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Apolipoprotein (apo) E modulates hepatic very low density lipoprotein (VLDL) assembly and secretion
158
Wednesday June 28,200O: Workshop Abstracts W:18 Proteolysis and Plaque Rupture
In vitro rates of hepatic FAS and CS were determined by incorporation of tritiated water. Results: Plasma levels of free fatty acids (NEFA), cholesterol, ketone bodies and triacylglycerol were increased in the (-/-) mice and did not follow the circadian variation seen in (+I+) animals. In the (+/+) mice, the rates of FAS and CS were significantly higher during the dark phase (D6) than during the light phase (L6). These differences were abolished in the (-/-) mice. The lower rate of FAS at D6 in the (-I-) mice was associated with a decreased expression of acetyl CoA carboxylase (ACC) mRNA and could be related to the increased level of plasma NEFA. Surprisingly, the increased rate of hepatic CS in the (-/-) mice was associated with a marked decline in the expression of hydroxymethylglutaryl CoA reductase (HMGR) mRNA. PPARa deletion did not appear to have any effect on the expression of LDL-receptor or cholesterol ‘I-alpha hydroxylase mRNAs. Conclusions: Deletion of PPARa attenuates or abolishes the circadian rhythm of hepatic and plasma lipid metabolism. The rate of hepatic CS is increased whilst that of FAS is diminished. The FAS change may be explained by a decreased expression of ACC mRNA. The effects on CS do not appear to result from changes in HMGR mRNA but may be related to changes in the demand for a common substrate, acetyl-CoA.
WeW6.17 Generation of adult LPL-deficient mice I Juliane G. Strauss ‘, Sasa Frank*, Gabriele Knipping2, Rudolf Zechner’ ‘Institutes of Biochemistry; 2Medical Biochemistry, Karl-Franzens University, A-8010 Graz, Austria Objective: The transient adenovirus-mediated expression of LPL during the suckling period to rescue LPL knock-out mice (LO) from neonatal death and generate adult LPL deficient mice. Methods: A total of 5 x lo9 plaque forming units (pfu) of LPL-expressing adenovirus was injected intraperitoneally into mouse litters from matings of heterozygous LPL knock-out mice immediatley after birth. Results: Over 90% of treated LO-mice survived the first days of life. In 3% of all cases LO-mice survived the suckling period and stayed alive for at least one year. Rescued LO-mice were smaller than their control littermates until 3 months of age and had strongly elevated triglyceride levels in the fed (5000 mg/dl) and in the fasted state (2000 mg/dl). Total cholesterol levels were also increased in both conditions due to increased VLDL-cholesterol. LO-mice lacked HDL-cholesterol indicating that LPL is essential for the HDL biogenesis. Glucose levels of LO-mice were normal, whereas ketone bodies and FFA were elevated. Conclusion: Adult LO-mice will provide a valuable model to study the role of LPL deficiency in rodents.
I
WeW7.17
Apolipoprotein (apo) E modulates hepatic very low density lipoprotein (VLDL) assembly and secretion
Y. Huanq’~*, W.J. Brecht’, X.Q. Liu’, Y. Wang’,*, J.M. Taylor’,*, S.C. Rall, Jr.‘, R.W. Mahlev’,*. t Gladstone Institute of Cardiovascular Disease; 2University of California, San Francisco, CA 94141-9100, USA Objective: To investigate apoE’s regulatory effect on hepatic VLDL assembly/secretion in transgenic animals and transfected hepatoma cells. Methods and Results: In transgenic mice (apoE-null background) and rabbits expressing medium (10-20 mg/dl) or high (720 mg/dl) levels of human apoE in the liver, hepatic VLDL triglyceride (TG) and VLDL-apoB production rates were determined with the Triton-WR1339 method. Compared with wild-type mice, apoE medium- and high-expressing mice had increases of 29% and 56% in VLDL-TG production and 20% and 42% in VLDL-apoB production, respectively; apoE-null mice had decreases of 47% in VLDL-TG and 53% in VLDL-apoB production. Compared with nontransgenie rabbits, apoE medium- and high-expressing rabbits had increases of two- and fourfold in VLDL-TG production and 1.5- and 3-fold in VLDL-apoB production, respectively. The increased hepatic VLDL production contributed largely to hypertriglyceridemia in high-expressing mice and combined hyperli idemia in high-expressing rabbits. After 2 h of incubation with 3H-glycerol, 3PS-methionine, and 0.4 mM oleic acid, stably transfected McA-RI-I7777 cells overexpressing apoE (1.2 bglmg cell rotein/h) secreted 2.5fold more VLDL-3H-TG and twofold more VLDL-3 PS-apoB into the medium than nontransfected cells; transfected cells also had 75% higher 3H-TG content, most of which accumulated in the lumen of the endoplasmic reticulum, where VLDL assembly occurs. Microsomal TG transfer protein activity was about twofold higher in transfected than nontransfected cells and may have helped to mobilize TG to the endoplasmic reticulum.
