A simple and accurate method to quantify real-time contraction of vascular smooth muscle cell in vitro

Abstract

Vascular smooth muscle cells (VSMCs) constitute the medial layer of the blood vessel wall. Their contractile state regulates blood flow in physiological and pathological conditions. Current methods for assessing the contractility of VSMCs are not amenable to the high-throughput screening of pharmaceutical compounds. This study aimed to develop a method to address this shortcoming in the field. Real-time contraction was visualized in living VSMCs using the exogenous expression of green fluorescent protein (GFP). Image-Pro Plus software (IPPS) was used to measure various morphological cell indices. In phenylephrine-treated VSMCs, GFP fluorescence imaging was more accurate than brightfield imaging or phalloidin staining in representing VSMC morphology, as measured using IPPS. Among the multiple indices of VSMC shape, area and mean-diameter were more sensitive than length in reflecting the morphological changes in VSMC. We developed a new index, compound length, by combining the mean-diameter and length to differentiate contracted and uncontracted VSMCs. Based on the compound length, we further generated a contraction index to define a single-VSMC contractile status as single-VSMC contraction-index (SVCI). Finally, compound length and SVCI were validated to effectively assess cell contraction in VSMCs challenged with U46619 and KCl. In conclusion, GFP-based indices of compound length and SVCI can accurately quantify the real-time contraction of VSMCs. In future, the new method will be applied to high-throughput drug screening or basic cardiovascular research.

Keywords: Cell morphology; Compound length; Green fluorescent protein; Single-VSMC contraction-index; Vascular smooth muscle cell.