- Email: [email protected]
The role of lipophagy in hepatic energy metabolism
Abstracts / Atherosclerosis 263 (2017) e1ee28
Conclusions: Our findings reveal a previously unsuspected athero-protective role for MZB cells that relies on the control of Tfh/GC response to a HCD, and uncover a new mechanism through which MZB cells can couple their unique metabolic and innate immune properties and use them to maintain a tolerogenic state.
LB2:4. MONOCYTIC GLUTAREDOXIN 1 PROTECTS MICE AGAINST OBESITY, HYPERGLYCEMIA AND ATHEROSCLEROSIS Reto Asmis1, Kevin Downs2, Sina Tavakoli3, John Short4, Huynh Nga Nguyen5. 1 Clinical Lab Sciences, UTHSCSA, San Antonio, USA; 2 Cell Systems and Anatonmy, UTHSCSA, San Antonio, USA; 3 Radiology, UTHSCSA, San Antonio, USA; 4 Pharmacology, UTHSCSA, San Antonio, USA; 5 Biochemistry, UTHSCSA, San Antonio, USA Aim: We aimed to determine the role of monocytic Grx1 in mice and in the development of atherogenesis and obesity. Methods: We transplanted bone marrow (BM) from either wild-type (WT) or Grx1-/- donor mice into atherosclerosis-prone LDLR-/- mice and fed these mice a high-fat diet (HFD) for up to 20 weeks. Results: Grx1Leuko -/- mice showed accelerated weight gain after 9 weeks followed by early onset of hyperglycemia. After 6 weeks on HFD, atherosclerotic lesions were slightly larger in Grx1Leuko -/- mice than in WT mice, but the differences did not reach statistical significance. However, after 20 weeks, Grx1Leuko -/- mice showed 36% larger lesions than WT-BM recipients, and monocyte chemotaxis in vivo was increased 1.6-fold. Adipose tissues and livers of Grx1Leuko -/- mice also showed increased macrophage content and elevated tissue inflammation as determined by IHC and qRTPCR-based gene array. Adipose tissue showed significant increases in the expression of proinflammatory genes in addition to an increased abundance of proinflammatory “crown-like” structures. In contrast, genes associated with inflammation resolving macrophages were significantly suppressed. Macrophages isolated from Grx1-/- mice and stimulated with INFg+TNFa also showed increased expression of pro-inflammatory M1-associated genes, whereas M2-associated genes were suppressed in Grx-1-/- macrophages activated with IL-4. Furthermore, macrophages from Grx1-/- mice exposed to metabolic stress also display increased protein S-glutathionylation, enhanced hypersensitization to chemokine, and impaired autophagy compared to macrophages from wild-type mice. Conclusions: We conclude that monocytic Grx1 is critical for maintaining macrophage function and immunometabolic homeostasis in mice and protects mice against obesity and atherogenesis. WORKSHOP 2.3 Hepatic lipid biology W2.3:1. REVEALING THE ROLE OF THE ENDOSMAL SORTING MACHINERY IN CHOLESTEROL HOMEOSTASIS Fedoseienko1, Karin Wolters1, Daphne Melinde Wijers1, Alina Nicolette Huijkman1, Sanne Wilbrink1, Niels Dekkers1, 1 2 Burstein , Daniel Billadeau3, Jan -Albert Kloosterhuis , Ezra Kuivenhoven1, Bart Van De Sluis1. 1 University of Groningen, University Medical Center Groningen, Department of Pediatrics, Molecular Genetics Section, Groningen, The Netherlands; 2 University of Texas Southwestern Medical Center, Dallas, USA; 3 Department of Immunology and Biochemistry, Division of Oncology Research, Mayo Clinic, Rochester, USA Aim: The CCC complex was recently identified as a pivotal multiprotein complex in LDLR trafficking. CCC colocalizes to the WASH complex, a crucial component of the endosomal sorting machinery. In this study, we further investigated the role of hepatic WASH in cholesterol homeostasis in mice. In addition, we used CRISPR/Cas9 genome editing technology to study the role of the endosomal sorting machinery in the liver. Methods: The WASH component WASH1 was specifically deleted in hepatocytes using the Cre-Lox system. Mice were fed either a chow or a high fat cholesterol diet (HFC), and subsequently plasma triglyceride (TG) and cholesterol levels were determined. LDL uptake and biotinylation assays were used to assess LDL uptake and investigate the surface proteome of
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WASH deficient primary hepatocytes. To further investigate the role of endosomal sorting machinery in cholesterol homeostasis in vivo, CRISPR/ Cas9 gene editing technology was used to edit genes specifically in hepatocytes of living mice. Results: WASH1 deficiency results in instability of the complete WASH complex. Hepatic WASH destabilization causes hypercholesterolemia in mice, and LDL uptake is adversely affected in WASH1-deficient hepatocytes. Interestingly, in addition to LDLR also LRP1 and SR-B1 are markedly reduced at the cell surface of WASH1 ablated primary hepatocytes. In contrast to elevated total plasma cholesterol levels plasma TG levels were significantly diminished in HFC-fed WASH1 knockout mice. In addition, HDL-production is likely diminished in WASH1-deficienct mice when fed a HFC-diet. Conclusions: Our results show that the endocytic system in hepatocytes is crucial to control LDL clearance but also HDL production.
