Endothelin signalling in the retina

Das vasoaktive Peptid Endothelin wird als drei Isoformen (Endothelin 1, 2, 3) exprimiert, die an zwei G-Protein gekoppelte Rezeptoren Endothelin Rezeptor Typ a (Eta) und Endothelin Rezeptor Typ b (Etb) binden können. Die Rolle des Endothelin-Signalwegs im Auge ist noch weitgehendst unbekannt und wird kontrovers diskutiert. Deshalb war es das Hauptziel dieser Arbeit die Rolle des Endothelin-Signalwegs im Auge zu untersuchen und besonders einem potenziellen neuroprotektiven Effekt von Etb für das Überleben von Photorezeptoren nach Licht-induzierter Schädigung nachzugehen. Außerdem sollten die zugrundeliegenden molekularen Mechanismen aufgeschlüsselt werden. Zunächst wurde die Lokalisation des Etb im Auge untersucht und durch immunhistochemische Doppelfärbungen dessen zelltypspezifische Lokalisation bestimmt. Außerdem wurde die retinale Expressionstärke von Etb im Vergleich zu Eta ermittelt. Des Weiteren sollte die Expression des gesamten Endothelin-Signalwegs in wildtypischen Retinae im Falle von okulärem Trauma, hervorgerufen durch alleinige Perforation des Auges, PBS-Injektion oder Lichtschaden, untersucht werden. In allen drei Schadensmodellen kam es zu einer Aktivierung des gesamten Endothelin-Signalwegs. Anschließend generierten wir mittels Cre-loxP-System eine Mauslinie mit einer Deletion von Etb im gesamten Auge (EtbΔeye) und eine weitere Mauslinie mit einer Etb Deletion in Müllerzellen und retinalen Neuronen (EtbΔOC). Darüber hinaus etablierten wir Photorezeptorzelllinien mit einer stabilen Deletion von Etb mittels des CRISPR/Cas9-System. Die Deletion von Etb in retinalen Neuronen und Müllerzellen führte zu keinen offensichtlichen Veränderungen der retinalen Morphologie und Gefäßstruktur. Allerdings war die Vulnerabilität der Photorezeptoren bei EtbΔOC Mäusen nach Licht-induzierten Photorezeptor Degeneration signifikant höher, einhergehend mit einer signifikant dünner äußeren Körnerschicht 14 Tage nach Lichtschaden. Auch die Etb-defizienten Photorezeptor-Zelllinien wiesen eine signifikant höhere Apoptoserate nach Zellstress, verursacht durch Serum-Entzug, auf. Weiterführende molekulare Analysen zeigten, eine signifikante Erhöhung der neuroprotektiven Faktoren leucaemia inhibitory factor (lif) und fibroblast growth factor (fgf2) bei lichtgeschädigten EtbΔOC Mäusen im Vergleich zu lichtgeschädigten Kontrolltieren. Überraschenderweise detektierten wir dennoch eine signifikante Erhöhung von Caspase 8 bei lichtgeschädigten EtbΔOC Mäusen im Vergleich zu lichtgeschädigten Kontrolltieren, was den Rückschluss nahelegt, dass die beobachtete verstärkte Degeneration der Photorezeptoren durch den extrinsischen, d.h. durch Todesrezeptoren vermittelten Signalweg ablief. Weiterführende Analysen identifizierten eine verminderte Aktivierung (Phosphorylierung) des neuroprotektiven Protein Kinase B (Akt) Signalwegs als den Signalweg worüber Etb sein neuroprotektives Signal vermittelt. Zusammenfassend zeigen die erhobenen Daten erstmals, dass das durch den Etb vermittelte Signal essenziell und weitaus potenter für das Überleben von Photorezeptoren ist als die Wirkung der neuroprotektiven Faktoren fibroblast growth factor 2 (Fgf2) und leukaemia inhibitory factor. Downstream von Etb wird die Neuroprotektion über den Akt-Signalweg vermittelt. Zukünftig kann dieser durch Etb vermittelte Signalweg einen vielversprechenden Ansatz für die Entwicklung von neuen Therapieansätzen zur Behandlung retinaler Degenerationen wie Retinopathia pigmentosa oder altersbedingte Makuladegeneration darstellen

Complement Components Showed a Time-Dependent Local Expression Pattern in Constant and Acute White Light-Induced Photoreceptor Damage

