5 research outputs found
Einfluss epigenetischer Modifikationen auf die Regulation des GefĂ€Ătonus
EinflĂŒsse epigenetischer Mechanismen auf die GefĂ€Ătonusregulation sind bisher kaum untersucht. Ziel der vorliegenden Arbeit war es, die Bedeutung bekannter epigenetischer Modifikatoren in der GefĂ€Ătonuskontrolle aufzuzeigen und molekulare Mechanismen zu identifizieren. Hierzu wurden VasoreaktivitĂ€tsstudien an MausgefĂ€Ăen im Organbad durchgefĂŒhrt und molekularbiologische Methoden eingesetzt. Verwendet wurde ein Mausstamm mit induzierbarem Knockout der Histondemethylase JARID1B (KDM5B) sowie verschiedene Inhibitoren von Histonmethylasen und -demethylasen.
Mittlerweile sind eine Vielzahl an Inhibitoren epigenetischer Modifikatoren kommerziell erhĂ€ltlich. Getestet wurde u.a. der Wirkstoff GSK343, welcher spezifisch die Funktion der H3K27me3-Methyltransferase Enhancer of zeste homolog 2 (EZH2) inhibiert. Bekannt ist eine Beteiligung von EZH2 in der Carcinogenese und Zellzykluskontrolle. Unter basalen Bedingungen sowie deutlich verstĂ€rkt nach mehrstĂŒndiger Inkubation mit murinem Lipopolysaccharid (mLPS) zeigte sich eine verzögerte Phenylephrin-induzierte Kontraktion von PrĂ€paraten der murinen Aorta thoracica. Da unter inflammatorischen Bedingungen die induzierbare NO-Synthase iNOS stark induziert wird, wurde die Hypothese formuliert, dass eine höhere BioverfĂŒgbarkeit von NO ursĂ€chlich fĂŒr diese Beobachtung ist. NO ist ein potenter Vasodilator und wird im GefĂ€Ăendothel produziert. Organbadversuche mit Inhibitoren der iNOS und eNOS konnten jedoch keine Differenzen in der NO-BioverfĂŒgbarkeit zeigen, weder vor noch nach Stimulation mit mLPS. Ăber schrittweise Depolarisation durch K+-Ionen konnte eine BeeintrĂ€chtigung des kontraktilen Apparats der glatten GefĂ€Ămuskelzellen ausgeschlossen werden. Auch die Thromboxan-induzierte Kontraktion, ausgelöst durch das Thromboxan-Analogon U46619, war nicht beeinflusst. Inhibition von EZH2 unter inflammatorischen Bedingungen, wie beispielsweise in der Sepsis, scheint einer GefĂ€Ădysfunktion vorzubeugen. Dies geschieht unabhĂ€ngig von der NO-BioverfĂŒgbarkeit und ohne BeeintrĂ€chtigung der KontraktilitĂ€t der glatten GefĂ€Ămuskelzellen sowie der Thromboxan-induzierten GefĂ€Ăkontraktion. Es ergibt sich die Hypothese, dass EZH2 nicht in die eigentliche GefĂ€Ătonuskontrolle, sondern in der Transduktion inflammatorischer Signale involviert ist und damit in der Entstehung einer GefĂ€Ădysfunktion. Diese Annahme sollte weiter untersucht werden, da sich durch Inhibition von EZH2 eine mögliche Therapieoption in der Sepsis bietet. Neuste Publikationen bestĂ€tigen eine Rolle von EZH2 in der inflammatorischen Signalkaskade.
