Die Rolle der Epoxyeicosatriensäuren (EETs) bei der nozizeptiven Verarbeitung

Im Rahmen dieser Arbeit wurden Epoxyeicosatriensäuren (EETs) hinsichtlich ihrer Beteiligung an der Verarbeitung nozizeptiver Information untersucht. Im ersten Teil der Arbeit lag der Fokus auf der löslichen Epoxidhydrolase (sEH) und der drei von ihr metabolisierten EETs, 8,9-, 11,12-, und 14,15-EET. Dabei stellte sich heraus, dass sEH-defiziente Mäuse eine verlängerte mechanische Hyperalgesie bei zymosan-induziertem pathophysiologischen Nozizeptorschmerz aufwiesen. Anhand von Lipidmessungen mittels LC-MS/MS konnte gezeigt werden, dass zum Zeitpunkt des stärksten Schmerzempfindens (48 Stunden nach Zymosan-Injektion) vorwiegend 8,9-EET in den Dorsalwurzelganglien der sEH-defizienten Mäuse akkumuliert. Zudem wurde anhand von Calcium-Imaging-Versuchen gezeigt, dass 8,9-EET Calcium-Einströme in primär afferenten Neuronen von Wildtyp-Mäusen hervorruft, und eine Stimulation von Ischiasnerven mit 8,9-EET zu erhöhter Freisetzung des pronozizeptiven Peptids CGRP führt. Schließlich konnte gezeigt werden, dass Wildtyp-Mäuse nach intraplantarer 8,9-EET-Injektion eine geringere mechanische Schmerzschwelle aufweisen. Die Resultate dieses Teils der Arbeit weisen darauf hin, dass die lösliche Epoxidhydrolase (sEH) eine wichtige Rolle in der späten Phase des pathophy-siologischen Nozizeptorschmerzes spielt, indem sie 8,9-EET zu seinem bioinaktiven Metaboliten 8,9-DHET umsetzt. Im zweiten Teil der Arbeit wurde 5,6-EET gesondert untersucht, da es nicht durch sEH metabolisiert wird. Dabei wurde beobachtet, dass 5,6-EET bei akutem Schmerz in DRGs freigesetzt wird. In Calcium-Imaging-Versuchen mit DRG-Neuronen aus Wildtyp- TRPV4- und TRPA1-defizienten Mäusen sowie transfizierten Zelllinien zeigte sich, dass schon geringe Konzentrationen an 5,6-EET den TRPA1- (transient receptor potetntial ankyrin 1-) Kanal aktivieren (EC50 193 nM) und den TRPV1-Kanal sensibilisieren können. Auch die CGRP-Freisetzung am Ischiasnerv ist nach 5,6-EET-Stimulation signifikant erhöht. Zudem konnte beobachtet werden dass eine periphere Injektion von 5,6-EET zu akuter mechanischer Hyperalgesie in Wildtyp-, aber nicht in TRPA1-defizienten Mäusen führt. Die Resultate dieses Teils der Arbeit weisen 5,6-EET als bisher potentesten endogenen TRPA1-Aktivator aus, und implizieren eine wichtige Rolle dieses Lipids beim Übergang von physiologischem zu pathophysiologischem Nozizeptorschmerz und zu neruogener Inflammation. Darüber hinaus leisten die Resultate einen Beitrag zum grundlegenden Verständnis endogener TRP-Kanal-Aktivatoren bei der Schmerzwahrnehmung

The FKBP51 Inhibitor SAFit2 Restores the Pain-Relieving C16 Dihydroceramide after Nerve Injury

Neuropathic pain is a pathological pain state with a broad symptom scope that affects patients after nerve injuries, but it can also arise after infections or exposure to toxic substances. Current treatment possibilities are still limited because of the low efficacy and severe adverse effects of available therapeutics, highlighting an emerging need for novel analgesics and for a detailed understanding of the pathophysiological alterations in the onset and maintenance of neuropathic pain. Here, we show that the novel and highly specific FKBP51 inhibitor SAFit2 restores lipid signaling and metabolism in nervous tissue after nerve injury. More specifically, we identify that SAFit2 restores the levels of the C16 dihydroceramide, which significantly reduces the sensitization of the pain-mediating TRPV1 channel and subsequently the secretion of the pro-inflammatory neuropeptide CGRP in primary sensory neurons. Furthermore, we show that the C16 dihydroceramide is capable of reducing acute thermal hypersensitivity in a capsaicin mouse model. In conclusion, we report for the first time the C16 dihydroceramide as a novel and crucial lipid mediator in the context of neuropathic pain as it has analgesic properties, contributing to the pain-relieving properties of SAFit2

