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Session Webcast
Introduction
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Intracellular cholesterol trafficking
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Impact of lipid droplets - implications for human disease
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Heterogeneity of LDL Uptake Responses in Individual Hypercholesterolemia Patients
Abstract
Background and Aims
Despite widespread genetic analyses, we understand little of how hypercholesterolemia evolves differently in individual patients. Cellular LDL receptor (LDLR) expression, translocation to the cell surface and ultimately LDL internalization governs clearance of blood LDL. Currently, we cannot readily measure these parameters from individual patients due to high cost and long turnaround times. We set up an automated platform that allows dissection of these cellular pathways in primary human cells with improved efficiency.
Methods
We established a multi-parametric imaging platform for primary human peripheral blood mononuclear cells. This allows us to quantify LDLR surface abundance, LDL uptake and lipid storage at the single cell level from the same samples. We studied individuals with verified LDLR mutations from the METSIM (METabolic Syndrome In Men) cohort as well as hypercholesterolemic individuals lacking LDLR mutations from the FINRISK 2012 cohort. Up to date, we have analysed >60 patient samples.
Results
First, we show that our analysis of primary cells is superior to their immortalized counterparts, which display altered cellular lipid metabolism. For each patient we can quantify multiple cell populations with different LDL internalization rates under basal and stimulated conditions. We observe substantial differences in LDL internalization and LDLR surface abundance in individuals bearing identical mutations in the LDLR gene. Additionally, we demonstrate that individuals without LDLR mutations may have decreased LDL uptake. Reduced LDL uptake is often accompanied by increased cellular lipid storage, indicative of reprogrammed lipid metabolism.
Conclusions
Automated profiling provides novel insights into disease mechanisms for individual patients and will be of diagnostic value.
Webcast
Defective lysosomal cholesteryl ester processing promotes the formation and altered phenotype of arterial smooth muscle but not macrophage foam cells in humans and mice
Abstract
Background and Aims
We previously reported that smooth muscle cells (SMCs) comprise the majority of foam cells in human and apoE-/- mouse atheromas. In the present study we investigated the potential role of lysosomal dysfunction and lysosomal acid lipase (LAL) in foam cell formation by SMCs and macrophages in vitro and in vivo.
Methods
Human and mouse arterial SMCs and macrophages treated with aggregated LDL (agLDL) to generate foam cells, and foam cells isolated from apoE-deficient mice, were investigated for lysosomal cholesterol metabolism and sites of lipid storage. Cultured foam cells and human and mouse atheromas were analyzed for LAL expression.
Results
Following agLDL loading, confocal microscopy revealed retention of cholesteryl ester droplets within lysosomal compartments in human and mouse SMCs, while macrophages showed most lipid droplets in cytosolic compartments. Macrophage foam cells isolated from apoE-/- mice showed higher cell granularity as determined by forward scatter on fluorescence-activated cell sorting when compared to SMC foam cells. SMC foam cells showed no defects in lysosomal acidification or proteolysis, however, LAL expression and activity was markedly reduced in SMCs compared to macrophages both in culture and in human and mouse atheromas.
Conclusions
Arterial SMC foam cells show a striking reduction in LAL activity and storage of lipoprotein cholesteryl esters primarily in lysosomal compartments when compared to macrophage foam cells both in vitro and in vivo. This provides a likely reason for the propensity of SMCs to form the majority of atheroma foam cells, and a novel target for the prevention and regression of atherosclerosis.
Webcast
Wnt5a promotes endosomal cholesterol trafficking to the ER and protects against atherosclerosis
Abstract
Background and Aims
The Wnt ligand, Wnt5a limits cellular cholesterol accumulation, however, its role in atherosclerosis is unknown.
Methods
To test whether Wnt5a protects against atherosclerosis, we generated SM22Cre+/Wnt5aflox/flox/LDLR-/- mice (smWnt5a-) and littermate controls SM22Cre-/Wnt5aflox/flox/LDLR-/- mice (smWnt5a+) using the Cre/lox system, and studied their vascular phenotype.
Results
We found that mice deleted for Wnt5a in vascular smooth muscle cells (VSMCs) developed atherosclerosis lesions when fed a cholesterol-rich diet. VSMCs accumulated cholesterol in enlarged endosome/lysosome (LELs), and lipofuscin containing vesicles. We identified Wnt5a, as a member of the nutrient/energy/stress sensor, mTORC1 scaffolding complex, which drives lysosomal function and promotes cholesterol trafficking. Treatment of VSMCs with mTORC1 inhibitors or with recombinant Wnt5a decreased activity of mTORC1 allowing endosomal exit of the cholesterol to the ER. This raised ER cholesterol and limited cholesterol accumulation.
