- Email: [email protected]
195 CHOLESTEROL EFFLUX POTENTIAL OF SERA FROM PATIENTS BEFORE AND AFTER LDL-APHERESIS
79th EAS Congress
Atherosclerosis Supplements 12, no. 1 (2011) 13–184
Objectives: This study was aimed to investigate the effect of aging on the antiinflammatory effect of HDL and of PON1. Also to elucidated the effect of 12 weeks of extra-virgin olive oil (EVOO) consumption on this activity. Methods: HDL and PON1 were isolated from healthy young (29±5.0 years) and elderly (71±4.0 years) volunteers before and after 12 weeks of EVOO consumption (25 ml/day). The ICAM in Eahy926 cells was measured by using FACScan technique. THP1 monocytes chemotaxis assay was measured using Boyden chamber. Results: As assessed by the ICAM expression, Y-HDL exhibited the highest anti-inflammatory activity when compared to E-HDL (28.72% versus 44.22%). Moreover, we showed an age impact on PON1 anti-inflammatory effect. Indeed, Y-PON1 incubated in presence of HDL, has a highest anti-inflammatory action compared to E-PON1 subjects (67.36% versus 87.31% respectively). These results are confirmed with those obtained following the measurement of THP-1 macrophages chemotaxis. In addition, we showed that twelve weeks of EVOO consumption increased significantly the anti-inflammatory effect of HDL (ICAM expression −23%) and particularly in Elderly (−29% versus 17% in Young). Conclusions: The anti-inflammatory effect of HDL and PON1 decreases with aging which may explain the increase of the incidence of cardio-vascular risk in elderly. Supplementation with EVOO may be a good strategy for preventing the reduction of the anti-inflammatory activity of HDL in the elderly. 193 COMPARISON OF THEORETICAL AND EXPERIMENTAL RELATIONSHIPS BETWEEN HDL SIZE AND THE RATIO OF HDL CHOLESTEROL (HDL-C) TO APOLIPOPROTEIN A-I (APOA-I) N.A. Mazer1 , S. Mora2 . 1 Translational Research Sciences, F. HoffmannLa Roche, Basel, Switzerland, 2 Divisions of Preventative and Cardiovascular Medicine, Brigham and Women’s Hospital/Harvard Medical School, Boston, MA, USA Introduction: Shen (PNAS 1977:74 (3); 837–841) proposed a theoretical model relating the size, structure and composition of lipoproteins. For HDL, the model predicts that particle size is correlated with the HDL-C to ApoA-I ratio. Objective: To compare predictions of Shen’s model with measured data on HDL size and HDL-C/ApoA-I ratio from the Women’s Health Study (WHS). Theoretical aspects: Shen’s model assumes that cholesterol esters (CE) and triglycerides (TG) form a spherical core of radius rc. Phospholipids (PL), unesterified cholesterol (C) and apolipoproteins (70% being ApoA-I) form a surface monolayer of thickness t (2.02 nm). The corresponding numbers n_CE. n_TG, n_PL, n_C and n_ApoA-I per particle are determined by geometric, compositional and thermodynamic relationships as a function of rc. The HDL-C/ ApoA-I ratio equals (n_CE + n_C)/n_ApoA-I multiplied by the molecular weight ratio (387/28,000). Particle diameter equals 2(rc + t). Experimental methods: 26,766 WHS subjects had mean HDL diameter measured by nuclear magnetic resonance (NMR); HDL-C was measured enzymatically and ApoA-I by immunoturbidometric assay.