W2.3:2. FGF15/19 UPREGULATES AND LOCALIZES ABCG5 AND ABCG8 TO THE CANALICULAR SURFACE OF HEPATOCYTES Sonja Pijut, Yuhuan Wang, Ailing Ji, Deneys Van Der Westhuyzen, Gregory Graf. University of Kentucky, Saha Cardiovascular Research Center, Lexington, USA Aim: To elucidate mechanisms by which therapeutic bile acids promote biliary cholesterol secretion and accelerate reverse cholesterol transport. Methods: C57Bl6/J mice were injected with two doses of carrier or FGF19 (1 ug/g bw) within an 8-hour treatment window. A second cohort of mice were fed a control diet or diet supplemented with ursodeoxycholic acid following pretreatment with a control antisense oligonucleotide (ASO) or an ASO directed against FGF4 receptor. Wild-type mice and mice lacking ABCG5 ABCG8 (G5G8) were treated with FGF19. Body weight, liver weight, bile flow rate and plasma, hepatic and biliary lipids were measured. Hepatic G5G8 was examined by immunoblotting, immunolocalizationm and real-time PCR. Results: Mice treated with FGF19 had increased gene expression of Shp and decreased Fxr, Cyp7a1, and Cyp8b1. Abcg8 mRNA was modestly increased while both ABCG5 and ABCG8 protein were increased by more than 2-fold. Increases in G5G8 were consistent with increases in biliary total cholesterol. Both UDCA and FGF19 localized G5G8 from discrete puncta dispersed in the cytosol to the canalicular surface of hepatocytes. The localization observed with UDCA was impaired in the presence of FGFR4 ASO, indicating that FGF15/19-FGFR4 signaling is required. The absence of G5G8 resulted in the diversion of cholesterol away from bile and back to the plasma compartment. Conclusions: FGF15/19 suppresses bile acid synthesis, post-transcriptionally upregulates G5G8, and localizes G5G8 to the canalicular surface of hepatocytes. UDCA causes a redistribution of G5G8, an effect that is dependent on FGF15/19-FGFR4 signaling. In the absence of G5G8, FGF15/ 19 did not acutely disrupt cholesterol metabolism.
