AG Genetische Diversität der kornealen (Lymph)angiogenese
A und B: Repräsentative Flachpräparate der Hornhaut von Mäusen aus C57BL/6N (A) und B6N-TyrcBrd (B), gefärbt für LYVE-1. Die eingekreisten Bereiche in A und B Oben sind Unten in höherer Vergrößerung dargestellt. Die gestrichelten Linien zeigen die Grenze zwischen dem Limbus und der Hornhaut.
C: Quantifizierung der lymphatisch vaskularisierten Fläche. D und E: Weitere Charakterisierung der Lymphgefäßarchitektur durch Bestimmung der Anzahl der Verzweigungspunkte (D) und der Anzahl der Endpunkte (E). Die Ergebnisse sind als Durchschnittswerte von C57BL/6N- und B6N-TyrcBrd-Mäusen dargestellt. F und G: Repräsentative Flachpräparate der Hornhaut nach entzündungsinduzierten Lymphangiogenese bei C57BL/6N (F) und B6N-TyrcBrd (G), gefärbt für LYVE-1. Die gestrichelten Linien zeigen die Grenze zwischen dem Limbus und der Hornhaut. H: Quantifizierung der entzündlichen, lymphatischen, vaskularisierten Fläche. Die statistische Signifikanz wurde mit einem zweiseitigen t-Test berechnet. Die Daten sind als Mittelwerte SEM angegeben. n = 7 C57BL/6N-Mäuse (A und C-E); n = 6 B6N-TyrcBrd-Mäuse (B und C-E); n = 6 C57BL/6N- und B6N-TyrcBrd-Mäuse (F-H). *p < 0,05, **p < 0,01, und ***p < 0,001. Skalenbalken: 500 mm (A und B, oben, F und G); 200 mm (A und B, unteren). Originalvergrößerung, x100 (A, B, F und G).
Copyright: Elsevier
Im Gegensatz zur Angiogenese, bei der in den letzten Jahrzehnten wesentliche Fortschritte beim Verständnis der molekularen Mechanismen und Regulationswege erzielt wurden, wurde die Erforschung der Lymphangiogenese lange Zeit durch das Fehlen spezifischer molekularer Marker behindert. Dies änderte sich mit der Entdeckung spezifischer molekularer Marker, wie LYVE-1, Podoplanin, Prox1 und VEGFR-3, und verschiedener in vitro- und in vivo-Modelle in den letzten Jahren.
Die Hornhaut stellt ein gut etabliertes Modell dar, um die Mechanismen der (Lymph-)Angiogenese zu untersuchen. Die Vorteile der Hornhaut als Modell sind ihre physiologische Avaskularität, ihre Transparenz und exponierte Lage. Nach einer experimentell ausgelösten Entzündung können sowohl Blut- als auch Lymphgefäße, die aus bereits bestehenden Gefäßen im Hornhautlimbus entstehen, in die Hornhaut einwachsen.
Unsere Gruppe konnte zeigen, dass sowohl in der Entwicklung als auch unter Entzündungsbedingungen Unterschiede in der lymphangiogenen Reaktion der Hornhaut bei verschiedenen Mausstämmen vom genetischen Hintergrund abhängen (wobei C57BL/6 "hoch" und BALB/c "niedrig-lymphangiogene" Stämme sind). Die zugrundeliegenden genetischen Ursachen für die interindividuellen Unterschiede sind jedoch nur teilweise bekannt. In den letzten Jahren konnten wir neue Modulatoren der Lymphangiogenese, wie Trail, tPa und Tyrosinase, identifizieren und verifizieren. Unsere Forschung konzentriert sich auf die Identifizierung weiterer Kandidatengene, Genmodule und Signalwege, die für die beobachteten stammabhängigen Unterschiede verantwortlich sind. Diese neuen identifizierten endogenen Regulatoren könnten dazu beitragen, neue therapeutische Ziele für die Behandlung der pathologischen Lymphangiogenese bei einer Reihe von okulären und extraokulären Erkrankungen wie Transplantatabstoßung oder Tumormetastasierung zu entwickeln.
