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Defective NOTCH signalling drives smooth muscle cell death and differentiation in bicuspid aortic valve aortopathy

Defective NOTCH signalling drives smooth muscle cell death and differentiation in bicuspid aortic valve aortopathy
Defective NOTCH signalling drives smooth muscle cell death and differentiation in bicuspid aortic valve aortopathy
OBJECTIVES: Bicuspid aortic valve disease is common and is associated with ascending aortic aneurysms. Vascular smooth muscle cell (VSMC) apoptosis is characteristic of the ascending aorta of bicuspid patients, and NOTCH1 gene mutations have also been linked to the disease. NOTCH signalling is a fundamental cell signalling pathway, which dictates cell fate decisions including apoptosis. Our objective was to elucidate the role of NOTCH signalling in VSMC apoptosis and differentiation in bicuspid aortopathy.

METHODS: Ascending aortic biopsies were obtained from 19 bicuspid and 12 tricuspid aortic valve patients and were sub-classified into 4 groups according to the maximum ascending aortic diameter (aneurysmal  ≥45 mm). Apoptotic VSMCs were counted by light microscopy using a TUNEL assay. Gene expression of key regulators of NOTCH signalling (NOTCH1 and HES1), apoptosis (BAX and BCL-2) and VSMC differentiation (MYH11, CNN1 and MYH10) were quantified using quantitative real-time PCR. Primary VSMCs were cultured from 2 tricuspid aortic valve and 2 bicuspid aortic valve patients, NOTCH signalling was inhibited with N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, and the gene expression was again quantified.

RESULTS: The apoptotic cell count was significantly higher in bicuspid aortic valve patients (3.2 cells/50 000 μm2 vs 1.1 cells/50 000 μm2; P = 0.033). There was a trend towards lower apoptotic cell count in the aneurysmal versus non-aneurysmal tricuspid and bicuspid groups and an increased ratio of proapoptotic gene expression, which was not statistically significant. This was associated with a 2.8-fold increase in contractile gene expression (P = 0.026) and a 2.0-fold increase in NOTCH signalling gene expression in bicuspid versus tricuspid aortic valve patients (P = 0.022). NOTCH inhibition in cultured VSMCs induced a similar pattern of increased proapoptotic and procontractile gene expressions.

CONCLUSIONS: This preliminary study suggests that NOTCH activation in the non-aneurysmal bicuspid aortas may underlie aortopathy by influencing VSMC apoptosis and differentiation. NOTCH signalling manipulation may provide a therapeutic target for preventing aneurysms in bicuspid patients. Further studies with larger sample sizes are needed to substantiate the present findings.
Bicuspid aortic valve, Ascending aortic aneurysm, Vascular smooth muscle cell, NOTCH signalling, Apoptosis, Cell differentiation
1010-7940
Harrison, Oliver J.
1feda5d5-8833-4587-9800-93135b68135a
Torrens, Christopher
15a35713-0651-4249-8227-5901e2cfcd22
Salhiyyah, Kareem
63ac0bb3-5b7d-4d9f-87e5-3a20ba738a6a
Moorjani, Narain
ce44e232-2a6e-4616-8341-837ad0446acb
Modi, Amit
3f1018c0-f808-4b9d-ba38-e9c64eb83929
Townsend, Paul A.
89300833-c898-4ae1-a3b2-03214c71da52
Ohri, Sunil K.
8aa5698c-78cf-4f59-a5af-5afa46f0348c
Cagampang, Felino
7cf57d52-4a65-4554-8306-ed65226bc50e
Harrison, Oliver J.
1feda5d5-8833-4587-9800-93135b68135a
Torrens, Christopher
15a35713-0651-4249-8227-5901e2cfcd22
Salhiyyah, Kareem
63ac0bb3-5b7d-4d9f-87e5-3a20ba738a6a
Moorjani, Narain
ce44e232-2a6e-4616-8341-837ad0446acb
Modi, Amit
3f1018c0-f808-4b9d-ba38-e9c64eb83929
Townsend, Paul A.
89300833-c898-4ae1-a3b2-03214c71da52
Ohri, Sunil K.
8aa5698c-78cf-4f59-a5af-5afa46f0348c
Cagampang, Felino
7cf57d52-4a65-4554-8306-ed65226bc50e