W2.3:3. THE ROLE OF LIPOPHAGY IN HEPATIC ENERGY METABOLISM Christina Leopold1, Douglas Mashek2, Dagmar Kratky1. 1 Medical University of Graz-Department of Biochemistry and Molecular Biology, Graz, Austria; 2 University of Minnesota, Department of Biochemistry, Molecular Biology and Biophysics, Minneapolis, USA Aim: Autophagy is a well-known recycling mechanism, which acts through delivery of cytoplasmic cargo into lysosomes and subsequent degradation to provide the cell with alternative energy during starvation. Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters (CE) and triglycerides (TG) in the lysosome to generate free fatty acids (FFA) and cholesterol. The aim of this study is to show that inhibiting different lysosomal functions affects hepatic energy metabolism and consequently autophagy-dependent pathways. Methods: To determine the effects of LAL on hepatic energy metabolism we isolated primary hepatocytes and treated them with a specific inhibitor of LAL (Lalistat-2) alone or in combination with chemical compounds that
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Abstracts / Atherosclerosis 263 (2017) e1ee28
either inhibit the Ca2+-dependent fusion of lysosomes with the cell membrane or the fatty acid re-uptake from the cytosol. We further tested a TRPML-channel agonist promoting Ca2+-dependent release of lipids from the lysosome. Results: Treatment of primary hepatocytes with Lalistat-2 reduced mitochondrial ß-oxidation as well as medium TG and FFA concentrations. Similar effects were obtained by treating cells with the Ca2+-dependent inhibitor for the fusion of lysosomes with the cell membrane or co-treatments of both. By inhibiting the re-uptake of FFA from the cytosol we observed reduced medium TG and FFA levels but increased cellular lipids after fluorescence staining. However, our approach to rescue the loss of LAL via a TRPM-channel agonist did not reduce cellular lipid stain but decreased medium TG and ß-oxidation. Conclusions: We therefore conclude that loss of LAL alone independent of Ca2+ availability has the capacity to alter hepatic energy metabolism and downstream autophagy-dependent pathways. Oral Communications: Novel Insights from Experimental Studies in Atherosclerosis CO2:1. PROTECTIVE EFFECT OF PERILIPIN1 OVEREXPRESSION MACROPHAGES ON PROGRESSION OF ATHEROSCLEROSIS
Methods: ApoE-/- mice transplanted with AT1aR-/- or WT control BM underwent left renal artery clipping to generate the 2K1C model (Ang IIdependent mouse model of advanced atherosclerosis and vulnerable plaques). Results: Four weeks after clipping, mice transplanted with AT1aR-/- BM exhibited significantly reduced plaque size in both the aortic sinus (-79%) and whole aorta (-71%) compared with controls. Moreover, plaques from AT1aR-/- BM 2K1C mice showed a more stable phenotype as evidenced by significantly reduced lipid core/fibrous cap and macrophage/smooth muscle cells ratios (-82% and -88%, respectively), and increased collagen content (+70%). Aortic mRNA levels of IL-12p35, IL-1b, and TNF-a were significantly reduced in AT1aR-/- BM 2K1C mice. No significant differences in either aortic M1 or M2 macrophage markers mRNA levels, or number of circulating Ly6Chigh inflammatory monocyte and Ly6Clow resident antiinflammatory monocyte subsets were observed between the 2 groups. Splenic mRNA levels of T cell subsets (Th1, Th2, Th17 and Treg) markers did not significantly differ among the 2 groups. Conclusions: Direct AT1R activation on bone marrow-derived cells is crucial for Ang II-induced advanced atherosclerosis and plaque destabilization. AT1R activation promotes vascular inflammatory response.
IN
Miyoshi, Kyu Yong Cho, Akinobu Kohei Yamamoto, Hideaki Nakamura, Tatsuya Atsumi. Hokkaido University, Department of Rheumatology, Endocrinology and Nephrology, Sapporo, Japan Aim: Perilipin1 (PLIN1), a lipid droplet associated protein has an important role in the regulation of lipolysis and lipid storage in adipocytes. PLIN1 is also expressed in macrophages at atheroma plaques and could therefore play a role in the accumulation of lipids in arterial wall and in the development of atherosclerosis. We previously generated transgenic mice that overexpressed PLIN1 in adipose tissue and macrophages using the aP2 promoter/enhancer. We now use these transgenic mice to assess the effect of PLIN1 overexpression on the progression of atherosclerosis. Methods: PLIN1 transgenic mice (PlinTg) were crossed with apolipoprotein E knockout mice (ApoeKO). C57BL/6J, ApoeKO and PlinTg/ApoeKO mice received a normal chow diet for 20 weeks. Body weight, gonadal fat mass and plasma lipid concentrations were measured. Aortas were collected at the end of the study for quantification of atheroma lesions and histology (Oil Red O staining). Results are presented as means ± sdm. Comparisons between groups were performed by Dunn’s test or by Kruskal-Wallis test using JMP Pro (version 12.0.1). Results: Body weight, gonadal adipose mass and plasma triglyceride concentrations were not significantly different among C57BL/6J, ApoeKO, or PlinTg/ApoeKO mice, respectively. In contrast, atherosclerotic lesion area was significantly (P<0.01) increased in ApoeKO mice (14.4 ± 3.0%) compared with C57BL/6J (3.3 ± 1.2%) and PlinTg/ApoeKO (5.6 ± 1.9%) mice. Conclusions: Overexpression of PLIN1 in macrophages protected against atheroma progression in apolipoprotein E knockout mice independent of changes in gonadal fat mass or plasma lipids levels.
