Experimentelle Untersuchungen zur Wirkung von erhitzten Fetten auf ausgewählte Parameter des Lipidstoffwechsels und der Atherogenese

Erhitzte Fette entstehen während der thermischen Behandlung fetthaltiger Lebensmittel. Sie enthalten biologisch hochaktive Bestandteile, die natürliche Aktivatoren des Transkriptionsfaktors PPARalpha sind. PPARalpha ist maßgeblich an der Regulation der Fettsäureverwertung beteiligt und seine Aktivierung geht im Tiermodell mit einer verminderten Triglyzeridkonzentration im Serum sowie der Leber einher. Neben der hypolipidämischen Wirkung ist bekannt, dass eine PPARalpha-Aktivierung in vaskulären Zellen zu einer Entzündungshemmung führt, den reversen Cholesteroltransport in Makrophagen aktiviert und dadurch die Atherosklerose hemmt. Ziel der vorliegenden Arbeit war daher die Hypothese zu bestätigen, dass erhitzte Fette durch eine PPARalpha-Aktivierung anti-atherosklerotisch wirken. Dafür wurde zunächst über einen Zeitraum von 14 Wochen ein Fütterungsversuch mit einem etablierten Tiermodell der Atheroskleroseforschung, der LDL Rezeptor -Knockout-Maus, durchgeführt. Im Ergebnis führte die Verabreichung erhitzter Fette, welche unter praxisrelevanten Bedingungen hergestellt wurden, zu verminderten Konzentrationen an Triglyzeriden und Cholesterol in Plasma und Lipoproteinen. Weiterhin wurde anhand erhöhter relativer mRNA-Konzentrationen bekannter Zielgene des PPARalpha dessen Aktivierung in der Leber nachgewiesen. Die Ergebnisse der immunhistologischen Auswertung zeigen erstmals, dass die Aufnahme erhitzter Fette die Ausbildung atherosklerotischer Läsionen im Aortenursprung der Tiere reduziert und mit einer erhöhten PPARalpha-Proteinexpression in den krankhaft veränderten Gefäßwandabschnitten einhergeht. Dabei ist anzunehmen, dass die erhöhte PPARalpha-Expression indirekt für die verminderte Expression der inflammatorischen Markerproteine VCAM-1 und smooth muscle actin sowie für die verminderten Lipid- und Kollageneinlagerungen in den Läsionen der Versuchstiere, die erhitztes Fett erhielten, verantwortlich ist. Insgesamt zeigen diese Ergebnisse, dass erhitzte Fette anti-atherosklerotisch wirken. Dieser Effekt beruht dabei voraussichtlich auf einer günstigen Beeinflussung des Blutlipidprofils auf Grund einer ligandenabhängigen Aktivierung des PPARalpha in der Leber sowie auf der erhöhten Expression des Transkriptionsfaktors in der Gefäßwand, die mit einer Hemmung pro-atherogener Mechanismen wie der Rekrutierung von Monozyten und der Proliferation von glatten Gefäßmuskelzellen einhergeht. Zusätzlich sollte in einer zweiten Studie geprüft werden, ob oxidierte Fettsäuren als Bestandteile erhitzter Fette und starke PPARalpha-Aktivatoren für die anti-atherogene Wirkung erhitzter Fette mitverantwortlich sind. Im Fokus standen Untersuchungen zur Wirkung von 13-Hydroxy-9,11-octadecadiensäure (13-HODE) auf die Cholesterolhomöostase in murinen RAW264.7-Makrophagen. Dabei zeigte sich, dass die Behandlung der Zellen mit 13-HODE zu einer gesteigerten PPAR-Transaktivierung führt und die Konzentration von Proteinen erhöht, die an der Regulation des zellulären Cholesteroltransports beteiligt sind. Darüber hinaus führte die Behandlung mit 13-HODE und dem Cholesterolakzeptor Apolipoprotein A-1 (Apo A-1) zu verminderten zellulären Cholesterolkonzentrationen in Makrophagen sowie zu erhöhten Cholesterolkonzentrationen im Kulturmedium. Beim Einsatz selektiver PPARalpha- und PPARgamma-Antagonisten blieben die Effekte von 13-HODE auf den Cholesterolefflux und die ermittelten Proteinexpressionen aus, was darauf hindeutet, dass beide PPAR-Isoformen für die beobachteten Effekte eine Rolle spielen. Die Befunde aus dieser Studie zeigen insgesamt, dass 13-HODE den Apo A-1-abhängigen Cholesterolefflux aus Makrophagen durch Aktivierung des PPAR-LXRalpha-Signalwegs stimuliert. Da eine Cholesterolüberladung in Makrophagen die Entwicklung atherosklerotischer Läsionen fördert, deuten die Ergebnisse