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W04-P-003 Elevated serum proinflammatory cytokines and extracellular matrix remodeling markers levels in patients with homozygous familial hypercholesterolemia
Workshops W4 Extra cellular matrix in vascular disease
18
"IT genotype in hypertensive group. Lowering effect on HDL cholesterol levels by 6A6A/GG combination of genotypes showed significance in normotensive group. Conclusions: Association of MMP-3 5A/6A polymorphism with hypertension should be thoroughly investigated in future studies. Results regarding association of certain genotypes of studied polymorphlsms with serum HDL cholesterol levels might have significance in prediction of greater cardiovascular risk and certainly should be confirmed in different well defined patients groups as well as in other populations.
I W04-P-003 I ELEVATED SERUM P R O I N F L A M M A T O R Y C Y T O K I N E S AND E X T R A C E L L U L A R MATRIX R E M O D E L I N G M A R K E R S LEVELS IN PATIENTS W I T H H O M O Z Y G O U S FAMILIAL HYPERCHOLESTEROLEMIA A. Drissi 1, P. Giral 2, M. E1 Messal 4, E. Bruckert 2, R. Chater I , K. Ait Chihab 4 , A. Hafidi 5, C. Abdelkhirane 6, O. Benabdesselam7 , A. Kettani 1, M.-J. Chapman 3, A. Adlouni 1, J.-L. Beaudeux 7 . ~Facult# des sciences Ben
M'Sik, Casablanca, Morocco; 2Service d'endocrinologie-m#tabolisme, H@ital Piti(-Saletr&re, Paris, France; 31NSERM U551, H@ital Piti#Saletriere, Paris, France; 4eacult( des Sciences Ain Chock, Casablanca, Morocco; 5Service d'Endocrinologie, CHU lbn Sina, Pabag Morocca; 6Centre de Cardiologie Maarif. Clinique Maarif,, Casablanca, Morocco; ZF(d(ration de Biochimie, Hrpital Piti#-Salpetriere, Paris, France Familial hypercholesterolemia (FH) is a common inherited disorder of lipoprotein metabolism, consisting of mutations in the gene coding for the low-density-lipoprotein-receptor protein. Although the cause of FH is monogenic, there is wide variation in the onset and severity of atherosclerotic disease in FH patients. Additional atherogenic risk factors, including a proinflammatory state, axe presumed to influence the clinical phenotype in FH. We determined serum levels of MMP-3, active MMP-9 and TIMP-1 as well as pro-inflammatory cytokines (TNF-a, IL-18) in 4 non treated homozygous FH Moroccan patients and compared it to those of healthy control subjects (n=5) and of heterozygous HF subjects (n=7). When compared to controls, homozygous patients exhibited significantly elevated levels of active MMP-9 and TIMP-1 levels (p<0.05) and an elevation of MMP-3 levels although not significant. In the same way, both serum hs-CRP and IL-18 levels were significantly higher in homozygous HF patients (p<0.05 and p<0.01. Heterozygous FI-I patients' group exhibited intermediate values for these markers, except MMP-9 activity which was significantly elevated (p<0.05). Finally, serum TNF-a remained unchanged in the three groups. Multivariate analysis revealed a positive correlation between TIMP-1, IL-18 and apolipoprotein B, and between hs-CRP and IL18 (p<0.01). Although size of the FH group was limited, our data suggest that nontreated homozygous FH moroccan patients, and at a lesser extent heterozygous FH patients, exhibited an increased vascular proinflammatory state as well as extracellular matrix remodeling, as evaluated by circulating levels of inflammatory cytokines and MMPs.