CO2:2. LACK OF ANGIOTENSIN II TYPE 1 RECEPTOR IN BONE MARROWDERIVED CELLS INHIBITS VULNERABLE ATHEROSCLEROTIC PLAQUE DEVELOPMENT IN 2-KIDNEY, 1-CLIP APOE-/- MICE Maxime Pellegrin1, Karima Bouzourene1, Jean -Francois Aubert1, Aimable Nahimana2, Michel A. Duchosal2, Lucia Mazzolai1. 1 Division of Angiology, University Hospital of Lausanne, Lausanne, Switzerland; 2 Central Laboratory of Hematology, University Hospital of Lausanne, Lausanne, Switzerland Aim: Angiotensin (Ang) II plays a major role in atherogenesis and vulnerable plaque development. It remains unclear, however, whether Ang II induces plaque vulnerability directly through AT1 receptor (AT1R) on bone marrow (BM)-derived cells. This study therefore examined the effects of AT1R deficiency within BM-derived cells on Ang IImediated vulnerable plaque in the 2-kidney, 1-clip [2K1C] ApoE-/- mouse model.
CO2:3. CLONAL HEMATOPOIESIS ASSOCIATED WITH TET2 DEFICIENCY ACCELERATES ATHEROSCLEROSIS IN HYPERLIPIDEMIC MICE Jose Javier Fuster, Susan Maclauchlan, Maria Angeles Zuriaga, Maya Ninan Polackal, Kenneth Walsh. Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, USA Aim: Human studies have shown that normal aging is associated with an increased frequency of somatic mutations in the hematopoietic system, which provide a competitive growth advantage to the mutant cell and allow its progressive clonal expansion (clonal hematopoiesis). These somatic mutations are associated with increased incidence of atherosclerotic CVD, suggesting an unexpected connection between mutations in blood cells and atherosclerosis. However, the existence of a cause-effect relationship remains to be established. This study tests the contribution to atherosclerosis of clonal hematopoiesis associated with ablation of TET2, the first gene reported to exhibit somatic mutations in blood cells in cancer-free individuals with clonal hematopoiesis. Methods: Competitive bone marrow transplantation (BMT) strategies with Tet2-/- donors and atherosclerosis-prone Ldlr-/- recipient mice were used to mimic the human scenario of expansion of a small number of TET2 mutant hematopoietic cells. Myeloid-specific Tet2-deficient mice and cell culture studies were used to test the contribution of Tet2-deficient macrophages to atherosclerosis. Results: BM reconstitution with 10% Tet2-/- donor cells accelerated aortic atherosclerosis in Ldlr-/- mice, mainly by generating a pool of macrophages with enhanced pro-inflammatory activity, as revealed by experiments with myeloid-restricted Tet2-deficient mice. Tet2-/- macrophages exhibited a pronounced increase in NLRP3 inflammasome-mediated IL1beta secretion. Treatment with a pharmacological inhibitor of NLRP3 abolished the atherogenic effects of clonal expansion of Tet2-deficient hematopoietic cells in mice. Conclusions: These results provide mouse genetic evidence supporting a causal connection between somatic TET2 mutations in blood cells and atherosclerotic CVD in humans and document a new mechanism of proinflammatory IL-1beta signaling regulation in atherosclerosis.
CO2:4. HOMEOSTATIC CYTOKINES INTERLEUKIN-7 (IL-7) AND IL-15 DRIVE THE EXPANSION AND ACTIVATION OF CD4+CD28NULL T CELLS IN PATIENTS WITH MYOCARDIAL INFARCTION Ingrid Elena Dumitriu, Jessica Bulenkamp, Ismita Chhetri. St. George's University of London, London, United Kingdom Aim: We have previously shown that CD4+CD28null (CD28null) T cells, a unique T lymphocyte subset with pro-inflammatory and cell-lytic
Conclusions: Our findings reveal a previously unsuspected athero-protective role for MZB cells that relies on the control of Tfh/GC response to a HCD, and uncover a new mechanism through which MZB cells can couple their unique metabolic and innate immune properties and use them to maintain a tolerogenic state.
