W30:51AEPISTATIC POLIPOPROTEIN E AND LIPOPROTEIN
LIPASE GENE VARIANTS ON HYPERTRIGLYCERIDEMIA HYPERAPOBETALIPOPROTEINEMIA EXPRESSION
E Perron I , D. Brisson I , M. Santure I , E Blackburn 1"2"3, J.E Despres 3, D. Gaudet 1 . 1CMGC, Chicoutimi Hospital, Quebec, Cancuda: 2 UQAC,
Quebec, Canwda: 3Quebec Heart b~stitute, Quebec, Canada The combination of hypertriglyceridemia (hyperTG) and hyperapobetalipoproteinemia (hyperapoB) is associated with an increased coronary artery disease (CAD) risk. Both apoE and lipoprotein lipase (LPL) genes are involved in the TG-rich apoB containing lipoproteins catabolic pathway. Several apoE and LPL gene variants, known to adfect TG or apoB concentrations, have an allelic frequency (AF) of at least 5% in the general population. Objective: This study examined the combined effect of frequent apoE and LPL gene variants on the expression of hyperTG-hyperapoB. M e t h o d s : ApoE and LPL genotypes, and lipid profile, were assessed among 1,441 French-Canadians from the Saguenay founder population. The population AF of the studied apoE and LPL variants has been previously estimated in a random sample of 2,000 individuals. Analyses were performed to estimate the relationship between apoE and LPL gene variants and the risk of hyperTG and hyperapoB. Results: The apoE4 increased the risk of expressing the hyperTGhyperapoB phenotype (odds ratio (OR)=1.96,p=0.014). This risk was exacerbated (OR=4.7, p=0.017) in presence of LPL gene variants. The apoE2 allele (E2-E3) was negatively associated with hyperTG-hyperapoB (OR=0.52,p=0.005) in absence LPL gene variants. The presence of LPL gene variants among E2 carriers shifts this association towards non-significance. Conclusion: These results suggest that epistasis is an important phenomenon to consider while assessing the CAD risk assodated with gene variants or the effect of frequent alleles on high-risk lipid profiles. Funding: This project was supported by ECOGENE-21 (CIHR #CAR43283).
IWe-W30:6
I
Ii T H E A P O A V G E N E IS A S S O C I A T E D W I T H F A M I L I A L
COMBINED HYPERLIPIDEMIA AND DYSLIPIDEMIA G.M. van der Vleuten 1, A. Isaacs ~, W. Zeng 3 , EH. Talmud 3, C.M. van Duijn ~, A.F.H. Stalenhoef 1 , J. de Graaf 1 . 1RcuJboud Universi~ Nijmegen,
Nijmegen, the Netherlands: 2Erasmus University Rotterdam, Rotterdam, the Netherlands." ~ Universi~ College London, London, United Kingdom Objective: Apolipoprotein AV (ApoAV) is a recently identified gene in the apolipoprotein AI-CIII-AIV-AV cluster and is associated with plasma triglyceride (TG) and HDL-cholesterol (HDL-c) levels. The apoAV gene resides on chromosome 11, where linkage analyses have identified a locus for familial combined hyperlipidemia (FCH), the most common genetic lipid disorder. The aim of the present study was to investigate whether the apoAV gene is associated with FCH and its related phenotypes. M e t h o d s : The study population included 36 families, compromising 620 individuals, of whom 158 individuals were diagnosed as FCH patient. The diagnosis of FCH was based on plasma TG, total cholesterol (TC) and apolipoprotein B (apoB) levels, using the recently published nomogram. Two polymorphisms in the apoAV gene ( - l l 3 1 T > C and S19W) were genotyped with PCR-RFLE The FBAT program was used for the analysis of each polymorphism and haplotype. Results: Haplotype analysis showed that apoAV was associated with FCH (p=0.03). Associations were also found with individual lipid and lipoprotein levels for the apoAV haplotypes (TC (p=0.03), TG (p<0.01), apoB (p=0.01), HDL-c (p=0.02), small dense LDL (p=0.01), VLDL-c (p<0.01) and VLDL-tg levels (p<0.01)). Furthermore, FCH patients carrying a -1131C allele or a 19W allele had higher TG (+25% and +27%), VLDL-c (+37% and +45%) and VLDL-tg levels (+54% and +43%), compared to wildtype homozygotes. C o n d u s i o n : In these Dutch FCH families, the apoAV gene is associated with FCH and dyslipidemia. ApoAV might be the gene underlying the linkage signal previously detected on chromosome 11 for FCH.
