- Email: [email protected]
[72] cDNA MICROARRAY APPROACH FOR THE DIAGNOSIS OF FAMILIAL HYPERCHOLESTEROLEMIA
S18
Nutrition, Metabolism & Cardiovascular Diseases (2009) S1–S32
Methods: We pooled published and unpublished data up to 67,687 individuals to evaluate the effect of CETP SNPs on lipids and BP The findings were compared to a pooled analysis of the effect of torcetrapib on the same traits obtained from randomised controlled trials. Results: SNPs in the CETP gene were associated with reduced CETP level and activity. Both torcetrapib treatment and CETP SNPs were associated with directionally concordant effects on total, LDL- and HDL-cholesterol, HDL2, HDL3, apolipoproteins A-I, and B, and triglycerides. The observed difference in HDL-cholesterol between subjects homozygous for different CETP alleles (0.13 mmol/L; 95% CI 0.11, 0.14) was in accordance with that expected (0.13 mmol/L; 0.10, 0.15) from a 10 mg dose of torcetrapib. Torcetrapib (60 mg daily) elevated systolic BP by 4.47 (4.10, 4.84) mmHg and diastolic blood pressure by 2.08 (1.84, 2.31) mmHg. However, the systolic difference of 0.16 mmHg ( 0.28, 0.60), and the diastolic BP difference of 0.04 mmHg ( 0.36, 0.28) observed between subjects homozygous for CETP gene variants was consistent with a null effect and significantly different from the differences of 0.72 mmHg (0.59, 0.86) for systolic and 0.34 mmHg (0.27, 0.41) for diastolic BP expected from a 10 mg dose of torcetrapib. Conclusions: Discordance in the effect of CETP SNPs and torcetrapib treatment on BP, despite the concordant effects on lipids indicates that the hypertensive effect of torcetrapib is unlikely to be due to CETP-inhibition. Using genetic studies as a type of natural trial could have wider application in drug development, helping to validate targets, model drug effects, and distinguish on and off-target effects. 70 EFFECT OF CONJUGATED LINOLEIC ACID ISOMERS ON TF EXPRESSION IN HUMAN MACROPHAGES E. Napoleone, G. Colavecchia, A. Cutrone, D. Cugino, L. Iacoviello, G. de Gaetano, O. Belton1 , M.B. Donati, R. Lorenzet. Research Laboratories, Center for High Technology Research and Education in Biomedical Sciences, Catholic University, Campobasso, Italy; 1 UCD Conway Institute, UCD, Dublin, Ireland E-mail: [email protected] Background: Conjugated linoleic acid (CLA) is a group of positional and geometric isomers of linoleic acid. CLA is a mixture of dietary fatty acids that exerts various beneficial effects including decrease in proliferation, atherogenesis, diabetes and inflammation in animal models. Conflicting data on the identity of the different CLA isomers responsible for their effect were reported. Tissue factor (TF), expressed mainly by infiltrating inflammatory cells, is considered one of the main contributors to the thrombogenicity associated with atheroma. In this study we investigated the effect of the main isomer of CLA, cis-9, trans-11 (c9,t11), and of the blend of isomers: 80% (c9, t11) and 20% (t10, c12) on TF expression in mononuclear cells (MNs) and macrophages (Ms). Methods: MNs from peripheral blood of healthy donors were incubated with (c9, t11) or blend with or without lipopolysaccharide (LPS) for 6 hours at 37ºC. Cells were then disrupted by freezing and thawing and procoagulant activity was assessed by a one-stage clotting time and expressed in arbitrary units (U) by comparison with a standard preparation of human brain thromboplastin. TF mRNA levels were assessed by real time RT-PCR. Results: Both (c9, t11) and blend inhibited TF activity from LPS-stimulated MNs in a dose dependent way. Down regulation of TF activity was accompanied by a decrease in TF mRNA levels. To test whether (c9, t11) and blend were effective also when agonists different from LPS were used to stimulate MNs, other known inducers of TF activity, namely IL-1b and TNF-a, were incubated with the cells. The decrease in TF activity was observed also in the presence of these inducing agents. The same TF downregulation was observed when CLA was tested on human macrophages obtained by spontaneous differentiation of blood monocytes in culture. Interestingly, TF inhibition was accompanied by a decrease in TNF-a release as assessed by ELISA. Conclusions: The suppression of TF in macrophages by CLA isomers indicate a potential mechanism by which these substances may interfere with the formation and progression of atherosclerotic plaques and their cardiovascular complications. 