LB2:4. MONOCYTIC GLUTAREDOXIN 1 PROTECTS MICE AGAINST OBESITY, HYPERGLYCEMIA AND ATHEROSCLEROSIS Reto Asmis1, Kevin Downs2, Sina Tavakoli3, John Short4, Huynh Nga Nguyen5. 1 Clinical Lab Sciences, UTHSCSA, San Antonio, USA; 2 Cell Systems and Anatonmy, UTHSCSA, San Antonio, USA; 3 Radiology, UTHSCSA, San Antonio, USA; 4 Pharmacology, UTHSCSA, San Antonio, USA; 5 Biochemistry, UTHSCSA, San Antonio, USA Aim: We aimed to determine the role of monocytic Grx1 in mice and in the development of atherogenesis and obesity. Methods: We transplanted bone marrow (BM) from either wild-type (WT) or Grx1-/- donor mice into atherosclerosis-prone LDLR-/- mice and fed these mice a high-fat diet (HFD) for up to 20 weeks. Results: Grx1Leuko -/- mice showed accelerated weight gain after 9 weeks followed by early onset of hyperglycemia. After 6 weeks on HFD, atherosclerotic lesions were slightly larger in Grx1Leuko -/- mice than in WT mice, but the differences did not reach statistical significance. However, after 20 weeks, Grx1Leuko -/- mice showed 36% larger lesions than WT-BM recipients, and monocyte chemotaxis in vivo was increased 1.6-fold. Adipose tissues and livers of Grx1Leuko -/- mice also showed increased macrophage content and elevated tissue inflammation as determined by IHC and qRTPCR-based gene array. Adipose tissue showed significant increases in the expression of proinflammatory genes in addition to an increased abundance of proinflammatory “crown-like” structures. In contrast, genes associated with inflammation resolving macrophages were significantly suppressed. Macrophages isolated from Grx1-/- mice and stimulated with INFg+TNFa also showed increased expression of pro-inflammatory M1-associated genes, whereas M2-associated genes were suppressed in Grx-1-/- macrophages activated with IL-4. Furthermore, macrophages from Grx1-/- mice exposed to metabolic stress also display increased protein S-glutathionylation, enhanced hypersensitization to chemokine, and impaired autophagy compared to macrophages from wild-type mice. Conclusions: We conclude that monocytic Grx1 is critical for maintaining macrophage function and immunometabolic homeostasis in mice and protects mice against obesity and atherogenesis. WORKSHOP 2.3 Hepatic lipid biology W2.3:1. REVEALING THE ROLE OF THE ENDOSMAL SORTING MACHINERY IN CHOLESTEROL HOMEOSTASIS Fedoseienko1, Karin Wolters1, Daphne Melinde Wijers1, Alina Nicolette Huijkman1, Sanne Wilbrink1, Niels Dekkers1, 1 2 Burstein , Daniel Billadeau3, Jan -Albert Kloosterhuis , Ezra Kuivenhoven1, Bart Van De Sluis1. 1 University of Groningen, University Medical Center Groningen, Department of Pediatrics, Molecular Genetics Section, Groningen, The Netherlands; 2 University of Texas Southwestern Medical Center, Dallas, USA; 3 Department of Immunology and Biochemistry, Division of Oncology Research, Mayo Clinic, Rochester, USA Aim: The CCC complex was recently identified as a pivotal multiprotein complex in LDLR trafficking. CCC colocalizes to the WASH complex, a crucial component of the endosomal sorting machinery. In this study, we further investigated the role of hepatic WASH in cholesterol homeostasis in mice. In addition, we used CRISPR/Cas9 genome editing technology to study the role of the endosomal sorting machinery in the liver. Methods: The WASH component WASH1 was specifically deleted in hepatocytes using the Cre-Lox system. Mice were fed either a chow or a high fat cholesterol diet (HFC), and subsequently plasma triglyceride (TG) and cholesterol levels were determined. LDL uptake and biotinylation assays were used to assess LDL uptake and investigate the surface proteome of
e13
WASH deficient primary hepatocytes. To further investigate the role of endosomal sorting machinery in cholesterol homeostasis in vivo, CRISPR/ Cas9 gene editing technology was used to edit genes specifically in hepatocytes of living mice. Results: WASH1 deficiency results in instability of the complete WASH complex. Hepatic WASH destabilization causes hypercholesterolemia in mice, and LDL uptake is adversely affected in WASH1-deficient hepatocytes. Interestingly, in addition to LDLR also LRP1 and SR-B1 are markedly reduced at the cell surface of WASH1 ablated primary hepatocytes. In contrast to elevated total plasma cholesterol levels plasma TG levels were significantly diminished in HFC-fed WASH1 knockout mice. In addition, HDL-production is likely diminished in WASH1-deficienct mice when fed a HFC-diet. Conclusions: Our results show that the endocytic system in hepatocytes is crucial to control LDL clearance but also HDL production.
