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Role of the soluble epoxide hydrolase and cytochrome P450-derived epoxyeicosatrienoic acids in hypoxic pulmonary vasoconstriction and hypoxia-induced pulmonary vascular remodelling

By Benjamin Keserü

Abstract

Hypoxic pulmonary vasoconstriction (HPV) redistributes pulmonary blood flow from areas of low oxygen partial pressure to areas of normal or relativity high oxygen availability, thus optimising the matching of perfusion to ventilation and preventing arterial hypoxemia. Generalised alveolar hypoxia results in a sustained increase in pulmonary artery pressure which in turn leads to structural changes in the walls of the pulmonary vasculature (pulmonary vascular remodelling). Recent findings have indicated a role for cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) in hypoxia-induced pulmonary vasoconstriction. Given that the intracellular concentration of EETs is determined by the soluble epoxide hydrolase (sEH), which metabolises EETs to their less active dihydroxyeicosatrienoic acids (DHETs), we assessed the influence of the sEH and EETs on pulmonary artery pressure, acute and chronic HPV, and pulmonary vascular remodelling in the mouse lung. In isolated lungs from wild-type mice, acute HPV was significantly increased by sEH inhibition, an effect abolished by pre-treatment with CYP epoxygenase inhibitors and the EET antagonist 14,15-EEZE. The acute hypoxia-induced vasoconstriction and EET production were greater in lungs from sEH-/- mice than from wild-type mice and sEH inhibition had no further effect on HPV in lungs from the former animals, while MSPPOH (CYP epoxygenase inhibitor) and 14,15-EEZE decreased the response. Exogenous application of 11,12-EET increased pulmonary artery pressure in a concentration-dependent manner and enhanced acute HPV in wild-type lungs, while 14,15-EET and 11,12-DHET were without significant effect on pulmonary artery pressure. 5-HT2A receptor antagonism or Rho kinase inhibition shifted the EET concentration-response curve to the right and abrogated the EET- and sEH inhibition-induced potentiation of acute hypoxic vasoconstriction. In lungs from wild-type and sEH-/- mice, hypoxic preconditioning (hypoxic ventilation for 10 minutes) enhanced the 5-HT response. 1-Adamantyl-3-cyclohexylurea (ACU), a sEH inhibitor, further amplified the hypoxia-induced 5-HT-hypersensitivity in wild-type mice. However, after hypoxic preconditioning, the sEH-/- lungs displayed a striking leftward shift in the 5-HT response. 11,12-EET can activate TRPC6 channels in endothelial cells by eliciting its translocation to the plasma membrane, more specifically to membrane domains enriched with the caveolae marker caveolin-1. This effect was also observed in rat pulmonary artery smooth muscle cells overexpressing the channel. Exposure of the latter cells to acute hypoxia also stimulated the intracellular translocation of TRPC6 to caveolae, an effect that was sensitive to the EET antagonist. The EET-induced translocation of TRPC6 channels was prevented by a 5-HT2A receptor antagonist but not by a Rho kinase inhibitor. Moreover, while acute hypoxia and 11,12-EET increased pulmonary pressure in lungs from TRPC6+/- mice, lungs from TRPC6-/- mice did not respond to either stimuli. These results indicate that the sEH and CYP-derived EETs are involved in acute HPV and that EET-induced pulmonary contraction under normoxic and hypoxic conditions involves a TRPC6 channel, a 5-HT2A receptor-dependent pathway and Rho kinase activation. In