I WO4-P-O04 I DIABETES
INCREASES EXPRESSION O F GENES FOR NATRIURETIC PEPTIDE R E C E P T O R S
C. Christoffersen, E.D. Barrels, L.B. Nielsen. Dept. Clinical Biochemistry,
Rigshospitaleg University of Copenhagen, Copenhagen, Denmark Objective: Diabetes confers vascular changes and fibrosis in the heart, but the mechanisms involved are not clear. Natriuretic poptides type A, B, and C (i.e. ANP, BNP, and CNP) have autocrine effects in the heart and vasculature through their binding to the natriuretic poptide receptors A and B (i.e. ANP and BNP binds to NPR-A and CNP bind to NPR-B). NPR-C function as clearance receptor for all three natriuretic poptides. The aim of this study was to investigate whether diabetes affects the expression of genes for the natriuretic poptide receptors in the heart. Remits: Twelve-week old ob/ob mice (n=10) with type II diabetes had mild cardiac dysfunction on echocardiography but no changes in expression of the ANP, BNP, CNP, or NPR-A genes compared to non-diabetic ob/+ mice (n=10). In contrast, the NPR-B (1.64- 0.1 vs 1.14-0.1, p=0.0008) and NPR-C (1.84-0.2 vs 1.0+0.1, p=0.002) genes were upregulated in ob/ob mice compared with ob/+ mice. Streptozotocin (STZ)-treated mice (n=27) with type I diabetes were examined 3, 6, 9, or 12 weeks after induction of diabetes. After 12 weeks, STZ-treated mice had cardiac dysfunction on echocardiogruphy and increased BNP and NPR-B gene expression. BNP and NPR-B gene expression were not affected 3, 6, or 9 weeks after
STZ-treatment. The expression of the ANP, CNP, NPR-A, and NPR-C genes was similar in STZ-treated and control mice (n=27) at all time points. Conclusion: The results suggest that diabetes increases heart expression of the receptor for CNP. Alterations in natriuretic poptide receptor expression may affect diabetic vasculopathy and cardiomyopathy.
W04-P-005 I E X T R A C E L L U L A R MATRIX-MEDIATED C O N T R O L OF S M O O T H M U S C L E C E L L G R O W T H AND M I G R A T I O N BY A COMBINATION OF ASCORBIC ACID, LYSINE, PROLINE AND CATECHINS V. Ivanov, S. Ivanova, M.W. Roomi, A. Niedzwiecki, M. Rath. Research
Institute, Matthias Path BV, Santa Clara, USA Objective: Extracellular matrix (ECM) function and structure are severely compromised at atherosclerotic lesion sites, contributing to initiation and progression of the disease. We investigated whether ECM biological propeaies would be beneficially affected by exposure to nutrients essential for collagen synthesis and post-translational modification. Methods: Confluent layers of human aortic smooth muscle cells (SMC) grown on collagen substrate were cultured in the presence of the tested compounds for seven to ten days. Pre-treated cells were removed from the ECM surface by differential treatment and replaced with secondary innocent SMC cultures. Secondary SMC growth rate and invasiveness were assayed in standard growth medium. ECM protein composition was assayed immunochemically. Results: ECM produced in the presence of ascorbic acid significantly reduced SMC proliferation as compared to the untreated control. Lysine and proline potentiated this growth inhibitory effect, and addition of green tea catechins maximized it. In addition, the ECM deposited by SMC in the presence of ascorbate, lysine, proline and green tea catechins inhibited SMC migration rate up to 70%. ECM composition showed increased deposition of collagens III and IV and decreased collagen I under treatment with the nutrients. Conclusion: ECM produced under conditions of chronic essential nutrient deficiency can support pro-atherosclerotic SMC behavior. These adverse effects can be counteracted by nutrient replenishment.
