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Influence of dietary fats on human monocyte activation in vitro
Abstracts / Atherosclerosis Supplements 9 (2008) 83–102
associated with differences in postprandial lipemia relates to the small sample size of postprandial studies. More recently, we have carried out postprandial study [Genetics of Lipid Lowering Drugs and Diet Network (GOLDN)] including over 1000 subjects that in addition to receiving a standardized initial fat load, they were subjected to a second one after receiving several weeks of fenofibrate treatment. Moreover, we used NMR to measure lipoprotein subclasses in the fasting and postprandial phases. Using this experimental design, we are currently examining a large number of candidate genes to characterize which ones act as significant modulators of lipemic response and other cardiovascular risk factors (i.e., inflammation biomarkers). Preliminary data for some of these candidate genes (i.e., perilipin) will be presented. doi:10.1016/j.atherosclerosissup.2008.09.426 Oxidation of chylomicron remnants and vascular dysfunction Kathleen Margaret Botham (PhD, DSc) Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London NW1 0TU, UK Recent evidence suggests that chylomicron remnants (CMR), the lipoproteins which carry dietary lipid in the blood, may play a direct role in the initiation of atherosclerosis by influencing vascular function. Unlike low-density lipoprotein (LDL), CMR do not require prior oxidation to bring about potentially pro-atherogenic effects on vascular endothelial cell function and macrophage foam cell formation. However, CMR carry oxidized lipids from the diet and may also become oxidized in the body, thus it is important to establish how the oxidative state of the particles may modulate these effects. Pharmacological studies have demonstrated that oxidation of CMR significantly enhances their inhibitory effects on endothelium-dependent vascular relaxation and their potentiation of vasoconstriction in rat and pig arteries. In striking contrast to the effects of LDL oxidation, however, the induction of macrophage foam cell formation has been found to be inversely related to the oxidative state of CMR. Thus, oxidation of CMR has potentially pro-atherogenic effects on endothelial function, but appears to protect against foam cell generation. These findings indicate that the oxidative state of CMR may cause important changes in the atherogenicity of the particles. doi:10.1016/j.atherosclerosissup.2008.09.427
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Influence of dietary fats on human monocyte activation in vitro C. Bentley, N. Hathaway, C. DePascale, M. Avella, C. Wheeler-Jones, K. Botham, C. Lawson ∗ Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London NW1 0TU, UK Background: In early atherosclerosis the frequency of activated monocytes in the peripheral circulation is amplified, and migration of monocytes into the walls of the aorta and large arteries is increased. There is evidence from our laboratory and others that lipoproteins of dietary origin including chylomicron remnants (CMR), are pro-atherogenic. Here we have measured activation of human monocytes by synthetic CMR-like particles (CRLP). Methods: Human monocytes were isolated from whole blood by negative selection, and incubated with CRLPs for 24 h. Generation of reactive oxygen species (ROS) was measured after pre-loading of monocytes with dihydrorhodamine-1,2,3 and analysis in a fluorescent microtitre plate reader. Constitutive and CRLP-mediated monocyte secretion of pro-inflammatory chemokines was measured using a commercial ELISA. Chemotaxis of monocytes towards CCL-2 after pre-exposure to CRLP was measured using transwell culture and analysis by flow cytometry. U0126 (×10−6 M), PDTC (×10−4 M) and apocynin (×10−4 M) were used to determine the roles of the MEKERK and NF-B pathways in chemokine secretion and ROS generation. Results: We previously showed that CRLP induced the generation of ROS in primary human monocytes within a few minutes. We have extended these studies and have detected ROS in monocytes for up to 48 h after the beginning of co-culture with CRLP. Furthermore, using pharmacological inhibitors we have shown that ROS generation is inhibited by NF-B inhibition (PDTC) but not MEK inhibition (UO126) or by NADPH oxidase inhibition (apocynin). CRLP induced a significant reduction in CCL2 secretion. U0126 or PDTC significantly reduced CCL2 secretion, with no further effect of co-incubation with CRLP. In contrast, whilst incubation with U0126 or PDTC significantly reduced CXCL8 secretion, the effect was reversed by CRLP. Chemotaxis towards CCL-2 was increased in monocytes preexposed to CRLP and this was reversed after addition of exogenous CCL-2 into the upper transwell chamber. Conclusions: CRLP-mediated inhibition of CCL2 secretion by monocytes enhances migration towards CCL-2. CRLP also stimulates CXCL8 secretion by monocytes, which as well as its function as a neutrophil chemoattractant, has recently been shown to increase monocyte firm adhesion to endothelium. Therefore, by inhibition of CCL2 and stimulation of CXCL8 secretion, CMR could augment monocyte trans-endothelial migration into a developing neointima in vivo. These findings lend support to the current hypothesis that post-prandial lipids contribute to the inflammatory milieu
