Induced pluripotent stem cell-derived vascular smooth muscle cells as an in vitro model of coronary spastic angina
Authors: Soma T , Shibutani S , Iwasaki T , Saito K , Washima S , Kato K , Konno Y , Hanada K , Yokoyama H , Tomita H 
Published: November 2023

Abstract

Background
Coronary spastic angina (CSA), also known as Prinzmetal’s variant angina, is a form of angina caused by coronary artery spasm. CSA is characterized by enhanced basal tone of coronary artery smooth muscle and hyperreactivity to vasoconstrictor stimuli. The vascular smooth muscle hyperreactivity is likely due to a post-receptorial cellular abnormality in the regulation of myofibril contraction, suggesting the possibility of multiple stimuli acting on various cellular receptors. However, its detailed mechanism is still unclear. To investigate it, various experiments using coronary artery smooth muscle cells obtained from the CSA patients is preferable, but it is impossible to do it clinically and ethically.

Purpose
The purpose of this study was to establish induced pluripotent stem cell (iPSC)-derived vascular smooth muscle cells (VSMCs) and to characterize this VSMC model for CSA research.

Methods
Three CSA patients with ST-segment elevation, which is a characteristic of variant angina, were recruited for this study. The isolated skin fibroblasts or peripheral blood mononuclear cells were reprogrammed into iPSCs through electroporation of episomal plasmids encoding the reprogramming genes OCT3/4, SOX2, KLF4, L-MYC, LIN28, and mouse p53DD. We differentiated mesodermal using Glycogen synthase kinase 3 inhibition and Bone morphogenetic protein 4 treatment from iPSCs and resulted in the differentiation of VSMCs by platelet-derived growth factor-BB and Activin A. The differentiation of VSMCs was confirmed by VSMC-specific gene expression in qRT–PCR and fluorescence-activated cell sorting (FACS). We examined biological features and intracellular calcium response to acetylcholine (ACh) in VSMCs obtained from 3 CSA patients and 3 healthy controls. Intracellular free Ca2+ concentration ([Ca2+]i; nM) was measured by fura-2.

Results
CSA iPSCs were confirmed to have normal karyotypes and pluripotency to differentiate. FACS showed that VSMCs differentiated from iPSCs exhibited smooth muscle cell marker expressions including alpha-smooth muscle actin and SM22-alpha. qRT–PCR showed that VSMCs from iPSCs had expressions of ACTA2 and TAGLN gene similar to the human coronary artery smooth muscle cells. Differentiation induction rate of VSMCs differentiated from iPSCs between CSA and control did not show significant difference. [Ca2+]i at baseline did not differ significantly between them (143 nM in CSA versus 120 nM in control, p=0.63, n=3), but peak increase in [Ca2+]i from baseline after ACh stimulation was significantly increased in CSA group compared with control (372 nM versus 177 nM, p&lt;0.05, n=3).

Conclusions
iPSC-derived VSMCs obtained from CSA patients showed an enhanced intracellular calcium response to ACh, which is likely responsible for CSA phenotype. Our iPSC-derived VSMC model can be useful for elucidating the mechanism of coronary spasm and developing therapeutic strategy such as drug efficacy.