Background: Photoreceptor cell death due to extensive light exposure and induced oxidative-stress are associated with retinal degeneration. A correlated dysregulation of the complement system amplifies the damaging effects, but the local and time-dependent progression of this mechanism is not thoroughly understood. Methods: Light-induced photoreceptor damage (LD) was induced in Balb/c mice with white light illumination either for 24 h with 1000 lux (constant model) or 0.5 h with 5000 lux (acute model). Complement protein and mRNA expression levels were compared at 1 and 3 days post-LD for C1s, complement factor B (CFB), mannose binding lectin A, mannose-binding protein-associated serine protease 1 (MASP-1), C3, C4, C9, and complement factor P in retina and RPE/choroid. Histological analyses visualized apoptosis, microglia/macrophage migration, gliosis and deposition of the complement activation marker C3d. Systemic anaphylatoxin serum concentrations were determined using an ELISA. Results: Apoptosis, gliosis and microglia/macrophage migration into the outer nuclear layer showed similar patterns in both models. Local complement factor expression revealed an early upregulation of complement factor mRNA in the acute and constant light regimen at 1 day post-treatment for c1s, cfb, masp-1, c3, c4 and c9 in the RPE/choroid. However, intraretinal complement mRNA expression for c1s, cfb, c3 and c4 was increased at 1 day in the constant and at 3 days in the acute model. A corresponding regulation on protein level in the retina following both LD models was observed for C3, which was upregulated at 1 day and correlated with increased C3d staining in the ganglion cell layer and at the RPE. In the RPE/choroid C1s-complex protein detection was increased at 3 days after LD irrespectively of the light intensities used. Conclusion: LD in mouse eyes is correlated with local complement activity. The time-dependent local progression of complement regulation on mRNA and protein levels were equivalent in the acute and constant LD model, except for the intraretinal, time-dependent mRNA expression. Knowing the relative time courses of local complement expression and cellular activity can help to elucidate novel therapeutic options in retinal degeneration indicating at which time point of disease complement has to be rebalanced

Isolation of Hox Cluster Genes from Insects Reveals an Accelerated Sequence Evolution Rate

Among gene families it is the Hox genes and among metazoan animals it is the insects (Hexapoda) that have attracted particular attention for studying the evolution of development. Surprisingly though, no Hox genes have been isolated from 26 out of 35 insect orders yet, and the existing sequences derive mainly from only two orders (61% from Hymenoptera and 22% from Diptera). We have designed insect specific primers and isolated 37 new partial homeobox sequences of Hox cluster genes (lab, pb, Hox3, ftz, Antp, Scr, abd-a, Abd-B, Dfd, and Ubx) from six insect orders, which are crucial to insect phylogenetics. These new gene sequences provide a first step towards comparative Hox gene studies in insects. Furthermore, comparative distance analyses of homeobox sequences reveal a correlation between gene divergence rate and species radiation success with insects showing the highest rate of homeobox sequence evolution

Cell Invasion by Neisseria meningitidis Requires a Functional Interplay between the Focal Adhesion Kinase, Src and Cortactin

Entry of Neisseria meningitidis (the meningococcus) into human brain microvascular endothelial cells (HBMEC) is mediated by fibronectin or vitronectin bound to the surface protein Opc forming a bridge to the respective integrins. This interaction leads to cytoskeletal rearrangement and uptake of meningococci. In this study, we determined that the focal adhesion kinase (FAK), which directly associates with integrins, is involved in integrin-mediated internalization of N. meningitidis in HBMEC. Inhibition of FAK activity by the specific FAK inhibitor PF 573882 reduced Opc-mediated invasion of HBMEC more than 90%. Moreover, overexpression of FAK mutants that were either impaired in the kinase activity or were not capable of autophosphorylation or overexpression of the dominant-negative version of FAK (FRNK) blocked integrin-mediated internalization of N. meningitidis. Importantly, FAK-deficient fibroblasts were significantly less invaded by N. meningitidis. Furthermore, N. meningitidis induced tyrosine phosphorylation of several host proteins including the FAK/Src complex substrate cortactin. Inhibition of cortactin expression by siRNA silencing and mutation of critical amino acid residues within cortactin, that encompass Arp2/3 association and dynamin binding, significantly reduced meningococcal invasion into eukaryotic cells suggesting that both domains are critical for efficient uptake of N. meningitidis into eukaryotic cells. Together, these results indicate that N. meningitidis exploits the integrin signal pathway for its entry and that FAK mediates the transfer of signals from activated integrins to the cytoskeleton. A cooperative interplay between FAK, Src and cortactin then enables endocytosis of N. meningitidis into host cells

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

SMAD7 deficiency stimulates Müller progenitor cell proliferation during the development of the mammalian retina

The transforming growth factor-beta (TGF-beta) pathway contributes to maintain the quiescence of adult neural stem and progenitor cells in the brain. In the retina, Muller cells are discussed to represent a glial cell population with progenitor-like characteristics. Here, we aimed to investigate if elevated TGF-beta signaling modulates the proliferation of Muller cells during retinal development. We generated mutant mice with a systemic, heterozygous up-regulation of TGF-beta signaling by deleting its inhibitor SMAD7. We investigated apoptosis, proliferation, and differentiation of Muller cells in the developing retina. We show that a heterozygous deletion of SMAD7 results in an increased proliferation of Muller cell progenitors in the central retina at postnatal day 4, the time window when Muller cells differentiate in the mouse retina. This in turn results in a thickened retina and inner nuclear layer and a higher number of differentiated Muller cells in the more developed retina. Muller cells in mutant mice contain higher amounts of nestin than those of control animals which indicates that the increase in TGF-beta signaling activity during retinal development contribute to maintain some progenitor-like characteristics in Muller cells even after their differentiation period. We conclude that TGF-beta signaling influences Muller cell proliferation and differentiation during retinal development