Figure 1 Results: The predicted relationship between HDL diameter and HDL-C/ApoA-I (shown for different molar ratios of TG/CE) is in reasonable agreement with experimental data from the WHS study (Pearson r = 0.574) (Figure 1). The model further predicts that n_ApoA-I varies from 2.9 to 4.7 as HDL diameter
43
increases from 8 to 11 nm, consistent with Kontush’s data (Pharmacol Rev 2006). Conclusions: As predicted by Shen’s model, HDL size is significantly correlated with the HDL-C/ApoA-I ratio. 194 HISTONE DEACETYLASE (HDAC) INHIBITORS UPREGULATE CLUSTERIN/APOLIPOPROTEIN J EXPRESSION IN VASCULAR SMOOTH MUSCLE CELLS (VSMCS) F.M. Yatsu1 , K. Ranganna2 , O.P. Mathew2 . 1 Neurology, University of Texas Health Science Center at Houston, 2 Texas Southern University, Houston, TX, USA Objective: Clusterin/Apolipoprotein J (CLU), a secreted heterodimeric glycoprotein, composed of alpha and beta subunits is induced in VSMCs during atherosclerosis and injury-induced neointimal hyperplasia, although its functional roles in VSMCs remain obscure and mysterious. Here our goal is to confirm and evaluate the significance of CLU upregulation in proliferation arrest of VSMCs by HDAC inhibitors, butyrate and trichostatin A (TSA). Methods: VSMC proliferation is assessed by cell counting. CLU, p21Cip1 and p15INK4B expression is evaluated by western analysis and immunostaining. CLU knockout is performed with siRNA. Involvement of PI3/AKT pathway is determined by western blotting. Results: Contrast to untreated proliferating VSMCs, butyrate and TSA treated VSMCs exhibit proliferation arrest and upregulation of both cellular and secretory CLU along with increased levels of cdk/cell cycle inhibitors, p21Cip1 and p15INK4B. Moreover, CLU-specific siRNA inhibits butyrate and TSAinduced upregulation of both cellular and secretory CLU confirming butyrate and TSA indeed induce clusterin expression and secretion. Furthermore, based on the phosphorylation state of Akt and impact of PI3/Akt pathway inhibitor, Ly294002, it appears butyrate- and TSA-induced CLU expression involves PI3/Akt signaling pathway. Conclusion: Taken together HDAC inhibitors appear to have antiatherogenic potential by arresting VSMC proliferation that may be associated with CLU upregulation, which is suggested to have multiple protective roles in atherosclerosis and restenosis. Support: G12RR003045−21 and C06RR012537 from NIH/NCRR and Adriana Blood Endowment 195 CHOLESTEROL EFFLUX POTENTIAL OF SERA FROM PATIENTS BEFORE AND AFTER LDL-APHERESIS E. Favari1 , M.P. Adorni1 , F. Sbrana2 , M. Puntoni2 , F. Bigazzi2 , F. Zimetti1 , F. Bernini1 , T. Sampietro2 . 1 Pharmacological and Biological Sciences and Applied Chemistries, University of Parma, Parma, 2 CNR Institute of Clinical Physiology, Pisa, Italy Efflux of free cholesterol from peripheral cells to serum is the first step in reverse cholesterol transport. Serum cholesterol efflux potential depends on the ability of individual lipoproteins to interact with specific transporters. LDL-apheresis may dramatically affect lipoprotein profile by lowering, in addition to LDL, triglycerides and HDL. Aims: To study the impact of LDL-apheresis on 1. efflux potential of sera 2. serum ability to induce cholesterol accumulation in macrophages. Sera were obtained from 15 patients with FH receiving bi-weekly LDL-apheresis. Methods: Serum cholesterol efflux potential was tested in pathway-specific, [3 H]-cholesterol cell models. Cholesterol mass was determined in THP-1 human macrophages by fluorimetric method. Results: LDL-apheresis reduced total cholesterol and triglycerides by 70% and HDL by 30%. Cholesterol efflux by SR-BI, passive diffusion and ABCA1 were reduced by 18.2% (±1.3%; p < 0.001), 23% (±1.3%; p < 0.0001), and 24% (±1.8%; p < 0.0001) respectively. Within 48h LDL increased about two times compare to the values reached soon after LDL-apheresis treatment, while HDL and sera efflux potential returned to pre-treatment levels. LDL-apheresis reduced about 50% serum-induced cholesterol accumulation in human macrophages. This effect was maintained in the 48h after treatment. Conclusions: LDL-apheresis: 1. reduces serum cholesterol efflux potential and HDL are the only responsible for this effect; 2. reduces serum ability to induce macrophage cholesterol accumulation by both affecting LDL and HDL levels.