IWe-W30:71 FOAVSETRIENXGP RI NEDS SUICNEGS
HYPERLIPIDEMIA PCSK9
IN M I C E
G. Lambert 1 , T. Pmeau ~, M. Krempf 1 , E Costet . b~serm U539, Nantes,
France:
2hlt'a,
Toulouse, France
A i m : PCSK9 mutations lead to familial hypercholesterolemia (FH) by virtue of its role as a negative modulator of the LDL receptor (LDLr) and thereby of plasma LDL clearance. But the exact role of PCSK9 on V L D L hepatic pro-
XIV bztentational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
We-W31
Wednesday, June 21, 2006: Workshop Diagnostic technologies for arterial disectses ( lstpart)
duction remains controversial. Here, we uncover that upon fasting, the down regulation of the LDLr is also the mechanism by which PCSK9 modulates the hepatic production of VLDL. Results: The adenoviral mediated overexpression of PCSK9 results in massive hyperlipaemia in 24-hours fasted but not in fed mice [TG=137-4-9, 111-4-18, 251-4-32", ll3-4-8mg/dL in fed Ad-PCSK9, fed Ad-Null, fasted Ad-PCSK9 & fasted Ad-Null, respectively, n=6 all, *P<0.01 vs. all other groups]. Using the triton method, we observed a 2.5-fold increase in VLDL apoB100 and TG hepatic output in the fasted Ad-PCSK9 mice [*P<0.02 vs. all groups]. We performed similar experiments in LDLr (-/-) mice showing that VLDL production is increased upon fasting irrespective of the PCSK9 expression status. This increased production of V L D L was associated with (i) a concomitant reduction of intrahepatic lipid stores as well as an increased flux of free fatty acids (FFA) to the liver of fasted mice overexpressing PCSK9 and with (ii) a lack of down-regulation of PPARa and target genes expression. Interrestingly, PCSK9 hepatic expression is inhibited by the hypotriglyceridemic PPARa agonist fenofibrate. Conclusion: The negative modulation of LDLr expression by PCSK9, that decreases plasma LDL clearance, is therefore a molecular mechanism by which PCSK9 may alter VLDL production in pathophysiological conditions mimicked by long-term fasting in mice.
