HTRA1: Ein Kandidatengen für die Altersbedingte Makuladegeneration?

Polymorphismen in der chromosomalen Region 10q26 sind stark mit einem erhöhten Erkrankungsrisiko der Altersabhängigen Makuladegeneration (AMD) assoziiert. In diesem Bereich liegt das Gen high temperature requirement protein A1 (HTRA1). Das Gen kodiert eine Serinprotease, die vor allem in der Qualitätskontrolle von Extrazellulärmatrix (EZM)-Proteinen eine Rolle spielt. Der single nucleotide polymorphism (SNP) rs11200638 im Promotor des HTRA1-Gens verursacht eine Überexpression von HTRA1 im retinalen Pigmentepithel (RPE) und erhöht das Erkrankungsrisiko der AMD deutlich. In AMD-Patienten wurde eine zwei- bis dreifache Überexpression an HTRA1-Protein in RPE-Zellen nachgewiesen. Bislang gab es jedoch keine funktionellen Studien zur Überexpression von HTRA1 im RPE, die Aufschluss über die Beteiligung von HTRA1 an der Entstehung der AMD geben könnten. In dieser Arbeit wurde die Rolle der HTRA1-Überexpression in der Pathogenese der AMD untersucht. Dazu wurden transgene Mäuse generiert, die HTRA1-Protein im RPE 2,7-fach überexprimierten. Die Htra1-transgenen Mäuse entwickelten keine spontane choroidale Neovaskularisation (CNV) in der Retina. Die CNV-Induktion mittels Laserkoagulation ließ in der Fluoreszenz-Angiographie (FAG), der spektralen optischen Kohärenztomographie (SD-OCT) und histologischen Untersuchungen nicht auf eine erhöhte Angiogenese durch die HTRA1-Überexpression schließen. Ebenfalls besaßen Htra1-transgene Mäuse im Vergleich zum Wildtypen keine Expressionsunterschiede vom transformierenden Wachstumsfaktor-beta (TGF-ß), vom Insulin-ähnlichen Wachstumsfaktors-1 (IGF-1) und vom vaskulären endothelialen Wachstumsfaktor (VEGF) im Auge. Unterdessen zeigte sich in Zellkulturüberständen Htra1-transfizierter Nierenzellen (HEK-293-EBNA) eine reduzierte Konzentration von VEGF, Endostatin und Angiogenin im Vergleich zu nicht-transfizierten Zellen und Kontrollen. Die Konzentration von Angiopoietin-1 war dagegen leicht erhöht. Anhand von Transmissonselektronenmikroskopie (TEM)-Bildern war eine Fragmentierung der elastischen Schicht (EL) der Bruchschen Membran (BrM) in Htra1-transgenen Mäusen zu beobachten. Zusätzlich kam es zu verminderten Expressionen von Fibulin-5 und Tropoelastin (TE) in Protein-Lysaten von RPE, BrM und Choroidea. Versuche mit rekombinantem HTRA1 bestätigten weiterhin den Abbau von Fibulin-5 durch HTRA1. Diese Daten deuten auf eine gestörte Elastogenese in der BrM von Htra1-transgenen Mäusen hin. Interessanterweise sind Mutationen im FBLN5 (Fibulin-5)-Gen, die zu einer reduzierten Sekretion von Fibulin-5 führen, mit der AMD assoziiert. Darüber hinaus war die Stabilität der BrM weiterhin durch den HTRA1-bedingten Abbau von Fibronektin gestört. So war die Expression von Fibronektin und Fibronektin-Fragmenten in den transgenen Mäusen erhöht und die Adhäsion Htra1-transfizierter Nieren- und RPE (ARPE-19)-Zellen an Fibronektin deutlich reduziert. Zudem wurden Fibronektin und Nidogen-1 und -2, aber nicht Laminin-1 und Kollagen IV von rekombinantem HTRA1-Protein proteolytisch gespalten. Die Immunfluoreszenzfärbungen auf Paraffinschnitten der Retinae Htra1-transgener und Wildtyp-Mäuse ließen allerdings keine Änderungen in der Farbintensität oder Verteilung von Nidogen-1 und -2 und auch Laminin-1 und Kollagen IV erkennen. Die Ergebnisse dieser Arbeit zeigen, dass die BrM eine wichtige Rolle bei der Entstehung der AMD hat. Eine HTRA1-Überexpression führt durch die Reduktion von Fibulin-5 und TE zu einer gestörten Elastogenese in der EL der BrM. Auf Grund dieser Ergebnisse kann HTRA1 auch mit anderen AMD-Risikogenen wie MMP-9 (Matrixmetalloproteinase-9), TIMP-3 (Gewebeinhibitor von Metalloptroteinasen-3) und FBLN-3, -5 und -6 in Verbindung gebracht werden

