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Vitamin D protects against atherosclerosis via regulation of macrophage foam cell formation and polarization in hypercholesterolemic swine
Abstracts / Atherosclerosis 241 (2015) e72ee148
Ox-LDL was given before Wnts their protective effects, both on uptake and apoptosis, were not observed. In summary this suggests that these Wnt proteins may have profound effects on foam cell formation and apoptosis, in particular modulating Ox-LDL uptake rather than lipid clearance. EAS-0365. TRANSCRIPTIONAL PROFILING OF THE HUMAN M-CSF-1-DRIVEN MONOCYTE-MACROPHAGE TRANSITION IDENTIFIES GENES REGULATING CHOLESTEROL EFFLUX AND SYNTHESIS S. Hamby 1, *, F. Cambien 2, W.H. Ouwehand 3, A.H. Goodall 1, 2, 3. on behalf of the CARDIOGENICS Consortium1 1 Cardiovascular Sciences, University of Leicester, Leicester, United Kingdom; 2 Cardiovascular Genomics, INSERM, Paris, France; 3 Department of Haematology, University of Cambridge, Cambridge, United Kingdom
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cores of less advanced plaques in coronary artery; whereas CCR7þ M1macrophages were more concentrated in the advanced atheromas, especially in the fibrous cap but absent from foam cell-accumulated cores. Vitamin D-deficiency decreased the expression of LXRa, ABCA1, ABCG1 and promoted cholesterol accumulation in THP-1-macrophage-derived foam cells via impaired cholesterol efflux. Elevated intercellular free cholesterol in 1,25(OH)2D3-deficient media polarized macrophages to M1phenotype with increased expression of IL-1b, IL-6, and MCP-1. Calcitriol markedly induced CYP27A1 expression and increased 27-hydroxycholesterol levels, which induced LXRa, ABCA1 and ABCG1 expression, stimulated cholesterol efflux, and reduced cholesterol accumulation and M1-polarization in THP-1-macrophage-derived foam cells. Effects of 1,25(OH)2D3 in macrophages were inhibited by VDR antagonist. Conclusion: Vitamin D inhibits atherosclerosis in hypercholesterolemic swine via controlling intercellular cholesterol efflux and macrophage polarization.
* Corresponding author. MCSF-1 induces differentiation of monocytes into “M4-2-like” macrophages and plays a role in the monocyte-to-macrophage transition within plaques. Here we have analysed changes in gene expression between monocytes from 458 healthy subjects and paired, monocyte-derived macrophages cultured for 7 days with M-CSF1 (50mg.L-1) in macrophage serum-free medium (Invitrogen). Gene expression was analysed on Illumina’s Human Ref-8 arrays, which contained 24,526 probes corresponding to 18,311 distinct genes. Patterns of gene expression were analyzed using QUSAGE software linked to the Reactome database to identify key pathways. Pathways were excluded if <3 or <40% of genes were detected, or if the mean log-fold change was >0.3 or <-0.3. The most highly up-regulated gene was ApoE and, the three most highly up-regulated pathways (log fold change >1.25; p<10-17) contained overlapping sets of 22 genes involved in HDL and lipid transport , and lipoprotein metabolism. The fourth highest pathway contained 66.7% of the genes involved in cholesterol synthesis; mostly catalases such as HMGCoA synthase, and dehydrocholesterol reductases (DHCR7 and DHCR24), while the next three pathways were linked to bile acid synthesis and metabolism , and contained genes involved in reverse cholesterol transport such as CYP27A1. Interestingly none of the pathways showed any significant differences in relation to gender, age or the BMI of the subjects. In summary QUSAGE analysis allows identification of key regulatory pathways in M-CSF-1 macrophages that fit with a cell that is both synthesizing and clearing cholesterol. It is important to note that these changes occurred in the absence of exogenous lipoproteins. EAS-0438. VITAMIN D PROTECTS AGAINST ATHEROSCLEROSIS VIA REGULATION OF MACROPHAGE FOAM CELL FORMATION AND POLARIZATION IN HYPERCHOLESTEROLEMIC SWINE D. Agrawal*, K. Yin, V. Swier, L. Tang, M. Radwan. Center for Clinical & Translational Science, Creighton University School of Medicine, Omaha, USA