dieser Studie darauf hin, dass die beobachteten anti-atherosklerotischen Effekte erhitzter Fette in LDL Rezeptor- Knockout-Mäusen zumindest teilweise durch eine Hemmung der Cholesterolakkumulierung und durch eine Stimulierung des reversen Cholesteroltransports in Makrophagen durch oxidierte Fettsäuren, wie 13-HODE, begründet sind.Dietary heated fats are generated during thermal treatment of fatty foods. They contain biologically active compounds which are natural activators of the transcription factor PPARalpha which is the main regulator of fatty acid metabolism. Thereby numerous observations from feeding experiments demonstrating triglyceride-lowering properties of heated fats in liver and plasma. Besides the hypolipidemic effects, PPARalpha regulates anti-atherogenic inflammatory processes in vascular cells and also activates the reverse cholesterol transport in macrophages. The aim of the present work was to investigate anti-atherogenic properties of heated fats. Therefore, we performed first of all a feeding experiment with low-density lipoprotein receptor (LDLR) knockout mice as a well-established experimental model of atherosclerosis. As a result of feeding dietary heated fat, there was a reduction of triglycerides and cholesterol in plasma and lipoproteins. Furthermore, we determined increased relative mRNA concentrations of PPARalpha target genes in the liver in animals with dietary heated fats as an indicative effect of its hepatic activation. It became apparent for the first time that feeding diets containing heated fats caused a significant reduction in cross-sectional lesion area and increased PPARalpha protein expression in the aortic root. It can be suggested that the reduction of the proinflammatory marker vascular cell adhesion molecule-1 (VCAM-1) and smooth muscle actin as well as the decrease in lesion lipid and collagen content in the aortic root of mice fed heated fat is a consequence of an enhanced PPARalpha expression in vascular cells. In conclusion, the results of the first experiment demonstrate anti-atherogenic effects of heated fats. Therefore, we assume that these effects are due to ligand–dependent activation of PPARalpha in the liver, which contributes to plasma lipid lowering but also due to increased PPARalpha expression in vascular cells which inhibits pro-atherogenic events like monocyte recruitment and proliferation of vascular smooth muscle cells. The aim of a second study was to verify whether oxidized fatty acids as inherent part of heated fats and strong PPARalpha activators are also responsible for the anti-atherogenic effects of heated fats. The focal point was to investigate the effects of 13-hydroxy-9,11-octadecadienoic acid (13-HODE) on cholesterol homeostasis in murine RAW264.7 macrophages. Treatment of cells with 13-HODE increased PPAR-transactivation activity and concentrations of proteins involved in cellular cholesterol transport. In addition, 13-HODE decreased cellular cholesterol concentration in macrophages during incubation with the extracellular lipid acceptor apolipoprotein A-1 as well as 13-HODE increased cholesterol concentration in the culture medium. Pre-treatment of macrophages with a selective PPARalpha or PPARgamma antagonist completely abolished the effects of 13-HODE on cholesterol efflux and protein levels of genes investigated, suggesting an involvement of both PPAR isotypes. The results indicate a stimulatory effect of 13-HODE on apolipoprotein A-1-dependent cholesterol efflux from macrophages due to PPAR-LXRalpha-pathway. Because extensive cholesterol accumulation by macrophages in the arterial wall promotes atherosclerotic lesion development, these findings suggest that the observed anti-atherogenic effects of heated fats in LDLR knockout mice might be, at least in part, due to inhibition of macrophage cholesterol accumulation and stimulation of reverse cholesterol transport caused by oxidized fatty acids such as 13-HODE