We-W31
DIAGNOSTIC TECHNOLOGIES DISEASES (lst PART)
FOR ARTERIAL
I We-W31:1 I A S S E S S M E N T OF S T R U C T U R A L M A R K E R S AS I
I
SURROGATE ENDPOINTS FOR ARTERIAL DISEASE
D.H. O'Leaxy. Department of RcMiology, T~ts Universi~ School of
Medicbw, Boston, MA, USA Objective: Overview of imaging technologies available for monitoring cardiovascular disease (CVD) progression. Methods: Review of the literature keyed to the following terms: carotid intima-media thickness (CIMT), intravascular ultrasound (IVUS), quantitative coronary angiography (QCA), coronary calcium scoring and electron beam computerized tomography (EBCT), multislice spiral computed tomography angiography (CTA), and magnetic resonance imaging (MRI). Results: Imaging tests that can directly image arterial wall structure may serve as substitutes for clinical endpoints. By direct measurement of change over time, such surrogates have the potential to increase greatly the efficacy of clinical trials by permitting the design of shorter studies involving fewer subjects. While QCA is the most established imaging technique for measuring CVD progression, it is invasive and is no longer employed in therapeutic trials. IVUS is also invasive, but because of its ability to circumferentially study the adjacent coronary artery wall with high-resolution ultrasound, it is considered promising as a surrogate and is currently being used in several clinical trials. CTA is a very new technique and is the early stages of gaining clinical acceptance. It potential for utilization as a surrogate is unclear. Of the noninvasive measures, CIMT as measured by B mode ultrasound is the most established method for measuring atherosclerosis progression and regression. Several large observational studies have demonstrated a graded response between increasing CIMT and increased risk of incident CVD events. While no single study has been powered to demonstrate that slowing of CIMT progression leads to a decrease in future events, in six clinical trials of HMG-CoA reductase inhibitors (statins) which reported a significant beneficial impact of statins on IMT progression, there were beneficial trends with respect to reported to reported CVD endpoints [1]. It is not clear that the results seen can be generalized to other theraputic agents, however. CIMT also remains handicapped in that there are no generally accepted standards for measuring CIMT change. EBCT, while useful in identifying advance atherosclerosis, seems to have little use as a tool for assessing disease progression. MRI, because of its unique potential to study plaque composition as well as wall structure, continues to be widely viewed as having great potential as a surrogate. However, it is a technique that continues to be developed and improved and its efficacy remains undefined. Conclusion: Imaging biomarkers can serve as valid surrogates for clinical endpoints. Of the noninvasive techniques available, CIMT is currently the most useful. IVUS, while limited because it is invasive, will likely have great value in limited circumstances. References [1] Espeland MA, O'Leaxy DH, Terry JG, Morgan T, Evans G, Mudra H. Carotid intimal-media thickness as a surrogate for cardiovascular disease
307
events in trials of HMG-CoA reductase inhibitors. Current Controlled Trials in Cardiovascular medicine 2005 ;6:3 l
We-W31:2 I INTIMA-MEDIA THICKNESS AS AN ADDITIVE MARKER OF CARDIOVASCULAR RISK J
D. Baldassaxre. Depat'tment of Pharntacological Sciences, Universi~ of Milan, and Cardiologico "Monzbto" Center; IRCCS, Milan, Italy
Objective: To elucidate whether carotid artery intima media thickness (IMT) detected by B-mode ultrasound can be considered as a clinically useful marker of cardiovascular risk. Methods: To address the study objectives the correlation between carotid IMT and vascular risk factors (VRFs), the IMT performance in the recognition of patients with and without history of vascular events, the association between carotid IMT and the extent of coronary atherosclerosis, and the carotid IMT capacity to predict new vascular events are discussed. Results: In large cross-sectional studies mainly performed in patients attending our Lipid Clinic, we have previously shown that carotid artery IMT correlates well with coronary VRFs [1,2]. We have also shown that carotid IMT discriminates well between groups of patients with and without a history of events [1]. In a later study, carried out by using Artificial neural networks as an innovative approach to perform statistical analyses, we have also shown that carotid IMT can significantly increase the capacity of VRFs to identify patients with and without a history of cardiovascular events even on an individual basis [3]. More recently we have also investigated the relationship between carotid atherosclerosis detected by external B-mode ultrasound and coronary atherosclerosis measured by both quantitative coronary angiography (QCA) and intravascular ultrasound (IVUS). In this study, the highest correlation coefficients between carotid and coronary atherosclerosis were observed when both vascular districts were measured with ultrasound methods (external carotid B-mode ultrasound vs coronary IVUS). In the same study, carotid ultrasonic variables proved more