* Corresponding author. Objective: Prevalence of Vitamin D-deficiency and its association with the risk of cardiovascular disease prompted us to evaluate the effect of vitamin status on macrophages in lipid metabolism and atherosclerosis in hypercholesterolemic swine. Methods: Yucatan microswine were fed with vitamin D-deficient, -sufficient (1,000 IU/d) or vitamin D-supplemented (3000IU/d) high cholesterol diet for 48 weeks. Serum lipids and 25(OH)D3 levels were regularly measured. M1/M2 macrophages and histology of coronary arteries were analyzed. Effect of vitamin-D on apolipoprotein A-I (Apo-AI) was examined in human THP-1 macrophage-derived foam cells. Results: Vitamin D-deficiency decreased plasma HDL and apo-AI levels, expression of liver-X-receptor-a (LXRa), ATP-binding cassette transporterA1 (ABCA1) and ABCG1, and promoted cholesterol accumulation and atherosclerosis in hypercholesterolemic microswine. The CD206þ M2macrophages were predominantly present in the foam cell-accumulated
EAS-0536. CHARACTERISING HUMAN MONOCYTE SUBSETS AS LIPID VEHICLES FOLLOWING DIETARY STIMULI M. Rahman 1, *, G. Frost 2, J.F. Scott 3, D.O. Haskard 3, K.J. Woollard 2. 1 Department of Medicine / National Heart & Lung Institute, Imperial College London, London, United Kingdom; 2 Department of Medicine, Imperial College London, London, United Kingdom; 3 National Heart & Lung Institute, Imperial College London, London, United Kingdom
* Corresponding author. An increased inflammatory response associated with recruitment of blood monocytes is thought to play a role in atherogenesis. The role of dietary lipids remains enigmatic, as has the mechanism through which monocytes/macrophages may process lipids within existing atherosclerotic plaques. Another important factor is monocyte heterogeneity that can exhibit subset-specific functions. Therefore, we explored the postprandial response of monocyte subsets to dietary lipids from intervention studies in healthy human donors. Healthy volunteers were fed a commercial mixed high fat meal (MFM) or high saturated fat meal (SFM). Monocytes were isolated from fasting and postprandial blood using FACS, then analysed for surface protein expression, intracellular lipid staining and gene expression. Monocytes from MFM exhibited highly heterogeneous subset-specific postprandial responses that were either due to donor-specific reactions or from multiple stimuli in the mixed meal. SFM on the other hand resulted in a moderate increase of CD11c expression, a downregulation of inflammatory gene expression and some difference in lipid handling genes in all monocyte subsets. Both diets exhibited postprandial accumulation of intracellular lipid vesicles in all monocytes, indicating blood monocytes act as potent carriers of neutral lipids, which may provide a lipid delivery target in atherosclerosis. We are now further characterising the postprandial molecular lipid responses and performing in-vivo tracking studies to understand the kinetics of monocyte lipid uptake following meals rich in saturated fats. In conclusion, dietary fat intake can modulate innate immune responses and this may have further implications on postprandial lipid transport and monocyte function in health and cardiovascular disease. EAS-0623. FOAM CELL FORMATION IN VIVO IS PRO-FIBROTIC A. Newby*, A. Thomas. School of Clinical Sciences, University of Bristol, Bristol, United Kingdom
* Corresponding author. Foam cell formation is a key step in the initiation of atherosclerosis but its role in lesion progression is unclear. To investigate this further, we compared the transcriptomes between mouse foam cell macrophages (FCMs) generated in subcutaneous sponges in ApoE null mice and nonfoamy macrophages (NFMs) from control C57Bl6 mice using the Illumina platform, validated and extended by quantitative RT-PCR and coupled with immunohistochemistry for proteins and signalling molecules in brachiocephalic artery plaques in ApoE null mice.