71 LEPTIN UPREGULATES HUMAN BREAST CANCER CELL LINE MCF7 EXPRESSION OF TISSUE FACTOR: A POSSIBLE LINK BETWEEN OBESITY AND RISK OF CANCER E. Napoleone, A. Cutrone, F. Zurlo, G. Colavecchia, M.C. Latella, D. Cugino, L. Iacoviello, G. de Gaetano, M.B. Donati, R. Lorenzet. Research Laboratories, Center for High Technology Research and Education in Biomedical Sciences, Catholic University, Campobasso, Italy E-mail: [email protected] Background: Epidemiological studies have shown an increased risk of cancer associated with obesity and adipose tissue mass. Breast tumors are surrounded by adipose tissue which synthesizes adipokines. Leptin, one of the adipokines, whose levels are found elevated in obese individuals, has been shown to act as a mitogen, transforming factor and migration factor for many different cell types. Tissue factor (TF), is often expressed by tumor cells, and for its involvement in tumor growth, angiogenesis, and metastasis is considered a hallmark of cancer progression. Since leptin has been shown to induce TF expression in human monocytes, (Napoleone et al. J Thromb Haemost 5:1462; 2007), we decided to investigate
whether leptin could modulate the constitutive expression of TF by the metastatic breast carcinoma estrogen-receptor-positive cell line MCF7. Methods: Confluent MCF7 cells were incubated with and the different reagents in various combination for different time-intervals at 37ºC in 7.5% CO2. At the end of incubation, cells were disrupted by freezing and thawing and procoagulant activity was assessed by a one-stage clotting assay and expressed in arbitrary units (U) by comparison with a standard preparation of human brain thromboplastin. TF antigen cellular expression was determined by ELISA and TF mRNA levels were assessed by real time RT-PCR. Leptin receptor (ObR) was detected by flow-cytometry. Results: In basal conditions MCF7 cells expressed high levels of constitutive TF activity. When the cells were exposed to leptin, A dose-dependent enhancement on TF activity was observed (p < 0.05). The activity was attributable to TF, since a MoAb against TF completely blocked the shortening of the clotting time. The increase in TF activity was accompanied by an increase in TF antigen (p < 0.03) and mRNA levels (p < 0.05). A strong inhibition of TF activity was observed when incubation was carried out in the presence of an inhibitory anti-leptin antibody. Similar results were obtained with an inhibitory anti-leptin receptor antibody, indicating that leptin exerted its effect by binding to its receptor, whose presence was detected on MCF7 membrane by flow cytometry. Experiments with specific inhibitors of MAPK signalling revealed the involvement of ERK1/2 kinase, but not p38 kinase, pathway. Conclusions: These data support the hypothesis that leptin, by its upregulation of TF, may contribute to processes underlying both cancer and vascular cell disorders. 72 cDNA MICROARRAY APPROACH FOR THE DIAGNOSIS OF FAMILIAL HYPERCHOLESTEROLEMIA G.D. Norata1,2 , K. Garlaschelli1 , L. Grigore1 , F. Pellegatta1,2 , S. Calandra3 , A.L. Catapano1,2 . 1 Center for the Study of Atherosclerosis S.I.S.A., H.Bassini, Cinisello Balsamo, Milan, Italy; 2 Department of Pharmacological Sciences, University of Milan, Italy; 3 Department of Biomedical Sciences, University of Modena and Reggio Emilia, Italy E-mail: [email protected] Familial Hypercholesterolemia (FH) is a genetic disorder characterized by increased levels of total cholesterol, LDL cholesterol and early cardiovascular events. FH is mainly related to mutation in the gene coding for LDL-R which results in a protein with reduced to null efficiency in promoting LDL clearance form plasma. In addition to mutation in this gene FH has been associated less frequently with mutation in the genes coding for apolipoprotein B (ApoB) or for proprotein convertase subtilisin Kexin-9 (PCSK9). In the clinical setting, FH patient is identified according to the MEDPED score which encompass mainly the evaluation of the presence of elevated LDL cholesterol levels, presence of xanthoma, xanthelasma, early cardiovascular events, family history of CHD. Although this approach is very informative, the genetic diagnosis is the gold standard in the determination of FH. As the gene coding for LDL-R has several exons with reported mutations, this approach requires the amplification and sequencing of several amplicons thus needing long time for processing. More recently, the development of high throughput technologies such