effective than QCA to identify patients with a positive IVUS test. Finally, in a longitudinal - observational study, we have also shown that the combined use of the Framingham Risk Score (FRS) and ultrasonic measurements of carotid IMT significantly increases the ability of VRFs to predict cardiovascular events in patients at low/intermediate risk (FRS <20%). A prospective, multicenter, longitudinal, long-term, observational study (The IMPROVE study) is currently ongoing. It aims to investigate the association between carotid IMT-progression and the rate of new vascular events in subjects at high risk. The patients enrolment ended in April 2005 and a total of 3746 patients were recruited in 6 European countries (1095 in Italy, 504 in France and 2140 in northern Europe). Condusions: Data will be presented clearly supporting the possibility of using carotid IMT not only as a marker of cardiovascular risk but also as a predictor of new events. These results, together with those reported by others, also support the possibility of using carotid IMT as a surrogate endpoint in pharmacological studies. Although this approach has been already used in a number of clinical trials to evaluate the effectiveness of anti atherosclerotic treatments, studies axe needed to fully demonstrate that IMT-progression, like IMT measured in cross-sectional studies, is a predictor of new vascular events. The currently ongoing IMPROVE study will address also this still missing piece of information; results axe expected for the end of 2007. References [1] Baldassarre D, Amato M, Bondioli A, Sirtori CR, Tremoli E. Carotid artery intima-media thickness measured by ultrasonography in normal clinical practice correlates well with atherosclerosis risk factors. Stroke 2000; 31:2426-2430. [2] Baldassaxre D, Amato M, Pustina L, Tremoli E, Sirtori CR, Calabresi L, Franceschini G. Increased carotid artery intima-media thickness in subjects with primary hypoalphalipoproteinemia. ArteHoscler Thromb Vasc Biol 2002;22:317-322. [3] Baldassaxre D, Grossi E, Buscema M, Intraligi M, Amato M, Tremoli E, Pustina L, Castelnuovo S, Sirtori CR. Recognition of patients with cardiovascular disease by artificial neural networks. Ann of Med. 2004;36:630640. Funding: Research describing correlations between carotid and coronary atherosclerosis is supported by the Italian Ministry of Health. The improve study is supported by European Union.
XIV bztentational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
Wednesday, June 21, 2006: Workshop We-W30 Familial hyper- and hypolipidemias
provide novel strategies for gene identification in FCHL. Linkage signals obtadned in the previous low-density genome-wide scans can be fine mapped by using high-density of single nucleotide polymorphisms (SNPs) for association analyses and monitoring gene expression differences between FCHL cases and controls. The human sequence variation data provided by the HapMap Project can be utilized in fine mapping to define the regional linkage disequilibrium patterns. The goal is to select the tag SNPs, capturing most of the genetic variation in the linked regions, for the association testing in extended FCHL families. Results: I will describe our recent studies aiming to identify FCHL genes using these kinds of approaches. We recently identified the upstream transcription factor 1 for FCHL and high triglycerides in Finnish FCHL families (Pajukanta et al. 2004). USF1 is a transcription factor regulating several genes in lipid and glucose metabolism. Variants of this gene axe also implicated in Mexican FCHL families (Huertas-Vazquez et al. 2005). Although genetic evidence is supportive of a role for USF1 in the etiology of FCHL, the mechanisms of causality for the identified variants still remain largely unknown. Therefore, we axe currently systematically investigating the associated variants for functional significance. We axe also in the process to fine map other chromosomal regions linked to FCHL or its component traits in several study samples. Accordingly, the linked regions on chromosomes l l p , 16q, and 20q are being studied to identify the underlying genes. As an alternative strategy, gene expression data from fat biopsies is utilized to identify regional, differentially expressed candidate genes for sequencing. Conclusions: High-resolution approaches with dense sets of SNPs can now be utilized in the linked regions and at the genomic level to identify novel genes for FCHL and its component traits. Furthermore, as interactions between DNA sequence variants are expected to play a crucial role in complex diseases, such as FCHL, identified variants should be investigated for the possibility of genetic interactions to identify the full spectrum of DNA sequence variants that contribute to the complex FCHL phenotype References [1] Pajukanta P et al. Familial combined hyperlipidemia is assodated with upstream transcription factor 1 (USF1). Nature Genetics, 2004, 36:371376. 2. Huertas-Vazquez A e t al. Familial Combined Hyperlipidemia in Mexicans: Association with Upstream Transcription Factor 1 and Linkage on Chromosome 16q24.1. Arteriosclerosis Thrombosis and Vascular Biology, 2005, 25:1985-1991. Funding: NIH grants HL-28481 and HL-70150, and A H A grant 0430180N.