Conclusions
Thus, absence of Wnt5a decoupled mTORC1 from variations in LELs sterol levels, and this resulted in accumulation of large intracellular inclusion bodies, large LELs, decreased cholesterol content in the ER, and activation of the sterol regulatory element-binding protein-2 (SREBP-2) a master regulator of cholesterol biosynthesis. These results reveal an unexpected function of the Wnt5a pathway, and provide a conceptually new basis for future drug development to prevent cholesterol accumulation, atherosclerosis, and lysosomal storage diseases.
Webcast
Effect of Monocytes on NLRP3 Inflammasome Activation in Vascular Smooth Muscle Cells Phenotypic Switch and Foam Cells Formation in Atherosclerosis
Abstract
Background and Aims
Atherosclerosis is a lipid driven, chronic inflammatory disease. The monocytes expansion is causally linked to the enlargement of the atherosclerotic lesion, while NLRP3 inflammasome activation has a major role in atherogenesis progression. We aim to reveal the effect of monocytes/macrophages on NLRP3 inflammasome activation in vascular smooth cells (VSMCs) and their phenotypic switch/foam cells formation.
Methods
Flow cytometry, qPCR, caspase 1 and pyroptosis assay showed the effect of oxLDL activated monocytes on VSMCs NLRP3 inflammasome activation and phenotypic switch in a co-culture system. Immunofluorescence quantification and ELISA illustrated the NLRP3 inflammasome activation in VSMCs in aortic roots of Apoe-/- mice on normal or high cholesterol diet (HCD).
Results
In the presence of oxLDL activated monocytes VSMCs contractile proteins - α-SMA and SM22α and the transcription factor Oct-4 preserving VSMCs contractile phenotype were downregulated, while the expression of the macrophages markers - MAC2, F4/80 and CD68 and KLF4 promoting atherosclerotic plaque pathogenesis were upregulated in VSMCs. In parallel, oxLDL activated monocytes conditioned medium promoted VSMCs caspase 1 activation and pyroptosis, programmed cell death. In vivo Apoe-/- mice fed HCD exhibited in the aortic roots an increased in α-SMA cells expressing CD68 in addition to NLRP3 and Caspase 1, Apoe-/- mice derived VSMCs secreted IL-1b (Figure 1).
Conclusions
These results show that oxLDL activated monocytes trigger VSMCs phenotypic switch, NLRP3 inflammasome activation, which in hypercholesteremia is linked to VSMCs phenotypic transformation in atherosclerosis. In conclusion, monocytes are a potent trigger of NLRP3 inflammasome activation, VSMCs phenotypic switch and atherosclerotic disease progression.
Webcast
Worldwide prevalence of familial hypercholesterolemia: Meta-analyses of 11 million subjects
Abstract
Background and Aims
Background: Despite the enrichment of familial hypercholesterolemia(FH) in subjects with ischemic heart disease(IHD), premature IHD, and hyperlipidemia, overall prevalence estimates are not available.
Aims: Provide worldwide estimates of FH prevalence in subjects with IHD, premature IHD, and hyperlipidemia compared with individuals in the general population.
Methods
Methods: In this systematic review and meta-analyses, Embase, Pubmed and Web of Science search until June 3, 2019 for peer-reviewed literature and conference abstracts reporting a heterozygous FH prevalence in non-founder populations revealed 104 studies eligible for inclusion.
Results
Results: Estimates of FH prevalence were pooled using random effects meta-analyses and were 0.32%(95%Confidence interval:0.26-0.39%,[corresponding to 1:313]) in 10,921,310 unique individuals in the general population(33,036 FH cases) based on 44 studies, 3.2%(2.2-4.3%,[1:31]) in 84,479 unique subjects with IHD(2,103 FH cases) based on 28 studies, 6.7%(4.9-8.7%,[1:15]) in 31,316 unique subjects with premature IHD(1,471 FH cases) based on 32 studies, and 15.4%(9.6-22.2%,[1:6]) in 27,271 unique subjects with hyperlipidemia(2,107 FH cases) based on 19 studies. Study-differences were investigated using subgroups based on publication year, geography, FH diagnostic criteria, and quality score. Seventeen out of 195 countries(9%) in the world have reported an FH prevalence for the general population, leaving 178(91%) countries in the world with an unknown prevalence.
Conclusions
Conclusion: Compared with 1:313 in individuals in the general population, FH prevalence is 10-fold higher in subjects with IHD, 20-fold higher in subjects with premature IHD, and 50-fold higher in subjects with hyperlipidemia. The prevalence of FH in the general population is unknown in 9 of 10 countries in the world.