as DNA microarray approches, allowed to investigate the presence of several DNA sequences in a reduced time frame. The Lipochip microarray encompass 300 hundred previously identified mutations mostly within the LDL-R but also for ApoB (exon 26) and PCSK9 and allows the identification of large insertions or deletion. We describe here the use of this approach for detecting the most frequent mutations associated with FH in the Italian population. First a set of validation experiments were performed on 14 DNA samples from FH patients previously identified following classical sequencing. The analysis resulted in the identification of all the 14 different mutation in 36 hours. Next 24 DNA samples form patients with clinically diagnosis of FH were investigated in a first version of the microarray chip. The analysis allowed the identification in 14 patients of 11 different mutations in the gene coding for LDL-R. Although we can not exclude that within the 10 patients which did not show the presence of mutation either are present non FH subjects or FH subjects with previously unidentified mutations, the most probably explanation is that the version of the microarray used encompass only part of the most frequent mutations in Italy and therefore a more specific version for the Italian population is under preparation and will be available at the beginning of 2010 with the aim of identifying 90 to 95% of Italian mutations. In summary this approach will parallel the actual genetic diagnosis of FH based on sequencing allowing the screening of a maximum of 48 samples in 36hours thus improving the efficiency of the screening for the most common mutations associated with FH. 73 DEFICIENCY OF THE LONG PENTRAXIN PTX3 PROMOTES VASCULAR INFLAMMATION AND ATHEROSCLEROSIS G.D. Norata, C. Garlanda, A. Mantovani, A.L. Catapano. Department of Pharmacological Sciences, University of Milan, Italy E-mail: [email protected] Background: Immune responses participate in several phases of atherosclerosis; there is, in fact, increasing evidence that both adaptive and innate immunity tightly regulate atherogenesis. Pentraxins are a superfamily of acute phase proteins which include short pentraxins such as CRP or long pentraxins such as PTX3, a molecule acting as the humoral arm of innate immunity. To
Nutrition, Metabolism & Cardiovascular Diseases (2009) S1–S32
Methods: We pooled published and unpublished data up to 67,687 individuals to evaluate the effect of CETP SNPs on lipids and BP The findings were compared to a pooled analysis of the effect of torcetrapib on the same traits obtained from randomised controlled trials. Results: SNPs in the CETP gene were associated with reduced CETP level and activity. Both torcetrapib treatment and CETP SNPs were associated with directionally concordant effects on total, LDL- and HDL-cholesterol, HDL2, HDL3, apolipoproteins A-I, and B, and triglycerides. The observed difference in HDL-cholesterol between subjects homozygous for different CETP alleles (0.13 mmol/L; 95% CI 0.11, 0.14) was in accordance with that expected (0.13 mmol/L; 0.10, 0.15) from a 10 mg dose of torcetrapib. Torcetrapib (60 mg daily) elevated systolic BP by 4.47 (4.10, 4.84) mmHg and diastolic blood pressure by 2.08 (1.84, 2.31) mmHg. However, the systolic difference of 0.16 mmHg ( 0.28, 0.60), and the diastolic BP difference of 0.04 mmHg ( 0.36, 0.28) observed between subjects homozygous for CETP gene variants was consistent with a null effect and significantly different from the differences of 0.72 mmHg (0.59, 0.86) for systolic and 0.34 mmHg (0.27, 0.41) for diastolic BP expected from a 10 mg dose of torcetrapib. Conclusions: Discordance in the effect of CETP SNPs and torcetrapib treatment on BP, despite the concordant effects on lipids indicates that the hypertensive effect of torcetrapib is unlikely to be due to CETP-inhibition. Using genetic studies as a type of natural trial could have wider application in drug development, helping to validate targets, model drug effects, and distinguish on and off-target effects. 