Pharmacological inhibition of protein tyrosine kinases axl and fyn reduces TNF-α-induced endothelial inflammatory activation in vitro

Sophie F Ellermann; Rianne M Jongman; Matthijs Luxen; Timara Kuiper; Josee Plantinga; Jill Moser; Thomas W L Scheeren; Gregor Theilmeier; Grietje Molema; Matijs Van Meurs

doi:10.3389/fphar.2022.992262

Pharmacological inhibition of protein tyrosine kinases axl and fyn reduces TNF-α-induced endothelial inflammatory activation in vitro

Front Pharmacol. 2022 Dec 1:13:992262. doi: 10.3389/fphar.2022.992262. eCollection 2022.

Authors

Sophie F Ellermann^{1

2

3}, Rianne M Jongman^{1

2}, Matthijs Luxen^{1

4}, Timara Kuiper¹, Josee Plantinga¹, Jill Moser^{1

4}, Thomas W L Scheeren², Gregor Theilmeier^{2

3}, Grietje Molema¹, Matijs Van Meurs^{1

4}

Affiliations

¹ Medical Biology Section, Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Groningen, Netherlands.
² Department of Anaesthesiology, University Medical Center Groningen, University of Groningen, Groningen, Netherlands.
³ Perioperative Inflammation and Infection, Department of Human Medicine, Faculty of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, Oldenburg, Germany.
⁴ Department of Critical Care, University Medical Center Groningen, University of Groningen, Groningen, Netherlands.

Abstract

Major surgery induces systemic inflammation leading to pro-inflammatory activation of endothelial cells. Endothelial inflammation is one of the drivers of postoperative organ damage, including acute kidney injury Tumour Necrosis Factor alpha (TNF-α) is an important component of surgery-induced pro-inflammatory activation of endothelial cells. Kinases, the backbone of signalling cascades, can be targeted by pharmacological inhibition. This is a promising treatment option to interfere with excessive endothelial inflammation. In this study, we identified activated kinases as potential therapeutic targets. These targets were pharmacologically inhibited to reduce TNF-α-induced pro-inflammatory signalling in endothelial cells. Kinome profiling using PamChip arrays identified 64 protein tyrosine kinases and 88 serine-threonine kinases, the activity of which was determined at various timepoints (5-240 min) following stimulation with 10 ng/ml TNF-α in Human umbilical vein endothelial cells in vitro. The PTKs Axl and Fyn were selected based on high kinase activity profiles. Co-localisation experiments with the endothelial-specific protein CD31 showed Axl expression in endothelial cells of glomeruli and Fyn in arterioles and glomeruli of both control and TNF-α-exposed mice. Pharmacological inhibition with Axl inhibitor BMS-777607 and Fyn inhibitor PP2 significantly reduced TNF-α-induced pro-inflammatory activation of E-selectin, VCAM-1, ICAM-1, IL-6 and IL-8 at mRNA and VCAM-1, ICAM-1, and IL-6 at protein level in HUVEC in vitro. Upon pharmacological inhibition with each inhibitor, leukocyte adhesion to HUVEC was also significantly reduced, however to a minor extent. In conclusion, pre-treatment of endothelial cells with kinase inhibitors BMS-777607 and PP2 reduces TNF-α-induced endothelial inflammation in vitro.

Keywords: BMS-777607; PP2; axl; endothelial inflammation; fyn; kinases; postoperative organ damage; tumour necrosis factor alpha (TNF-α).