Cyp2c44 epoxygenase-derived epoxyeicosatrienoic acids in vascular smooth muscle cells elicit vasoconstriction of the murine ophthalmic artery

Jiong Hu,Marco Sisignano,Caroline Manicam,Roman Brecht,Ingrid Fleming,Beate Fisslthaler,Natarajan Perumal,Carlo Angioni,Norbert Pfeiffer,Iris-Sofia Bibli,Hartmut Kleinert

doi:10.1038/s41598-021-98236-w

Abstract

Cytochrome P450 (CYP) signalling pathway has been shown to play a vital role in the vasoreactivity of wild type mouse ophthalmic artery. In this study, we determined the expression, vascular responses and potential mechanisms of the CYP-derived arachidonic acid metabolites. The expression of murine CYP (Cyp2c44) and soluble epoxide hydrolase (sEH) in the wild type ophthalmic artery was determined with immunofluorescence, which showed predominant expression of Cyp2c44 in the vascular smooth muscle cells (VSMC), while sEH was found mainly in the endothelium of the wild type ophthalmic artery. Artery of Cyp2c44−/− and sEH−/− mice were used as negative controls. Targeted mass spectrometry-based lipidomics analysis of endogenous epoxide and diols of the wild type artery detected only 14, 15-EET. Vasorelaxant responses of isolated vessels in response to selective pharmacological blockers and agonist were analysed ex vivo. Direct antagonism of epoxyeicosatrienoic acids (EETs) with a selective inhibitor caused partial vasodilation, suggesting that EETs may behave as vasoconstrictors. Exogenous administration of synthetic EET regioisomers significantly constricted the vessels in a concentration-dependent manner, with the strongest responses elicited by 11, 12- and 14, 15-EETs. Our results provide the first experimental evidence that Cyp2c44-derived EETs in the VSMC mediate vasoconstriction of the ophthalmic artery.

Highlights

Cytochrome P450 (CYP) signalling pathway has been shown to play a vital role in the vasoreactivity of wild type mouse ophthalmic artery
Epoxyeicosatrienoic acids are synthesized as four distinct regioisomers composed of 5,6, 8,9, 11,12- and 14,15-epoxyeicosatrienoic acids (EETs), which are reported to elicit vasodilation in an equipotent manner in some vascular beds such as the renal, coronary and pulmonary arteries of different species including h uman[10,16,17,18,19,20,21], while they are shown to behave in a regio-selective manner in others[22,23]
The evidence implicating a role for the CYP-soluble epoxide hydrolase (sEH) pathway in regulating the tone of the wild type (WT) mouse ophthalmic artery has been obtained in our previous study using non-specific pharmacological inhibitors in vitro[3]

Summary

Introduction

Cytochrome P450 (CYP) signalling pathway has been shown to play a vital role in the vasoreactivity of wild type mouse ophthalmic artery. The largely unknown aspects of the Cyp-sEH signalling pathway in the ophthalmic artery provided the impetus to further explore the vasoreactivity of the regioisomeric EETs, as well as to determine the expression and localization of Cyp2c44 and sEH in the present study For this purpose, apart from the use of more selective pharmacological tools, the state-of-the-art mass spectrometry-based epoxide and diol profiling, Western blot analysis and immunofluorescence staining using mice with targeted deletion of the CYP and sEH genes (Cyp2c44−/− and sEH−/−) were instrumental to assess the role of this pathway in the regulation of ophthalmic arterial tone

Methods

Results

Conclusion