the second part of the study the role of the sEH in the development of pulmonary hypertension and vascular remodelling induced in mice by exposure to hypoxia (10% O2) for 21 days was analysed. In wild-type mice, chronic hypoxia decreased the pulmonary expression/activity of the sEH, induced right heart hypertrophy and erythropoiesis, and increased the number of partially and fully muscularised pulmonary resistance arteries (by 3-fold). Moreover, in HEK 293 cells, hypoxia (1% O2 up to 24 h) decreased sEH promoter activity by 50%. In isolated lungs, pre-exposure to chronic hypoxia significantly increased baseline perfusion pressures and potentiated the acute HPV. While an sEH inhibitor, ACU, potentiated acute HPV in lungs from mice maintained in normoxic conditions, it had no effect on HPV in lungs from mice exposed to hypoxia. The EET antagonist, 14,15-EEZE, abolished the sEH inhibitor-dependent increase in acute HPV in normoxic lungs and decreased HPV in chronic hypoxic lungs. Hypoxia-induced right heart hypertrophy and erythropoiesis were more pronounced in sEH-/- than in wild-type mice. Under normoxic and hypoxic conditions the muscularisation of resistance pulmonary arteries was greater in lungs from sEH-/- mice than in lungs from wild-type mice. sEH-/- mice also displayed an enhanced acute HPV, compared to that observed in wild-type mice and chronic exposure to hypoxia did not further potentiate acute HPV. However, in the presence of 14,15-EEZE responses returned to levels observed in normoxic lungs from wild-type animals. Furthermore, immunohistochemistry demonstrated an extensive expression of the sEH in the medial wall of pulmonary arteries from human donor lungs. Whereas sEH expression was not detectable in samples from pulmonary hypertension patients, indicating that the sEH is involved in hypoxia-induced pulmonary vascular remodelling and hypoxic pulmonary vasoconstriction. Taken together, the results presented in this thesis indicate that the expression/activity of the sEH is an important determinant of the magnitude of acute and chronic hypoxia-induced pulmonary vasoconstriction and pulmonary vascular remodelling by inactivating vasoconstrictor CYP-derived EETs. As sEH inhibitors are currently being developed for the treatment of human systemic hypertension, it should be noted that these compounds may even promote the development of pulmonary hypertension.Die akute hypoxische pulmonale Vasokonstriktion (HPV) ist ein physiologischer Mechanismus, der es erlaubt, den lokalen Blutfluss der Lunge an alveolaren O2- Mangel anzupassen. Durch die Hypoxie-bedingte Widerstandserhöhung besteht die Möglichkeit, die Durchblutung schlecht ventilierter Lungenbezirke einzuschränken und den Blutstrom in gut ventilierte Gebiete zu leiten. Die HPV wurde erstmals im Jahr 1946 durch von Euler und Liljestrand beschrieben und ist bis heute Gegenstand intensiver Forschung. Dennoch sind die Signaltransduktionswege und die Sauerstoff-Sensoren, die in die HPV involviert sind, weitgehend unbekannt. Als Sensoren werden unter anderem die Mitochondrien und die NADPH-Oxidasen diskutiert. Allerdings konnten neuere Studien zeigen, dass die glatten Muskelzellen der pulmonalen Widerstandsgefäße Effektorzellen darstellen. Des Weiteren konnte nachgewiesen werden, dass Mediatoren - wie 5-Hydroxytryptamin (5-HT) und Endothelin-1 - die akute HPV modulieren und ein nicht selektiver Kationen-Kanal (TRPC6-Kanal) sowie die Aktivierung der Rho-Kinase wesentlich an der akuten HPV beteiligt sind. Bei chronischer Hypoxie, wie