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Characterization of Lymphatic Vessels and Lymphatic Endothelial Cells in Type 2 Diabetes Mellitus

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Characterization of Lymphatic Vessels and Lymphatic Endothelial Cells in Type 2 Diabetes Mellitus CHARACTERIZATION OF LYMPHATIC VESSELS AND LYMPHATIC ENDOTHELIAL CELLS IN TYPE 2 DIABETES MELLITUS Structural, morphological and molecular analysis DOCTORAL THESIS for obtaining the academic degree of Doctor of Philosophy (Ph.D.) submitted by Monika Hämmerle, MD within the thematic program: Cell communication in health and disease (CCHD) supervised by Prof. Dr. Dontscho Kerjaschki & Dr. Brigitte Hantusch Clinical Institute of Pathology Vienna, August 2012 Acknowledgements First of all, I would like to thank my supervisors Prof. Dontscho Kerjaschki und Dr. Brigitte Hantusch for giving me the opportunity to do my PhD in the research laboratory of the Clinical Institute of Pathology and who supported me throughout the years. I would like to thank my cooperation partners at the Department of Rheumatology, Carl‐Walter Steiner, for excellent technical assistence in FACS sorting and at the Department of General Surgery, Dr. Christoph Neumayer, for guaranteeing me that I could use my material as fresh as possible. Moreover, I would like to thank Dr. Stefan Thurner and especially Dejan Stokic for helping me with the bioinformatical data analysis. I thank all my friends and colleagues from the CCHD PhD program, especially my lab mate Tom without whom the day would not have been so much fun. I would like to announce a big thank to Bernhard Höfle, who triggered my enthusiasm for science. Last but not least, I would like to thank my family and friends for their incessant support, love and motivation. Die Wissenschaft, richtig verstanden, heilt den Menschen von seinem Stolz; denn sie zeigt ihm seine Grenzen. Albert Schweitzer Summary Background ‐ Small vessel disease of kidney, nerves, retina and skin, referred to as microangiopathy, is a major cause of morbidity in type 2 diabetes mellitus (T2DM). While characteristic changes in blood capillary walls and endothelial dysfunction of blood vessels are well studied in type 2 diabetes, examination of lymphatic endothelial cells (LECs) and lymphatic vessels (LVs) is scarcely done. However, complications seen in type 2 diabetes, e.g. increased risk for infections, wound healing defects and obesity, may be related to lymphatic dysfunction. Therefore, we aimed at comprehensively analyzing potential morphological and structural differences of lymphatic endothelial cells and lymphatic vessels in the skin of type 2 diabetes mellitus patients. Further, we wanted to identify gene expression signatures that are deregulated in human dermal lymphatic vessels to define mechanisms that are linked with microvascular complications observed in type 2 diabetes. Methods – By immunohistochemistry, basement membranes of lymph vessels were analyzed and blood and lymph vessel densities of diabetic versus normoglycemic skin was evaluated. Further, we identified signs of inflammation, e.g. macrophage infiltration and TNFα expression. We compared the gene expression profiles of ex vivo isolated dermal LECs retrieved from normoglycemic versus type 2 diabetic patients using microarrays and subsequent intensive bioinformatical analysis. The up‐ or downregulated expression of selected candidate genes was confirmed by quantitative real‐time PCR and immunofluorescence stainings. Further, we focused on two differentially regulated genes and performed macrophage adhesion, transmigration and chemotatic assays as well as siRNA‐mediated knockdown experiments to identify their specific function in lymphatic endothelial cells. Results ‐ Neither prominent alterations in extracellular matrix (ECM) protein deposition, nor morphological BM changes of lymphatic capillaries and collecting LVs were found in the skin of T2DM patients. This excluded the occurrence of diabetic lymphangiopathy comparable to that of blood vessels. However, the evaluation of lymph vessel counts revealed a prominent enhanced lymph vessel density in type 2 diabetic patient's skin. Further, we traced a strong macrophage infiltration in the dermis of type 2 diabetic patients. These macrophages produced vascular endothelial growth factors VEGF‐A and VEGF‐C, as well as the pro‐inflammatory cytokine TNFα. I Transcriptomal analysis of ex vivo isolated diabetic versus non‐diabetic LECs resulted in a list of 180 differently expressed genes. Consistent with earlier studies, we identified several genes that have already been linked to genetic susceptibility for type 2 diabetes, including HP, APOD, HHEX, CD55, ANXA1, LMNA and FABP4. Essentially, we observed multiple changes related to altered LEC proliferation, adhesion and migration. Further, in line with increased TNFα abundance, we observed expression changes of CXCL10, VCAM1, CYR61, CXADR, SDC1 