A and B: Representative whole mounts of the murine cornea from C57BL/6N (A) and albino C57BL/6N (B6N-TyrcBrd) (B) stained for LYVE-1. The boxed areas in the top panels are shown in higher magnification in the bottom panels. Dashed lines show the border between the limbus and the cornea. C: Quantification of the lymphatic vascularized area of the whole mounts. D and E: Further characterization of the lymphatic vessel architecture by determination of the number of branching points (D) and the number of end points (E). Results are presented as the average of C57BL/6N and B6N-TyrcBrd mice. F and G: Representative corneal whole mounts of inflammation-induced lymphangiogenesis of C57BL/6N (F) and B6N-TyrcBrd (G) stained for LYVE-1. Dashed lines show the border between the limbus and the cornea. H: Quantification of the inflammatory lymphatic vascularized area of the whole mounts. Statistical significance was calculated by two-tailed t-test. Data are expressed as means SEM. n = 7 C57BL/6N mice (A and C-E); n = 6 B6N-TyrcBrd mice (B and C-E); n = 6 C57BL/6N and B6N-TyrcBrd mice (F-H). *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bars: 500 mm (A and B, top panels, F, and G); 200 mm (A and B, bottom panels). Original magnification, x100 (A, B, F, and G).
Copyright: Elsevier
Contrary to angiogenesis, where substantial progress in understanding the molecular mechanisms and regulation-pathways was gained in the last decades, lymphangiogenesis research was long hampered by the absence of specific molecular markers. This changed with the discovery of specific molecular markers, such as LYVE-1, Podoplanin, Prox1, and VEGFR-3 and various in vitro and in vivo models in the last few years.
The cornea is a well-established model to analyze the mechanisms underlying (lymph)angiogenesis. Advantages of the cornea as a model are its physiological avascularity, transparency and exposed position. Following experimentally induced inflammation, both blood and lymphatic vessels arising from pre-existing vessels in the corneal limbus can grow into the cornea. Our group was able to show that both developmentally as well as under inflammatory conditions, differences in the lymphangiogenic response of the cornea in different mouse strains depend on the genetic background (with C57BL/6 being “high-” and BALB/c being “low-lymphangiogenic” strains). However, the underlying genetic causes of the inter-individual differences are only partly understood. Over the last years we were able to identify and verify novel modulators of lymphangiogenesis, like Trail, tPa, and tyrosinase.
Our research focuses on the identification of additional candidate genes, gene modules, and signaling pathways responsible for the observed strain-dependent differences. These newly identified endogenous regulators may help to develop new therapeutic targets for the treatment of pathological lymphangiogenesis in a variety of ocular and extraocular diseases such as graft rejection or tumor metastasis.
Projects
Identification of novel endogenous modulators of developmental and inflammatory lymphangiogenesis by analyzing mouse strain-specific differences
The focus lies in the identification of further candidate genes, gene modules and pathways, which are responsible for the observed strain-dependent differences by using different inbred strains and Collaborative Cross Lines and the subsequent functional analysis of molecular pathways of the novel lymphangioregulatory candidates. Novel endogenous regulators identified in this project may help to develop new therapeutic targets for the treatment of pathological lymphangiogenesis in a variety of ocular and extraocular diseases such as transplant rejection or tumor metastasis.
Functional relevance and molecular mechanisms of tyrosinase in controlling lymphangiogenesis in different ocular diseases
Tyrosinase is primarily expressed in melanocytes in the skin but is also found in different compartments of the eye, such as the uvea, retinal pigment epithelium, and in the limbal area. Furthermore, tyrosinase is also significantly involved in the developmental process of the eye and has a putative function as modifier of the drainage structure phenotype in primary congenital glaucoma. The functional relevance of tyrosinase in regulating ocular lymphatic vessels at different locations is yet unclear. Therefore, this project analyzes the functional relevance and underlying molecular mechanism of tyrosinase in regulating ocular lymphangiogenesis using different ocular disease models.
Our findings will provide novel therapeutic strategies to modulate (pathological) corneal lymphangiogenesis / lymphatic vessel function in corneal transplantation, glaucoma, and limbal stem cell disease.