Conclusion: ApoE overexpression stimulates the synthesis and mobilization of TG to the endoplasmic reticulum and increases hepatic assembly/secretion of TG-rich VLDL. Thus, apoE appears to be a powerful modulator of hepatic VLDL assembly and secretion.
w:18
IWeWl .18
PROTEOLYSIS
AND PLAQUE RUPTURE
Divergent roles of extracellular proteases in plaque stabilization and rupture; what are the molecular switches?
A. Newbv, T. Izzard, M. Bond, S. Hussein, A. Chase. Bristol Heart Institute, Bristol Royal lnfinnary, Bristol BS2 8Hw UK Basement membrane degrading metalloproteinases (MMPs-2 and -9) may be essential to allow vascular smooth muscle cells (VSMC) to migrate and proliferate in response to injury and inflammation. The molecular events which need to take place before quiescent VSMC in the artery wall can migrate and proliferate are however ill-defined. The presentation will identify the important events taking place during the Gl phase of the cell cycle, which have been compared in isolated rat VSMC and segments of rat aorta. The studies identify molecular switches that may be controlled by injury and extracellular proteolysis, which permit proliferation. MMPs with activity against interstitial matrix, MMPs-1, -3, -7, -12 and -13, for example, have been associated with plaque rupture rather than stabilisation. We have sought to identify common and divergent features of the regulation of the MMP genes in VSMC. These studies may identify whether plaques prone to rupture produce an unfavourable complement of MMPs, and, if so, by what mechanisms. We also hope to identify potential inhibitors so as to stabilise plaques.
WeW2 18 L._..d
Why is it that some plaques rupture and others don’t?
A.E. Becker, A.C. van der Wal, O.J. de Boer, C.M. van der Loos. Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Plaque rupture is defined as the condition in which the fibrous cap of an atherosclerotic (AS) plaque is tom apart producing a fissure into the lipid core, associated with intraplaque hemorrhage and adherent mural thrombosis. This is the most common pathology underlying acute myocardial infarction (AMI). Over the past decade it has been shown that plaques prone to rupture are characterized by a large lipid core, an attenuated fibrous cap and a dense inflammatory infiltrate composed of macrophages and T lymphocytes and, to a lesser extent, mast cells. It is presently acknowledged that the immune system is involved; AS plaques contain an inflammatory cell-mediated (Thl)-like response, responsible for the release of a variety of cytokines, growth factors and enzymes. It appears as if the balance between these products may be responsible for the eventual effects on the tissues. The release of a cascade of matrix metalloproteinases, for instance, may lead to excessive collagen breakdown and this process, in the presence of high tissue tensile forces and rheological factors, may produce plaque rupture. Why then is it that some AS plaques with characteristics alluded to above remain intact and others rupture? Atherectomy specimens from patients with AM1 show recent onset activation of T lymphocytes. This fits with clinical observations of increased inflammatory markers in peripheral blood. We have isolated T cells from AS plaques, which were tested against particular antigens, such as C. pneumoniae. Our preliminary results have shown that in some patients T cells cloned from plaques respond to C. pneumoniae. This boosts our belief that an additional stimulus serves as “the last straw that breaks the camel’s back”.