Enolase 1 (ENO1) and protein disulfide-isomerase associated 3 (PDIA3) regulate Wnt/beta-catenin-driven trans-differentiation of murine alveolar epithelial cells

The alveolar epithelium represents a major site of tissue destruction during lung injury. It consists of alveolar epithelial type I (ATI) and type II (ATII) cells. ATII cells are capable of self-renewal and exert progenitor function for ATI cells upon alveolar epithelial injury. Cell differentiation pathways enabling this plasticity and allowing for proper repair, however, are poorly understood. Here, we applied proteomics, expression analysis and functional studies in primary murine ATII cells to identify proteins and molecular mechanisms involved in alveolar epithelial plasticity. Mass spectrometry of cultured ATII cells revealed a reduction of carbonyl reductase 2 (CBR2) and an increase in enolase 1 (ENO1) and protein disulfide-isomerase associated 3 (PDIA3) protein expression during ATII-to-ATI cell trans-differentiation. This was accompanied by increased Wnt/beta-catenin signaling, as analyzed by qRT-PCR and immunoblotting. Notably, ENO1 and PDIA3, along with T1 alpha (podoplanin;an ATI cell marker),exhibited decreased protein expression upon pharmacological and molecular Wnt/beta-catenin inhibition in cultured ATII cells, whereas CBR2 levels were stabilized. Moreover, we analyzed primary ATII cells from mice with bleomycin-induced lung injury, a model exhibiting activated Wnt/beta-catenin signaling in vivo. We observed reduced CBR2 significantly correlating with surfactant protein C (SFTPC),whereas ENO1 and PDIA3 along with T1 alpha were increased in injured ATII cells. Finally, siRNA-mediated knockdown of ENO1, as well as PDIA3, in primary ATII cells led to reduced T1 alpha expression, indicating diminished cell trans-differentiation. Our data thus identified proteins involved in ATII-to-ATI cell trans-differentiation and suggest a Wnt/beta-catenin-driven functional role of ENO1 and PDIA3 in alveolar epithelial cell plasticity in lung injury and repair

Overexpression of HTRA1 Leads to Ultrastructural Changes in the Elastic Layer of Bruch's Membrane via Cleavage of Extracellular Matrix Components

Variants in the chromosomal region 10q26 are strongly associated with an increased risk for age-related macular degeneration (AMD). Two potential AMD genes are located in this region: ARMS2 and HTRA1 (high-temperature requirement A1). Previous studies have suggested that polymorphisms in the promotor region of HTRA1 result in overexpression of HTRA1 protein. This study investigated the role of HTRA1 overexpression in the pathogenesis of AMD. Transgenic Htra1 mice overexpressing the murine protein in the retinal pigment epithelium (RPE) layer of the retina were generated and characterized by transmission electron microscopy, immunofluorescence staining and Western Blot analysis. The elastic layer of Bruch's membrane (BM) in the Htra1 transgenic mice was fragmented and less continuous than in wild type (WT) controls. Recombinant HTRA1 lacking the N-terminal domain cleaved various extracellular matrix (ECM) proteins. Subsequent Western Blot analysis revealed an overexpression of fibronectin fragments and a reduction of fibulin 5 and tropoelastin in the RPE/choroid layer in transgenic mice compared to WT. Fibulin 5 is essential for elastogenesis by promoting elastic fiber assembly and maturation. Taken together, our data implicate that HTRA1 overexpression leads to an altered elastogenesis in BM through fibulin 5 cleavage. It highlights the importance of ECM related proteins in the development of AMD and links HTRA1 to other AMD risk genes such as fibulin 5, fibulin 6, ARMS2 and TIMP3

Upregulation of TGF-1 in experimental proliferative vitreoretinopathy is accompanied by epithelial to mesenchymal transition

