CRC1366

Chronic hypoxia causes detrimental structural alterations in the lung, which are partially evoked by stress responses of the endothelium. In this context, in vitro analyses revealed that hypoxia-exposed murine lung endothelial cells (MLEC) activate nuclear factor of activated T-cells 5 (NFAT5/TonEBP) - a transcription factor that adjusts the cellular transcriptome to cope with multiple environmental stressors. In vivo, targeted ablation of Nfat5 in endothelial cells did not evoke phenotypic abnormalities in normoxia-exposed mice. However, Nfat5-deficient MLEC reinforced energy- and protein-metabolism-associated gene expression under normobaric hypoxia (10% O2) for seven days as evidenced by microarray- and scRNA-seq-based analyses. This was accompanied by reinforced release of platelet-derived growth factor B (PDGFB) from alveolar endothelial cells, intensified coverage of distal pulmonary arterioles by vascular smooth muscle cells, increased pulmonary vascular resistance and impaired right ventricular contractility.
Besides these general effects, we identified Hspa1a and Hspa1b as transcriptional targets of NFAT5 in hypoxic MLEC encoding subunits of heat shock protein 70 (HSP70). Considering the important role of HSP70 in protein folding, trafficking and degradation, we assume that shortage of HSP70 as observed in Nfat5-deficient MLEC impairs adaptation of their protein and energy metabolism in response to hypoxia. Further preparatory work on that topic suggested that hypoxia-exposed Nfat5-deficient (vs. control) MLEC (i) fail to increase HSP70 levels, (ii) consume more oxygen and (iii) show impaired mitochondrial isocitratedehydrogenase 3 (IDH3) activity and metabolite generation, including 2-hydroxyglutatarate – an important determinant of endothelial cell quiescence.
Considering these results, we intend to investigate the relevance of NFAT5 for regulating energy and protein metabolism of MLEC in a hypoxic environment. Specifically, we plan (i) to determine the role of HSP70 in adapting energy and protein metabolism in this context and (ii) to analyze the impact of endothelial metabolite generation on these parameters. The corresponding experimental approaches will be based on a large portfolio of in vivo and in vitro tools and techniques including a mouse model for the conditional EC-specific knockout of Nfat5 and induction of reporter gene expression. Perspectively, this project may delineate and eventually (therapeutically) exploit mechanisms supporting adaptation of the lung endothelium to hypoxia in the lung as caused by ventilation disorders or chronic obstructive pulmonary diseases to limit their severity and progression.