Harrison, Oliver J., Torrens, Christopher, Salhiyyah, Kareem, Moorjani, Narain, Modi, Amit, Townsend, Paul A., Ohri, Sunil K. and Cagampang, Felino (2019) Defective NOTCH signalling drives smooth muscle cell death and differentiation in bicuspid aortic valve aortopathy. European Journal of Cardio-thoracic Surgery. (doi:10.1093/ejcts/ezy464).

Record type: Article

Abstract

OBJECTIVES: Bicuspid aortic valve disease is common and is associated with ascending aortic aneurysms. Vascular smooth muscle cell (VSMC) apoptosis is characteristic of the ascending aorta of bicuspid patients, and NOTCH1 gene mutations have also been linked to the disease. NOTCH signalling is a fundamental cell signalling pathway, which dictates cell fate decisions including apoptosis. Our objective was to elucidate the role of NOTCH signalling in VSMC apoptosis and differentiation in bicuspid aortopathy.

METHODS: Ascending aortic biopsies were obtained from 19 bicuspid and 12 tricuspid aortic valve patients and were sub-classified into 4 groups according to the maximum ascending aortic diameter (aneurysmal  ≥45 mm). Apoptotic VSMCs were counted by light microscopy using a TUNEL assay. Gene expression of key regulators of NOTCH signalling (NOTCH1 and HES1), apoptosis (BAX and BCL-2) and VSMC differentiation (MYH11, CNN1 and MYH10) were quantified using quantitative real-time PCR. Primary VSMCs were cultured from 2 tricuspid aortic valve and 2 bicuspid aortic valve patients, NOTCH signalling was inhibited with N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, and the gene expression was again quantified.

RESULTS: The apoptotic cell count was significantly higher in bicuspid aortic valve patients (3.2 cells/50 000 μm2 vs 1.1 cells/50 000 μm2; P = 0.033). There was a trend towards lower apoptotic cell count in the aneurysmal versus non-aneurysmal tricuspid and bicuspid groups and an increased ratio of proapoptotic gene expression, which was not statistically significant. This was associated with a 2.8-fold increase in contractile gene expression (P = 0.026) and a 2.0-fold increase in NOTCH signalling gene expression in bicuspid versus tricuspid aortic valve patients (P = 0.022). NOTCH inhibition in cultured VSMCs induced a similar pattern of increased proapoptotic and procontractile gene expressions.

CONCLUSIONS: This preliminary study suggests that NOTCH activation in the non-aneurysmal bicuspid aortas may underlie aortopathy by influencing VSMC apoptosis and differentiation. NOTCH signalling manipulation may provide a therapeutic target for preventing aneurysms in bicuspid patients. Further studies with larger sample sizes are needed to substantiate the present findings.

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More information

Accepted/In Press date: 10 December 2018
e-pub ahead of print date: 25 January 2019
Published date: 25 January 2019
Keywords: Bicuspid aortic valve, Ascending aortic aneurysm, Vascular smooth muscle cell, NOTCH signalling, Apoptosis, Cell differentiation

Identifiers

Local EPrints ID: 430247
URI: https://eprints.soton.ac.uk/id/eprint/430247
ISSN: 1010-7940
PURE UUID: 1dba082a-5cfb-47e6-8403-9e0a9ff5726d
ORCID for Felino Cagampang: ORCID iD orcid.org/0000-0003-4404-9853

Catalogue record

Date deposited: 23 Apr 2019 16:30
Last modified: 22 May 2019 00:35

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Contributors

Author: Oliver J. Harrison
Author: Kareem Salhiyyah
Author: Narain Moorjani
Author: Amit Modi
Author: Paul A. Townsend
Author: Sunil K. Ohri

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