Strain- and metabolism-dependent genetic variations of (lymph)angiogenesis: identification of candidates and influence on corneal wound healing
There are about 60 million patients with diabetes in the European Union and 4.6 million in Germany. Diabetics not only show impaired (corneal) wound healing, they also develop various other eye complications such as diabetic retinopathy. The disease affects all corneal layers, corneal nerves and tear film. Diabetics have a stronger inflammatory reaction after eye surgery, which can lead to a higher rate of immune rejections after corneal transplantation. In this context, impaired inflammatory cell function, reduced secretion of cytokines/growth factors, and a prolonged inflammatory phase could be observed. Therefore, CC lines with metabolic disease will be investigated using quantitative trait locus (QTL) mapping of the lymphatic vessel phenotypes. In addition, RNA-sequence analysis and expression QTL (eQTL) mapping in these CC lines under metabolic disease conditions will be performed and compare them with corresponding healthy lines in order to determine differences in the regulation of the modulators. The novel endogenous factors identified in this project will contribute to the understanding of disease mechanisms. When translated to humans, they will help identification of new therapeutic targets involved in pathological lymphangiogenesis and impaired wound healing, with and without metabolic disorder. They will also be of importance for numerous other projects in the CRC, such as the development of personalized anti(lymph)angiogenic therapies.
Clahsen, T., K. Hadrian, M. Notara, S.L. Schlereth, A. Howaldt, V. Prokosch, T. Volatier, D. Hos, F. Schroedl, A. Kaser-Eichberger, L.M. Heindl, P. Steven, J.J. Bosch, A. Steinkasserer, A.C. Rokohl, H. Liu, M. Mestanoglu, H. Kashkar, B. Schumacher, F. Kiefer, S. Schulte-Merker, M. Matthaei, Y. Hou, S. Fassbender, J. Jantsch, W. Zhang, P. Enders, B. Bachmann, F. Bock, and C. Cursiefen. 2023. The novel role of lymphatic vessels in the pathogenesis of ocular diseases. Prog Retin Eye Res 101157.
Howaldt A, W. Zhang, S. Deng, M. Becker, Y. Hou, T. Clahsen, F. Bock, C. Cursiefen. 2023 Corneal crosslinking ameliorates the extent of corneal oedema in subsequent acute keratoconus in an ex vivo model. Acta Ophthalmol. 2023 May 31
Hatami, N., C. Buttner, F. Bock, S. Simfors, G. Musial, A. Reis, C. Cursiefen, and T. Clahsen. 2022. Cystathionine beta-synthase as novel endogenous regulator of lymphangiogenesis via modulating VEGF receptor 2 and 3. Commun Biol 5:950.
Ozer, O., M. Mestanoglu, A. Howaldt, T. Clahsen, P. Schiller, S. Siebelmann, N. Reinking, C. Cursiefen, B. Bachmann, and M. Matthaei. 2022. Correlation of Clinical Fibrillar Layer Detection and Corneal Thickness in Advanced Fuchs Endothelial Corneal Dystrophy. J Clin Med 11:
Hribek, A., M. Mestanoglu, T. Clahsen, N. Reinking, F. Frentzen, A. Howaldt, S. Siebelmann, B.O. Bachmann, C. Cursiefen, and M. Matthaei. 2022. Scheimpflug Backscatter Imaging of the Fibrillar Layer in Fuchs Endothelial Corneal Dystrophy. Am J Ophthalmol 235:63-70.
Hribek, A., T. Clahsen, J. Horstmann, S. Siebelmann, N. Loreck, L.M. Heindl, B.O. Bachmann, C. Cursiefen, and M. Matthaei. 2021. Fibrillar Layer as a Marker for Areas of Pronounced Corneal Endothelial Cell Loss in Advanced Fuchs Endothelial Corneal Dystrophy. Am J Ophthalmol 222:292-301.
Clahsen, T., C. Buttner, N. Hatami, A. Reis, and C. Cursiefen. 2020. Role of Endogenous Regulators of Hem- And Lymphangiogenesis in Corneal Transplantation. J Clin Med 9:
Hayashi, T., D. Hos, S. Schrittenlocher, S. Siebelmann, M. Matthaei, J. Franklin, T. Clahsen, F. Bock, B. Bachmann, and C. Cursiefen. 2020. Effect of Iris Color on the Outcome of Descemet Membrane Endothelial Keratoplasty. Cornea 39:846-850.