sie z. B. bei dauerhaftem Aufenthalt in großen Höhen entsteht, kommt es zu einer globalen Vasokonstriktion der Lunge. Die Folge ist ein pulmonaler Bluthochdruck mit einer Hypertrophie der glatten Muskulatur der Pulmonalgefäße („pulmonales vaskuläres Remodelling“). Hinzu kommt, dass eine dauerhafte Hypoxie zur Produktion von endothelialen Substanzen führt, die sowohl die Kontraktilität als auch die Proliferation der glatten Muskelzellen beeinflussen. Es konnte in mehreren Studien nachgewiesen werden, dass eine chronische Hypoxie zu einer verminderten Bildung von Stickstoffmonoxid und Prostazyklinen sowie zu einer vermehrten Produktion von 5-HT, Endothelin-1 und verschiedenen Wachstumsfaktoren führt. Über eine Beteiligung des 5-HT-Transporters, der TRPC-Kanäle und der Rho-Kinase in der chronischen Hypoxie-bedingten pulmonalen Hypertension wird ebenfalls diskutiert. Enzyme der Cytochrom P450 (CYP)-Epoxygenase-Familie werden extrahepatisch vor allem im Herz, dem Gefäßsystem, dem Gastrointestinaltrakt, der Niere und der Lunge exprimiert. Diese liefern durch Metabolisierung von Arachidonsäure vier verschiedene Isomere der Epoxyeicosatriensäuren (5,6-, 8,9-, 11,12-, und 14,15-EETs), die sowohl den Gefäßtonus modulieren als auch antiinflammatorische und angiogenetische Eigenschaften haben. Nach ihrer Synthese stehen den EETs zwei Wege offen: Zum einen können sie in Phospholipide inkorporiert, zum anderen durch die Cyclooxygenase (5,6-EET) und die lösliche Epoxidhydrolase (sEH; 8,9-, 11,12- und 14,15-EET) zu ihren weniger aktiven Dihydroxyeicosatriensäuren (DHETs) metabolisiert werden. In Koronar- und Nierenarterien unterschiedlicher Spezies stellen EETs den sogenannten endothelialen hyperpolarisierenden Faktor dar, der zur Hyperpolarisation und Relaxation der glatten Gefäßmuskulatur führt. Kürzlich konnte unsere Arbeitsgruppe nachweisen, dass 11,12-EET die Agonisten-induzierte endotheliale Hyperpolarisation durch Translokation des TRRPC6-Kanals in mit Caveolin-1 angereicherte Membrangebiete, erhöht. Des Weiteren legen verschiedene Untersuchungen eine Verbindung zwischen der sEH und kardiovaskulären Erkrankungen nahe, da eine Hemmung dieses Enzyms der Entstehung einer Hypertonie entgegenwirkt. Dagegen ist die Rolle von CYP-Epoxygenase-generierten EETs und der sEH in der pulmonalen Zirkulation weitgehend unbekannt. Hierzu wurden in den letzten Jahren widersprüchliche Ergebnisse publiziert. Jedoch konnte unsere Arbeitsgruppe vor kurzem nachweisen, dass eine CYP-Epoxygenase in die HPV und das chronische Hypoxie-induzierte pulmonale vaskuläre Remodelling involviert ist. Ziel der vorliegenden Arbeit war es daher, ausführlich die Rolle der sEH und der CYP-Epoxygenase-generierten EETs in der akuten und chronischen HPV sowie im Hypoxie-bedingten pulmonalen vaskulären Remodelling zu analysieren. Dazu wurden CYP-Epoxygenase-Inhibitoren, ein EET-Antagonist und sEH-Inhibitoren verwendet. Um den molekularen Mechanismus der Hypoxie- und EET-induzierten pulmonalen Vasokonstriktion und des pulmonalen vaskulären Remodelling zu untersuchen, wurden kultivierte pulmonale glatte Muskelzellen, Promotor-Aktivitäts-Assays und genetisch veränderte Tiere (sEH-/- und TRPC6-/- Mäusen) verwendet. Zunächst wurde die Rolle der sEH und EETs in der akuten HPV anhand der isolierten perfundierten Mauslunge untersucht. In Lungen von Wildtyp-Mäusen führte eine Hemmung der sEH zu einer deutlichen Steigerung der Amplitude der akuten HPV. Darüber hinaus konnte nachgewiesen werden, dass eine vorherige Inkubation mit CYP-Epoxygenase-Inhibitoren