and AQP3. TNFα treatment of cultured LECs led to deregulated expression of selected genes, recapitulating the array results, indicating that TNFα is one major contributor to diabetes‐specific gene expression signatures in lymphatic endothelial cells. CXCL10 was confirmed as one important candidate gene only expressed in chronically inflamed lymphatic vessels, contributing to adhesion and transmigration of macrophages and possibly intending to resolute the dermal inflammation. Further, the fatty acid transporter FABP4 was specifically upregulated in LECs and lymphatic vessels in type 2 diabetes in comparison to blood endothelial cells (BECs) and blood vessels. FABP4 was shown to regulate LEC proliferation and permeability in vitro, and pointed out the crucial role of lymphatic vessels in fatty acid transport and metabolism. Conclusion ‐ These data reveal gene sets highlighting the dramatically altered milieu skin lymphatic vessels have to cope with during type 2 diabetes mellitus. Further, we discovered that skin lymphatics show a chronic subacute inflammatory phenotype characterized by macrophage recruitment and de novo lymphangiogenesis. We provide evidence for a paracrine crosstalk, mainly via TNFα and CXCL10, fostering macrophage recruitment to LECs as one pathophysiological process that might contribute to persistent inflammation and consecutively, aberrant lymphangiogenesis in the skin. II Zusammenfassung Hintergrund ‐ Mikroangiopathie, eine Erkrankung der kleinen Gefäße der Nieren, der Nerven, der Retina und der Haut, ist eine häufige Komplikation im Verlauf des Typ 2 Diabetes mellitus. Während diabetische Veränderungen der Blutgefäße in der Haut sehr gut charakterisiert sind, ist über mögliche morphologische, strukturelle und molekulare Veränderungen von Lymphgefäßen wenig bekannt. Trotzdem besteht die Vermutung, dass Komplikationen des Diabetes wie erhöhtes Infektionsrisiko, Wundheilungsstörungen und Veränderungen des Fettstoffwechsels auch die Folge einer Lymphgefäßdysfunktion sind. Das Ziel dieser Arbeit war es, potentielle Veränderungen des Lymphgefäßsystems in der Haut auf morphologischer, zellulärer und molekularer Ebene zu beschreiben. Darüber hinaus war es das Ziel, deregulierte Genexpressionsmuster zu erkennen, um sie mit den oben genannten Komplikationen in Verbindung zu bringen. Methoden – In dieser Arbeit wird eine umfassende immunhistochemische Analyse der diabetischen Haut, inklusive einer Analyse der Basalmembranen der Gefäße, der Gefäßdichte und von Zeichen einer Entzündung, präsentiert. Zusätzlich wurde mit Hilfe der Genechip Microarray‐Technologie und nachfolgender intensiver bioinformatischen Analyse das mRNA‐ Expressionsprofil der diabetischen im Vergleich zu nicht‐diabetischen Lymphendothelzellen, die ex vivo aus Patientenhaut isoliert wurden, analysiert. Die Expression wichtiger Kandidatengene wurde mit Hilfe von quantitativen PCR‐Analysen sowie Immunfluoreszenzfärbungen bestätigt. Im weiteren Verlauf haben wir funktionell auf zwei Gene fokussiert. Es wurden Makrophagenadhäsions‐, ‐transmigrations und –chemotaxis‐Experimente durchgeführt, um ihre Rolle in der Interaktion mit Makrophagen zu analysieren. In siRNA‐mediierten knockdown Studien wurde versucht, die spezifische Funktion dieser Gene in lymphatischen Endothelzellen herauszufinden. Ergebnisse ‐ Signifikante Veränderungen der Basalmembranen von, sowie erhöhte Expression von Extrazellulärmatrixproteinen rund um diabetische Lymphgefäße konnten nicht gefunden werden, was die Existenz einer sogenannten diabetischen Lymphangiopathie ausschloss. Dennoch zeigte ein Vergleich der Lymphgefäßdichte von diabetischer und nicht‐diabetischer Haut eine signifikant erhöhte Dichte der Lymphgefäße beim diabetischen Patienten. Zusätzlich konnte in der diabetischen Haut eine starke Infiltration mit Makrophagen nachgewiesen III werden. Diese Makrophagen produzierten vaskuläre Wachstumsfaktoren wie VEGF‐A und VEGF‐C, sowie das pro‐inflammatorische Zytokin TNFα. Der transkriptionelle Vergleich des mRNA‐Profils von diabetischen und nicht‐diabetischen Lymphendothelzellen führte zur Identifikation von 180 differentiell regulierten Genen. Neben Genen, die als mögliche Suszeptibilitätsgene für die Entwicklung des Typ 2 Diabetes gelten, wie z.B. HP, APOD, HHEX, CD55, ANXA1, LMNA und FABP4, wurden Transkripte gefunden, die mit der Proliferation von Lymphendothelzellen, sowie mit der Adhäsion und Migration von inflammatorischen Zellen in Gefäßen assoziiert sind, was mit dem immunhistochemischen Befund korrelierte. Es wurden besonders prominente Expressionsunterschiede von CXCL10, VCAM1, CYR61, CXADR, SDC1 und AQP3 detektiert. Diese konnten durch eine Stimulation von Lymphendothelzellen mit TNFα in vitro spezifisch rekapituliert werden. CXCL10 wurde als ein wichtiges Chemokin identifiziert, das wahrscheinlich nur im Rahmen einer Entzündung auf dermalen Lymphgefäßen exprimiert
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