Notch3/Hes5 induces vascular dysfunction in hypoxia-induced pulmonary hypertension through ER stress and redox-sensitive pathways

Morris, H. E., Neves, K. B. , Nilsen, M., Montezano, A. C., MacLean, M. R. and Touyz, R. M. (2023) Notch3/Hes5 induces vascular dysfunction in hypoxia-induced pulmonary hypertension through ER stress and redox-sensitive pathways. Hypertension, 80(8), pp. 1683-1696. (doi: 10.1161/hypertensionaha.122.20449) (PMID:37254738)

Abstract

BACKGROUND: Notch3 (neurogenic locus notch homolog protein 3) is implicated in vascular diseases, including pulmonary hypertension (PH)/pulmonary arterial hypertension. However, molecular mechanisms remain elusive. We hypothesized increased Notch3 activation induces oxidative and endoplasmic reticulum (ER) stress and downstream redox signaling, associated with procontractile pulmonary artery state, pulmonary vascular dysfunction, and PH development. METHODS: Studies were performed in TgNotch3R169C mice (harboring gain-of-function [GOF] Notch3 mutation) exposed to chronic hypoxia to induce PH, and examined by hemodynamics. Molecular and cellular studies were performed in pulmonary artery smooth muscle cells from pulmonary arterial hypertension patients and in mouse lung. Notch3-regulated genes/proteins, ER stress, ROCK (Rho-associated kinase) expression/activity, Ca2+ transients and generation of reactive oxygen species, and nitric oxide were measured. Pulmonary vascular reactivity was assessed in the presence of fasudil (ROCK inhibitor) and 4-phenylbutyric acid (ER stress inhibitor). RESULTS: Hypoxia induced a more severe PH phenotype in TgNotch3R169C mice versus controls. TgNotch3R169C mice exhibited enhanced Notch3 activation and expression of Notch3 targets Hes Family BHLH Transcription Factor 5 (Hes5), with increased vascular contraction and impaired vasorelaxation that improved with fasudil/4-phenylbutyric acid. Notch3 mutation was associated with increased pulmonary vessel Ca2+ transients, ROCK activation, ER stress, and increased reactive oxygen species generation, with reduced NO generation and blunted sGC (soluble guanylyl cyclase)/cGMP signaling. These effects were ameliorated by N-acetylcysteine. pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension recapitulated Notch3/Hes5 signaling, ER stress and redox changes observed in PH mice. CONCLUSIONS: Notch3 GOF amplifies vascular dysfunction in hypoxic PH. This involves oxidative and ER stress, and ROCK. We highlight a novel role for Notch3/Hes5-redox signaling and important interplay between ER and oxidative stress in PH.

• Item Type: Articles
• Additional Information: This work was funded by grants from the British Heart Foundation (BHF; RG_F_21_110047) and Medical Research Council (MRC; MR/R502327/1 and MR/T015713/1). A.C. Montezano was supported by a University of Glasgow Walton Fellowship. R.M. Touyz was supported through a BHF Chair award (CH/4/29762), the Dr Phil Gold Chair, McGill University, Montreal, and a Canada Research Chair, Canadian Institutes of Health Research.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Morris, Ms Hannah and Montezano, Dr Augusto and Touyz, Professor Rhian and Neves, Dr Karla
• Authors: Morris, H. E., Neves, K. B., Nilsen, M., Montezano, A. C., MacLean, M. R., and Touyz, R. M.
• College/School: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Journal Name: Hypertension
• Publisher: American Heart Association
• ISSN: 0194-911X
• ISSN (Online): 1524-4563
• Published Online: 31 May 2023
• Copyright Holders: Copyright © 2023 The Authors
• First Published: First published in Hypertension 80(8):1683-1696
• Publisher Policy: Reproduced under a Creative Commons license

Project Code	Award No	Project Name	Principal Investigator	Funder's Name	Funder Ref	Lead Dept
313949		National Productivity Investment Fund Studentships	George Baillie	Medical Research Council (MRC)	MR/R502327/1	MVLS - Education Hub