We-W30:4 ] GENOME-WIDE EXPRESSION ANALYSIS OF CELLS EXPRESSING WILD-TYPE OR GAIN OF FUNCTION M U T A N T (D374Y) P C S K 9 A. Chester, X.M. Sun, A.K. Soutar. Lipoproteb~ Group, MRC Cl#ueal
Sciences Centre, Hanwrsmith Hospital, London, United Kingdom Objective: Mutations in PCSK9 can cause familial hypercholesterolemia (FH), however, loss of function mutations result in low LDL cholesterol. PCSK9 is a proprotein convertase which undergoes self cleavage but its role in cholesterol metabolism is unclear. Microarray studies have linked PCSK9 to sterol metabolism, overexpression in rat liver causes hypercholesterolemia by reducing LDL receptor (LDLR) protein, but gain of function mutations increases secretion of apoB containing lipoproteins without changes in LDLR. Comparison of gene expression from cells expressing wild-type (WT) PCSK9 or a naturally occurring D374Y mutation will identify genes regulated by PCSK9. M e t h o d s : Rat McArdleRH7777 hepatoma liver cells stably expressing WT, D374Y PCSK9 or empty vector were generated. Total RNA extracted from these cells in triplicate was used to generate biotin-labelled cRNA which was hybridised to Affymetrix GeneChip ® Rat 230 arrays. Results: Data was normalised and analysed using Rosetta Resolver software. An error weighted, one-way ANOVA was performed on the 3 groups of combined data. 1647 genes were determined to have altered expression levels in at least one of the groups. Pair wise comparison reveals 116 genes that are differentially regulated between W T and D374Y with a 2.0 or greater fold change between the means of the two groups. This greatly exceeds the differences between W T and vector (36) or D374Y and vector (15) analysed using the same criteria. Conclusions: Gene expression analysis has identified a number of genes that axe differentially regulated in cells expressing W T or D374Y PCSK9. Funding: BHF (UK)
EFFECT OF FREQUENT IWe-W30:51AEPISTATIC POLIPOPROTEIN E AND LIPOPROTEIN
LIPASE GENE VARIANTS ON HYPERTRIGLYCERIDEMIA HYPERAPOBETALIPOPROTEINEMIA EXPRESSION
E Perron I , D. Brisson I , M. Santure I , E Blackburn 1"2"3, J.E Despres 3, D. Gaudet 1 . 1CMGC, Chicoutimi Hospital, Quebec, Cancuda: 2 UQAC,
Quebec, Canwda: 3Quebec Heart b~stitute, Quebec, Canada The combination of hypertriglyceridemia (hyperTG) and hyperapobetalipoproteinemia (hyperapoB) is associated with an increased coronary artery disease (CAD) risk. Both apoE and lipoprotein lipase (LPL) genes are involved in the TG-rich apoB containing lipoproteins catabolic pathway. Several apoE and LPL gene variants, known to adfect TG or apoB concentrations, have an allelic frequency (AF) of at least 5% in the general population. Objective: This study examined the combined effect of frequent apoE and LPL gene variants on the expression of hyperTG-hyperapoB. M e t h o d s : ApoE and LPL genotypes, and lipid profile, were assessed among 1,441 French-Canadians from the Saguenay founder population. The population AF of the studied apoE and LPL variants has been previously estimated in a random sample of 2,000 individuals. Analyses were performed to estimate the relationship between apoE and LPL gene variants and the risk of hyperTG and hyperapoB. Results: The apoE4 increased the risk of expressing the hyperTGhyperapoB phenotype (odds ratio (OR)=1.96,p=0.014). This risk was exacerbated (OR=4.7, p=0.017) in presence of LPL gene variants. The apoE2 allele (E2-E3) was negatively associated with hyperTG-hyperapoB (OR=0.52,p=0.005) in absence LPL gene variants. The presence of LPL gene variants among E2 carriers shifts this association towards non-significance. Conclusion: These results suggest that epistasis is an important phenomenon to consider while assessing the CAD risk assodated with gene variants or the effect of frequent alleles on high-risk lipid profiles. Funding: This project was supported by ECOGENE-21 (CIHR #CAR43283).
