Rüdiger Popp

Epoxyeicosatrienoic acids and the soluble epoxide hydrolase are determinants of pulmonary artery pressure and the acute hypoxic pulmonary vasoconstrictor response

Recent findings have indicated a role for cytochrome P‐450 (CYP) epoxygenase‐derived epoxyeicosatrienoic acids (EETs) in acute hypoxic pulmonary vasoconstriction (HPV). Given that the intracellular concentration of EETs is determined by the soluble epoxide hydrolase (sEH), we assessed the influence of the sEH and 11,12‐EET on pulmonary artery pressure and HPV in the isolated mouse lung. In lungs from wild‐type mice, HPV was significantly increased by sEH inhibition, an effect abolished by pretreatment with CYP epoxygenase inhibitors and the EET antagonist 14,15‐EEZE. HPV and EET production were greater in lungs from sEH‐/‐ mice than from wild‐type mice and sEH inhibition had no further effect on HPV, while MSPPOH and 14,15‐EEZE decreased the response. 11,12‐EET increased pulmonary artery pressure in a concentration‐dependent manner and enhanced HPV via a Rho‐dependent mechanism. Both 11,12‐EET and hypoxia elicited the membrane translocation of a transient receptor potential (TRP) C6‐V5 fusion protein, the latter effect was sensitive to 14,15‐ EEZE. 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 data demonstrate that CYP‐derived EETs are involved in HPV and that EET‐induced pulmonary contraction under normoxic and hypoxic conditions involves a TRPC6‐dependent pathway.— Keserü, B., Barbosa‐Sicard, E., Popp, R., Fisslthaler, B., Dietrich, A., Gudermann, T., Hammock, B. D., Falck, J. R., Weissmann, N., Busse, R., Fleming, I. Epoxyeicosatrienoic acids and the soluble epoxide hydrolase are determinants of pulmonary artery pressure and the acute hypoxic pulmonary vasoconstrictor response. FASEB J. 22, 4306–4315 (2008)

Aged Spontaneously Hypertensive Rats Exhibit a Selective Loss of EDHF-Mediated Relaxation in the Renal Artery

Abstract—Endothelium-dependent relaxation is frequently attenuated in hypertension. We hypothesized that the contribution of the endothelium-derived hyperpolarizing factor (EDHF) to the acetylcholine (ACh)-induced, endothelium-dependent relaxation is attenuated with aging in the renal artery of spontaneously hypertensive rats (SHR) compared with age-matched Wistar-Kyoto (WKY) rats. ACh-induced, NO-mediated relaxation was identical in young (8-week-old) WKY and SHR, whereas EDHF-mediated relaxations (assessed in the presence of N&ohgr;-nitro-l-arginine and diclofenac) were much more pronounced in SHR than WKY. KCl-induced relaxations were more pronounced in vessels from young WKY rats than from young SHR. The cytochrome P450 inhibitor sulfaphenazole significantly inhibited EDHF-mediated relaxation in vessels from young SHR but not WKY. Vessels from old (22 months) SHR exhibited a slightly reduced NO-mediated relaxation but a complete loss of EDHF-mediated responses. In contrast, aging did not affect EDHF-mediated responses in WKY. Moreover, ACh-induced hyperpolarization and resting membrane potential were decreased in old SHR but not in WKY. KCl-induced relaxation increased with age in WKY, whereas no response to KCl was recorded in arteries from aged SHR. In vessels from old WKY but not old SHR, mRNA expression of the Na-K-ATPase subunit &agr;2 was increased by 2-fold compared with young animals. These data indicate that the increase in EDHF responses in renal arteries from aged WKY can be attributed to the release of K+ ions from the endothelium, whereas increased EDHF responses in renal arteries from young SHR can be attributed to a sulfaphenazole-sensitive cytochrome P450-dependent EDHF.

Bradykinin‐induced relaxation of coronary microarteries: S‐nitrosothiols as EDHF?

