Räumliche, molekulare und funktionale Charakterisierung von organspezifischen lymphatischen Endothelvorläuferzellen
Zellbiologie
Zusammenfassung der Projektergebnisse
The lymphatic vasculature controls fluid homeostasis, transport of lipids, antigens, and immune cells. Hence it plays central roles in lipid metabolism, tumor metastasis as well as immunity and consequently the lymphatic system is implicated in the pathogenesis of common diseases such as obesity, autoimmunity, atherosclerosis and cancer. Recently, novel, nonvenous-derived lymphatic endothelial progenitor cells (LEPCs) that contribute to the formation of organ-specific lymphatic vasculatures, e.g. in the mesentery, have been identified. Based on Tie2-Cre-, Pdgfb-CreERT2- and cKit-CreERT2-, but not Vav-Cre-positive lineage tracing it has been suggested that mesenteric LEPCs likely derive from hemogenic endothelium. This fellowship proposal aimed to (i) define the precise origin of mesenteric non-venous LEPCs using genetic lineage tracing; (ii) to characterize the molecular identity of mesenteric LEPCs by single cell RNA sequencing; and (iii) to characterize the functional properties of lymphatic endothelial cells (LECs) from non-venous- and venous sources using mouse models for selected progenitor signature genes. (i) Based on the suggested hemogenic endothelial origin of mesenteric LEPCs, novel inducible Runx1-CreERT2 and Gfi1-CreERT2 Cre lines were generated and used in lineage tracing experiments. Both strains efficiently traced hematopoietic cells, whereas only for the Runx1- CreERT2 strain recombination in hemogenic endothelial clusters within the dorsal aorta and vitelline as well as umbilical artery were observed. However, lineage tracing of endothelial cells (ECs) was just observed using the Runx1-CreERT2 strain and limited to few cells in the yolk sac vasculature, while mesenteric LEPCs could not be traced. Based on these results we speculate that Runx1 and its target Gfi1 might be too far downstream in endothelial-hematopoietic transition and therefore predominantly label hematopoietic cells but not LEPCs. A blood EC-specific Flt1- CreERT2 strain on the other hand specifically recombined in endothelial but not hematopoietic cells thus indicating lack of recombination in hemogenic ECs. Currently, additional inducible Cre strains, such as Cdh5-CreERT2 and Pdgfb-CreERT2 are being used to shed further light on the origin of mesenteric LEPCs. (ii) To decipher the molecular identity of mesenteric LEPCs single cell RNA sequencing (Smart-seq2) of E14 (isolated LEC/LEPC clusters) and E16 (lumenized lymphatic vessels exposed to flow) mesenteric blood and lymphatic ECs was successfully performed and different LEC subclusters were identified. A detailed characterization of these clusters using the identified cluster-specific genes is currently ongoing by immunostaining and RNAscope. In addition, the E14 dataset is being compared to the dataset derived from E16 mesenteries which might allow for the definition of a molecular LEPCs signature. (iii) In parallel, cytoskeletal/cytoskeleton-related lymphatic signature genes were selected using a RNA sequencing and Affymetrix dataset of freshly isolated murine dermal blood and lymphatic ECs. With this 11 LEC-enriched cytoskeletal/cytoskeleton-associated genes were identified and validated in vitro. Based on this the roles of three genes (Mmrn1, Itgb3, and Apold1) in lymphangiogenesis and endothelial homeostasis were further characterized in vivo using different knockout mouse models and partially correlated to human pathologies. These studies identified a potential novel therapeutic target for anti- or pro-lymphangiogenic therapy as well as revealed a new candidate gene for a vasculature-based bleeding diathesis.
Projektbezogene Publikationen (Auswahl)
- Twinfilin 2a regulates platelet reactivity and turnover in mice. Blood 2017
Stritt S, Beck S, Becker IC, Vögtle T, Hakala M, Heinze KG, Du X, Bender M, Braun A, Lappalainen P, Nieswandt B
(Siehe online unter https://doi.org/10.1182/blood-2017-02-770768) - Profilin 1-mediated cytoskeletal rearrangements regulate integrin function in mouse platelets. Blood Advances 2018
Stritt S, Birkholz I, Beck S, Sorrentino S, Sapra KT, Viaud J, Heck J, Gaits-Iacovoni F, Schulze H, Du X, Hartwig JH, Braun A, Bender M, Medalia O, Nieswandt B
(Siehe online unter https://doi.org/10.1182/bloodadvances.2017014001) - Transient loss of venous integrity during developmental vascular remodeling leads to red blood cell extravasation and clearance by lymphatic vessels. Development 2018
Zhang Y, Daubel N, Stritt S, Mäkinen T
(Siehe online unter https://doi.org/10.1242/dev.156745)