IWe-W30:6
I
Ii T H E A P O A V G E N E IS A S S O C I A T E D W I T H F A M I L I A L
COMBINED HYPERLIPIDEMIA AND DYSLIPIDEMIA G.M. van der Vleuten 1, A. Isaacs ~, W. Zeng 3 , EH. Talmud 3, C.M. van Duijn ~, A.F.H. Stalenhoef 1 , J. de Graaf 1 . 1RcuJboud Universi~ Nijmegen,
Nijmegen, the Netherlands: 2Erasmus University Rotterdam, Rotterdam, the Netherlands." ~ Universi~ College London, London, United Kingdom Objective: Apolipoprotein AV (ApoAV) is a recently identified gene in the apolipoprotein AI-CIII-AIV-AV cluster and is associated with plasma triglyceride (TG) and HDL-cholesterol (HDL-c) levels. The apoAV gene resides on chromosome 11, where linkage analyses have identified a locus for familial combined hyperlipidemia (FCH), the most common genetic lipid disorder. The aim of the present study was to investigate whether the apoAV gene is associated with FCH and its related phenotypes. M e t h o d s : The study population included 36 families, compromising 620 individuals, of whom 158 individuals were diagnosed as FCH patient. The diagnosis of FCH was based on plasma TG, total cholesterol (TC) and apolipoprotein B (apoB) levels, using the recently published nomogram. Two polymorphisms in the apoAV gene ( - l l 3 1 T > C and S19W) were genotyped with PCR-RFLE The FBAT program was used for the analysis of each polymorphism and haplotype. Results: Haplotype analysis showed that apoAV was associated with FCH (p=0.03). Associations were also found with individual lipid and lipoprotein levels for the apoAV haplotypes (TC (p=0.03), TG (p<0.01), apoB (p=0.01), HDL-c (p=0.02), small dense LDL (p=0.01), VLDL-c (p<0.01) and VLDL-tg levels (p<0.01)). Furthermore, FCH patients carrying a -1131C allele or a 19W allele had higher TG (+25% and +27%), VLDL-c (+37% and +45%) and VLDL-tg levels (+54% and +43%), compared to wildtype homozygotes. C o n d u s i o n : In these Dutch FCH families, the apoAV gene is associated with FCH and dyslipidemia. ApoAV might be the gene underlying the linkage signal previously detected on chromosome 11 for FCH.
IWe-W30:71 FOAVSETRIENXGP RI NEDS SUICNEGS
HYPERLIPIDEMIA PCSK9
IN M I C E
G. Lambert 1 , T. Pmeau ~, M. Krempf 1 , E Costet . b~serm U539, Nantes,
France:
2hlt'a,
Toulouse, France
A i m : PCSK9 mutations lead to familial hypercholesterolemia (FH) by virtue of its role as a negative modulator of the LDL receptor (LDLr) and thereby of plasma LDL clearance. But the exact role of PCSK9 on V L D L hepatic pro-
XIV bztentational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
We-W31
Wednesday, June 21, 2006: Workshop Diagnostic technologies for arterial disectses ( lstpart)
duction remains controversial. Here, we uncover that upon fasting, the down regulation of the LDLr is also the mechanism by which PCSK9 modulates the hepatic production of VLDL. Results: The adenoviral mediated overexpression of PCSK9 results in massive hyperlipaemia in 24-hours fasted but not in fed mice [TG=137-4-9, 111-4-18, 251-4-32", ll3-4-8mg/dL in fed Ad-PCSK9, fed Ad-Null, fasted Ad-PCSK9 & fasted Ad-Null, respectively, n=6 all, *P<0.01 vs. all other groups]. Using the triton method, we observed a 2.5-fold increase in VLDL apoB100 and TG hepatic output in the fasted Ad-PCSK9 mice [*P<0.02 vs. all groups]. We performed similar experiments in LDLr (-/-) mice showing that VLDL production is increased upon fasting irrespective of the PCSK9 expression status. This increased production of V L D L was associated with (i) a concomitant reduction of intrahepatic lipid stores as well as an increased flux of free fatty acids (FFA) to the liver of fasted mice overexpressing PCSK9 and with (ii) a lack of down-regulation of PPARa and target genes expression. Interrestingly, PCSK9 hepatic expression is inhibited by the hypotriglyceridemic PPARa agonist fenofibrate. Conclusion: The negative modulation of LDLr expression by PCSK9, that decreases plasma LDL clearance, is therefore a molecular mechanism by which PCSK9 may alter VLDL production in pathophysiological conditions mimicked by long-term fasting in mice.
