Clin Res Cardiol (2021). 10.1007/s00392-021-01933-9

Bioinformatics-based transcription factor profiling in hypoxic human endothelial cells highlights pro-angiogenic signaling

A. Schmidt1, M. Fuchs2, S. Stojanović1, C. Liang2, K. Schmidt1, J. Weusthoff3, M. Jung1, K. Xiao1, A. Just1, A. Pfanne1, M. Kunz2, T. Thum1, für die Studiengruppe: IMTTS

1Institut für Molekulare und Translationale Therapiestrategien, OE-8886, Medizinische Hochschule Hannover, Hannover; 2Institut für Medizininformatik, Biometrie und Epidemiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen; 3Institut für molekulare und translationale Therapiestrategien, Medizinische Hochschule Hannover, Hannover;

Background: Constant supply of oxygen is a key event for cellular and tissue homeostasis regulating energy metabolism in cardiac tissue. As a first response to acute hypoxia, endothelial cells promote recruitment and adherence of immune cells to the dysbalanced endothelium by releasing inflammatory mediators and growth factors, whereas longterm hypoxia leads to the activation of a transcription factor battery (e.g. HIF-1α), that potently induces expression of growth factors and cytokines including platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). Herein, we apply a novel bioinformatic approach aiming to identify transcription factors that regulate short-term angiogenic signaling. Methods and Results: Initially, a previously reported RNA-Seq dataset derived from human ECs subjected to normoxic or hypoxic conditions was selected to identify significantly regulated genes based on (A) fold change (normoxia vs hypoxia) and (B) relative abundancy. Transcriptional regulation of this gene set was confirmed via qPCR in validation experiments where HUVECs were incubated for 24 h under hypoxic conditions. Screening the promotor and upstream regulatory elements of these genes identified two transcription factors: KLF5 and SP1, both with a possible binding site within these regions of selected target genes. In vitro, siRNA experiments confirmed SP-1 and KLF5-mediated regulation of identified hypoxia-sensitive endothelial genes. Next to angiogenic signaling, we also validated impact of endothelial transcription factors on inflammatory and autophagic signaling, both key events in hypoxic sensing. Additionally, we characterized SP1- and KLF5-deficient endothelial cells in terms of their angiogenic capabilities by examining proliferation, migration and tube formation. Conclusion: By detailed in silico analysis of the promotor region and upstream regulatory elements for a list of preselected hypoxia-sensitive genes, our software approach can identify putative binding sites for transcription factors of SP or KLF family in vitro. This strategy highlights which transcription factors are functionally involved in human angiogenic signaling and therefore serves as a base for identifying novel drug entities either supporting or inhibiting these networks in a therapeutic setting.

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