Die H3K4me3-Histondemethylase JARID1B wird in GefĂ€Ăendothelzellen stark exprimiert, bekannt sind eine Rolle in der Embryogenese, Cancerogenese sowie Angiogenese. Die Arteria mesenterica superior der Tamoxifen-induzierbaren globalen Jarid1b-Knockout Maus zeigte eine verstĂ€rkte Acetylcholin-induzierte Vasorelaxation. Diese Beobachtung konnte durch Behandlung von Wildtyp- ArterienprĂ€paraten mit dem JARID1B-Inhibitor 2â4(4âmethylphenyl)â1,2âBenziso-thiazolâ3(2H)âon (PBIT) reproduziert werden. Ein spezifischer Knockout von Jarid1b in Makrophagen zeigte keinen vergleichbaren PhĂ€notyp. Untersucht wurde auch hier die Hypothese, dass VerĂ€nderungen der NO-BioverfĂŒgbarkeit Ursache des beobachteten PhĂ€notyps sind. Unterschiede in der NO-BioverfĂŒgbarkeit, der Expression oder des Aktivierungsgrades der eNOS konnten in Versuchen mit Inhibitoren der Synthasen sowie mittels Proteinisolation nicht festgestellt werden. Neben NO wirken Metabolite der ArachidonsĂ€ure als Vasorelaxantien. Die Inkubation mit ArachidonsĂ€ure im Organbad ergab zunĂ€chst keine Unterschiede im Relaxationsverhalten zwischen Knockout- und WildtypgefĂ€Ăen. Zur weiteren Untersuchung der Hypothese verĂ€nderter Prostanoid-Signalwege sind weitere Studien notwendig. Nach Abschluss der Arbeit konnte gezeigt werden, dass der JARID1B-Knockout die lösliche Epoxid-Hydroxylase (sEH) destabilisiert und damit ĂŒber verminderten Abbau von EpoxyeicosatriensĂ€uren (EETs) relaxierend und unter Angiotensin II-Einfluss gefĂ€Ăprotektiv wirkt.
Modifikationen der epigenetischen Regulation in GefĂ€Ăzellen wirken sich auf die GefĂ€Ătonusregulation aus. Die Experimente in dieser Arbeit zeigen, dass dies abseits der hĂ€ufigsten vasoaktiven Autacoid-Signalwege und unter bestimmten Voraussetzungen stattfinden kann. Epigenetische Regulation ermöglicht es, die GefĂ€Ătonuskontrolle den Umgebungsbedingungen anzupassen und spielt in der Pathophysiologie von GefĂ€Ăerkrankungen eine entscheidende Rolle.The role of epigenetic mechanisms in the control of the vessel tone are incompletely understood. The aim of this study was to unravel the relevance of known epigenetic modifiers in the regulation of vessel tone and to reveal underlying molecular mechanisms. Therefore, vessel reactivity studies were performed in an organ bath setting. A mouse strain harboring an inducible knockout of histone-demethylase JARID1B (KDM5a) and different inhibitors of histone-methylases and demethylases were used.
Today, a variety of inhibitors of epigenetic modifying enzymes are commercially available. In this study, the agent GSK343, which specifically inhibits the function of the epigenetic repressor and H3K27me3-methyltransferase Enhancer of zeste homolog 2 (EZH2), was used. EZH2 is known to play a role in carcinogenesis as well as cell cycle control. Preparations of the murine thoracic aorta incubated with GSK343 showed a delayed phenylephrine-induced contraction. This effect was strongly increased after incubation with murine lipopolysaccharide (mLPS). Since inducible NO synthase (iNOS) is upregulated in inflammatory conditions it was proposed that an increased bioavailability of nitric oxide (NO) is responsible for the delayed contraction. NO is a potent vasodilator and is produced in the vascular endothelium. Nevertheless, organ bath studies with inhibitors of endothelial NO synthase (eNOS) and iNOS showed no differences for the bioavailability of NO, both before and after mLPS stimulation. Impairment of the contractile apparatus within vascular smooth muscle cells was excluded by induction of vascular contraction with low concentrations of potassium ions. Also, thromboxane-induced contraction caused by the thromboxane analogue U46619 was not affected. Inhibition of EZH2 under inflammatory conditions, like in sepsis, seemed to prevent vessel dysfunction. This was independent from NO bioavailability and not caused by impairment of contractile apparatus. Also, thromboxane-induced contraction was not affected. It can be hypothesized that EZH2 might not directly interfere with the control of vessel tone but is rather involved in inflammatory signaling which causes vessel dysfunction. This hypothesis should be further investigated, as inhibition of EZH2 might prove valuable in therapy of sepsis and septic shock. Current publications prove an involvement of EZH2 in the inflammatory signaling cascade.