We-W31
DIAGNOSTIC TECHNOLOGIES DISEASES (lst PART)
FOR ARTERIAL
I We-W31:1 I A S S E S S M E N T OF S T R U C T U R A L M A R K E R S AS I
I
SURROGATE ENDPOINTS FOR ARTERIAL DISEASE
D.H. O'Leaxy. Department of RcMiology, T~ts Universi~ School of
Medicbw, Boston, MA, USA Objective: Overview of imaging technologies available for monitoring cardiovascular disease (CVD) progression. Methods: Review of the literature keyed to the following terms: carotid intima-media thickness (CIMT), intravascular ultrasound (IVUS), quantitative coronary angiography (QCA), coronary calcium scoring and electron beam computerized tomography (EBCT), multislice spiral computed tomography angiography (CTA), and magnetic resonance imaging (MRI). Results: Imaging tests that can directly image arterial wall structure may serve as substitutes for clinical endpoints. By direct measurement of change over time, such surrogates have the potential to increase greatly the efficacy of clinical trials by permitting the design of shorter studies involving fewer subjects. While QCA is the most established imaging technique for measuring CVD progression, it is invasive and is no longer employed in therapeutic trials. IVUS is also invasive, but because of its ability to circumferentially study the adjacent coronary artery wall with high-resolution ultrasound, it is considered promising as a surrogate and is currently being used in several clinical trials. CTA is a very new technique and is the early stages of gaining clinical acceptance. It potential for utilization as a surrogate is unclear. Of the noninvasive measures, CIMT as measured by B mode ultrasound is the most established method for measuring atherosclerosis progression and regression. Several large observational studies have demonstrated a graded response between increasing CIMT and increased risk of incident CVD events. While no single study has been powered to demonstrate that slowing of CIMT progression leads to a decrease in future events, in six clinical trials of HMG-CoA reductase inhibitors (statins) which reported a significant beneficial impact of statins on IMT progression, there were beneficial trends with respect to reported to reported CVD endpoints [1]. It is not clear that the results seen can be generalized to other theraputic agents, however. CIMT also remains handicapped in that there are no generally accepted standards for measuring CIMT change. EBCT, while useful in identifying advance atherosclerosis, seems to have little use as a tool for assessing disease progression. MRI, because of its unique potential to study plaque composition as well as wall structure, continues to be widely viewed as having great potential as a surrogate. However, it is a technique that continues to be developed and improved and its efficacy remains undefined. Conclusion: Imaging biomarkers can serve as valid surrogates for clinical endpoints. Of the noninvasive techniques available, CIMT is currently the most useful. IVUS, while limited because it is invasive, will likely have great value in limited circumstances. References [1] Espeland MA, O'Leaxy DH, Terry JG, Morgan T, Evans G, Mudra H. Carotid intimal-media thickness as a surrogate for cardiovascular disease
307
events in trials of HMG-CoA reductase inhibitors. Current Controlled Trials in Cardiovascular medicine 2005 ;6:3 l
We-W31:2 I INTIMA-MEDIA THICKNESS AS AN ADDITIVE MARKER OF CARDIOVASCULAR RISK J
D. Baldassaxre. Depat'tment of Pharntacological Sciences, Universi~ of Milan, and Cardiologico "Monzbto" Center; IRCCS, Milan, Italy