13-hydroxy linoleic acid increases expression of the cholesterol transporters ABCA1, ABCG1 and SR-BI and stimulates apoA-I-dependent cholesterol efflux in RAW264.7 macrophages

<p>Abstract</p> <p>Background</p> <p>Synthetic activators of peroxisome proliferator-activated receptors (PPARs) stimulate cholesterol removal from macrophages through PPAR-dependent up-regulation of liver × receptor α (LXRα) and subsequent induction of cholesterol exporters such as ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type 1 (SR-BI). The present study aimed to test the hypothesis that the hydroxylated derivative of linoleic acid (LA), 13-HODE, which is a natural PPAR agonist, has similar effects in RAW264.7 macrophages.</p> <p>Methods</p> <p>RAW264.7 macrophages were treated without (control) or with LA or 13-HODE in the presence and absence of PPARα or PPARγ antagonists and determined protein levels of LXRα, ABCA1, ABCG1, SR-BI, PPARα and PPARγ and apolipoprotein A-I mediated lipid efflux.</p> <p>Results</p> <p>Treatment of RAW264.7 cells with 13-HODE increased PPAR-transactivation activity and protein concentrations of LXRα, ABCA1, ABCG1 and SR-BI when compared to control treatment (P < 0.05). In addition, 13-HODE enhanced cholesterol concentration in the medium but decreased cellular cholesterol concentration during incubation of cells with the extracellular lipid acceptor apolipoprotein A-I (P < 0.05). Pre-treatment of cells with a selective PPARα or PPARγ antagonist completely abolished the effects of 13-HODE on cholesterol efflux and protein levels of genes investigated. In contrast to 13-HODE, LA had no effect on either of these parameters compared to control cells.</p> <p>Conclusion</p> <p>13-HODE induces cholesterol efflux from macrophages via the PPAR-LXRα-ABCA1/SR-BI-pathway.</p

Ecto-Nucleotide Triphosphate Diphosphohydrolase-2 (NTPDase2) Deletion Increases Acetaminophen-Induced Hepatotoxicity

Ecto-nucleotidase triphosphate diphosphohydrolase-2 (NTPDase2) is an ecto-enzyme that is expressed on portal fibroblasts in the liver that modulates P2 receptor signaling by regulating local concentrations of extracellular ATP and ADP. NTPDase2 has protective properties in liver fibrosis and may impact bile duct epithelial turnover. Here, we study the role of NTPDase2 in acute liver injury using an experimental model of acetaminophen (APAP) intoxication in mice with global deletion of NTPDase2. Acute liver toxicity was caused by administration of acetaminophen in wild type (WT) and NTPDase2-deficient (Entpd2 null) mice. The extent of liver injury was compared by histology and serum alanine transaminase (ALT). Markers of inflammation, regeneration and fibrosis were determined by qPCR). We found that Entpd2 expression is significantly upregulated after acetaminophen-induced hepatotoxicity. Entpd2 null mice showed significantly more necrosis and higher serum ALT compared to WT. Hepatic expression of IL-6 and PDGF-B are higher in Entpd2 null mice. Our data suggest inducible and protective roles of portal fibroblast-expressed NTPDase2 in acute necrotizing liver injury. Further studies should investigate the relevance of these purinergic pathways in hepatic periportal and sinusoidal biology as such advances in understanding might provide possible therapeutic targets

Article Ecto-Nucleotide Triphosphate Diphosphohydrolase-2 (NTPDase2) Deletion Increases Acetaminophen-Induced Hepatotoxicity

