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PO2-28 SERUM FROM TYPE IV HYPERTRIGLYCERIDEMIC SUBJECTS ENHANCED GREATER CHOLESTEROL REMOVAL FROM ABCA1-EXPRESSING AND CHOLESTEROL LOADED J774 MACROPHAGES
Poster Sessions PO2 Lipoprotein and cholesterol metabolism deadliest complications—the cardiovascular diseases. Type 2 diabetes and cardiovascular diseases are increasing in prevalence and represent a major public health burden of the 21st century. We have focused on elucidation of the mechanism by which lipid droplets form. We have previously described that the formation of lipid droplets is dependent on PLD1 and Erk2 which both occurs in the insulin signaling pathway. Moreover, we have shown that the lipid droplet grove in size by fusion, and that this fusion process is dependent on a SNARE protein; SNAP23. We have now aimed at identifying more proteins that are important in the assembly of lipid droplets. Using proteomics we have identified a number of proteins on the lipid droplet, i. e. known lipid droplet proteins such as ADRP, TIP47 and Vimentin as well as proteins involved in lipid metabolism and membrane traffic i.e. cPLA2, lanosterol synthase, CGI58, HSL, Rab7, Rab11, Rab18 and valosin containg protein, VCP. The role of these proteins in the assembly of lipid droplets will be investigated using knockdown and overexpression experiments. Knockdown of VCP has shown a decrease in the amount of produced lipid droplets as well as in the amount of total triglycerides. These results indicate that VCP is important for the assembly of lipid droplets. PO2-26
K. Ikewaki, Y. Nakada, Y. Inoue, S. Mochizuki. Division of Cardiology, Department of Internal Medicine, Jikei University School of Medicine, Tokyo, Japan Background and Aims: Small dense LDL (sdLDL) is an emerging risk factor for coronary heart disease. However, detailed in vivo metabolism of sdLDL has been poorly understood. In order to assess sdLDL metabolism, we performed in vivo kinetic studies utilizing stable isotopically labeled leucine in 10 hypercholesterolemic patients and 5 healthy controls. Effects of statin on sdLDL metabolism were also investigated. Methods: Deuterated leucine was injected and blood samples were collected up to 48hours. sdLDL was isolated by heparin-magnesium (JLR 44:2193, 2003), followed by ultracentrifugation (d=1.019–1.063g/dL). VLDL, IDL, and LDL were also isolated by sequential ultracentrifugation. ApoB was precipitated by isopropanol method, then hydrolyzed and derivatized to determine tracer/tracee ratios of apoB by gas-chromatography mass spectrometry. Fractional catabolic rate (FCR) was estimated by SAAMII software. Results: The FCR of sdLDL apoB was 0.23 ± 0.06 pools/day, which was 35% lower than that of LDL apoB of 0.35 ± 0.12 pools/day (p<0.001). Furthermore, statin therapy significantly increased sdLDL apoB FCR by 67% (p<0.01), in addition to 94% increase in LDL apoB FCR. Conclusions: This is the first in vivo kinetic evidence of the delayed catabolism of sdLDL which was improved by statin therapy. Therefore, the impaired catabolism is translated into the longer residence time, thus supporting the proatherogenic nature of sdLDL.