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Abstracts / Atherosclerosis Supplements 9 (2008) 83–102
seen in susceptible areas of the artery wall in early atherosclerosis, and suggest that it could be in part due to interactions with monocytes or other leukocytes within the blood. Acknowledgements This work was supported in part by grants from The British heart Foundation and The University of London Central Research Fund. doi:10.1016/j.atherosclerosissup.2008.09.428 Exacerbated production of intestinal lymph chylomicrons is associated with increased intestinal transport of cholesterol during hyper-insulinemia in the JCR:LA-cp rodent D.F. Vine 1,∗ , S.E. Kelly 1 , K. MacNaughton 1 , Z. 1 1 1,2 Hassanali , R. Mangat , K. Madsen , S.D. Proctor 1 1 Metabolic
and Cardiovascular Diseases Laboratory, Alberta Institute for Human Nutrition, Canada 2 Department of Gastroenterology, University of Alberta, Edmonton, Alberta, Canada Background: The role of the intestine in the absorption of dietary and endogenous production of lipids has emerged in recent years as a contributor to the first-pass metabolism of lipids, and indeed to whole body lipid metabolism. At present, there remains a limited understanding of the physiological mechanisms and regulatory factors involved in the ‘absorption to chylomicron secretion’ axis of lipid transport in the intestine, particularly during conditions of obesity and insulin resistance (IR). Emerging evidence suggests insulin plays a direct role in the expression of intestinal lipid transporters and modulating the lipidogenic pathway of chylomicron (CM) production. Recent findings suggest CM apolipoprotein-B48 (apo-B48) secretion and the mRNA expression of cholesterol transporters, such as NPC1L1 and ABCG5/8, are altered in different insulin states. However the simultaneous functional assessment of cholesterol transport and chylomicron synthesis in these conditions remains unexplored. Objective: To determine the production of intestinal lymph chylomicrons in the fasted and fed state, and the intestinal flux of cholesterol under normo- and hyper-insulinemic conditions in the JCR:LA-cp rodent. Methods: Chylomicron production was determined directly by sampling lymph from donor normo- and hyperinsulinemic JCR:LA-cp rats following cannulation of the mesenteric lymphatic duct during conditions of fasted (saline infusion) and fat-fed states (20% Intralipid infusion). Lymph was collected during both the fasted phase and the fat-fed phase from each animal. Apo-B48 was quantified using western immuno-blot methods. Intestinal cholesterol transport was assessed in vitro using segments of jejunum isolated from fasted animals, and then mounted in modified Ussing chambers. An emulsion containing unlabelled cholesterol
substrate and [3− H]-cholesterol was used, and the apparent permeability of cholesterol was calculated (%/cm2 ). Results: Hyper-insulinemic JCR:LA-cp animals were observed to have 2.5-fold greater production of intestinal apo-B48 during the fasted state relative to control normo-insulinemic animals (1917 ± 346 g/ml vs. 750 ± 102 g/ml, respectively, p < 0.01). The net amount of cholesterol and TG in lymph was not different between normo- and hyper-insulinemic animals collected during the fasted phase. However, the same hyper-insulinemic animals had a further net increase (×1.8-fold; p < 0.05) in apoB48 following a lipid infusion relative to control animals. In contrast, to the fasted state, cholesterol in lymph collected from hyper-insulinemic animals during the fat-fed state increased 2-fold (p < 0.05) relative to normo-insulinemic animals (161.9 ± 27.7 mg/dL vs. 79.7 ± 10.2 mg/dL). Consistent with fasted data, preliminary findings have shown the absorptive flux of cholesterol is increased (mucosal to serosal flux: 32.5 ± 2.0 %/cm2 vs. 25.5 ± 3.2 %/cm2 ), and the luminal efflux of cholesterol is increased 2-fold (serosal to mucosal flux: 26.2 ± 5.0 vs. 64.5 ± 5.2) in the hyper-insulinemic compared to normo-insulinemic animals, respectively. Conclusions: These results suggest that hyper-insulinemia alters cholesterol transport with perhaps an adaptive response to increase the luminal flux of cholesterol, at least in the fasted state. Moreover, our mesenteric lymphatic data indicates that under hyper-insulinemic conditions, the intestine oversecretes smaller and less lipidogenic CM particles during the fasted state, and the over-production of CM is exacerbated further following a lipid load. Collectively, our results support the concept that during conditions of hyper-insulinemia the intestine has altered lipid transport and CM production, which may contribute significantly to dyslipidemia in the fasted and post-prandial state. Keywords: Intestinal cholesterol transport; Ussing chamber; apo-B48; Chylomicrons; Post-prandial lipemia; Insulin resistance; Obesity; Metabolic syndrome.
