Sepsis downregulates aortic Notch signaling to produce vascular hyporeactivity in mice

Vandana Singh,Neeraj Gangwar,Amit Shukla,Shyama N Prabhu,Raut Akash,Satish K Garg,Soumen Choudhury,Rajneesh Singh,Gaurav Chaudhary

doi:10.1038/s41598-022-06949-3

Abstract

Inhibition of Notch signaling in macrophages is known to reduce inflammation, however, its role in regulating vascular hyporeactivity in sepsis is unknown. Thus we aimed to evaluate the effect of sepsis on vascular Notch signaling. Polymicrobial sepsis was induced by caecal ligation and puncture (CLP) in mice. mRNA expressions of Notch receptors (Notch1,3) and ligands (Jag1, Dll4), and downstream effector genes (Hey1, MLCK, MYPT1) were assessed by RT-qPCR. Protein level of activated Notch (NICD) was assessed by Western blot and immuno-histochemistry. Isometric tension in isolated aortic rings was measured by wire myography.CLP down-regulated aortic expression of Notch3, Jag1 and Dll4 as compared to control mice. Additionally, the protein level of NICD was found to be lesser in aortic tissue sections from CLP mice. Expression of Hey1 and MLCK were attenuated whereas MYPT1 expression was increased in septic mouse aorta. DAPT pretreatment did not improve CLP-induced vascular hyporeactivity to NA, CaCl2 and high K+ (80 mM), rather significantly attenuated the aortic response to these vasoconstrictors in control mice. Treatment with 1400 W reversed attenuated Notch3 (but not Jag1 and MLCK) expression in septic mouse aorta. In conclusion, sepsis significantly attenuated the Notch (especially Notch3) signaling in mouse aorta along with reduction in contractile gene expression and vasoconstriction response. Further, iNOS/NO pathway was involved in sepsis-induced down-regulation of Notch3 receptor. Thus systemic inhibition of Notch signaling during sepsis may have serious impact on sepsis-induced vascular hyporeactivity.

Highlights

Inhibition of Notch signaling in macrophages is known to reduce inflammation, its role in regulating vascular hyporeactivity in sepsis is unknown
The septic animals were survived for around 20 h, all the aortic samples for the present study were collected at 19 ± 1 h post-caecal ligation and puncture (CLP) or surgery
Major findings of the present study were (1) Notch[3] was significantly down-regulated in mouse aorta following CLP, (2) Down-regulation of aortic Notch signalling in sepsis was associated with corresponding decrease in aortic myosin light chain kinase (MLCK) and increase in myosin phosphatase targeting subunit-1 (MYPT1) expression, (3) pre-treatment with DAPT did not improve sepsis-induced vascular hyporeactivity to NA or CaCl2, rather significantly attenuated the response to these vasoconstrictors in SO mice, (4) treatment with 1400 W reversed attenuated Notch[3] expression without significantly altering Jag[1] and MLCK expression in septic mouse aorta

Summary

Introduction

Inhibition of Notch signaling in macrophages is known to reduce inflammation, its role in regulating vascular hyporeactivity in sepsis is unknown. MRNA expressions of Notch receptors (Notch1,3) and ligands (Jag[1], Dll4), and downstream effector genes (Hey[1], MLCK, MYPT1) were assessed by RT-qPCR. DAPT pretreatment did not improve CLP-induced vascular hyporeactivity to NA, CaCl2 and high K+ (80 mM), rather significantly attenuated the aortic response to these vasoconstrictors in control mice. Sepsis significantly attenuated the Notch (especially Notch3) signaling in mouse aorta along with reduction in contractile gene expression and vasoconstriction response. The canonical Notch signalling is an evolutionary conserved pathway that governs cell fate, proliferation, differentiation, apoptosis, angiogenesis and vascular remodelling[9] In this cell to cell communication system, a membrane-tethered Notch ligand on one cell interacts with transmembrane Notch receptor on a juxtaposed cell. We evaluated the effect of sepsis on vascular Notch signalling and associated vascular dysfunction

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