isopropyl alcohol-hexane and the distribution of [3 H]cholesterol between free cholesterol and cholesteryl ester was determined by TLC. Results: Overexpression of CETP in transgenic mice decreased radiolabeled HDL-bound [3 H]cholesterol 24 and 48 hours after the label injection. However, the magnitude of P388D1 macrophage-derived [3 H]cholesterol in liver and feces did not differ between CETP-Tg and control mice on either diet. We also found reduced plasma [3 H]HDL cholesterol after the injection of [3 H]cholesterol-labeled endogenous PEM into the Tg mice fed the chow diet. However, the injection of CETP-expressing PEM did not alter macrophage RCT in CETP-Tg or control mice. Conclusion: CETP overexpression in transgenic mice does not affect RCT from macrophages to feces in vivo. PO2-28
N. Fournier 1,2 , N. Attia-Skhiri 1 , M. Cambillau 2 , A. Simon 3 , J-L. Paul 1,2 . de Biochimie, UMR INRA 1154, Faculte Des Sciences Pharmaceutiques, Chatenay-Malabry, France; 2 Service de Biochimie Cardio Vasculaire, Hopital Europeen Georges Pompidou, AP-HP, Paris, France; 3 Centre de Medecine Preventive Cardio Vasculaire, Hopital Broussais, AP-HP, Paris, France 1 Laboratoire
Hypertriglyceridemia being an independent cardiovascular risk factor, we have compared both the free cholesterol (C) efflux potential and C mass variation by exposing loaded J774 macrophages to sera from asymptomatic hypertriglyceridemic type IIb, type IV or normolipidemic (NLP) subjects (n=10/group). The cells loaded with C by incubation with acetyl LDL (75 μg/mL, 30 h) were incubated in the absence or presence of cAMP (0.3 mmol/L, 12 h), which upregulates ABCA1 (ATP Binding Cassette Transporter A1), and then incubated for 24 h with 1% serum. The cellular total C content was determined enzymatically; free C and cholesteryl ester were quantified after separation by Thin Layer Chromatography. Compared with NLP, both type IIb and IV exhibited higher ABCA1-mediated C efflux capacities, partly because higher serum pre-beta HDL levels: a positive correlation was indeed observed between C efflux and pre-beta HDL levels (r=0.46; p=0.02). The major finding was that the sera from type IV induced higher total C and cholesteryl ester mass depletion from ABCA1 overexpressing cells compared with other groups. Moreover, a positive correlation was established between C mass depletion and serum pre-beta HDL levels (r=0.41; p=0.04). In conclusion, we demonstrated for the first time that the serum pre-beta HDL present in high proportions in type IV hypertriglyceridemic subjects are not only responsible for higher C efflux potential but also for increased abilities to promote net clearance from C loaded J774 macrophages. These results reinforce thus the concept that ABCA1 macrophage overexpression could be an important pharmaceutical target to reduce cardiovascular risk. PO2-29
PO2-27
CETP OVEREXPRESSION IN TRANSGENIC MICE DOES NOT AFFECT MACROPHAGE-SPECIFIC REVERSE CHOLESTEROL TRANSPORT IN VIVO
N. Rotllan 1 , L. Calpe-Berdiel 1 , S. Suren-Castillo 1 , F. Blanco-Vaca 2 , J.C. Escola-Gil 1 . 1 Department of Biochemistry, Institut de Recerca Hospital de La Santa Creu I Sant Pau, Barcelona, Spain; 2 Department of Biochemistry, Hospital de La Santa Creu I Sant Pau, Barcelona, Spain Background and Aims: CETP expression results in a heteroexchange between HDL-cholesteryl esters and triglycerides from chylomicrons and VLDL. The impact of CETP activity variation on macrophage-specific reverse cholesterol transport (RCT) remain unknown. In this study, we assessed the effects of overexpressing CETP in transgenic (Tg) mice on macrophage-specific RCT. Methods: P388D1 and endogenous peritoneal exudates macrophages (PEM) were cultured in 75-cm tissue culture plaques at 5 million cells per plaque and incubated for 48 hours in RPMI 1640 supplemented with 5 mCi/ml of [1α,2α(n)-3 H]cholesterol. [3 H]cholesterol-labeled macrophages were injected intraperitoneally into mice maintained on a chow diet or an atherogenic diet. Mice were then individually housed in metabolic cages and stools were collected over the next two days. HDL and non-HDL-associated [3 H]cholesterol were measured after precipitation with phosphotungstic acid and magnesium ions. Liver and fecal lipids were extracted with
SERUM FROM TYPE IV HYPERTRIGLYCERIDEMIC SUBJECTS ENHANCED GREATER CHOLESTEROL REMOVAL FROM ABCA1-EXPRESSING AND CHOLESTEROL LOADED J774 MACROPHAGES
DEFICIENCY OF MACROPHAGE PHOSPHOLIPID TRANSFER PROTEIN PROTECTS AGAINST ATHEROSCLEROTIC LESION DEVELOPMENT