The H3K4me3-histondemethylase JARID1B is strongly expressed in human umbilical vein endothelial cells. It is known for its role in embryogenesis, angiogenesis, and carcinogenesis. Preparations of superior mesenteric arteries from tamoxifen-inducible JARID1B-knockout mice showed an increased acetylcholine-induced relaxation. This was reproduced by treatment with JARID1B-inhibitor PBIT. Specific knockout in endothelial cells and macrophages did not show this phenotype. Again, it was hypothesized that altered bioavailability of NO causes this finding. However, differences in NO-bioavailability, eNOS activation or expression level between knockout- and wildtype-preparations could not be found using inhibitors of NO-synthases in organ bath as well as on protein level. Besides NO, metabolites of arachidonic acid act as vasorelaxant autacoids. A first approach of incubating both knockout- and wildtype mesenteric artery in organ-bath did not show differences in relaxation between the two groups. Further experiments should be performed to support the hypothesis of an influence of JARID1B-knockout on prostanoid metabolism. After finishing this experimental study, JARID1B-knockout was proven to destabilize soluble epoxide hydrolase (sEH), resulting in higher bioavailability of vasorelaxant Epoxyeicosatrienoic acids (EETs) and therefore has a vasoprotective function under challenge with Angiotensin II.
Modifications of epigenetic mechanisms in the vasculature appear to have direct effects on the control of vessel tone. The results of this experimental study indicate that this is different to the common vasoactive autacoid signaling pathways and exists under certain preconditions. Epigenetic regulation enables the vasculature to adapt to specific conditions and plays a decisive role in pathophysiology of vessel diseases
Epigenetic control of microsomal prostaglandin E synthase-1 by HDAC-mediated recruitment of p300
Nonsteroidal anti-inflammatory drugs are the most widely used medicine to treat pain and inflammation, and to inhibit platelet function. Understanding the expression regulation of enzymes of the prostanoid pathway is of great medical relevance. Histone acetylation crucially controls gene expression. We set out to identify the impact of histone deacetylases (HDACs) on the generation of prostanoids and examine the consequences on vascular function. HDAC inhibition (HDACi) with the pan-HDAC inhibitor, vorinostat, attenuated prostaglandin (PG)E2 generation in the murine vasculature and in human vascular smooth muscle cells. In line with this, the expression of the key enzyme for PGE2 synthesis, microsomal PGE synthase-1 (PTGES1), was reduced by HDACi. Accordingly, the relaxation to arachidonic acid was decreased after ex vivo incubation of murine vessels with HDACi. To identify the underlying mechanism, chromatin immunoprecipitation (ChIP) and ChIP-sequencing analysis were performed. These results suggest that HDACs are involved in the recruitment of the transcriptional activator p300 to the PTGES1 gene and that HDACi prevented this effect. In line with the acetyltransferase activity of p300, H3K27 acetylation was reduced after HDACi and resulted in the formation of heterochromatin in the PTGES1 gene. In conclusion, HDAC activity maintains PTGES1 expression by recruiting p300 to its gene
The histone demethylase Jarid1b mediates angiotensin II-induced endothelial dysfunction by controlling the 3'UTR of soluble epoxide hydrolase.
The NADPH organizers NoxO1 and p47phox are both mediators of diabetes-induced vascular dysfunction in mice
Aim: NADPH oxidases are important sources of reactive oxygen species (ROS). Several Nox homologues are present together in the vascular system but whether they exhibit crosstalk at the activity level is unknown. To address this, vessel function of knockout mice for the cytosolic Nox organizer proteins p47phox, NoxO1 and a p47phox-NoxO1-double knockout were studied under normal condition and during streptozotocin-induced diabetes.
Results: In the mouse aorta, mRNA expression for NoxO1 was predominant in smooth muscle and endothelial cells, whereas p47phox was markedly expressed in adventitial cells comprising leukocytes and tissue resident macrophages. Knockout of either NoxO1 or p47phox resulted in lower basal blood pressure. Deletion of any of the two subunits also prevented diabetes-induced vascular dysfunction. mRNA expression analysis by MACE (Massive Analysis of cDNA ends) identified substantial gene expression differences between the mouse lines and in response to diabetes. Deletion of p47phox induced inflammatory activation with increased markers of myeloid cells and cytokine and chemokine induction. In contrast, deletion of NoxO1 resulted in an attenuated interferon gamma signature and reduced expression of genes related to antigen presentation. This aspect was also reflected by a reduced number of circulating lymphocytes in NoxO1-/- mice.
Innovation and conclusion: ROS production stimulated by NoxO1 and p47phox limit endothelium-dependent relaxation and maintain blood pressure in mice. However, NoxO1 and p47phox cannot substitute each other despite their similar effect on vascular function. Deletion of NoxO1 induced an anti-inflammatory phenotype, whereas p47phox deletion rather elicited a hyper-inflammatory response