I W04-P-006 I H M G - C O A REDUCTASE INHIBITORS, VIA R H O PROTEINS, DECREASE ANGIOTENSIN II-INDUCED CONNECTIVE TISSUE G R O W T H FACTOR IN VASCULAR S M O O T H M U S C L E CELLS M. Ruprrez, L.M. Blanco-Colio, J. Rodriguez-Vita, E. Sanchez-Lopez, V. Esteban, J. Egido, M. Ruiz-Ortega. Vascular and Renal Research
Laboratory, Fundacirn Jim(nez Dfaz, Universidad Autrnoma, Madrid, Spain Objectives: To investigate the role of HMG-CoA reductase inhibitors and the Rho/Rho Kinase pathway in AngII-induced vascular responses, studying CTGF regulation. AngII (Angiotensin If) has been implicated in the pathegenesis of cardiovascular diseases, such as atherosclerosis, and induce the expression of Connective tissue growth factor (CTGP'), a novel profibrotic factor. AngII activates Rho proteins via AT1 receptor. Methods: AngII-induced CTGF regulation was evaluated in vitro in cultured vascular smooth muscle cells (VSMC), and in vivo using the model of systemic infusion of AngII (100ng/kg/min) into normal rats, treating some animals with Atorvastatin (5 mg/kg/day) and the Rho-kinase inhibitor Y-27632 (30mg/kg/day). Results: Treatment of VSMC with HMG-CoA reductase inhibitors (atorvastatin and simvastatin) dose-dependently inhibited AngII-induced CTGF production. This effect was reversed by mevalonate and gerauylgeranylpyrophosphate. Statins inhibited AngII-induced RhoA cellular membrane localization. Treatment with C3 exoenzyme diminished AngIIinduced CTGF production. Transfection with a constitutively active form of RhoA increased CTGF production and overexpmssion of a dominant negative vector of RhoA suppressed CTGF synthesis caused by AngII. Treatment with two Rho-kiuase inhibitors (Y-27632 and fasudil) decreased CTGF gene expression and protein production. AngII increased CTGF promoter activity that was diminished by Y-27632 and by overexpression of the dominant negative RhoA. Ovemxpmssion of an active form of RhoA increased CTGF promoter activity. In AngII-infused rats, Rho-kinase inhibitior or Atorvastatin treatment diminished CTGF overexpmssion (gene and protein) observed in the aorta.
75th EAS Congress, 23-26 April 2005, Prague, Czech Republic
18
"IT genotype in hypertensive group. Lowering effect on HDL cholesterol levels by 6A6A/GG combination of genotypes showed significance in normotensive group. Conclusions: Association of MMP-3 5A/6A polymorphism with hypertension should be thoroughly investigated in future studies. Results regarding association of certain genotypes of studied polymorphlsms with serum HDL cholesterol levels might have significance in prediction of greater cardiovascular risk and certainly should be confirmed in different well defined patients groups as well as in other populations.
I W04-P-003 I ELEVATED SERUM P R O I N F L A M M A T O R Y C Y T O K I N E S AND E X T R A C E L L U L A R MATRIX R E M O D E L I N G M A R K E R S LEVELS IN PATIENTS W I T H H O M O Z Y G O U S FAMILIAL HYPERCHOLESTEROLEMIA A. Drissi 1, P. Giral 2, M. E1 Messal 4, E. Bruckert 2, R. Chater I , K. Ait Chihab 4 , A. Hafidi 5, C. Abdelkhirane 6, O. Benabdesselam7 , A. Kettani 1, M.-J. Chapman 3, A. Adlouni 1, J.-L. Beaudeux 7 . ~Facult# des sciences Ben