R. Vikstedt 1 , J. Metso 1 , D. Ye 2 , R. Hildebrand 2 , T. Van Berkel 2 , C. Ehnholm 1 , M. Jauhiainen 1 , M. Van Eck 2 . 1 National Public Health Institute, Helsinki, Finland; 2 Division of Biopharmaceutics, Leiden University, Leiden, The Netherlands Objective: Phospholipid transfer protein (PLTP) deficiency in mice is associated with a decreased susceptibility to atherosclerosis, suggesting a pro-atherogenic role for PLTP. Interestingly, PLTP is also expressed by macrophages in human atherosclerotic lesions, but its function in atherogenesis is not known. Methods: To investigate the role of macrophage PLTP in atherosclerosis, PLTP was selectively disrupted in hematopoietic cells, including macrophages, by transplantation of bone marrow from PLTP knockout (PLTP-/-) mice into LDLr receptor knockout mice. Atherosclerotic lesion formation was induced by feeding a Western-type diet for 9 wks. Results: Selective deficiency of macrophage PLTP resulted in a 1.4-fold (p<0.01) reduction in aortic root lesion area compared to mice with functional macrophage PLTP (384000±36000 μm2 in the PLTP-/- group (n=10) vs. 539000±35000 μm2 in the PLTP+/+ group (n=14)). The decreased lesion size in the PLTP-/- group coincided with a significant reduction in total cholesterol and triglyceride levels. The relative amount
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland
POSTER SESSIONS
DELAYED IN VIVO CATABOLISM OF SMALL DENSE LDL: A STABLE ISOTOPE STUDY
25
K. Ikewaki, Y. Nakada, Y. Inoue, S. Mochizuki. Division of Cardiology, Department of Internal Medicine, Jikei University School of Medicine, Tokyo, Japan Background and Aims: Small dense LDL (sdLDL) is an emerging risk factor for coronary heart disease. However, detailed in vivo metabolism of sdLDL has been poorly understood. In order to assess sdLDL metabolism, we performed in vivo kinetic studies utilizing stable isotopically labeled leucine in 10 hypercholesterolemic patients and 5 healthy controls. Effects of statin on sdLDL metabolism were also investigated. Methods: Deuterated leucine was injected and blood samples were collected up to 48hours. sdLDL was isolated by heparin-magnesium (JLR 44:2193, 2003), followed by ultracentrifugation (d=1.019–1.063g/dL). VLDL, IDL, and LDL were also isolated by sequential ultracentrifugation. ApoB was precipitated by isopropanol method, then hydrolyzed and derivatized to determine tracer/tracee ratios of apoB by gas-chromatography mass spectrometry. Fractional catabolic rate (FCR) was estimated by SAAMII software. Results: The FCR of sdLDL apoB was 0.23 ± 0.06 pools/day, which was 35% lower than that of LDL apoB of 0.35 ± 0.12 pools/day (p<0.001). Furthermore, statin therapy significantly increased sdLDL apoB FCR by 67% (p<0.01), in addition to 94% increase in LDL apoB FCR. Conclusions: This is the first in vivo kinetic evidence of the delayed catabolism of sdLDL which was improved by statin therapy. Therefore, the impaired catabolism is translated into the longer residence time, thus supporting the proatherogenic nature of sdLDL.
isopropyl alcohol-hexane and the distribution of [3 H]cholesterol between free cholesterol and cholesteryl ester was determined by TLC. Results: Overexpression of CETP in transgenic mice decreased radiolabeled HDL-bound [3 H]cholesterol 24 and 48 hours after the label injection. However, the magnitude of P388D1 macrophage-derived [3 H]cholesterol in liver and feces did not differ between CETP-Tg and control mice on either diet. We also found reduced plasma [3 H]HDL cholesterol after the injection of [3 H]cholesterol-labeled endogenous PEM into the Tg mice fed the chow diet. However, the injection of CETP-expressing PEM did not alter macrophage RCT in CETP-Tg or control mice. Conclusion: CETP overexpression in transgenic mice does not affect RCT from macrophages to feces in vivo. PO2-28
N. Fournier 1,2 , N. Attia-Skhiri 1 , M. Cambillau 2 , A. Simon 3 , J-L. Paul 1,2 . de Biochimie, UMR INRA 1154, Faculte Des Sciences Pharmaceutiques, Chatenay-Malabry, France; 2 Service de Biochimie Cardio Vasculaire, Hopital Europeen Georges Pompidou, AP-HP, Paris, France; 3 Centre de Medecine Preventive Cardio Vasculaire, Hopital Broussais, AP-HP, Paris, France 1 Laboratoire
