Impact of co-activation of AT1-, thromboxane A2- and EGF-receptors in primary human endothelial cells

Abstract Background In vivo vascular cells are simultaneously exposed to a dynamic microenvironment of multiple vasoactive mediators that act through distinct receptor systems, such as angiotensin II type 1 (AT1R), thromboxane A2 (TP), and epidermal growth factor (EGFR) receptors. Those are thus often activated concurrently, modulating complex signaling networks that regulate e.g., vascular tone, remodeling, and inflammation, and can contribute to vascular pathology development. In contrast, in vitro models are often used to study the effect of single stimuli, thereby creating simplistic cellular environments. But they also allow controlled combined receptor stimulation, offering then a unique platform to dissect complex pathways that may underlie pathological conditions, such as endothelial dysfunction. Purpose We investgated the functional interactions between AT1R, TP and EGFR in primary human vascular endothelial cells (EC) from various female donors. Specifically, we aimed to determine whether simultaneous receptor activation exerts a synergistic (over- or under-additive) action on EC, leading to qualitatively different outcomes compared to single-receptor activation. Methods EC were incubated for 48h with specific ligands of AT1R (angiotensin II), TP (U46619, thromboxane A2 analog) and EGFR (EGF), applied individually or in defined combinations. Cellular responses were assessed through analyses of viability, proliferation, glucose consumption and lipid accumulation, and the measurement of endothelial- and dedifferentiation-markers expression (including vWF, eNOS, IL-6, and ICAM-1). Results Co-activation of AT1R, TP and EGFR elicited responses in EC that were mostly not predictable based on individual receptor effects. (1) Single AT1R or TP activation promoted cell viability, but these effects were gone when combined with EGFR activation. (2) EGFR activation increased cell proliferation, and co-activation of AT1R or TP did not affect this response. (3) Receptor co-activations increased glucose consumption without proportionally increasing lactate production, suggesting a redirection of glucose toward non-glycolytic pathways. (4) Neutral lipid accumulation was largely unchanged, except under AT1R-EGFR co-activation, indicating here again metabolic rerouting. (5) IL-6 and ICAM-1 were additively induced by EGFR–TP co-stimulation, while EGFR–AT1R co-activation synergistically upregulated IL-6. In contrast, TP-induced regulation of eNOS expression and phosphorylation was limited by EGFR co-stimulation. Conclusion These findings demonstrate that EC respond to simultaneous AT1R, TP and EGFR activation by integrating their signals through mechanisms ranging from additive to synergistic. These findings underscore the complexity of receptor cross-talk in EC and highlight how simultaneous engagement of multiple vasoactive pathways can reshape endothelial behavior in ways not predictable from single-receptor activation alone.