To investigate whether S‐nitrosothiols, in addition to NO, mediate bradykinin‐induced vasorelaxation, porcine coronary microarteries (PCMAs) were mounted in myographs. Following preconstriction, concentration–response curves (CRCs) were constructed to bradykinin, the NO donors S‐nitroso‐N‐penicillamine (SNAP) and diethylamine NONOate (DEA‐NONOate) and the S‐nitrosothiols L‐S‐nitrosocysteine (L‐SNC) and D‐SNC. All agonists relaxed PCMAs. L‐SNC was ∼5‐fold more potent than D‐SNC. The guanylyl cyclase inhibitor ODQ and the NO scavenger hydroxocobalamin induced a larger shift of the bradykinin CRC than the NO synthase inhibitor L‐NAME, although all three inhibitors equally suppressed bradykinin‐induced cGMP responses. Complete blockade of bradykinin‐induced relaxation was obtained with L‐NAME in the presence of the large‐ and intermediate‐conductance Ca2+‐activated K+‐channel (BKCa, IKCa) blocker charybdotoxin and the small‐conductance Ca2+‐activated K+‐channel (SKCa) channel blocker apamin, but not in the presence of L‐NAME, apamin and the BKCa channel blocker iberiotoxin. Inhibitors of cytochrome P450 epoxygenase, cyclooxygenase, voltage‐dependent K+ channels and ATP‐sensitive K+ channels did not affect bradykinin‐induced relaxation. SNAP‐, DEA‐NONOate‐ and D‐SNC‐induced relaxations were mediated entirely by the NO‐guanylyl cyclase pathway. L‐SNC‐induced relaxations were partially blocked by charybdotoxin+apamin, but not by iberiotoxin+apamin, and this blockade was abolished following endothelium removal. ODQ, but not hydroxocobalamin, prevented L‐SNC‐induced increases in cGMP, and both drugs shifted the L‐SNC CRC 5–10‐fold to the right. L‐SNC hyperpolarized intact and endothelium‐denuded coronary arteries. Our results support the concept that bradykinin‐induced relaxation is mediated via de novo synthesized NO and a non‐NO, endothelium‐derived hyperpolarizing factor (EDHF). S‐nitrosothiols, via stereoselective activation of endothelial IKCa and SKCa channels, and through direct effects on smooth muscle cells, may function as an EDHF in porcine coronary microarteries.

Cathepsin D and H2O2 Stimulate Degradation of Thioredoxin-1 IMPLICATION FOR ENDOTHELIAL CELL APOPTOSIS

Cathepsin D (CatD) is a lysosomal aspartic proteinase and plays an important role in the degradation of proteins and in apoptotic processes induced by oxidative stress, cytokines, and aging. All of these stimuli are potent inducers of endothelial cell apoptosis. Therefore, we investigated the role of CatD in endothelial cell apoptosis and determined the underlying mechanisms. Incubation with 100–500 μm H2O2 for 12 h induced apoptosis in endothelial cells. To determine a role for CatD, we co-incubated endothelial cells with the CatD inhibitor pepstatin A. Pepstatin A as well as genetic knock down of CatD abolished H2O2-induced apoptosis. In contrast, overexpression of CatD wild type but not a catalytically inactive mutant of CatD (CatDD295N) induced apoptosis under basal conditions. To gain insights into the underlying mechanisms, we investigated the effect of CatD on reactive oxygen species (ROS) formation. Indeed, knocking down CatD expression reduced H2O2-induced ROS formation and apoptosis. The major redox regulator in endothelial cells is thioredoxin-1 (Trx), which plays a crucial role in apoptosis inhibition. Thus, we hypothesized that CatD may alter Trx protein levels and thereby promote formation of ROS and apoptosis. Incubation with 100 μm H2O2 for 6 h decreased Trx protein levels, whereas Trx mRNA was not altered. H2O2-induced Trx degradation was inhibited by pepstatin A and genetic knock down of CatD but not by other protease inhibitors. Incubation of unstimulated cell lysates with recombinant CatD significantly reduced Trx protein levels in vitro, which was completely blocked by pepstatin A pre-incubation. Overexpression of CatD reduced Trx protein in cells. Moreover, H2O2 incubation led to a translocation of Trx to the lysosomes prior to the induction of apoptosis. Taken together, CatD induces apoptosis via degradation of Trx protein, which is an essential anti-apoptotic and reactive oxygen species scavenging protein in endothelial cells.

MicroRNA-223 Antagonizes Angiogenesis by Targeting β1 Integrin and Preventing Growth Factor Signaling in Endothelial Cells