W2.3:2. FGF15/19 UPREGULATES AND LOCALIZES ABCG5 AND ABCG8 TO THE CANALICULAR SURFACE OF HEPATOCYTES Sonja Pijut, Yuhuan Wang, Ailing Ji, Deneys Van Der Westhuyzen, Gregory Graf. University of Kentucky, Saha Cardiovascular Research Center, Lexington, USA Aim: To elucidate mechanisms by which therapeutic bile acids promote biliary cholesterol secretion and accelerate reverse cholesterol transport. Methods: C57Bl6/J mice were injected with two doses of carrier or FGF19 (1 ug/g bw) within an 8-hour treatment window. A second cohort of mice were fed a control diet or diet supplemented with ursodeoxycholic acid following pretreatment with a control antisense oligonucleotide (ASO) or an ASO directed against FGF4 receptor. Wild-type mice and mice lacking ABCG5 ABCG8 (G5G8) were treated with FGF19. Body weight, liver weight, bile flow rate and plasma, hepatic and biliary lipids were measured. Hepatic G5G8 was examined by immunoblotting, immunolocalizationm and real-time PCR. Results: Mice treated with FGF19 had increased gene expression of Shp and decreased Fxr, Cyp7a1, and Cyp8b1. Abcg8 mRNA was modestly increased while both ABCG5 and ABCG8 protein were increased by more than 2-fold. Increases in G5G8 were consistent with increases in biliary total cholesterol. Both UDCA and FGF19 localized G5G8 from discrete puncta dispersed in the cytosol to the canalicular surface of hepatocytes. The localization observed with UDCA was impaired in the presence of FGFR4 ASO, indicating that FGF15/19-FGFR4 signaling is required. The absence of G5G8 resulted in the diversion of cholesterol away from bile and back to the plasma compartment. Conclusions: FGF15/19 suppresses bile acid synthesis, post-transcriptionally upregulates G5G8, and localizes G5G8 to the canalicular surface of hepatocytes. UDCA causes a redistribution of G5G8, an effect that is dependent on FGF15/19-FGFR4 signaling. In the absence of G5G8, FGF15/ 19 did not acutely disrupt cholesterol metabolism.
