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Apolipoprotein a-i mimetic peptide 4f blocks sphingomyelinase-induced ldl aggregation: Interaction of the amphipathic helical peptide with ldl particles
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Abstracts / Atherosclerosis 235 (2014) e11–e26
isolated from WT mice showed lower levels of smooth muscle a-actin (-946%; ***P¼0.001 ) and calponin (-7418%; ***P¼0.001 ), and increased Col1a1 mRNA levels (2.300.3 fold ***P¼0.001) after stimulation with PDGF-BB. Finally, the proliferation rate of PCSK9-/- SMCs was significantly lower compared to PCSK9-/- SMCs reconstituted with PCSK9 encoding plasmid (doubling time 41.21.9 h vs. 32.23.1 h; ***P¼0.01). Conclusion: The present results suggest a favorable action of PCSK9 on neointima formation in response to perivascular carotid, probably facilitating the phenotypic switch of medial SMCs and their proliferation. The present work was supported by the grant 2012-0549 from Fondazione Cariplo, ITALY Novel protective effects of HDL EAS-0182. APOLIPOPROTEIN A-I MIMETIC PEPTIDE 4F BLOCKS SPHINGOMYELINASE-INDUCED LDL AGGREGATION: INTERACTION OF THE AMPHIPATHIC HELICAL PEPTIDE WITH LDL PARTICLES S. Nguyena, P. Terob, M. Navabc, A. Fogelmanc, P. Kovanena, O. Katariinaa a
Biomedicum Helsinki, Wihuri Research Institute, Helsinki, Finland; Program in Structural Biology and Biophysics Institute of Biotechnology, University of Helsinki, Helsinki, Finland; c Division of Cardiology Department of Medicine David Geffen School of Medicine, University of California, Los Angeles CA 90095, USA b
be significantly decreased (50%) in Apoe-/- mice compared to wild-type controls, which may be a function of the absence of HDL in these mice. Using photoactivatable-ribonucleoside-crosslinked-immunoprecipitation-high-throughput-sequencing, we found that HDL transfer multiple miR-92a isomiR variants from macrophages to HCAECs, which were found in association with Argonaute2 RNA-induced silencing complexes. Using whole-genome arrays, we found that HDL alters the expression of many genes in HCAECs, including the significant down-regulation of 18 putative miR-92a targets. Strikingly, miR-92a was found to be significantly increased 8.5-fold on HDL from CKD subjects. Results: Interleukin-33 (IL-33) is a cytokine and nuclear transcription factor largely expressed in endothelial cells. HDL from both normal and CKD subjects significantly decreased IL-33 expression in HCAECs, as determined by real-time PCR and microarrays. In a mouse model of CKD (Apoe-/-; 5/6 nephrectomy), IL-33 expression was increased in aortic endothelial cells; however, a single HDL injection (4mg) reduced IL-33 mRNA back to sham levels at 7d post-injection. Most interestingly, HDL loaded with locked-nucleic acid miR-92a inhibitors blocked HDL’s ability to suppress CKD induced IL-33 expression in the aortic endothelium. IL-33 may represent a novel marker of endothelial dysfunction, as IL-33 mRNA levels were also found to be increased in HCAECs under disturbed flow conditions. Conclusion: Results from this study suggest that HDL suppression of IL-33 in CKD requires miR-92a, which is also transferred to endothelial cells by HDL. Novel protective effects of HDL
Objectives: Lipolytic modification of LDL by sphingomyelinase (SMase) generates aggregated particles having a strong affinity for human arterial proteoglycans, a factor underlying LDL retention in the arterial wall. Here, we evaluated the effects of apoA-I mimetic peptide 4F on structural and functional properties of the SMase-modified LDL particles. Methods: LDL and LDL pretreated with 4F were incubated with SMase for various periods of time, after which their aggregation, structure, and proteoglycan binding were analyzed. Results: 4F blocked SMase-induced LDL aggregation in a dose-dependent fashion when present in the incubation mixture or even when added to SMase-premodified LDL particles. Pretreatment of LDL with 4F rendered LDL particles less susceptible to SMase hydrolysis, prevented SMaseinduced LDL aggregation, and