Proliferative vitreoretinopathy (PVR) is characterized by epithelial to mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells and consecutive formation of fibrous membranes, leading to retinal redetachment. Transforming growth factor beta (TGF-) has been suggested to play an important role in this process, but the role of TGF- isoforms is unknown. In pigmented rabbits (n = 14), PVR was induced by cryopexy and a full-thickness limbus-parallel incision. PVR was evaluated by indirect ophthalmoscopy. Concentrations of TGF- isoforms were determined by multiplex bead assay analysis in aqueous humor (AH) and vitreous samples. EMT marker vimentin was analyzed by western blot. Masson's-trichrome, haematoxilin and eosine (H&E), and immunohistochemical analysis for EMT marker alpha SMA were performed on cross-sections of eyes. PVR was induced in all treated eyes. The number of quadrants affected by PVR was 1 (n = 5), 2 (n = 2), 3 (n = 2), 4 (n = 5). Vimentin and alpha SMA were expressed during PVR development. During PVR development, both TGF-1 levels (AH: p = 0.001; vitreous: p = 0.002) and TGF-2 levels increased (AH: p = 0.027; vitreous: p = 0.02), while TGF-3 was not detected at any timepoint. The increase was more pronounced for TGF-1 than for TGF- 2 (AH: p = 0.002; vitreous: p = 0.0005), and only TGF-1 correlated with the amount of PVR (p = 0.024, r = 0,723). Development of PVR membranes was accompanied by a pronounced upregulation of TGF-1, rather than TGF-2. Therefore TGF-1 could be a promising target for inhibition of PVR

In-vivo and ex-vivo characterization of laser-induced choroidal neovascularization variability in mice

Background Retinal argon laser coagulation is an established procedure for induction of choroidal neovascularization (CNV) in rodents. This study aimed to evaluate the in-vivo and ex-vivo morphology and variability of laser-induced CNV spots over time. Methods Female C57/Bl/6 mice, 3-6 months of age, were treated with five spots of retinal argon laser coagulation per eye (150 mW, 100 ms, 50 mu m). In-vivo fluorescein angiography (FA) and standard high-resolution spectral-domain optical coherence tomography (SD-OCT) were performed on day (d) 0, d1, d4, d7, d14 and d21. Ex-vivo histology, CD31 immunostaining, flatmount and confocal microscopy were also conducted. CNV size in the retinal and choroidal focus, CNV morphology, central retinal thickness (CRT) and FA CNV activity grading were assessed in-vivo at all times and compared to the ex-vivo assessments. Results SD-OCT revealed sub-retinal and intra-retinal fluid, and permitted evaluation of longitudinal morphologic changes of the induced CNV. Laser spot area in FA and CRT in SD-OCT did not differ in longitudinal evaluation. CNV could not be consistently outlined on SD-OCT images, and CNV volume as assessed on SD-OCT did not change over time. Significant CNV activity changes were only found in FA CNV activity grading, peaking on d4 and decreasing by d7. Conclusions Non-invasive SD-OCT provides additional morphological information on laser-induced CNV. However, reliable evaluation of CNV requires FA. Spontaneous regression of CNV activity within 14 days after induction has to be taken into account when utilizing this model for testing the efficacies of potential future treatments

Additional file 1: Table S1. of BARD1 mediates TGF-β signaling in pulmonary fibrosis

Table S1 shows a list of transcription factors that bind to the BARD1 promoter sequence and are relevant to TGF-β signaling. Figure S1. BARD1 is overexpressed in response to hypoxia. Lung epithelial cells (human A549) and fibroblasts (mouse L929) were cultured in normal condition or in hypoxia, and cell extracts were prepared after 24 and 48 hours. Western Blot using anti-BARD1 BL (mapping exon 4). In hypoxia conditions, both BARD1 FL and BARD1β are upregulated in epithelial A549 cells. In fibroblasts L929, we can observe an upregulation of BARD1β at 48h whereas FL BARD1 is only weakly expressed. Figure S2. No staining observed in the absence of primary antibody. IHC was performed on adjacent tissue sections from NSIP patients without primary antibody (a-c) or with anti-BARD1 BL antibody (d-f). Figure S3. BARD1 expression in non-malignant and malignant cell lines. BARD1 expression is stronger in the murine immortalized but non-tumorigenic cell line derived from alveolar type II pneumocytes (E10) than in tumour cell lines (MLE12, murine tumorigenic transformed lung epithelial cells and A549, human adenocarcinomic alveolar basal epithelial cells). Figure S4. FL BARD1 and BARD1β overexpression in A549 cells. (A) Exon structures of mRNAs of FL BARD1 and BARD1β. Arrows indicate position of forward and reverse primers used for RT-PCR. (B) Epithelial cells (human lung cancer cells A549) were transfected with FL BARD1 (FL and FL2) or BARD1β (β ) expressing plasmids, or control plasmid (Co), and cell extracts were prepared after 48 hours. Reverse transcription and PCR was performed using primers from human exon1 to 11 of FL BARD1 (ex1-ex11) and from human exon 1/4 junction (BARD1β -specific) to exon 11of BARD1β (ex1/4-ex11). GAPDH PCR was performed as a control for cDNA quantity. Detailed Description of Materials and Methods. (PDF 1516 kb