W2.3:3. THE ROLE OF LIPOPHAGY IN HEPATIC ENERGY METABOLISM Christina Leopold1, Douglas Mashek2, Dagmar Kratky1. 1 Medical University of Graz-Department of Biochemistry and Molecular Biology, Graz, Austria; 2 University of Minnesota, Department of Biochemistry, Molecular Biology and Biophysics, Minneapolis, USA Aim: Autophagy is a well-known recycling mechanism, which acts through delivery of cytoplasmic cargo into lysosomes and subsequent degradation to provide the cell with alternative energy during starvation. Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters (CE) and triglycerides (TG) in the lysosome to generate free fatty acids (FFA) and cholesterol. The aim of this study is to show that inhibiting different lysosomal functions affects hepatic energy metabolism and consequently autophagy-dependent pathways. Methods: To determine the effects of LAL on hepatic energy metabolism we isolated primary hepatocytes and treated them with a specific inhibitor of LAL (Lalistat-2) alone or in combination with chemical compounds that
e14
Abstracts / Atherosclerosis 263 (2017) e1ee28
either inhibit the Ca2+-dependent fusion of lysosomes with the cell membrane or the fatty acid re-uptake from the cytosol. We further tested a TRPML-channel agonist promoting Ca2+-dependent release of lipids from the lysosome. Results: Treatment of primary hepatocytes with Lalistat-2 reduced mitochondrial ß-oxidation as well as medium TG and FFA concentrations. Similar effects were obtained by treating cells with the Ca2+-dependent inhibitor for the fusion of lysosomes with the cell membrane or co-treatments of both. By inhibiting the re-uptake of FFA from the cytosol we observed reduced medium TG and FFA levels but increased cellular lipids after fluorescence staining. However, our approach to rescue the loss of LAL via a TRPM-channel agonist did not reduce cellular lipid stain but decreased medium TG and ß-oxidation. Conclusions: We therefore conclude that loss of LAL alone independent of Ca2+ availability has the capacity to alter hepatic energy metabolism and downstream autophagy-dependent pathways. Oral Communications: Novel Insights from Experimental Studies in Atherosclerosis CO2:1. PROTECTIVE EFFECT OF PERILIPIN1 OVEREXPRESSION MACROPHAGES ON PROGRESSION OF ATHEROSCLEROSIS
Methods: ApoE-/- mice transplanted with AT1aR-/- or WT control BM underwent left renal artery clipping to generate the 2K1C model (Ang IIdependent mouse model of advanced atherosclerosis and vulnerable plaques). Results: Four weeks after clipping, mice transplanted with AT1aR-/- BM exhibited significantly reduced plaque size in both the aortic sinus (-79%) and whole aorta (-71%) compared with controls. Moreover, plaques from AT1aR-/- BM 2K1C mice showed a more stable phenotype as evidenced by significantly reduced lipid core/fibrous cap and macrophage/smooth muscle cells ratios (-82% and -88%, respectively), and increased collagen content (+70%). Aortic mRNA levels of IL-12p35, IL-1b, and TNF-a were significantly reduced in AT1aR-/- BM 2K1C mice. No significant differences in either aortic M1 or M2 macrophage markers mRNA levels, or number of circulating Ly6Chigh inflammatory monocyte and Ly6Clow resident antiinflammatory monocyte subsets were observed between the 2 groups. Splenic mRNA levels of T cell subsets (Th1, Th2, Th17 and Treg) markers did not significantly differ among the 2 groups. Conclusions: Direct AT1R activation on bone marrow-derived cells is crucial for Ang II-induced advanced atherosclerosis and plaque destabilization. AT1R activation promotes vascular inflammatory response.
IN
Miyoshi, Kyu Yong Cho, Akinobu Kohei Yamamoto, Hideaki Nakamura, Tatsuya Atsumi. Hokkaido University, Department of Rheumatology, Endocrinology and Nephrology, Sapporo, Japan Aim: Perilipin1 (PLIN1), a lipid droplet associated protein has an important role in the regulation of lipolysis and lipid storage in adipocytes. PLIN1 is also expressed in macrophages at atheroma plaques and could therefore play a role in the accumulation of lipids in arterial wall and in the development of atherosclerosis. We previously generated transgenic mice that overexpressed PLIN1 in adipose tissue and macrophages using the aP2 promoter/enhancer. We now use these transgenic mice to assess the effect of PLIN1 overexpression on the progression of atherosclerosis. Methods: PLIN1 transgenic mice (PlinTg) were crossed with apolipoprotein E knockout mice (ApoeKO). C57BL/6J, ApoeKO and PlinTg/ApoeKO mice received a normal chow diet for 20 weeks. Body weight, gonadal fat mass and plasma lipid concentrations were measured. Aortas were collected at the end of the study for quantification of atheroma lesions and histology (Oil Red O staining). Results are presented as means ± sdm. Comparisons between groups were performed by Dunn’s test or by Kruskal-Wallis test using JMP Pro (version 12.0.1). Results: Body weight, gonadal adipose mass and plasma triglyceride concentrations were not significantly different among C57BL/6J, ApoeKO, or PlinTg/ApoeKO mice, respectively. In contrast, atherosclerotic lesion area was significantly (P<0.01) increased in ApoeKO mice (14.4 ± 3.0%) compared with C57BL/6J (3.3 ± 1.2%) and PlinTg/ApoeKO (5.6 ± 1.9%) mice. Conclusions: Overexpression of PLIN1 in macrophages protected against atheroma progression in apolipoprotein E knockout mice independent of changes in gonadal fat mass or plasma lipids levels.