IWeW3.18
A novel adipocyte-derived plasma protein, adiponectin, suppresses lipid accumulation and class a scavenger receptor expression in human macrophages
N. Ouchi, S. Kihara, Y. Arita, A. Matsuyama, M. Nishida, Y. Okamoto, T. Nakamura, S. Yamashita, T. Funahashi, Y. Matsuzawa. Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Objectives: Obesity is the most common nutritional disorder and one of the major risk factors of atherosclerosis. However the molecular basis for the link between obesity and atherosclerosis has not been fully elucidated. We found
XIIth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
Wednesday June 28,200O: Workshop Abstracts W:18 Proteolysis and Plaque Rupture
In vitro rates of hepatic FAS and CS were determined by incorporation of tritiated water. Results: Plasma levels of free fatty acids (NEFA), cholesterol, ketone bodies and triacylglycerol were increased in the (-/-) mice and did not follow the circadian variation seen in (+I+) animals. In the (+/+) mice, the rates of FAS and CS were significantly higher during the dark phase (D6) than during the light phase (L6). These differences were abolished in the (-/-) mice. The lower rate of FAS at D6 in the (-I-) mice was associated with a decreased expression of acetyl CoA carboxylase (ACC) mRNA and could be related to the increased level of plasma NEFA. Surprisingly, the increased rate of hepatic CS in the (-/-) mice was associated with a marked decline in the expression of hydroxymethylglutaryl CoA reductase (HMGR) mRNA. PPARa deletion did not appear to have any effect on the expression of LDL-receptor or cholesterol ‘I-alpha hydroxylase mRNAs. Conclusions: Deletion of PPARa attenuates or abolishes the circadian rhythm of hepatic and plasma lipid metabolism. The rate of hepatic CS is increased whilst that of FAS is diminished. The FAS change may be explained by a decreased expression of ACC mRNA. The effects on CS do not appear to result from changes in HMGR mRNA but may be related to changes in the demand for a common substrate, acetyl-CoA.
WeW6.17 Generation of adult LPL-deficient mice I Juliane G. Strauss ‘, Sasa Frank*, Gabriele Knipping2, Rudolf Zechner’ ‘Institutes of Biochemistry; 2Medical Biochemistry, Karl-Franzens University, A-8010 Graz, Austria Objective: The transient adenovirus-mediated expression of LPL during the suckling period to rescue LPL knock-out mice (LO) from neonatal death and generate adult LPL deficient mice. Methods: A total of 5 x lo9 plaque forming units (pfu) of LPL-expressing adenovirus was injected intraperitoneally into mouse litters from matings of heterozygous LPL knock-out mice immediatley after birth. Results: Over 90% of treated LO-mice survived the first days of life. In 3% of all cases LO-mice survived the suckling period and stayed alive for at least one year. Rescued LO-mice were smaller than their control littermates until 3 months of age and had strongly elevated triglyceride levels in the fed (5000 mg/dl) and in the fasted state (2000 mg/dl). Total cholesterol levels were also increased in both conditions due to increased VLDL-cholesterol. LO-mice lacked HDL-cholesterol indicating that LPL is essential for the HDL biogenesis. Glucose levels of LO-mice were normal, whereas ketone bodies and FFA were elevated. Conclusion: Adult LO-mice will provide a valuable model to study the role of LPL deficiency in rodents.
I
WeW7.17
Apolipoprotein (apo) E modulates hepatic very low density lipoprotein (VLDL) assembly and secretion
Y. Huanq’~*, W.J. Brecht’, X.Q. Liu’, Y. Wang’,*, J.M. Taylor’,*, S.C. Rall, Jr.‘, R.W. Mahlev’,*. t Gladstone Institute of Cardiovascular Disease; 2University of California, San Francisco, CA 94141-9100, USA Objective: To investigate apoE’s regulatory effect on hepatic VLDL assembly/secretion in transgenic animals and transfected hepatoma cells. Methods and Results: In transgenic mice (apoE-null background) and rabbits expressing medium (10-20 mg/dl) or high (720 mg/dl) levels of human apoE in the liver, hepatic VLDL triglyceride (TG) and VLDL-apoB production rates were determined with the Triton-WR1339 method. Compared with wild-type mice, apoE medium- and high-expressing mice had increases of 29% and 56% in VLDL-TG production and 20% and 42% in VLDL-apoB production, respectively; apoE-null mice had decreases of 47% in VLDL-TG and 53% in VLDL-apoB production. Compared with nontransgenie rabbits, apoE medium- and high-expressing rabbits had increases of two- and fourfold in VLDL-TG production and 1.5- and 3-fold in VLDL-apoB production, respectively. The increased hepatic VLDL production contributed largely to hypertriglyceridemia in high-expressing mice and combined hyperli idemia in high-expressing rabbits. After 2 h of incubation with 3H-glycerol, 3PS-methionine, and 0.4 mM oleic acid, stably transfected McA-RI-I7777 cells overexpressing apoE (1.2 bglmg cell rotein/h) secreted 2.5fold more VLDL-3H-TG and twofold more VLDL-3 PS-apoB into the medium than nontransfected cells; transfected cells also had 75% higher 3H-TG content, most of which accumulated in the lumen of the endoplasmic reticulum, where VLDL assembly occurs. Microsomal TG transfer protein activity was about twofold higher in transfected than nontransfected cells and may have helped to mobilize TG to the endoplasmic reticulum.