Ox-LDL was given before Wnts their protective effects, both on uptake and apoptosis, were not observed. In summary this suggests that these Wnt proteins may have profound effects on foam cell formation and apoptosis, in particular modulating Ox-LDL uptake rather than lipid clearance. EAS-0365. TRANSCRIPTIONAL PROFILING OF THE HUMAN M-CSF-1-DRIVEN MONOCYTE-MACROPHAGE TRANSITION IDENTIFIES GENES REGULATING CHOLESTEROL EFFLUX AND SYNTHESIS S. Hamby 1, *, F. Cambien 2, W.H. Ouwehand 3, A.H. Goodall 1, 2, 3. on behalf of the CARDIOGENICS Consortium1 1 Cardiovascular Sciences, University of Leicester, Leicester, United Kingdom; 2 Cardiovascular Genomics, INSERM, Paris, France; 3 Department of Haematology, University of Cambridge, Cambridge, United Kingdom
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cores of less advanced plaques in coronary artery; whereas CCR7þ M1macrophages were more concentrated in the advanced atheromas, especially in the fibrous cap but absent from foam cell-accumulated cores. Vitamin D-deficiency decreased the expression of LXRa, ABCA1, ABCG1 and promoted cholesterol accumulation in THP-1-macrophage-derived foam cells via impaired cholesterol efflux. Elevated intercellular free cholesterol in 1,25(OH)2D3-deficient media polarized macrophages to M1phenotype with increased expression of IL-1b, IL-6, and MCP-1. Calcitriol markedly induced CYP27A1 expression and increased 27-hydroxycholesterol levels, which induced LXRa, ABCA1 and ABCG1 expression, stimulated cholesterol efflux, and reduced cholesterol accumulation and M1-polarization in THP-1-macrophage-derived foam cells. Effects of 1,25(OH)2D3 in macrophages were inhibited by VDR antagonist. Conclusion: Vitamin D inhibits atherosclerosis in hypercholesterolemic swine via controlling intercellular cholesterol efflux and macrophage polarization.
* Corresponding author. MCSF-1 induces differentiation of monocytes into “M4-2-like” macrophages and plays a role in the monocyte-to-macrophage transition within plaques. Here we have analysed changes in gene expression between monocytes from 458 healthy subjects and paired, monocyte-derived macrophages cultured for 7 days with M-CSF1 (50mg.L-1) in macrophage serum-free medium (Invitrogen). Gene expression was analysed on Illumina’s Human Ref-8 arrays, which contained 24,526 probes corresponding to 18,311 distinct genes. Patterns of gene expression were analyzed using QUSAGE software linked to the Reactome database to identify key pathways. Pathways were excluded if <3 or <40% of genes were detected, or if the mean log-fold change was >0.3 or <-0.3. The most highly up-regulated gene was ApoE and, the three most highly up-regulated pathways (log fold change >1.25; p<10-17) contained overlapping sets of 22 genes involved in HDL and lipid transport , and lipoprotein metabolism. The fourth highest pathway contained 66.7% of the genes involved in cholesterol synthesis; mostly catalases such as HMGCoA synthase, and dehydrocholesterol reductases (DHCR7 and DHCR24), while the next three pathways were linked to bile acid synthesis and metabolism , and contained genes involved in reverse cholesterol transport such as CYP27A1. Interestingly none of the pathways showed any significant differences in relation to gender, age or the BMI of the subjects. In summary QUSAGE analysis allows identification of key regulatory pathways in M-CSF-1 macrophages that fit with a cell that is both synthesizing and clearing cholesterol. It is important to note that these changes occurred in the absence of exogenous lipoproteins. EAS-0438. VITAMIN D PROTECTS AGAINST ATHEROSCLEROSIS VIA REGULATION OF MACROPHAGE FOAM CELL FORMATION AND POLARIZATION IN HYPERCHOLESTEROLEMIC SWINE D. Agrawal*, K. Yin, V. Swier, L. Tang, M. Radwan. Center for Clinical & Translational Science, Creighton University School of Medicine, Omaha, USA