Hypertriglyceridemia being an independent cardiovascular risk factor, we have compared both the free cholesterol (C) efflux potential and C mass variation by exposing loaded J774 macrophages to sera from asymptomatic hypertriglyceridemic type IIb, type IV or normolipidemic (NLP) subjects (n=10/group). The cells loaded with C by incubation with acetyl LDL (75 μg/mL, 30 h) were incubated in the absence or presence of cAMP (0.3 mmol/L, 12 h), which upregulates ABCA1 (ATP Binding Cassette Transporter A1), and then incubated for 24 h with 1% serum. The cellular total C content was determined enzymatically; free C and cholesteryl ester were quantified after separation by Thin Layer Chromatography. Compared with NLP, both type IIb and IV exhibited higher ABCA1-mediated C efflux capacities, partly because higher serum pre-beta HDL levels: a positive correlation was indeed observed between C efflux and pre-beta HDL levels (r=0.46; p=0.02). The major finding was that the sera from type IV induced higher total C and cholesteryl ester mass depletion from ABCA1 overexpressing cells compared with other groups. Moreover, a positive correlation was established between C mass depletion and serum pre-beta HDL levels (r=0.41; p=0.04). In conclusion, we demonstrated for the first time that the serum pre-beta HDL present in high proportions in type IV hypertriglyceridemic subjects are not only responsible for higher C efflux potential but also for increased abilities to promote net clearance from C loaded J774 macrophages. These results reinforce thus the concept that ABCA1 macrophage overexpression could be an important pharmaceutical target to reduce cardiovascular risk. PO2-29
PO2-27
CETP OVEREXPRESSION IN TRANSGENIC MICE DOES NOT AFFECT MACROPHAGE-SPECIFIC REVERSE CHOLESTEROL TRANSPORT IN VIVO
N. Rotllan 1 , L. Calpe-Berdiel 1 , S. Suren-Castillo 1 , F. Blanco-Vaca 2 , J.C. Escola-Gil 1 . 1 Department of Biochemistry, Institut de Recerca Hospital de La Santa Creu I Sant Pau, Barcelona, Spain; 2 Department of Biochemistry, Hospital de La Santa Creu I Sant Pau, Barcelona, Spain Background and Aims: CETP expression results in a heteroexchange between HDL-cholesteryl esters and triglycerides from chylomicrons and VLDL. The impact of CETP activity variation on macrophage-specific reverse cholesterol transport (RCT) remain unknown. In this study, we assessed the effects of overexpressing CETP in transgenic (Tg) mice on macrophage-specific RCT. Methods: P388D1 and endogenous peritoneal exudates macrophages (PEM) were cultured in 75-cm tissue culture plaques at 5 million cells per plaque and incubated for 48 hours in RPMI 1640 supplemented with 5 mCi/ml of [1α,2α(n)-3 H]cholesterol. [3 H]cholesterol-labeled macrophages were injected intraperitoneally into mice maintained on a chow diet or an atherogenic diet. Mice were then individually housed in metabolic cages and stools were collected over the next two days. HDL and non-HDL-associated [3 H]cholesterol were measured after precipitation with phosphotungstic acid and magnesium ions. Liver and fecal lipids were extracted with
SERUM FROM TYPE IV HYPERTRIGLYCERIDEMIC SUBJECTS ENHANCED GREATER CHOLESTEROL REMOVAL FROM ABCA1-EXPRESSING AND CHOLESTEROL LOADED J774 MACROPHAGES
DEFICIENCY OF MACROPHAGE PHOSPHOLIPID TRANSFER PROTEIN PROTECTS AGAINST ATHEROSCLEROTIC LESION DEVELOPMENT
R. Vikstedt 1 , J. Metso 1 , D. Ye 2 , R. Hildebrand 2 , T. Van Berkel 2 , C. Ehnholm 1 , M. Jauhiainen 1 , M. Van Eck 2 . 1 National Public Health Institute, Helsinki, Finland; 2 Division of Biopharmaceutics, Leiden University, Leiden, The Netherlands Objective: Phospholipid transfer protein (PLTP) deficiency in mice is associated with a decreased susceptibility to atherosclerosis, suggesting a pro-atherogenic role for PLTP. Interestingly, PLTP is also expressed by macrophages in human atherosclerotic lesions, but its function in atherogenesis is not known. Methods: To investigate the role of macrophage PLTP in atherosclerosis, PLTP was selectively disrupted in hematopoietic cells, including macrophages, by transplantation of bone marrow from PLTP knockout (PLTP-/-) mice into LDLr receptor knockout mice. Atherosclerotic lesion formation was induced by feeding a Western-type diet for 9 wks. Results: Selective deficiency of macrophage PLTP resulted in a 1.4-fold (p<0.01) reduction in aortic root lesion area compared to mice with functional macrophage PLTP (384000±36000 μm2 in the PLTP-/- group (n=10) vs. 539000±35000 μm2 in the PLTP+/+ group (n=14)). The decreased lesion size in the PLTP-/- group coincided with a significant reduction in total cholesterol and triglyceride levels. The relative amount
76th Congress of the European Atherosclerosis Society, June 10–13, 2007, Helsinki, Finland
POSTER SESSIONS
DELAYED IN VIVO CATABOLISM OF SMALL DENSE LDL: A STABLE ISOTOPE STUDY
25