Atherosclerosis Supplements 12, no. 1 (2011) 13–184
Objectives: This study was aimed to investigate the effect of aging on the antiinflammatory effect of HDL and of PON1. Also to elucidated the effect of 12 weeks of extra-virgin olive oil (EVOO) consumption on this activity. Methods: HDL and PON1 were isolated from healthy young (29±5.0 years) and elderly (71±4.0 years) volunteers before and after 12 weeks of EVOO consumption (25 ml/day). The ICAM in Eahy926 cells was measured by using FACScan technique. THP1 monocytes chemotaxis assay was measured using Boyden chamber. Results: As assessed by the ICAM expression, Y-HDL exhibited the highest anti-inflammatory activity when compared to E-HDL (28.72% versus 44.22%). Moreover, we showed an age impact on PON1 anti-inflammatory effect. Indeed, Y-PON1 incubated in presence of HDL, has a highest anti-inflammatory action compared to E-PON1 subjects (67.36% versus 87.31% respectively). These results are confirmed with those obtained following the measurement of THP-1 macrophages chemotaxis. In addition, we showed that twelve weeks of EVOO consumption increased significantly the anti-inflammatory effect of HDL (ICAM expression −23%) and particularly in Elderly (−29% versus 17% in Young). Conclusions: The anti-inflammatory effect of HDL and PON1 decreases with aging which may explain the increase of the incidence of cardio-vascular risk in elderly. Supplementation with EVOO may be a good strategy for preventing the reduction of the anti-inflammatory activity of HDL in the elderly. 193 COMPARISON OF THEORETICAL AND EXPERIMENTAL RELATIONSHIPS BETWEEN HDL SIZE AND THE RATIO OF HDL CHOLESTEROL (HDL-C) TO APOLIPOPROTEIN A-I (APOA-I) N.A. Mazer1 , S. Mora2 . 1 Translational Research Sciences, F. HoffmannLa Roche, Basel, Switzerland, 2 Divisions of Preventative and Cardiovascular Medicine, Brigham and Women’s Hospital/Harvard Medical School, Boston, MA, USA Introduction: Shen (PNAS 1977:74 (3); 837–841) proposed a theoretical model relating the size, structure and composition of lipoproteins. For HDL, the model predicts that particle size is correlated with the HDL-C to ApoA-I ratio. Objective: To compare predictions of Shen’s model with measured data on HDL size and HDL-C/ApoA-I ratio from the Women’s Health Study (WHS). Theoretical aspects: Shen’s model assumes that cholesterol esters (CE) and triglycerides (TG) form a spherical core of radius rc. Phospholipids (PL), unesterified cholesterol (C) and apolipoproteins (70% being ApoA-I) form a surface monolayer of thickness t (2.02 nm). The corresponding numbers n_CE. n_TG, n_PL, n_C and n_ApoA-I per particle are determined by geometric, compositional and thermodynamic relationships as a function of rc. The HDL-C/ ApoA-I ratio equals (n_CE + n_C)/n_ApoA-I multiplied by the molecular weight ratio (387/28,000). Particle diameter equals 2(rc + t). Experimental methods: 26,766 WHS subjects had mean HDL diameter measured by nuclear magnetic resonance (NMR); HDL-C was measured enzymatically and ApoA-I by immunoturbidometric assay.