reduced the binding of the modified LDL particles to human aortic proteoglycans. Incubation of LDL with 4F induced structural changes in apolipoprotein (apo) B-100 of LDL particles by increasing the alpha helix content of the protein and inhibited the conformational changes of apoB-100 induced by SMase modification. Conclusion: 4F stabilizes LDL particles by altering the conformation of apoB-100 and so blocks SMase-induced LDL aggregation. The present results provide evidence for a novel antiatherogenic role of 4F. Novel protective effects of HDL EAS-0835. NOVEL HDL FUNCTIONS ASSOCIATED WITH INTERCELLULAR COMMUNICATION, ATHEROSCLEROSIS, AND CHRONIC KIDNEY DISEASE C.B. Weisea, C.L. Totha, S.R. Landstreeta, R.C. Taylora, F. Tabetb, K.A. Ryeb, L.H. Hofmeistera, D.G. Harrisona, V. Kona, K. Vickersa a Medicine, Vanderbilt University School of Medicine, Nashville, USA; b Centre for Vascular Research, The University of New South Wales, Sydney NSW, Australia
Objectives: Chronic kidney disease (CKD) is associated with endothelial dysfunction and atherosclerosis. High-density lipoproteins (HDL) serve as a general cargo carrier for a wide-variety of proteins, nucleic acids, and small molecules which likely confer many of HDL’s alternative functions. Methods: Human coronary artery endothelial cells (HCAEC) treated with native HDL were found to have increased intracellular levels of miR-92a, a miRNA recently found to protect against endothelial dysfunction and atherosclerosis. Moreover, aortic endothelial miR-92a levels were found to
EAS-0836. A LOSS-OF-FUNCTION MUTATION IN SCARB1 SEVERELY IMPAIRS SR-BI PROCESSING AND ACTIVITY IN HUMANS P. Zanonia, D.B. Laracha, S.A. Khetarpala, W.F. Hancock-Ceruttia, G.M. Pelosob, A. Rodriguesa, J. Tohyamaa, J.T. Billheimera, S. Kathiresanb, M. Cuchela, D.J. Radera a Division of Translational Medicine and Human Genetics Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA; b Center for Human Genetic Research, Massachusetts General Hospital, Boston, USA
Objectives: SR-BI, encoded by SCARB1, is the main receptor for HDL in humans. Through sequencing of subjects with elevated HDL-C, we identified 2 homozygous and 16 heterozygous subjects for the Pro376Leu variant in SCARB1. This is, to our knowledge, the first identification of humans homozygous for a missense variant in SCARB1. We assessed the functional impact of this variant on SR-BI function and its clinical consequences. Methods: We analyzed lipid profiles and clinical characteristics of carriers. We tested the effect of the mutation on SR-BI expression, processing, and HDL-C uptake in vitro using transfected COS7 cells and iPSC-derived human hepatocytes. Additionally, we investigated the impact of the mutation through AAV-mediated hepatic overexpression in Scarb1 KO mice. Results: Mean HDL-C levels for the 2 homozygotes were 161 and 95 mg/dL, respectively, while the average for the heterozygotes was 8720 mg/dl. Likewise, homozygotes and heterozygotes had elevated plasma Apo A-I levels compared with controls. In iPSC-derived hepatocytes and transfected COS7 cells, the mutation conferred a w 80% reduction in selective cholesterol uptake from HDL relative to control cells. Similarly, inScarb1 KO mice, overexpression of the Pro376Leu mutant failed to reduce HDL-C levels and to restore hepatic selective cholesterol uptake of from HDL. Furthermore, in these three models we found decreased SR-BI levels and impaired N-glycosylation of the mutant protein. Also, the Pro376Leu mutation impaired cell-surface translocation of SR-BI, thus severely reducing its activity. Conclusion: The Pro376Leu mutation inhibits post-translational processing and maturation of SR-BI, impairing its translocation to the cell surface. This results in a profound reduction in SR-BI activity in vitro and in vivo. Consequently, this mutation results in greatly elevated HDL-C levels in humans. These subjects, the first reported homozygotes for a deleterious mutation in SCARB1, offer an unprecedented opportunity to study SR-BI’s role in HDL metabolism and CHD in humans.