Soluble epoxide hydrolase limits mechanical hyperalgesia during inflammation.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: Cytochrome-P450 (CYP450) epoxygenases metabolise arachidonic acid (AA) into four different biologically active epoxyeicosatrienoic acid (EET) regioisomers. Three of the EETs (i.e., 8,9-, 11,12- and 14,15-EET) are rapidly hydrolysed by the enzyme soluble epoxide hydrolase (sEH). Here, we investigated the role of sEH in nociceptive processing during peripheral inflammation. RESULTS: In dorsal root ganglia (DRG), we found that sEH is expressed in medium and large diameter neurofilament 200-positive neurons. Isolated DRG-neurons from sEH(-/-) mice showed higher EET and lower DHET levels. Upon AA stimulation, the largest changes in EET levels occurred in culture media, indicating both that cell associated EET concentrations quickly reach saturation and EET-hydrolyzing activity mostly effects extracellular EET signaling. In vivo, DRGs from sEH-deficient mice exhibited elevated 8,9-, 11,12- and 14,15-EET-levels. Interestingly, EET levels did not increase at the site of zymosan-induced inflammation. Cellular imaging experiments revealed direct calcium flux responses to 8,9-EET in a subpopulation of nociceptors. In addition, 8,9-EET sensitized AITC-induced calcium increases in DRG neurons and AITC-induced calcitonin gene related peptide (CGRP) release from sciatic nerve axons, indicating that 8,9-EET sensitizes TRPA1-expressing neurons, which are known to contribute to mechanical hyperalgesia. Supporting this, sEH(-/-) mice showed increased nociceptive responses to mechanical stimulation during zymosan-induced inflammation and 8,9-EET injection reduced mechanical thresholds in naive mice. CONCLUSION: Our results show that the sEH can regulate mechanical hyperalgesia during inflammation by inactivating 8,9-EET, which sensitizes TRPA1-expressing nociceptors. Therefore we suggest that influencing the CYP450 pathway, which is actually highly considered to treat cardiovascular diseases, may cause pain side effects.Peer Reviewe

The G2A Receptor Controls Polarization of Macrophage by Determining Their Localization Within the Inflamed Tissue

Macrophages are highly versatile cells, which acquire, depending on their microenvironment, pro- (M1-like), or antiinflammatory (M2-like) phenotypes. Here, we studied the role of the G-protein coupled receptor G2A (GPR132), in chemotactic migration and polarization of macrophages, using the zymosan-model of acute inflammation. G2A-deficient mice showed a reduced zymosan-induced thermal hyperalgesia, which was reversed after macrophage depletion. Fittingly, the number of M1-like macrophages was reduced in the inflamed tissue in G2A-deficient mice. However, G2A activation was not sufficient to promote M1-polarization in bone marrow-derived macrophages. While the number of monocyte-derived macrophages in the inflamed paw was not altered, G2A-deficient mice had less macrophages in the direct vicinity of the origin of inflammation, an area marked by the presence of zymosan, neutrophil accumulation and proinflammatory cytokines. Fittingly neutrophil efferocytosis was decreased in G2A-deficient mice and several lipids, which are released by neutrophils and promote G2A-mediated chemotaxis, were increased in the inflamed tissue. Taken together, G2A is necessary to position macrophages in the proinflammatory microenvironment surrounding the center of inflammation. In absence of G2A the macrophages are localized in an antiinflammatory microenvironment and macrophage polarization is shifted toward M2-like macrophages

Oxidized lipids in persistent pain states

Chemotherapy, nerve injuries, or diseases like multiple sclerosis can cause pathophysiological processes of persistent and neuropathic pain. Thereby, the activation threshold of ion channels is reduced in peripheral sensory neurons to normally noxious stimuli like heat, cold, acid, or mechanical due to sensitization processes. This leads to enhanced neuronal activity, which can result in mechanical allodynia, cold allodynia, thermal hyperalgesia, spontaneous pain, and may initiate persistent and neuropathic pain. The treatment options for persistent and neuropathic pain patients are limited; for about 50% of them, current medication is not efficient due to severe side effects or low response to the treatment. Therefore, it is of special interest to find additional treatment strategies. One approach is the control of neuronal sensitization processes. Herein, signaling lipids are crucial mediators and play an important role during the onset and maintenance of pain. As preclinical studies demonstrate, lipids may act as endogenous ligands or may sensitize transient receptor potential (TRP)-channels. Likewise, they can cause enhanced activity of sensory neurons by mechanisms involving G-protein coupled receptors and activation of intracellular protein kinases. In this regard, oxidized metabolites of the essential fatty acid linoleic acid, 9- and 13-hydroxyoctadecadienoic acid (HODE), their dihydroxy-metabolites (DiHOMEs), as well as epoxides of linoleic acid (EpOMEs) and of arachidonic acid (EETs), as well as lysophospholipids, sphingolipids, and specialized pro-resolving mediators (SPMs) have been reported to play distinct roles in pain transmission or inhibition. Here, we discuss the underlying molecular mechanisms of the oxidized linoleic acid metabolites and eicosanoids. Furthermore, we critically evaluate their role as potential targets for the development of novel analgesics and for the treatment of persistent or neuropathic pain