70 EFFECT OF CONJUGATED LINOLEIC ACID ISOMERS ON TF EXPRESSION IN HUMAN MACROPHAGES E. Napoleone, G. Colavecchia, A. Cutrone, D. Cugino, L. Iacoviello, G. de Gaetano, O. Belton1 , M.B. Donati, R. Lorenzet. Research Laboratories, Center for High Technology Research and Education in Biomedical Sciences, Catholic University, Campobasso, Italy; 1 UCD Conway Institute, UCD, Dublin, Ireland E-mail: [email protected] Background: Conjugated linoleic acid (CLA) is a group of positional and geometric isomers of linoleic acid. CLA is a mixture of dietary fatty acids that exerts various beneficial effects including decrease in proliferation, atherogenesis, diabetes and inflammation in animal models. Conflicting data on the identity of the different CLA isomers responsible for their effect were reported. Tissue factor (TF), expressed mainly by infiltrating inflammatory cells, is considered one of the main contributors to the thrombogenicity associated with atheroma. In this study we investigated the effect of the main isomer of CLA, cis-9, trans-11 (c9,t11), and of the blend of isomers: 80% (c9, t11) and 20% (t10, c12) on TF expression in mononuclear cells (MNs) and macrophages (Ms). Methods: MNs from peripheral blood of healthy donors were incubated with (c9, t11) or blend with or without lipopolysaccharide (LPS) for 6 hours at 37ºC. Cells were then disrupted by freezing and thawing and procoagulant activity was assessed by a one-stage clotting time and expressed in arbitrary units (U) by comparison with a standard preparation of human brain thromboplastin. TF mRNA levels were assessed by real time RT-PCR. Results: Both (c9, t11) and blend inhibited TF activity from LPS-stimulated MNs in a dose dependent way. Down regulation of TF activity was accompanied by a decrease in TF mRNA levels. To test whether (c9, t11) and blend were effective also when agonists different from LPS were used to stimulate MNs, other known inducers of TF activity, namely IL-1b and TNF-a, were incubated with the cells. The decrease in TF activity was observed also in the presence of these inducing agents. The same TF downregulation was observed when CLA was tested on human macrophages obtained by spontaneous differentiation of blood monocytes in culture. Interestingly, TF inhibition was accompanied by a decrease in TNF-a release as assessed by ELISA. Conclusions: The suppression of TF in macrophages by CLA isomers indicate a potential mechanism by which these substances may interfere with the formation and progression of atherosclerotic plaques and their cardiovascular complications. 71 LEPTIN UPREGULATES HUMAN BREAST CANCER CELL LINE MCF7 EXPRESSION OF TISSUE FACTOR: A POSSIBLE LINK BETWEEN OBESITY AND RISK OF CANCER E. Napoleone, A. Cutrone, F. Zurlo, G. Colavecchia, M.C. Latella, D. Cugino, L. Iacoviello, G. de Gaetano, M.B. Donati, R. Lorenzet. Research Laboratories, Center for High Technology Research and Education in Biomedical Sciences, Catholic University, Campobasso, Italy E-mail: [email protected] Background: Epidemiological studies have shown an increased risk of cancer associated with obesity and adipose tissue mass. Breast tumors are surrounded by adipose tissue which synthesizes adipokines. Leptin, one of the adipokines, whose levels are found elevated in obese individuals, has been shown to act as a mitogen, transforming factor and migration factor for many different cell types. Tissue factor (TF), is often expressed by tumor cells, and for its involvement in tumor growth, angiogenesis, and metastasis is considered a hallmark of cancer progression. Since leptin has been shown to induce TF expression in human monocytes, (Napoleone et al. J Thromb Haemost 5:1462; 2007), we decided to investigate
whether leptin could modulate the constitutive expression of TF by the metastatic breast carcinoma estrogen-receptor-positive cell line MCF7. Methods: Confluent MCF7 cells were incubated with and the different reagents in various combination for different time-intervals at 37ºC in 7.5% CO2. At the end of incubation, cells were disrupted by freezing and thawing and procoagulant activity was assessed by a one-stage clotting assay and expressed in arbitrary units (U) by comparison with a standard preparation of human brain thromboplastin. TF antigen cellular expression was determined by ELISA and TF mRNA levels were assessed by real time RT-PCR. Leptin receptor (ObR) was detected by flow-cytometry. Results: In basal conditions MCF7 cells expressed high levels of constitutive TF activity. When the cells were exposed to leptin, A dose-dependent enhancement on TF activity was observed (p < 0.05). The activity was attributable to TF, since a MoAb against TF completely blocked the shortening of the clotting time. The increase in TF activity was accompanied by an increase in TF antigen (p < 0.03) and mRNA levels (p < 0.05). A strong inhibition of TF activity was observed when incubation was carried out in the presence of an inhibitory anti-leptin antibody. Similar results were obtained with an inhibitory anti-leptin receptor antibody, indicating that leptin exerted its effect by binding to its receptor, whose presence was detected on MCF7 membrane by flow cytometry. Experiments with specific inhibitors of MAPK signalling revealed the involvement of ERK1/2 kinase, but not p38 kinase, pathway. Conclusions: These data support the hypothesis that leptin, by its upregulation of TF, may contribute to processes underlying both cancer and vascular cell disorders. 