Ecto-nucleotidase triphosphate diphosphohydrolase-2 (NTPDase2) is an ecto-enzyme that is expressed on portal fibroblasts in the liver that modulates P2 receptor signaling by regulating local concentrations of extracellular ATP and ADP. NTPDase2 has protective properties in liver fibrosis and may impact bile duct epithelial turnover. Here, we study the role of NTPDase2 in acute liver injury using an experimental model of acetaminophen (APAP) intoxication in mice with global deletion of NTPDase2. Acute liver toxicity was caused by administration of acetaminophen in wild type (WT) and NTPDase2-deficient (Entpd2 null) mice. The extent of liver injury was compared by histology and serum alanine transaminase (ALT). Markers of inflammation, regeneration and fibrosis were determined by qPCR). We found that Entpd2 expression is significantly upregulated after acetaminophen-induced hepatotoxicity. Entpd2 null mice showed significantly more necrosis and higher serum ALT compared to WT. Hepatic expression of IL-6 and PDGF-B are higher in Entpd2 null mice. Our data suggest inducible and protective roles of portal fibroblast-expressed NTPDase2 in acute necrotizing liver injury. Further studies should investigate the relevance of these purinergic pathways in hepatic periportal and sinusoidal biology as such advances in understanding might provide possible therapeutic targets

Immunosuppressive protocol with delayed use of low-dose tacrolimus after aortic transplantation suppresses donor-specific anti-MHC class I and class II antibody production in rats

Background: Arterial allografts are used as vascular conduits in the treatment of prosthetic graft infection. Immunosuppression decreases their rupture risk rate. However, immunosuppression can be unprofitable in florid infection. Previously, we confirmed inhibition of cell-mediated destruction of rat aortic grafts by delayed use of tacrolimus. In this work, we studied the influence of this protocol on the antibody-mediated rejection

Ecto-Nucleotide Triphosphate Diphosphohydrolase-2 (NTPDase2) Deletion Increases Acetaminophen-Induced Hepatotoxicity

Ecto-nucleotidase triphosphate diphosphohydrolase-2 (NTPDase2) is an ecto-enzyme that is expressed on portal fibroblasts in the liver that modulates P2 receptor signaling by regulating local concentrations of extracellular ATP and ADP. NTPDase2 has protective properties in liver fibrosis and may impact bile duct epithelial turnover. Here, we study the role of NTPDase2 in acute liver injury using an experimental model of acetaminophen (APAP) intoxication in mice with global deletion of NTPDase2. Acute liver toxicity was caused by administration of acetaminophen in wild type (WT) and NTPDase2-deficient (Entpd2 null) mice. The extent of liver injury was compared by histology and serum alanine transaminase (ALT). Markers of inflammation, regeneration and fibrosis were determined by qPCR). We found that Entpd2 expression is significantly upregulated after acetaminophen-induced hepatotoxicity. Entpd2 null mice showed significantly more necrosis and higher serum ALT compared to WT. Hepatic expression of IL-6 and PDGF-B are higher in Entpd2 null mice. Our data suggest inducible and protective roles of portal fibroblast-expressed NTPDase2 in acute necrotizing liver injury. Further studies should investigate the relevance of these purinergic pathways in hepatic periportal and sinusoidal biology as such advances in understanding might provide possible therapeutic targets

Ecto-Nucleotide Triphosphate Diphosphohydrolase-2 (NTPDase2) Deletion Increases Acetaminophen-Induced Hepatotoxicity

Ecto-nucleotidase triphosphate diphosphohydrolase-2 (NTPDase2) is an ecto-enzyme that is expressed on portal fibroblasts in the liver that modulates P2 receptor signaling by regulating local concentrations of extracellular ATP and ADP. NTPDase2 has protective properties in liver fibrosis and may impact bile duct epithelial turnover. Here, we study the role of NTPDase2 in acute liver injury using an experimental model of acetaminophen (APAP) intoxication in mice with global deletion of NTPDase2. Acute liver toxicity was caused by administration of acetaminophen in wild type (WT) and NTPDase2-deficient (Entpd2 null) mice. The extent of liver injury was compared by histology and serum alanine transaminase (ALT). Markers of inflammation, regeneration and fibrosis were determined by qPCR). We found that Entpd2 expression is significantly upregulated after acetaminophen-induced hepatotoxicity. Entpd2 null mice showed significantly more necrosis and higher serum ALT compared to WT. Hepatic expression of IL-6 and PDGF-B are higher in Entpd2 null mice. Our data suggest inducible and protective roles of portal fibroblast-expressed NTPDase2 in acute necrotizing liver injury. Further studies should investigate the relevance of these purinergic pathways in hepatic periportal and sinusoidal biology as such advances in understanding might provide possible therapeutic targets