Objective: To elucidate whether carotid artery intima media thickness (IMT) detected by B-mode ultrasound can be considered as a clinically useful marker of cardiovascular risk. Methods: To address the study objectives the correlation between carotid IMT and vascular risk factors (VRFs), the IMT performance in the recognition of patients with and without history of vascular events, the association between carotid IMT and the extent of coronary atherosclerosis, and the carotid IMT capacity to predict new vascular events are discussed. Results: In large cross-sectional studies mainly performed in patients attending our Lipid Clinic, we have previously shown that carotid artery IMT correlates well with coronary VRFs [1,2]. We have also shown that carotid IMT discriminates well between groups of patients with and without a history of events [1]. In a later study, carried out by using Artificial neural networks as an innovative approach to perform statistical analyses, we have also shown that carotid IMT can significantly increase the capacity of VRFs to identify patients with and without a history of cardiovascular events even on an individual basis [3]. More recently we have also investigated the relationship between carotid atherosclerosis detected by external B-mode ultrasound and coronary atherosclerosis measured by both quantitative coronary angiography (QCA) and intravascular ultrasound (IVUS). In this study, the highest correlation coefficients between carotid and coronary atherosclerosis were observed when both vascular districts were measured with ultrasound methods (external carotid B-mode ultrasound vs coronary IVUS). In the same study, carotid ultrasonic variables proved more effective than QCA to identify patients with a positive IVUS test. Finally, in a longitudinal - observational study, we have also shown that the combined use of the Framingham Risk Score (FRS) and ultrasonic measurements of carotid IMT significantly increases the ability of VRFs to predict cardiovascular events in patients at low/intermediate risk (FRS <20%). A prospective, multicenter, longitudinal, long-term, observational study (The IMPROVE study) is currently ongoing. It aims to investigate the association between carotid IMT-progression and the rate of new vascular events in subjects at high risk. The patients enrolment ended in April 2005 and a total of 3746 patients were recruited in 6 European countries (1095 in Italy, 504 in France and 2140 in northern Europe). Condusions: Data will be presented clearly supporting the possibility of using carotid IMT not only as a marker of cardiovascular risk but also as a predictor of new events. These results, together with those reported by others, also support the possibility of using carotid IMT as a surrogate endpoint in pharmacological studies. Although this approach has been already used in a number of clinical trials to evaluate the effectiveness of anti atherosclerotic treatments, studies axe needed to fully demonstrate that IMT-progression, like IMT measured in cross-sectional studies, is a predictor of new vascular events. The currently ongoing IMPROVE study will address also this still missing piece of information; results axe expected for the end of 2007. References [1] Baldassarre D, Amato M, Bondioli A, Sirtori CR, Tremoli E. Carotid artery intima-media thickness measured by ultrasonography in normal clinical practice correlates well with atherosclerosis risk factors. Stroke 2000; 31:2426-2430. [2] Baldassaxre D, Amato M, Pustina L, Tremoli E, Sirtori CR, Calabresi L, Franceschini G. Increased carotid artery intima-media thickness in subjects with primary hypoalphalipoproteinemia. ArteHoscler Thromb Vasc Biol 2002;22:317-322. [3] Baldassaxre D, Grossi E, Buscema M, Intraligi M, Amato M, Tremoli E, Pustina L, Castelnuovo S, Sirtori CR. Recognition of patients with cardiovascular disease by artificial neural networks. Ann of Med. 2004;36:630640. Funding: Research describing correlations between carotid and coronary atherosclerosis is supported by the Italian Ministry of Health. The improve study is supported by European Union.
XIV bztentational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006