Transcriptional Profiling Identifies Upregulation of Neuroprotective Pathways in Retinitis Pigmentosa

Hereditary retinal degenerations like retinitis pigmentosa (RP) are among the leading causes of blindness in younger patients. To enable in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death and to allow testing of therapeutic strategies that could prevent retinal degeneration, animal models have been created. In this study, we deeply characterized the transcriptional profile of mice carrying the transgene rhodopsin V20G/P23H/P27L (VPP), which is a model for autosomal dominant RP. We examined the degree of photoreceptor degeneration and studied the impact of the VPP transgene-induced retinal degeneration on the transcriptome level of the retina using next generation RNA sequencing (RNASeq) analyses followed by weighted correlation network analysis (WGCNA). We furthermore identified cellular subpopulations responsible for some of the observed dysregulations using in situ hybridizations, immunofluorescence staining, and 3D reconstruction. Using RNASeq analysis, we identified 9256 dysregulated genes and six significantly associated gene modules in the subsequently performed WGCNA. Gene ontology enrichment showed, among others, dysregulation of genes involved in TGF-β regulated extracellular matrix organization, the (ocular) immune system/response, and cellular homeostasis. Moreover, heatmaps confirmed clustering of significantly dysregulated genes coding for components of the TGF-β, G-protein activated, and VEGF signaling pathway. 3D reconstructions of immunostained/in situ hybridized sections revealed retinal neurons and Müller cells as the major cellular population expressing representative components of these signaling pathways. The predominant effect of VPP-induced photoreceptor degeneration pointed towards induction of neuroinflammation and the upregulation of neuroprotective pathways like TGF-β, G-protein activated, and VEGF signaling. Thus, modulation of these processes and signaling pathways might represent new therapeutic options to delay the degeneration of photoreceptors in diseases like RP

Deficiency in Retinal TGFβ Signaling Aggravates Neurodegeneration by Modulating Pro-Apoptotic and MAP Kinase Pathways

Transforming growth factor β (TGFβ) signaling has manifold functions such as regulation of cell growth, differentiation, migration, and apoptosis. Moreover, there is increasing evidence that it also acts in a neuroprotective manner. We recently showed that TGFβ receptor type 2 (Tgfbr2) is upregulated in retinal neurons and Müller cells during retinal degeneration. In this study we investigated if this upregulation of TGFβ signaling would have functional consequences in protecting retinal neurons. To this end, we analyzed the impact of TGFβ signaling on photoreceptor viability using mice with cell type-specific deletion of Tgfbr2 in retinal neurons and Müller cells (Tgfbr2ΔOC) in combination with a genetic model of photoreceptor degeneration (VPP). We examined retinal morphology and the degree of photoreceptor degeneration, as well as alterations of the retinal transcriptome. In summary, retinal morphology was not altered due to TGFβ signaling deficiency. In contrast, VPP-induced photoreceptor degeneration was drastically exacerbated in double mutant mice (Tgfbr2ΔOC; VPP) by induction of pro-apoptotic genes and dysregulation of the MAP kinase pathway. Therefore, TGFβ signaling in retinal neurons and Müller cells exhibits a neuroprotective effect and might pose promising therapeutic options to attenuate photoreceptor degeneration in humans

Deficiency in retinal TGFβ signaling aggravates neurodegeneration by modulating pro-apoptotic and MAP kinase pathways

Transforming growth factor β (TGFβ) signaling has manifold functions such as regulation of cell growth, differentiation, migration, and apoptosis. Moreover, there is increasing evidence that it also acts in a neuroprotective manner. We recently showed that TGFβ receptor type 2 (Tgfbr2) is upregulated in retinal neurons and Müller cells during retinal degeneration. In this study we investigated if this upregulation of TGFβ signaling would have functional consequences in protecting retinal neurons. To this end, we analyzed the impact of TGFβ signaling on photoreceptor viability using mice with cell type-specific deletion of Tgfbr2 in retinal neurons and Müller cells (Tgfbr2$_{ΔOC}$) in combination with a genetic model of photoreceptor degeneration (VPP). We examined retinal morphology and the degree of photoreceptor degeneration, as well as alterations of the retinal transcriptome. In summary, retinal morphology was not altered due to TGFβ signaling deficiency. In contrast, VPP-induced photoreceptor degeneration was drastically exacerbated in double mutant mice (Tgfbr2$_{ΔOC}$; VPP) by induction of pro-apoptotic genes and dysregulation of the MAP kinase pathway. Therefore, TGFβ signaling in retinal neurons and Müller cells exhibits a neuroprotective effect and might pose promising therapeutic options to attenuate photoreceptor degeneration in humans