oder dem EET-Antagonisten 14,15-EEZE den Effekt der sEH-Inhibitoren auf die akute HPV signifikant reduzierte. Im Vergleich zu Lungen von Wildtyp-Mäusen zeigten sEH-/--Lungen eine deutlich erhöhte akute Hypoxie-bedingte Vasokonstriktion. Erwartungsgemäß hatte eine Hemmung der sEH in Lungen von sEH-/--Mäusen keine Wirkung, während MSPPOH, ein CYP-Epoxygenase-Inhibitor, und 14,15-EEZE die gesteigerte HPV signifikant reduzierten. Weiterhin konnte in der vorliegenden Arbeit bewiesen werden, dass eine hypoxische Inkubation (1% O2, 10 Minuten) von Maus-Lungenmikrosomen die EET-Produktion erheblich steigerte. Dieser Effekt war deutlich potenziert in Lungenmikrosomen von sEH-/--Mäusen. Eine exogene Applikation von 11,12-EET in der isolierten perfundierten Mauslunge steigerte signifikant den basalen pulmonalarteriellen Druck und die akute HPV, während 14,15-EET und 11,12-DHET keine Effekte auf den pulmonalarteriellen Druck ausübten. Eine Inhibition des 5-HT2A-Rezeptors oder der Rho-Kinase führte sowohl zu einer deutlichen Reduktion der 11,12-EET-induzierten Vasokonstriktion, als auch zu einer signifikanten Hemmung der durch die EET und sEH-Inhibition gesteigerten akuten HPV. In isolierten Lungen von Wildtyp- und sEH-/--Mäusen führte eine hypoxische Präkonditionierung (hypoxische Ventilation der isolierten Lunge für 10 Minuten) zu einer Verstärkung der 5-HT-induzierten Vasokonstriktion. Der Verlust der sEH-Aktivität, entweder durch Einsatz spezifische Hemmstoffe oder durch genetische Deletion, führte zu einer weiteren deutlichen Verstärkung der 5-HT Dosiswirkungskurve nach hypoxischer Vorbehandlung. In pulmonalarteriellen glatten Muskelzellen, die TRPC6 überexprimieren, konnte nachgewiesen werden, dass 11,12-EET diesen Kanal durch Translokation in mit Caveolin-1 angereicherte Membrangebiete, aktiviert. Darüber hinaus führte eine akute Hypoxie in TRPC6-überexprimierenden glatten Muskelzellen ebenfalls zu einer TRPC6-Kanal-Translokation. Dieser Effekt wurde durch den EET-Antagonisten deutlich gehemmt. Ferner konnte gezeigt werden, dass die EET-induzierte TRPC6-Translokation durch einen 5-HT2A-Rezeptor-Antagonisten reduziert wurde, wogegen ein Rho-Kinase-Inhibitor ohne Effekt war. Außerdem bewirkte eine akute Hypoxie und die Zugabe von 11,12-EET einen Anstieg des pulmonalarteriellen Drucks in isolierten perfundierten Lungen von TRPC6+/--Mäusen. TRPC6-/--Mäuse reagierten auf keinen der beiden Stimuli. Diese Befunde zeigen, dass die Aktivität der sEH und CYP-Epoxygenase-generierte EETs die akute HPV modulieren und die EET-induzierte Kontraktion unter normoxischen und hypoxischen Bedingungen über einen TRPC6-Kanal-, einen 5-HT2A-Rezeptor-abhängigen Signaltransduktionsweg und die Aktivierung der Rho-Kinase verläuft. Ein weiteres Ziel der vorliegenden Arbeit war, die Rolle der sEH in der Entwicklung einer Hypoxie-bedingten pulmonalen Hypertension und des pulmonalen vaskulären Remodelling aufzuklären. Dafür wurden Mäuse 21 Tage in Hypoxie-Kammern hypoxischen Bedingungen (10% O2) ausgesetzt oder unter normoxischen Bedingungen gehalten. Eine chronische Hypoxie führte in Wildtyp-Mäusen zu einer verminderten pulmonalen Expression und folglich auch Aktivität der sEH. Zusätzlich induzierte eine dauerhafte Hypoxie eine Rechtsherzhypertrophie und Hämatopoese in Wildtyp-Mäusen und erhöhte signifikant die Anzahl der teil- und vollmuskularisierten pulmonalen Widerstandsgefäße. In isolierten perfundierten Lungen aus chronisch hypoxischen Mäusen war der Basisdruck signifikant erhöht und die akute HPV deutlich potenziert. Während