M'Sik, Casablanca, Morocco; 2Service d'endocrinologie-m#tabolisme, H@ital Piti(-Saletr&re, Paris, France; 31NSERM U551, H@ital Piti#Saletriere, Paris, France; 4eacult( des Sciences Ain Chock, Casablanca, Morocco; 5Service d'Endocrinologie, CHU lbn Sina, Pabag Morocca; 6Centre de Cardiologie Maarif. Clinique Maarif,, Casablanca, Morocco; ZF(d(ration de Biochimie, Hrpital Piti#-Salpetriere, Paris, France Familial hypercholesterolemia (FH) is a common inherited disorder of lipoprotein metabolism, consisting of mutations in the gene coding for the low-density-lipoprotein-receptor protein. Although the cause of FH is monogenic, there is wide variation in the onset and severity of atherosclerotic disease in FH patients. Additional atherogenic risk factors, including a proinflammatory state, axe presumed to influence the clinical phenotype in FH. We determined serum levels of MMP-3, active MMP-9 and TIMP-1 as well as pro-inflammatory cytokines (TNF-a, IL-18) in 4 non treated homozygous FH Moroccan patients and compared it to those of healthy control subjects (n=5) and of heterozygous HF subjects (n=7). When compared to controls, homozygous patients exhibited significantly elevated levels of active MMP-9 and TIMP-1 levels (p<0.05) and an elevation of MMP-3 levels although not significant. In the same way, both serum hs-CRP and IL-18 levels were significantly higher in homozygous HF patients (p<0.05 and p<0.01. Heterozygous FI-I patients' group exhibited intermediate values for these markers, except MMP-9 activity which was significantly elevated (p<0.05). Finally, serum TNF-a remained unchanged in the three groups. Multivariate analysis revealed a positive correlation between TIMP-1, IL-18 and apolipoprotein B, and between hs-CRP and IL18 (p<0.01). Although size of the FH group was limited, our data suggest that nontreated homozygous FH moroccan patients, and at a lesser extent heterozygous FH patients, exhibited an increased vascular proinflammatory state as well as extracellular matrix remodeling, as evaluated by circulating levels of inflammatory cytokines and MMPs.
I WO4-P-O04 I DIABETES
INCREASES EXPRESSION O F GENES FOR NATRIURETIC PEPTIDE R E C E P T O R S
C. Christoffersen, E.D. Barrels, L.B. Nielsen. Dept. Clinical Biochemistry,
Rigshospitaleg University of Copenhagen, Copenhagen, Denmark Objective: Diabetes confers vascular changes and fibrosis in the heart, but the mechanisms involved are not clear. Natriuretic poptides type A, B, and C (i.e. ANP, BNP, and CNP) have autocrine effects in the heart and vasculature through their binding to the natriuretic poptide receptors A and B (i.e. ANP and BNP binds to NPR-A and CNP bind to NPR-B). NPR-C function as clearance receptor for all three natriuretic poptides. The aim of this study was to investigate whether diabetes affects the expression of genes for the natriuretic poptide receptors in the heart. Remits: Twelve-week old ob/ob mice (n=10) with type II diabetes had mild cardiac dysfunction on echocardiography but no changes in expression of the ANP, BNP, CNP, or NPR-A genes compared to non-diabetic ob/+ mice (n=10). In contrast, the NPR-B (1.64- 0.1 vs 1.14-0.1, p=0.0008) and NPR-C (1.84-0.2 vs 1.0+0.1, p=0.002) genes were upregulated in ob/ob mice compared with ob/+ mice. Streptozotocin (STZ)-treated mice (n=27) with type I diabetes were examined 3, 6, 9, or 12 weeks after induction of diabetes. After 12 weeks, STZ-treated mice had cardiac dysfunction on echocardiogruphy and increased BNP and NPR-B gene expression. BNP and NPR-B gene expression were not affected 3, 6, or 9 weeks after
STZ-treatment. The expression of the ANP, CNP, NPR-A, and NPR-C genes was similar in STZ-treated and control mice (n=27) at all time points. Conclusion: The results suggest that diabetes increases heart expression of the receptor for CNP. Alterations in natriuretic poptide receptor expression may affect diabetic vasculopathy and cardiomyopathy.