Abstracts / Atherosclerosis 235 (2014) e11–e26
isolated from WT mice showed lower levels of smooth muscle a-actin (-946%; ***P¼0.001 ) and calponin (-7418%; ***P¼0.001 ), and increased Col1a1 mRNA levels (2.300.3 fold ***P¼0.001) after stimulation with PDGF-BB. Finally, the proliferation rate of PCSK9-/- SMCs was significantly lower compared to PCSK9-/- SMCs reconstituted with PCSK9 encoding plasmid (doubling time 41.21.9 h vs. 32.23.1 h; ***P¼0.01). Conclusion: The present results suggest a favorable action of PCSK9 on neointima formation in response to perivascular carotid, probably facilitating the phenotypic switch of medial SMCs and their proliferation. The present work was supported by the grant 2012-0549 from Fondazione Cariplo, ITALY Novel protective effects of HDL EAS-0182. APOLIPOPROTEIN A-I MIMETIC PEPTIDE 4F BLOCKS SPHINGOMYELINASE-INDUCED LDL AGGREGATION: INTERACTION OF THE AMPHIPATHIC HELICAL PEPTIDE WITH LDL PARTICLES S. Nguyena, P. Terob, M. Navabc, A. Fogelmanc, P. Kovanena, O. Katariinaa a
Biomedicum Helsinki, Wihuri Research Institute, Helsinki, Finland; Program in Structural Biology and Biophysics Institute of Biotechnology, University of Helsinki, Helsinki, Finland; c Division of Cardiology Department of Medicine David Geffen School of Medicine, University of California, Los Angeles CA 90095, USA b
be significantly decreased (50%) in Apoe-/- mice compared to wild-type controls, which may be a function of the absence of HDL in these mice. Using photoactivatable-ribonucleoside-crosslinked-immunoprecipitation-high-throughput-sequencing, we found that HDL transfer multiple miR-92a isomiR variants from macrophages to HCAECs, which were found in association with Argonaute2 RNA-induced silencing complexes. Using whole-genome arrays, we found that HDL alters the expression of many genes in HCAECs, including the significant down-regulation of 18 putative miR-92a targets. Strikingly, miR-92a was found to be significantly increased 8.5-fold on HDL from CKD subjects. Results: Interleukin-33 (IL-33) is a cytokine and nuclear transcription factor largely expressed in endothelial cells. HDL from both normal and CKD subjects significantly decreased IL-33 expression in HCAECs, as determined by real-time PCR and microarrays. In a mouse model of CKD (Apoe-/-; 5/6 nephrectomy), IL-33 expression was increased in aortic endothelial cells; however, a single HDL injection (4mg) reduced IL-33 mRNA back to sham levels at 7d post-injection. Most interestingly, HDL loaded with locked-nucleic acid miR-92a inhibitors blocked HDL’s ability to suppress CKD induced IL-33 expression in the aortic endothelium. IL-33 may represent a novel marker of endothelial dysfunction, as IL-33 mRNA levels were also found to be increased in HCAECs under disturbed flow conditions. Conclusion: Results from this study suggest that HDL suppression of IL-33 in CKD requires miR-92a, which is also transferred to endothelial cells by HDL. Novel protective effects of HDL
Objectives: Lipolytic modification of LDL by sphingomyelinase (SMase) generates aggregated particles having a strong affinity for human arterial proteoglycans, a factor underlying LDL retention in the arterial wall. Here, we evaluated the effects of apoA-I mimetic peptide 4F on structural and functional properties of the SMase-modified LDL particles. Methods: LDL and LDL pretreated with 4F were incubated with SMase for various periods of time, after which their aggregation, structure, and proteoglycan binding were analyzed. Results: 4F blocked SMase-induced LDL aggregation in a dose-dependent fashion when present in the incubation mixture or even when added to SMase-premodified LDL particles. Pretreatment of LDL with 4F rendered LDL particles less susceptible to SMase hydrolysis, prevented SMaseinduced LDL aggregation, and reduced the binding of the modified LDL particles to human aortic proteoglycans. Incubation of LDL with 4F induced structural