Acknowledgments Funding for this work has been provided in part by Heart and Stroke Foundation of Canada and the Natural Science and Engineering Research Council of Canada. doi:10.1016/j.atherosclerosissup.2008.09.429
associated with differences in postprandial lipemia relates to the small sample size of postprandial studies. More recently, we have carried out postprandial study [Genetics of Lipid Lowering Drugs and Diet Network (GOLDN)] including over 1000 subjects that in addition to receiving a standardized initial fat load, they were subjected to a second one after receiving several weeks of fenofibrate treatment. Moreover, we used NMR to measure lipoprotein subclasses in the fasting and postprandial phases. Using this experimental design, we are currently examining a large number of candidate genes to characterize which ones act as significant modulators of lipemic response and other cardiovascular risk factors (i.e., inflammation biomarkers). Preliminary data for some of these candidate genes (i.e., perilipin) will be presented. doi:10.1016/j.atherosclerosissup.2008.09.426 Oxidation of chylomicron remnants and vascular dysfunction Kathleen Margaret Botham (PhD, DSc) Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London NW1 0TU, UK Recent evidence suggests that chylomicron remnants (CMR), the lipoproteins which carry dietary lipid in the blood, may play a direct role in the initiation of atherosclerosis by influencing vascular function. Unlike low-density lipoprotein (LDL), CMR do not require prior oxidation to bring about potentially pro-atherogenic effects on vascular endothelial cell function and macrophage foam cell formation. However, CMR carry oxidized lipids from the diet and may also become oxidized in the body, thus it is important to establish how the oxidative state of the particles may modulate these effects. Pharmacological studies have demonstrated that oxidation of CMR significantly enhances their inhibitory effects on endothelium-dependent vascular relaxation and their potentiation of vasoconstriction in rat and pig arteries. In striking contrast to the effects of LDL oxidation, however, the induction of macrophage foam cell formation has been found to be inversely related to the oxidative state of CMR. Thus, oxidation of CMR has potentially pro-atherogenic effects on endothelial function, but appears to protect against foam cell generation. These findings indicate that the oxidative state of CMR may cause important changes in the atherogenicity of the particles. doi:10.1016/j.atherosclerosissup.2008.09.427
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Influence of dietary fats on human monocyte activation in vitro C. Bentley, N. Hathaway, C. DePascale, M. Avella, C. Wheeler-Jones, K. Botham, C. Lawson ∗ Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London NW1 0TU, UK Background: In early atherosclerosis the frequency of activated monocytes in the peripheral circulation is amplified, and migration of monocytes into the walls of the aorta and large arteries is increased. There is evidence from our laboratory and others that lipoproteins of dietary origin including chylomicron remnants (CMR), are pro-atherogenic. Here we have measured activation of human monocytes by synthetic CMR-like particles (CRLP). Methods: Human monocytes were isolated from whole blood by negative selection, and incubated with CRLPs for 24 h. Generation of reactive oxygen species (ROS) was measured after pre-loading of monocytes with dihydrorhodamine-1,2,3 and analysis in a fluorescent microtitre plate reader. Constitutive and CRLP-mediated monocyte secretion of pro-inflammatory chemokines was measured using a commercial ELISA. Chemotaxis of monocytes towards CCL-2 after pre-exposure to CRLP was measured using transwell culture and analysis by flow cytometry. U0126 (×10−6 M), PDTC (×10−4 M) and apocynin (×10−4 M) were used to determine the roles of the MEKERK and NF-B pathways in chemokine secretion and ROS generation. Results: We previously showed that CRLP induced the generation of ROS in primary human monocytes within a few minutes. We have extended these studies and have detected ROS in monocytes for up to 48 h after the beginning of co-culture with CRLP. Furthermore, using pharmacological inhibitors we have shown that ROS generation is inhibited by NF-B inhibition (PDTC) but not MEK inhibition (UO126) or by NADPH oxidase inhibition (apocynin). CRLP induced a significant reduction in CCL2 secretion. U0126 or PDTC significantly reduced CCL2 secretion, with no further effect of co-incubation with CRLP. In contrast, whilst incubation with U0126 or PDTC significantly reduced CXCL8 secretion, the effect was reversed by CRLP. Chemotaxis towards CCL-2 was increased in monocytes preexposed to CRLP and this was reversed after addition of exogenous CCL-2 into the upper transwell chamber. Conclusions: CRLP-mediated inhibition of CCL2 secretion by monocytes enhances migration towards CCL-2. CRLP also stimulates CXCL8 secretion by monocytes, which as well as its function as a neutrophil chemoattractant, has recently been shown to increase monocyte firm adhesion to endothelium. Therefore, by inhibition of CCL2 and stimulation of CXCL8 secretion, CMR could augment monocyte trans-endothelial migration into a developing neointima in vivo. These findings lend support to the current hypothesis that post-prandial lipids contribute to the inflammatory milieu