Conclusion: ApoE overexpression stimulates the synthesis and mobilization of TG to the endoplasmic reticulum and increases hepatic assembly/secretion of TG-rich VLDL. Thus, apoE appears to be a powerful modulator of hepatic VLDL assembly and secretion.
w:18
IWeWl .18
PROTEOLYSIS
AND PLAQUE RUPTURE
Divergent roles of extracellular proteases in plaque stabilization and rupture; what are the molecular switches?
A. Newbv, T. Izzard, M. Bond, S. Hussein, A. Chase. Bristol Heart Institute, Bristol Royal lnfinnary, Bristol BS2 8Hw UK Basement membrane degrading metalloproteinases (MMPs-2 and -9) may be essential to allow vascular smooth muscle cells (VSMC) to migrate and proliferate in response to injury and inflammation. The molecular events which need to take place before quiescent VSMC in the artery wall can migrate and proliferate are however ill-defined. The presentation will identify the important events taking place during the Gl phase of the cell cycle, which have been compared in isolated rat VSMC and segments of rat aorta. The studies identify molecular switches that may be controlled by injury and extracellular proteolysis, which permit proliferation. MMPs with activity against interstitial matrix, MMPs-1, -3, -7, -12 and -13, for example, have been associated with plaque rupture rather than stabilisation. We have sought to identify common and divergent features of the regulation of the MMP genes in VSMC. These studies may identify whether plaques prone to rupture produce an unfavourable complement of MMPs, and, if so, by what mechanisms. We also hope to identify potential inhibitors so as to stabilise plaques.
WeW2 18 L._..d
Why is it that some plaques rupture and others don’t?
A.E. Becker, A.C. van der Wal, O.J. de Boer, C.M. van der Loos. Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Plaque rupture is defined as the condition in which the fibrous cap of an atherosclerotic (AS) plaque is tom apart producing a fissure into the lipid core, associated with intraplaque hemorrhage and adherent mural thrombosis. This is the most common pathology underlying acute myocardial infarction (AMI). Over the past decade it has been shown that plaques prone to rupture are characterized by a large lipid core, an attenuated fibrous cap and a dense inflammatory infiltrate composed of macrophages and T lymphocytes and, to a lesser extent, mast cells. It is presently acknowledged that the immune system is involved; AS plaques contain an inflammatory cell-mediated (Thl)-like response, responsible for the release of a variety of cytokines, growth factors and enzymes. It appears as if the balance between these products may be responsible for the eventual effects on the tissues. The release of a cascade of matrix metalloproteinases, for instance, may lead to excessive collagen breakdown and this process, in the presence of high tissue tensile forces and rheological factors, may produce plaque rupture. Why then is it that some AS plaques with characteristics alluded to above remain intact and others rupture? Atherectomy specimens from patients with AM1 show recent onset activation of T lymphocytes. This fits with clinical observations of increased inflammatory markers in peripheral blood. We have isolated T cells from AS plaques, which were tested against particular antigens, such as C. pneumoniae. Our preliminary results have shown that in some patients T cells cloned from plaques respond to C. pneumoniae. This boosts our belief that an additional stimulus serves as “the last straw that breaks the camel’s back”.
IWeW3.18
A novel adipocyte-derived plasma protein, adiponectin, suppresses lipid accumulation and class a scavenger receptor expression in human macrophages
N. Ouchi, S. Kihara, Y. Arita, A. Matsuyama, M. Nishida, Y. Okamoto, T. Nakamura, S. Yamashita, T. Funahashi, Y. Matsuzawa. Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Objectives: Obesity is the most common nutritional disorder and one of the major risk factors of atherosclerosis. However the molecular basis for the link between obesity and atherosclerosis has not been fully elucidated. We found
XIIth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000