72 cDNA MICROARRAY APPROACH FOR THE DIAGNOSIS OF FAMILIAL HYPERCHOLESTEROLEMIA G.D. Norata1,2 , K. Garlaschelli1 , L. Grigore1 , F. Pellegatta1,2 , S. Calandra3 , A.L. Catapano1,2 . 1 Center for the Study of Atherosclerosis S.I.S.A., H.Bassini, Cinisello Balsamo, Milan, Italy; 2 Department of Pharmacological Sciences, University of Milan, Italy; 3 Department of Biomedical Sciences, University of Modena and Reggio Emilia, Italy E-mail: [email protected] Familial Hypercholesterolemia (FH) is a genetic disorder characterized by increased levels of total cholesterol, LDL cholesterol and early cardiovascular events. FH is mainly related to mutation in the gene coding for LDL-R which results in a protein with reduced to null efficiency in promoting LDL clearance form plasma. In addition to mutation in this gene FH has been associated less frequently with mutation in the genes coding for apolipoprotein B (ApoB) or for proprotein convertase subtilisin Kexin-9 (PCSK9). In the clinical setting, FH patient is identified according to the MEDPED score which encompass mainly the evaluation of the presence of elevated LDL cholesterol levels, presence of xanthoma, xanthelasma, early cardiovascular events, family history of CHD. Although this approach is very informative, the genetic diagnosis is the gold standard in the determination of FH. As the gene coding for LDL-R has several exons with reported mutations, this approach requires the amplification and sequencing of several amplicons thus needing long time for processing. More recently, the development of high throughput technologies such as DNA microarray approches, allowed to investigate the presence of several DNA sequences in a reduced time frame. The Lipochip microarray encompass 300 hundred previously identified mutations mostly within the LDL-R but also for ApoB (exon 26) and PCSK9 and allows the identification of large insertions or deletion. We describe here the use of this approach for detecting the most frequent mutations associated with FH in the Italian population. First a set of validation experiments were performed on 14 DNA samples from FH patients previously identified following classical sequencing. The analysis resulted in the identification of all the 14 different mutation in 36 hours. Next 24 DNA samples form patients with clinically diagnosis of FH were investigated in a first version of the microarray chip. The analysis allowed the identification in 14 patients of 11 different mutations in the gene coding for LDL-R. Although we can not exclude that within the 10 patients which did not show the presence of mutation either are present non FH subjects or FH subjects with previously unidentified mutations, the most probably explanation is that the version of the microarray used encompass only part of the most frequent mutations in Italy and therefore a more specific version for the Italian population is under preparation and will be available at the beginning of 2010 with the aim of identifying 90 to 95% of Italian mutations. In summary this approach will parallel the actual genetic diagnosis of FH based on sequencing allowing the screening of a maximum of 48 samples in 36hours thus improving the efficiency of the screening for the most common mutations associated with FH. 73 DEFICIENCY OF THE LONG PENTRAXIN PTX3 PROMOTES VASCULAR INFLAMMATION AND ATHEROSCLEROSIS G.D. Norata, C. Garlanda, A. Mantovani, A.L. Catapano. Department of Pharmacological Sciences, University of Milan, Italy E-mail: [email protected] Background: Immune responses participate in several phases of atherosclerosis; there is, in fact, increasing evidence that both adaptive and innate immunity tightly regulate atherogenesis. Pentraxins are a superfamily of acute phase proteins which include short pentraxins such as CRP or long pentraxins such as PTX3, a molecule acting as the humoral arm of innate immunity. To