W04-P-005 I E X T R A C E L L U L A R MATRIX-MEDIATED C O N T R O L OF S M O O T H M U S C L E C E L L G R O W T H AND M I G R A T I O N BY A COMBINATION OF ASCORBIC ACID, LYSINE, PROLINE AND CATECHINS V. Ivanov, S. Ivanova, M.W. Roomi, A. Niedzwiecki, M. Rath. Research
Institute, Matthias Path BV, Santa Clara, USA Objective: Extracellular matrix (ECM) function and structure are severely compromised at atherosclerotic lesion sites, contributing to initiation and progression of the disease. We investigated whether ECM biological propeaies would be beneficially affected by exposure to nutrients essential for collagen synthesis and post-translational modification. Methods: Confluent layers of human aortic smooth muscle cells (SMC) grown on collagen substrate were cultured in the presence of the tested compounds for seven to ten days. Pre-treated cells were removed from the ECM surface by differential treatment and replaced with secondary innocent SMC cultures. Secondary SMC growth rate and invasiveness were assayed in standard growth medium. ECM protein composition was assayed immunochemically. Results: ECM produced in the presence of ascorbic acid significantly reduced SMC proliferation as compared to the untreated control. Lysine and proline potentiated this growth inhibitory effect, and addition of green tea catechins maximized it. In addition, the ECM deposited by SMC in the presence of ascorbate, lysine, proline and green tea catechins inhibited SMC migration rate up to 70%. ECM composition showed increased deposition of collagens III and IV and decreased collagen I under treatment with the nutrients. Conclusion: ECM produced under conditions of chronic essential nutrient deficiency can support pro-atherosclerotic SMC behavior. These adverse effects can be counteracted by nutrient replenishment.
I W04-P-006 I H M G - C O A REDUCTASE INHIBITORS, VIA R H O PROTEINS, DECREASE ANGIOTENSIN II-INDUCED CONNECTIVE TISSUE G R O W T H FACTOR IN VASCULAR S M O O T H M U S C L E CELLS M. Ruprrez, L.M. Blanco-Colio, J. Rodriguez-Vita, E. Sanchez-Lopez, V. Esteban, J. Egido, M. Ruiz-Ortega. Vascular and Renal Research
Laboratory, Fundacirn Jim(nez Dfaz, Universidad Autrnoma, Madrid, Spain Objectives: To investigate the role of HMG-CoA reductase inhibitors and the Rho/Rho Kinase pathway in AngII-induced vascular responses, studying CTGF regulation. AngII (Angiotensin If) has been implicated in the pathegenesis of cardiovascular diseases, such as atherosclerosis, and induce the expression of Connective tissue growth factor (CTGP'), a novel profibrotic factor. AngII activates Rho proteins via AT1 receptor. Methods: AngII-induced CTGF regulation was evaluated in vitro in cultured vascular smooth muscle cells (VSMC), and in vivo using the model of systemic infusion of AngII (100ng/kg/min) into normal rats, treating some animals with Atorvastatin (5 mg/kg/day) and the Rho-kinase inhibitor Y-27632 (30mg/kg/day). Results: Treatment of VSMC with HMG-CoA reductase inhibitors (atorvastatin and simvastatin) dose-dependently inhibited AngII-induced CTGF production. This effect was reversed by mevalonate and gerauylgeranylpyrophosphate. Statins inhibited AngII-induced RhoA cellular membrane localization. Treatment with C3 exoenzyme diminished AngIIinduced CTGF production. Transfection with a constitutively active form of RhoA increased CTGF production and overexpmssion of a dominant negative vector of RhoA suppressed CTGF synthesis caused by AngII. Treatment with two Rho-kiuase inhibitors (Y-27632 and fasudil) decreased CTGF gene expression and protein production. AngII increased CTGF promoter activity that was diminished by Y-27632 and by overexpression of the dominant negative RhoA. Ovemxpmssion of an active form of RhoA increased CTGF promoter activity. In AngII-infused rats, Rho-kinase inhibitior or Atorvastatin treatment diminished CTGF overexpmssion (gene and protein) observed in the aorta.
75th EAS Congress, 23-26 April 2005, Prague, Czech Republic