PhD Scientific Days 2022

Budapest, 6-7 July 2022

Molecular Sciences II.

Angiotensin II Upregulates Cholesterol-25-hydroxylase Gene Expression in Vascular Smooth Muscle Cells

Department of Physiology, Semmelweis University, Budapest, Hungary

Text of the abstract

Angiotensin II (AngII) is a vasoactive peptide hormone and the effector of renin-angiotensin-aldosterone system. It exerts its main physiological effects through type 1 AngII receptor (AT1R), but it can also contribute to the development of cardiovascular diseases. Likewise, oxysterols such as 25-hydroxycholesterol (25-HC), the product of cholesterol-25-hydroxylase (CH25H), can have harmful effects on the vasculature since they affect vascular smooth muscle cells (VSMCs) negatively. However, there is no established connection between AngII and 25-HC production in VSMCs.
Our study aims to explore how AngII affects Ch25h gene expression in rat primary VSMCs. RNA-sequencing was used to determine which genes are differentially expressed in VSMCs stimulated with AngII compared to vehicle-stimulated cells. We also performed qRT-PCR measurements to asses Ch25h mRNA levels in our VSMC samples. VSMCs were stimulated with AngII for various time spans to determine Ch25h expression changes over time. We used multiple inhibitors to specify which signaling pathways are involved in the AngII-induced Ch25h expression changes. LC-MS/MS was used to measure 25-HC levels in the supernatant of AngII-stimulated VSMCs.
Our data show that Ch25h expression was robustly upregulated in response to AngII stimulus, Ch25h mRNA levels were at a peak one hour after stimulation. The AT1R, Gq/11, and p38 MAP kinase inhibitors prevented the AngII-induced Ch25h upregulation, whereas the β-arrestin biased-agonist of AT1R did not cause Ch25h upregulation. JNK, MEK and NOX inhibitors did not have any effect on AngII-induced Ch25h upregulation. LC-MS/MS results demonstrated that 25-HC is present in the supernatant of AngII-stimulated VSMCs. 25-HC concentration was highest (on average 8.2 ng/ml) four hours after AngII stimulation.
Based on our results we conclude that AngII-induced Ch25h upregulation is AT1R, Gq/11 and p38 MAP kinase signaling dependent in primary rat VSMCs. The elevation of 25-HC levels in AngII-stimulated VSMC supernatants means that CH25H enzyme is active and functional in primary rat VSMCs. Our study shows that AngII stimulus induces Ch25h upregulation and subsequently 25-HC production in VSMCs. Our findings enables the better understanding of mechanisms in the pathogenesis of cardiovascular diseases.
Funding : NKFI K116954, NVKP_16-1-2016-0039 and VEKOP-2.3.2-16-2016-00002.