changes in apolipoprotein (apo) B-100 of LDL particles by increasing the alpha helix content of the protein and inhibited the conformational changes of apoB-100 induced by SMase modification. Conclusion: 4F stabilizes LDL particles by altering the conformation of apoB-100 and so blocks SMase-induced LDL aggregation. The present results provide evidence for a novel antiatherogenic role of 4F. Novel protective effects of HDL EAS-0835. NOVEL HDL FUNCTIONS ASSOCIATED WITH INTERCELLULAR COMMUNICATION, ATHEROSCLEROSIS, AND CHRONIC KIDNEY DISEASE C.B. Weisea, C.L. Totha, S.R. Landstreeta, R.C. Taylora, F. Tabetb, K.A. Ryeb, L.H. Hofmeistera, D.G. Harrisona, V. Kona, K. Vickersa a Medicine, Vanderbilt University School of Medicine, Nashville, USA; b Centre for Vascular Research, The University of New South Wales, Sydney NSW, Australia
Objectives: Chronic kidney disease (CKD) is associated with endothelial dysfunction and atherosclerosis. High-density lipoproteins (HDL) serve as a general cargo carrier for a wide-variety of proteins, nucleic acids, and small molecules which likely confer many of HDL’s alternative functions. Methods: Human coronary artery endothelial cells (HCAEC) treated with native HDL were found to have increased intracellular levels of miR-92a, a miRNA recently found to protect against endothelial dysfunction and atherosclerosis. Moreover, aortic endothelial miR-92a levels were found to
EAS-0836. A LOSS-OF-FUNCTION MUTATION IN SCARB1 SEVERELY IMPAIRS SR-BI PROCESSING AND ACTIVITY IN HUMANS P. Zanonia, D.B. Laracha, S.A. Khetarpala, W.F. Hancock-Ceruttia, G.M. Pelosob, A. Rodriguesa, J. Tohyamaa, J.T. Billheimera, S. Kathiresanb, M. Cuchela, D.J. Radera a Division of Translational Medicine and Human Genetics Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA; b Center for Human Genetic Research, Massachusetts General Hospital, Boston, USA
Objectives: SR-BI, encoded by SCARB1, is the main receptor for HDL in humans. Through sequencing of subjects with elevated HDL-C, we identified 2 homozygous and 16 heterozygous subjects for the Pro376Leu variant in SCARB1. This is, to our knowledge, the first identification of humans homozygous for a missense variant in SCARB1. We assessed the functional impact of this variant on SR-BI function and its clinical consequences. Methods: We analyzed lipid profiles and clinical characteristics of carriers. We tested the effect of the mutation on SR-BI expression, processing, and HDL-C uptake in vitro using transfected COS7 cells and iPSC-derived human hepatocytes. Additionally, we investigated the impact of the mutation through AAV-mediated hepatic overexpression in Scarb1 KO mice. Results: Mean HDL-C levels for the 2 homozygotes were 161 and 95 mg/dL, respectively, while the average for the heterozygotes was 8720 mg/dl. Likewise, homozygotes and heterozygotes had elevated plasma Apo A-I levels compared with controls. In iPSC-derived hepatocytes and transfected COS7 cells, the mutation conferred a w 80% reduction in selective cholesterol uptake from HDL relative to control cells. Similarly, inScarb1 KO mice, overexpression of the Pro376Leu mutant failed to reduce HDL-C levels and to restore hepatic selective cholesterol uptake of from HDL. Furthermore, in these three models we found decreased SR-BI levels and impaired N-glycosylation of the mutant protein. Also, the Pro376Leu mutation impaired cell-surface translocation of SR-BI, thus severely reducing its activity. Conclusion: The Pro376Leu mutation inhibits post-translational processing and maturation of SR-BI, impairing its translocation to the cell surface. This results in a profound reduction in SR-BI activity in vitro and in vivo. Consequently, this mutation results in greatly elevated HDL-C levels in humans. These subjects, the first reported homozygotes for a deleterious mutation in SCARB1, offer an unprecedented opportunity to study SR-BI’s role in HDL metabolism and CHD in humans.