* Corresponding author. Objective: Prevalence of Vitamin D-deficiency and its association with the risk of cardiovascular disease prompted us to evaluate the effect of vitamin status on macrophages in lipid metabolism and atherosclerosis in hypercholesterolemic swine. Methods: Yucatan microswine were fed with vitamin D-deficient, -sufficient (1,000 IU/d) or vitamin D-supplemented (3000IU/d) high cholesterol diet for 48 weeks. Serum lipids and 25(OH)D3 levels were regularly measured. M1/M2 macrophages and histology of coronary arteries were analyzed. Effect of vitamin-D on apolipoprotein A-I (Apo-AI) was examined in human THP-1 macrophage-derived foam cells. Results: Vitamin D-deficiency decreased plasma HDL and apo-AI levels, expression of liver-X-receptor-a (LXRa), ATP-binding cassette transporterA1 (ABCA1) and ABCG1, and promoted cholesterol accumulation and atherosclerosis in hypercholesterolemic microswine. The CD206þ M2macrophages were predominantly present in the foam cell-accumulated
EAS-0536. CHARACTERISING HUMAN MONOCYTE SUBSETS AS LIPID VEHICLES FOLLOWING DIETARY STIMULI M. Rahman 1, *, G. Frost 2, J.F. Scott 3, D.O. Haskard 3, K.J. Woollard 2. 1 Department of Medicine / National Heart & Lung Institute, Imperial College London, London, United Kingdom; 2 Department of Medicine, Imperial College London, London, United Kingdom; 3 National Heart & Lung Institute, Imperial College London, London, United Kingdom
* Corresponding author. An increased inflammatory response associated with recruitment of blood monocytes is thought to play a role in atherogenesis. The role of dietary lipids remains enigmatic, as has the mechanism through which monocytes/macrophages may process lipids within existing atherosclerotic plaques. Another important factor is monocyte heterogeneity that can exhibit subset-specific functions. Therefore, we explored the postprandial response of monocyte subsets to dietary lipids from intervention studies in healthy human donors. Healthy volunteers were fed a commercial mixed high fat meal (MFM) or high saturated fat meal (SFM). Monocytes were isolated from fasting and postprandial blood using FACS, then analysed for surface protein expression, intracellular lipid staining and gene expression. Monocytes from MFM exhibited highly heterogeneous subset-specific postprandial responses that were either due to donor-specific reactions or from multiple stimuli in the mixed meal. SFM on the other hand resulted in a moderate increase of CD11c expression, a downregulation of inflammatory gene expression and some difference in lipid handling genes in all monocyte subsets. Both diets exhibited postprandial accumulation of intracellular lipid vesicles in all monocytes, indicating blood monocytes act as potent carriers of neutral lipids, which may provide a lipid delivery target in atherosclerosis. We are now further characterising the postprandial molecular lipid responses and performing in-vivo tracking studies to understand the kinetics of monocyte lipid uptake following meals rich in saturated fats. In conclusion, dietary fat intake can modulate innate immune responses and this may have further implications on postprandial lipid transport and monocyte function in health and cardiovascular disease. EAS-0623. FOAM CELL FORMATION IN VIVO IS PRO-FIBROTIC A. Newby*, A. Thomas. School of Clinical Sciences, University of Bristol, Bristol, United Kingdom
* Corresponding author. Foam cell formation is a key step in the initiation of atherosclerosis but its role in lesion progression is unclear. To investigate this further, we compared the transcriptomes between mouse foam cell macrophages (FCMs) generated in subcutaneous sponges in ApoE null mice and nonfoamy macrophages (NFMs) from control C57Bl6 mice using the Illumina platform, validated and extended by quantitative RT-PCR and coupled with immunohistochemistry for proteins and signalling molecules in brachiocephalic artery plaques in ApoE null mice.