Drug repurposing to target neuroinflammation and sensory neuron-dependent pain

Around 20% of the American population have chronic pain and estimates in other Western countries report similar numbers. This represents a major challenge for global health care systems. Additional problems for the treatment of chronic and persistent pain are the comparably low efficacy of existing therapies, the failure to translate effects observed in preclinical pain models to human patients and related setbacks in clinical trials from previous attempts to develop novel analgesics. Drug repurposing offers an alternative approach to identify novel analgesics as it can bypass various steps of classical drug development. In recent years, several approved drugs were attributed analgesic properties. Here, we review available data and discuss recent findings suggesting that the approved drugs minocycline, fingolimod, pioglitazone, nilotinib, telmisartan, and others, which were originally developed for the treatment of different pathologies, can have analgesic, antihyperalgesic, or neuroprotective effects in preclinical and clinical models of inflammatory or neuropathic pain. For our analysis, we subdivide the drugs into substances that can target neuroinflammation or substances that can act on peripheral sensory neurons, and highlight the proposed mechanisms. Finally, we discuss the merits and challenges of drug repurposing for the development of novel analgesics

Proton-sensing GPCRs in health and disease

The group of proton-sensing G-protein coupled receptors (GPCRs) consists of the four receptors GPR4, TDAG8 (GPR65), OGR1 (GPR68), and G2A (GPR132). These receptors are cellular sensors of acidification, a property that has been attributed to the presence of crucial histidine residues. However, the pH detection varies considerably among the group of proton-sensing GPCRs and ranges from pH of 5.5 to 7.8. While the proton-sensing GPCRs were initially considered to detect acidic cellular environments in the context of inflammation, recent observations have expanded our knowledge about their physiological and pathophysiological functions and many additional individual and unique features have been discovered that suggest a more differentiated role of these receptors in health and disease. It is known that all four receptors contribute to different aspects of tumor biology, cardiovascular physiology, and asthma. However, apart from their overlapping functions, they seem to have individual properties, and recent publications identify potential roles of individual GPCRs in mechanosensation, intestinal inflammation, oncoimmunological interactions, hematopoiesis, as well as inflammatory and neuropathic pain. Here, we put together the knowledge about the biological functions and structural features of the four proton-sensing GPCRs and discuss the biological role of each of the four receptors individually. We explore all currently known pharmacological modulators of the four receptors and highlight potential use. Finally, we point out knowledge gaps in the biological and pharmacological context of proton-sensing GPCRs that should be addressed by future studies

Next-generation sequencing of the human TRPV1 gene and the regulating co-players LTB4R and LTB4R2 based on a custom AmpliSeq™ panel

Background: Transient receptor potential cation channel subfamily V member 1 (TRPV1) are sensitive to heat, capsaicin, pungent chemicals and other noxious stimuli. They play important roles in the pain pathway where in concert with proinflammatory factors such as leukotrienes they mediate sensitization and hyperalgesia. TRPV1 is the target of several novel analgesics drugs under development and therefore, TRPV1 genetic variants might represent promising candidates for pharmacogenetic modulators of drug effects. Methods: A next-generation sequencing (NGS) panel was created for the human TRPV1 gene and in addition, for the leukotriene receptors BLT1 and BLT2 recently described to modulate TRPV1 mediated sensitisation processes rendering the coding genes LTB4R and LTB4R2 important co-players in pharmacogenetic approaches involving TRPV1. The NGS workflow was based on a custom AmpliSeq™ panel and designed for sequencing of human genes on an Ion PGM™ Sequencer. A cohort of 80 healthy subjects of Western European descent was screened to evaluate and validate the detection of exomic sequences of the coding genes with 25 base pair exon padding. Results: The amplicons covered approximately 97% of the target sequence. A median of 2.81 x 10 6 reads per run was obtained. This identified approximately 140 chromosome loci where nucleotides deviated from the reference sequence GRCh37 hg19 comprising the three genes TRPV1, LTB4R and LTB4R2. Correspondence between NGS and Sanger derived nucleotide sequences was 100%. Conclusions: Results suggested that the NGS approach based on AmpliSeq™ libraries and Ion Personal Genome Machine (PGM) sequencing is a highly efficient mutation detection method. It is suitable for large-scale sequencing of TRPV1 and functionally related genes. The method adds a large amount of genetic information as a basis for complete analysis of TRPV1 ion channel genetics and its functional consequences

Oxidized lipids in the treatment of chronic or neuropathic pain

Neuropathic pain is a debilitating disease with poor treatment options. Clinical investigations conclude that early therapeutic intervention is crucial for increasing the therapeutic success and for ameliorating neuropathic pain. However, in patients with diabetes or patients that suffer from adverse events of chemotherapy, the onset of neuropathic pain is difficult to estimate. For this reason, biomarkers represent important diagnostic markers that may be used for therapeutic strategies and, in an ideal case, for the prediction of onset, intensity and duration of neuropathic pain even before the first symptoms arise in patients. Using animal models of neuropathic pain, we observe that oxidized lipids and epoxylipids are generated in nervous tissue and plasma even before neuropathic pain arises in the animals. We therefore suggest determination of the concentrations of these lipids by mass spectrometry (LC-MS/MS) in the plasma of patients as potential biomarkers for neuropathic pain. Using this method, high-risk patients may be identified early and pharmacological treatment may start before neuropathic pain is established. The early treatment may lead to a reduction or even prevention of neuropathic pain in these patients