Figure 1 Results: The predicted relationship between HDL diameter and HDL-C/ApoA-I (shown for different molar ratios of TG/CE) is in reasonable agreement with experimental data from the WHS study (Pearson r = 0.574) (Figure 1). The model further predicts that n_ApoA-I varies from 2.9 to 4.7 as HDL diameter
43
increases from 8 to 11 nm, consistent with Kontush’s data (Pharmacol Rev 2006). Conclusions: As predicted by Shen’s model, HDL size is significantly correlated with the HDL-C/ApoA-I ratio. 194 HISTONE DEACETYLASE (HDAC) INHIBITORS UPREGULATE CLUSTERIN/APOLIPOPROTEIN J EXPRESSION IN VASCULAR SMOOTH MUSCLE CELLS (VSMCS) F.M. Yatsu1 , K. Ranganna2 , O.P. Mathew2 . 1 Neurology, University of Texas Health Science Center at Houston, 2 Texas Southern University, Houston, TX, USA Objective: Clusterin/Apolipoprotein J (CLU), a secreted heterodimeric glycoprotein, composed of alpha and beta subunits is induced in VSMCs during atherosclerosis and injury-induced neointimal hyperplasia, although its functional roles in VSMCs remain obscure and mysterious. Here our goal is to confirm and evaluate the significance of CLU upregulation in proliferation arrest of VSMCs by HDAC inhibitors, butyrate and trichostatin A (TSA). Methods: VSMC proliferation is assessed by cell counting. CLU, p21Cip1 and p15INK4B expression is evaluated by western analysis and immunostaining. CLU knockout is performed with siRNA. Involvement of PI3/AKT pathway is determined by western blotting. Results: Contrast to untreated proliferating VSMCs, butyrate and TSA treated VSMCs exhibit proliferation arrest and upregulation of both cellular and secretory CLU along with increased levels of cdk/cell cycle inhibitors, p21Cip1 and p15INK4B. Moreover, CLU-specific siRNA inhibits butyrate and TSAinduced upregulation of both cellular and secretory CLU confirming butyrate and TSA indeed induce clusterin expression and secretion. Furthermore, based on the phosphorylation state of Akt and impact of PI3/Akt pathway inhibitor, Ly294002, it appears butyrate- and TSA-induced CLU expression involves PI3/Akt signaling pathway. Conclusion: Taken together HDAC inhibitors appear to have antiatherogenic potential by arresting VSMC proliferation that may be associated with CLU upregulation, which is suggested to have multiple protective roles in atherosclerosis and restenosis. Support: G12RR003045−21 and C06RR012537 from NIH/NCRR and Adriana Blood Endowment 195 CHOLESTEROL EFFLUX POTENTIAL OF SERA FROM PATIENTS BEFORE AND AFTER LDL-APHERESIS E. Favari1 , M.P. Adorni1 , F. Sbrana2 , M. Puntoni2 , F. Bigazzi2 , F. Zimetti1 , F. Bernini1 , T. Sampietro2 . 1 Pharmacological and Biological Sciences and Applied Chemistries, University of Parma, Parma, 2 CNR Institute of Clinical Physiology, Pisa, Italy Efflux of free cholesterol from peripheral cells to serum is the first step in reverse cholesterol transport. Serum cholesterol efflux potential depends on the ability of individual lipoproteins to interact with specific transporters. LDL-apheresis may dramatically affect lipoprotein profile by lowering, in addition to LDL, triglycerides and HDL. Aims: To study the impact of LDL-apheresis on 1. efflux potential of sera 2. serum ability to induce cholesterol accumulation in macrophages. Sera were obtained from 15 patients with FH receiving bi-weekly LDL-apheresis. Methods: Serum cholesterol efflux potential was tested in pathway-specific, [3 H]-cholesterol cell models. Cholesterol mass was determined in THP-1 human macrophages by fluorimetric method. Results: LDL-apheresis reduced total cholesterol and triglycerides by 70% and HDL by 30%. Cholesterol efflux by SR-BI, passive diffusion and ABCA1 were reduced by 18.2% (±1.3%; p < 0.001), 23% (±1.3%; p < 0.0001), and 24% (±1.8%; p < 0.0001) respectively. Within 48h LDL increased about two times compare to the values reached soon after LDL-apheresis treatment, while HDL and sera efflux potential returned to pre-treatment levels. LDL-apheresis reduced about 50% serum-induced cholesterol accumulation in human macrophages. This effect was maintained in the 48h after treatment. Conclusions: LDL-apheresis: 1. reduces serum cholesterol efflux potential and HDL are the only responsible for this effect; 2. reduces serum ability to induce macrophage cholesterol accumulation by both affecting LDL and HDL levels.