der sEH-Inhibitor 1-Adamantyl-3-Cyclohexylurea die akute HPV in Lungen von Tieren, die unter normoxischen Bedingungen gehalten wurden, steigerte, hatte er keinen Effekt auf die Antwort in Lungen von chronisch hypoxischen Mäusen. Der EET-Antagonist 14,15-EEZE hemmte signifikant den sEH-Inhibitor-induzierten Anstieg der akuten HPV in normoxischen Lungen und reduzierte die potenzierte akute HPV in Lungen von chronisch hypoxischen Tieren. sEH-/--Mäuse zeigten im Vergleich zu Wildtyp-Mäusen eine verstärkte Hypoxie-induzierte Rechtsherzhypertrophie und Hämatopoese. Darüber hinaus konnte in Lungen von hypoxischen und normoxischen sEH-/--Mäusen eine erhöhte Muskularisierung der pulmonalen Widerstandsgefäße nachgewiesen werden. Außerdem zeigten sEH-/--Lungen eine verstärkte akute HPV. Eine vorherige hypoxische Exposition hatte keinen potenzierenden Effekt auf die akute HPV. Allerdings reduzierte 14,15-EEZE die akute HPV in sEH-/--Lungen auf das Niveau der HPV von normoxischen Wildtyp-Lungen. Überdies führte Hypoxie (1% O2, 24 Stunden) in humanen embryonalen Nierenzellen (HEK 293), die mit sEH Promotor-Konstrukten transfiziert wurden, zu einer verminderten sEH-Promotor-Aktivität. Über die Expression der sEH in Lungen von Patienten mit pulmonaler Hypertonie ist bisher wenig bekannt. In pulmonalen Gewebeschnitten des lungengesunden Kontrollkollektivs konnte eine starke sEH-Expression in der Media der Pulmonalarterien nachgewiesen werden, während in Gewebeschnitten von Patienten mit pulmonaler Hypertonie keine sEH-Expression detektiert werden konnte. Zusammenfassend legen die Resultate des zweiten Teils dieser Arbeit nahe, dass die sEH in die Entstehung der Hypoxie-bedingten pulmonalen Hypertension und des daraus folgenden pulmonalen vaskulären Remodellings involviert ist. Die Ergebnisse der vorliegenden Arbeit zeigen, dass die Aktivität/Expression der sEH die akute hypoxische Vasokonstriktion sowie die chronische HPV und die damit einhergehenden Veränderungen der pulmonalen Gefäße beeinflusst. Ihren Effekt vermittelt die sEH über den Abbau der vasokonstringierenden EETs - vor allem ist hier 11,12-EET relevant - zu weniger aktiven Diolen. Außerdem konnte hier nachgewiesen werden, dass an der EET-induzierten Vasokonstriktion unter normoxischen und unter akut hypoxischen Bedingungen der TRPC6-Kanal, der 5-HT2A-Rezeptor und die Rho-Kinase an der Signaltransduktion beteiligt sind. Wie chronische Hypoxie die Aktivität/Expression der sEH und folglich auch die Entwicklung einer pulmonalen Hypertension und des pulmonalen vaskulären Remodelling beeinflusst, bleibt offen. Gleiches gilt für eine Involvierung des für die akute HPV beschriebenen Signaltransduktionsweges (5-HT2A-Rezeptor, TRPC6-Kanal) in diese Prozesse. In verschiedenen Studien wurde nachgewiesen, dass TRPC6-Kanäle in pulmonaler Hypertension verstärkt exprimiert werden. TRPC6-/--Mäuse zeigten im Vergleich zu Wildtyp-Mäusen jedoch keine Unterschiede in der Entwicklung einer chronisch Hypoxie-induzierten pulmonalen Hypertension. sEH-Inhibitoren werden momentan für den Einsatz in der Therapie der Hypertonie entwickelt. Angesichts der Tatsache, dass eine CYP-Epoxygenase, EETs und die Aktivität/Expression der sEH in die HPV und in das chronische Hypoxie-induzierte pulmonale vaskuläre Remodelling involviert sind, besteht die Möglichkeit, dass diese Substanzen die Entwicklung einer pulmonalen Hypertension fördern

Topics: ddc:540
Year: 2009
OAI identifier: oai:publikationen.ub.uni-frankfurt.de:6172

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