CO2:2. LACK OF ANGIOTENSIN II TYPE 1 RECEPTOR IN BONE MARROWDERIVED CELLS INHIBITS VULNERABLE ATHEROSCLEROTIC PLAQUE DEVELOPMENT IN 2-KIDNEY, 1-CLIP APOE-/- MICE Maxime Pellegrin1, Karima Bouzourene1, Jean -Francois Aubert1, Aimable Nahimana2, Michel A. Duchosal2, Lucia Mazzolai1. 1 Division of Angiology, University Hospital of Lausanne, Lausanne, Switzerland; 2 Central Laboratory of Hematology, University Hospital of Lausanne, Lausanne, Switzerland Aim: Angiotensin (Ang) II plays a major role in atherogenesis and vulnerable plaque development. It remains unclear, however, whether Ang II induces plaque vulnerability directly through AT1 receptor (AT1R) on bone marrow (BM)-derived cells. This study therefore examined the effects of AT1R deficiency within BM-derived cells on Ang IImediated vulnerable plaque in the 2-kidney, 1-clip [2K1C] ApoE-/- mouse model.
CO2:3. CLONAL HEMATOPOIESIS ASSOCIATED WITH TET2 DEFICIENCY ACCELERATES ATHEROSCLEROSIS IN HYPERLIPIDEMIC MICE Jose Javier Fuster, Susan Maclauchlan, Maria Angeles Zuriaga, Maya Ninan Polackal, Kenneth Walsh. Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, USA Aim: Human studies have shown that normal aging is associated with an increased frequency of somatic mutations in the hematopoietic system, which provide a competitive growth advantage to the mutant cell and allow its progressive clonal expansion (clonal hematopoiesis). These somatic mutations are associated with increased incidence of atherosclerotic CVD, suggesting an unexpected connection between mutations in blood cells and atherosclerosis. However, the existence of a cause-effect relationship remains to be established. This study tests the contribution to atherosclerosis of clonal hematopoiesis associated with ablation of TET2, the first gene reported to exhibit somatic mutations in blood cells in cancer-free individuals with clonal hematopoiesis. Methods: Competitive bone marrow transplantation (BMT) strategies with Tet2-/- donors and atherosclerosis-prone Ldlr-/- recipient mice were used to mimic the human scenario of expansion of a small number of TET2 mutant hematopoietic cells. Myeloid-specific Tet2-deficient mice and cell culture studies were used to test the contribution of Tet2-deficient macrophages to atherosclerosis. Results: BM reconstitution with 10% Tet2-/- donor cells accelerated aortic atherosclerosis in Ldlr-/- mice, mainly by generating a pool of macrophages with enhanced pro-inflammatory activity, as revealed by experiments with myeloid-restricted Tet2-deficient mice. Tet2-/- macrophages exhibited a pronounced increase in NLRP3 inflammasome-mediated IL1beta secretion. Treatment with a pharmacological inhibitor of NLRP3 abolished the atherogenic effects of clonal expansion of Tet2-deficient hematopoietic cells in mice. Conclusions: These results provide mouse genetic evidence supporting a causal connection between somatic TET2 mutations in blood cells and atherosclerotic CVD in humans and document a new mechanism of proinflammatory IL-1beta signaling regulation in atherosclerosis.
CO2:4. HOMEOSTATIC CYTOKINES INTERLEUKIN-7 (IL-7) AND IL-15 DRIVE THE EXPANSION AND ACTIVATION OF CD4+CD28NULL T CELLS IN PATIENTS WITH MYOCARDIAL INFARCTION Ingrid Elena Dumitriu, Jessica Bulenkamp, Ismita Chhetri. St. George's University of London, London, United Kingdom Aim: We have previously shown that CD4+CD28null (CD28null) T cells, a unique T lymphocyte subset with pro-inflammatory and cell-lytic