100
Abstracts / Atherosclerosis Supplements 9 (2008) 83–102
seen in susceptible areas of the artery wall in early atherosclerosis, and suggest that it could be in part due to interactions with monocytes or other leukocytes within the blood. Acknowledgements This work was supported in part by grants from The British heart Foundation and The University of London Central Research Fund. doi:10.1016/j.atherosclerosissup.2008.09.428 Exacerbated production of intestinal lymph chylomicrons is associated with increased intestinal transport of cholesterol during hyper-insulinemia in the JCR:LA-cp rodent D.F. Vine 1,∗ , S.E. Kelly 1 , K. MacNaughton 1 , Z. 1 1 1,2 Hassanali , R. Mangat , K. Madsen , S.D. Proctor 1 1 Metabolic
and Cardiovascular Diseases Laboratory, Alberta Institute for Human Nutrition, Canada 2 Department of Gastroenterology, University of Alberta, Edmonton, Alberta, Canada Background: The role of the intestine in the absorption of dietary and endogenous production of lipids has emerged in recent years as a contributor to the first-pass metabolism of lipids, and indeed to whole body lipid metabolism. At present, there remains a limited understanding of the physiological mechanisms and regulatory factors involved in the ‘absorption to chylomicron secretion’ axis of lipid transport in the intestine, particularly during conditions of obesity and insulin resistance (IR). Emerging evidence suggests insulin plays a direct role in the expression of intestinal lipid transporters and modulating the lipidogenic pathway of chylomicron (CM) production. Recent findings suggest CM apolipoprotein-B48 (apo-B48) secretion and the mRNA expression of cholesterol transporters, such as NPC1L1 and ABCG5/8, are altered in different insulin states. However the simultaneous functional assessment of cholesterol transport and chylomicron synthesis in these conditions remains unexplored. Objective: To determine the production of intestinal lymph chylomicrons in the fasted and fed state, and the intestinal flux of cholesterol under normo- and hyper-insulinemic conditions in the JCR:LA-cp rodent. Methods: Chylomicron production was determined directly by sampling lymph from donor normo- and hyperinsulinemic JCR:LA-cp rats following cannulation of the mesenteric lymphatic duct during conditions of fasted (saline infusion) and fat-fed states (20% Intralipid infusion). Lymph was collected during both the fasted phase and the fat-fed phase from each animal. Apo-B48 was quantified using western immuno-blot methods. Intestinal cholesterol transport was assessed in vitro using segments of jejunum isolated from fasted animals, and then mounted in modified Ussing chambers. An emulsion containing unlabelled cholesterol
substrate and [3− H]-cholesterol was used, and the apparent permeability of cholesterol was calculated (%/cm2 ). Results: Hyper-insulinemic JCR:LA-cp animals were observed to have 2.5-fold greater production of intestinal apo-B48 during the fasted state relative to control normo-insulinemic animals (1917 ± 346 g/ml vs. 750 ± 102 g/ml, respectively, p < 0.01). The net amount of cholesterol and TG in lymph was not different between normo- and hyper-insulinemic animals collected during the fasted phase. However, the same hyper-insulinemic animals had a further net increase (×1.8-fold; p < 0.05) in apoB48 following a lipid infusion relative to control animals. In contrast, to the fasted state, cholesterol in lymph collected from hyper-insulinemic animals during the fat-fed state increased 2-fold (p < 0.05) relative to normo-insulinemic animals (161.9 ± 27.7 mg/dL vs. 79.7 ± 10.2 mg/dL). Consistent with fasted data, preliminary findings have shown the absorptive flux of cholesterol is increased (mucosal to serosal flux: 32.5 ± 2.0 %/cm2 vs. 25.5 ± 3.2 %/cm2 ), and the luminal efflux of cholesterol is increased 2-fold (serosal to mucosal flux: 26.2 ± 5.0 vs. 64.5 ± 5.2) in the hyper-insulinemic compared to normo-insulinemic animals, respectively. Conclusions: These results suggest that hyper-insulinemia alters cholesterol transport with perhaps an adaptive response to increase the luminal flux of cholesterol, at least in the fasted state. Moreover, our mesenteric lymphatic data indicates that under hyper-insulinemic conditions, the intestine oversecretes smaller and less lipidogenic CM particles during the fasted state, and the over-production of CM is exacerbated further following a lipid load. Collectively, our results support the concept that during conditions of hyper-insulinemia the intestine has altered lipid transport and CM production, which may contribute significantly to dyslipidemia in the fasted and post-prandial state. Keywords: Intestinal cholesterol transport; Ussing chamber; apo-B48; Chylomicrons; Post-prandial lipemia; Insulin resistance; Obesity; Metabolic syndrome.
Acknowledgments Funding for this work has been provided in part by Heart and Stroke Foundation of Canada and the Natural Science and Engineering Research Council of Canada. doi:10.1016/j.atherosclerosissup.2008.09.429