Rationale: Endothelial cells in situ are largely quiescent, and their isolation and culture are associated with the switch to a proliferative phenotype. Objective: To identify antiangiogenic microRNAs expressed by native endothelial cells that are altered after isolation and culture, as well as the protein targets that regulate responses to growth factors. Methods and Results: Profiling studies revealed that miR-223 was highly expressed in freshly isolated human, murine, and porcine endothelial cells, but those levels decreased in culture. In primary cultures of endothelial cells, vascular endothelial cell growth factor and basic fibroblast growth factor further decreased miR-223 expression. The overexpression of precursor-miR-223 did not affect basal endothelial cell proliferation but abrogated vascular endothelial cell growth factor–induced and basic fibroblast growth factor–induced proliferation, as well as migration and sprouting. Inhibition of miR-223 in vivo using specific antagomirs potentiated postnatal retinal angiogenesis in wild-type mice, whereas recovery of perfusion after femoral artery ligation and endothelial sprouting from aortic rings from adult miR-223−/y animals were enhanced. MiR-223 overexpression had no effect on the growth factor–induced activation of ERK1/2 but inhibited the vascular endothelial cell growth factor–induced and basic fibroblast growth factor–induced phosphorylation of their receptors and activation of Akt. &bgr;1 integrin was identified as a target of miR-223 and its downregulation reproduced the defects in growth factor receptor phosphorylation and Akt signaling seen after miR-223 overexpression. Reintroduction of &bgr;1 integrin into miR-223–ovexpressing cells was sufficient to rescue growth factor signaling and angiogenesis. Conclusions: These results indicate that miR-223 is an antiangiogenic microRNA that prevents endothelial cell proliferation at least partly by targeting &bgr;1 integrin.

Macrophages programmed by apoptotic cells promote angiogenesis via prostaglandin E2

Macrophages contribute to tissue homeostasis in the developing as well as the adult organism. They promote tissue regeneration and remodeling after injury, which requires efficient neoangiogenesis. Signaling pathways activating an angiogenic program in macrophages are still poorly defined. We report that apoptotic cells (ACs), which originate from stressed or damaged tissues, can induce angiogenic properties in primary human macrophages. The signal originating from ACs is the lipid mediator sphingosine‐1‐phosphate (S1P), which activates S1P1/3 on macrophages to upregulate cyclooxygenase‐2. The formation and liberation of prostaglandin E2 (PGE2) then stimulates migration of endothelial cells. This is demonstrated by using PGE2 receptor antagonists or a neutralizing PGE2 antibody in vitro, thereby attenuating endothelial cell migration using a Boyden chamber assay. In vivo, neutralization of PGE2 from proangiogenic macrophage supernatants blocked vessel formation into Matrigel plugs. In particular, apoptotic cancer cells shifted prostanoid formation in macrophages selectively toward PGE2 by up‐regulating cyclooxygenase‐2 and microsomal prostaglandin E synthase‐1 (mPGES1), while down‐regulating the PGE2‐degrading enzyme 15‐hydroxyprostaglandin dehydrogenase (15‐PGDH) or prostaglandin‐D synthase (PGDS). Angiogenic programming of macrophages by ACs, therefore, may control responses to tissue stress such as in tumors, where macrophages support cancer progression.—Brecht, K., Weigert, A., Hu, J., Popp, R., Fisslthaler, B., Korff, T., Fleming, I., Geisslinger, G., Brüne, B. Macrophages programmed by apoptotic cells promote angiogenesis via prostaglandin E2. FASEB J. 25, 2408–2417 (2011). www.fasebj.org

Soluble epoxide hydrolase regulates hematopoietic progenitor cell function via generation of fatty acid diols

Fatty acid epoxides are important lipid signaling molecules involved in the regulation of vascular tone and homeostasis. Tissue and plasma levels of these mediators are determined by the activity of cytochrome P450 epoxygenases and the soluble epoxide hydrolase (sEH), and targeting the latter is an effective way of manipulating epoxide levels in vivo. We investigated the role of the sEH in regulating the mobilization and proliferation of progenitor cells with vasculogenic/reparative potential. Our studies revealed that sEH down-regulation/inhibition impaired the development of the caudal vein plexus in zebrafish, and decreased the numbers of lmo2/cmyb-positive progenitor cells therein. In mice sEH inactivation attenuated progenitor cell proliferation (spleen colony formation), but the sEH products 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME) and 11,12- dihydroxyeicosatrienoic acid stimulated canonical Wnt signaling and rescued the effects of sEH inhibition. In murine bone marrow, the epoxide/diol content increased during G-CSF–induced progenitor cell expansion and mobilization, and both mobilization and spleen colony formation were reduced in sEH−/− mice. Similarly, sEH−/− mice showed impaired functional recovery following hindlimb ischemia, which was rescued following either the restoration of bone marrow sEH activity or treatment with 12,13-DiHOME. Thus, sEH activity is required for optimal progenitor cell proliferation, whereas long-term sEH inhibition is detrimental to progenitor cell proliferation, mobilization, and vascular repair.

