Publikationsdatenbank

Expression of VIM, TPI and MAT2A and their impact on in vitro angiogenesis in human dermal microvascular endothelial cells (2024)

Art

Hochschulschrift

Autor

Herre, Christina Nicole (WE 1)

Quelle

Berlin: Mensch & Buch Verlag, 2024 — VI, 96 Seiten

ISBN: 978-3-96729-258-9

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/44959

Kontakt

Institut für Veterinär-Anatomie

Koserstr. 20
14195 Berlin
+49 30 838 75784
anatomie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Angiogenesis is the dynamic process of building new blood vessels from pre-existing ones. During sprouting angiogenesis, tip cells are mainly responsible for migration, stalk cells for proliferation and phalanx cells for maturation of the new vessel. Major research effort was directed to anti- and pro-angiogenic therapy. Therefore, in vitro models are frequently used. However, these assays face problems regarding reproducibility based on single stage assays and the inhomogeneous character of endothelial cells (ECs). When establishing and performing the all-in-one assay, which covers all stages of in vitro angiogenesis, differences regarding angiogenic potency was detected resulting in a classification of ECs into angiogenic and non-angiogenic. Proteome expression profiles of both classes exhibited one protein only found in non-angiogenic ECs, i.e. adenosylmethionine synthetase isoform type 2 (MAT2A), and seven proteins exclusively in angiogenic ECs. MAT2A represents a highly conserved enzyme being mainly involved in regulatory functions and suggested to have anti-angiogenic effects. Among the seven proteins found in angiogenic ECs, vimentin (VIM) and triosephosphate isomerase (TPI) are hypothesized to have pro-angiogenic impact on ECs. By VIM being a type III intermediate filament protein, it is highly involved in cell shape and motility. TPI is a glycolic enzyme generating energy and mainly influencing cell proliferation. Both studies aimed to determine whether the expression of VIM, TPI and MAT2A is related to angiogenesis in vitro in human dermal microvascular endothelial cells (HDMECs). Therefore, two batches of HDMECs were long-term cultivated using pro-angiogenic media. Quantification of in vitro angiogenesis was carried out using phase-contrast microscopy twice a week. Knockdown groups got infected with lentiviral particles initiating a knockdown of VIM or TPI respectively. Additionally, a non-coding sequence was used for the infection of control groups. At days 1, 5, 25 and 50, cells of all groups were harvested and used for mRNA and Protein expression analysis. The mRNA expression of vascular endothelial growth factor 1 (VEGFR-1), vascular endothelial growth factor 2 (VEGFR-2), and mRNA and protein expression of VIM, TPI and MAT2A were determined by RT-qPCR and Western Blot. In native cells, VIM was shown to be expressed mainly in beginning stages of sprouting and migrating, which are suggested to be enabled by VIM’s influence on the cytoskeleton. By knocking down VIM, cell death and a deceleration of in vitro angiogenesis was observed leading to the conclusion of VIM being an essential protein for HDMECs survival and having pro-angiogenic effects on ECs. For TPI, native cells showed an overall increase in Expression over the cultivation period. By TPI providing energy for cells, it is suggested to be essential for most angiogenic stages, i.e. cell migration, proliferation, and tube formation. By knocking down TPI, a deceleration of in vitro Angiogenesis was observed, leading to the assumption of TPI increasing the angiogenic potency of ECs. Furthermore, native TPI expression was shown to be consistent. The single decrease of TPI expression was connected to cell death, showing the essential character of TPI for HDMECs survival. Native MAT2A expression was highest at the beginning of cultivation, followed by a significant decrease. Moreover, a final increase was detected. With MAT2A owning regulatory functions via methylations, it is suggested to have anti-angiogenic effects, mainly expressed in quiescent cells and involved in maturation of HDMECs. In both knockdown studies, MAT2A displayed an overall tendency of being increased when lowering the angiogenic potency, supporting the assumption of MAT2A impacting anti-angiogenic events. By characterizing both batches of HDMECs via VEGFR-1 and VEGFR-2 expression, it was shown that HD1 comprised a higher amount of stalk cells. The difference in distribution of cell differentiation led to a divergence in angiogenic, compensatory and expressional behaviour of the batches. HD1 inherited a higher proliferative power which was visible in them having a higher cell density and higher values of sums of assigned stages. With HD1 owning more stalk cells, VIM expression was lower and TPI was significantly higher in contrast to HD2. Furthermore, HD2 displayed an acceleration of in vitro angiogenesis, which was connected to higher MAT2A Ct-values. Additionally, knockdown cells of HD1 were able to recover from infection by increasing their cell density and progressing to further angiogenic stages, while knockdown cells of HD2 were either stagnating their differentiation or displaying cell death. Further, control group of HD2 displayed a deceleration of in vitro angiogenesis while control group of HD1 was unaffected. Overall, it has been shown that the characterization of cell differentiations is of immense importance for EC application and the interpretation of in vitro angiogenesis assays. Experiments should get repeated including cells from different distributors involving nonangiogenic cells, cell characterization should be extended by an additional detection method and specific assays should get employed for validating protein effects. For comprehension to living systems, subsequent experiments should involve more than one cell type, followed by in vivo studies. MAT2A knockdown should be initiated and further proteins influencing the angiogenic potency should be examined. Furthermore, upcoming investigations should Focus on developing and optimizing the characterization of EC populations to increase reproducibility and reliability of studies in the field of in vitro angiogenesis.