The F-BAR protein NOSTRIN participates in FGF signal transduction and vascular development

F‐BAR proteins are multivalent adaptors that link plasma membrane and cytoskeleton and coordinate cellular processes such as membrane protrusion and migration. Yet, little is known about the function of F‐BAR proteins in vivo. Here we report, that the F‐BAR protein NOSTRIN is necessary for proper vascular development in zebrafish and postnatal retinal angiogenesis in mice. The loss of NOSTRIN impacts on the migration of endothelial tip cells and leads to a reduction of tip cell filopodia number and length. NOSTRIN forms a complex with the GTPase Rac1 and its exchange factor Sos1 and overexpression of NOSTRIN in cells induces Rac1 activation. Furthermore, NOSTRIN is required for fibroblast growth factor 2 dependent activation of Rac1 in primary endothelial cells and the angiogenic response to fibroblast growth factor 2 in the in vivo matrigel plug assay. We propose a novel regulatory circuit, in which NOSTRIN assembles a signalling complex containing FGFR1, Rac1 and Sos1 thereby facilitating the activation of Rac1 in endothelial cells during developmental angiogenesis.

The biological actions of 11,12-epoxyeicosatrienoic acid in endothelial cells are specific to the R/S-enantiomer and require the G(s) protein.

Cytochrome P450–derived epoxides of arachidonic acid [i.e., the epoxyeicosatrienoic acids (EETs)] are important lipid signaling molecules involved in the regulation of vascular tone and angiogenesis. Because many actions of 11,12-cis-epoxyeicosatrienoic acid (EET) are dependent on the activation of protein kinase A (PKA), the existence of a cell-surface Gs-coupled receptor has been postulated. To assess whether the responses of endothelial cells to 11,12-EET are enantiomer specific and linked to a potential G protein–coupled receptor, we assessed 11,12-EET-induced, PKA-dependent translocation of transient receptor potential (TRP) C6 channels, as well as angiogenesis. In primary cultures of human endothelial cells, (±)-11,12-EET led to the rapid (30 seconds) translocation a TRPC6-V5 fusion protein, an effect reproduced by 11(R),12(S)-EET, but not by 11(S),12(R)-EET or (±)-14,15-EET. Similarly, endothelial cell migration and tube formation were stimulated by (±)-11,12-EET and 11(R),12(S)-EET, whereas 11(S),12(R)-EET and 11,12-dihydroxyeicosatrienoic acid were without effect. The effects of (±)-11,12-EET on TRP channel translocation and angiogenesis were sensitive to EET antagonists, and TRP channel trafficking was also prevented by a PKA inhibitor. The small interfering RNA-mediated downregulation of Gs in endothelial cells had no significant effect on responses stimulated by vascular endothelial growth or a PKA activator but abolished responses to (±)-11,12-EET. The downregulation of Gq/11 failed to prevent 11,12-EET–induced TRPC6 channel translocation or the formation of capillary-like structures. Taken together, our results suggest that a Gs-coupled receptor in the endothelial cell membrane responds to 11(R),12(S)-EET and mediates the PKA-dependent translocation and activation of TRPC6 channels, as well as angiogenesis.

Müller glia cells regulate Notch signaling and retinal angiogenesis via the generation of 19,20-dihydroxydocosapentaenoic acid

Cytochrome P450 (CYP) epoxygenases generate bioactive lipid epoxides which can be further metabolized to supposedly less active diols by the soluble epoxide hydrolase (sEH). As the role of epoxides and diols in angiogenesis is unclear, we compared retinal vasculature development in wild-type and sEH−/− mice. Deletion of the sEH significantly delayed angiogenesis, tip cell, and filopodia formation, a phenomenon associated with activation of the Notch signaling pathway. In the retina, sEH was localized in Muller glia cells, and Muller cell–specific sEH deletion reproduced the sEH−/− retinal phenotype. Lipid profiling revealed that sEH deletion decreased retinal and Muller cell levels of 19,20–dihydroxydocosapentaenoic acid (DHDP), a diol of docosahexenoic acid (DHA). 19,20-DHDP suppressed endothelial Notch signaling in vitro via inhibition of the γ-secretase and the redistribution of presenilin 1 from lipid rafts. Moreover, 19,20-DHDP, but not the parent epoxide, was able to rescue the defective angiogenesis in sEH−/− mice as well as in animals lacking the Fbxw7 ubiquitin ligase, which demonstrate strong basal activity of the Notch signaling cascade. These studies demonstrate that retinal angiogenesis is regulated by a novel form of neuroretina–vascular interaction involving the sEH-dependent generation of a diol of DHA in Muller cells.