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Vascular Smooth Muscle Cell Isolation and Culture from Mouse Aorta   

Edited by
Jia Li
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Anonymous reviewer
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In this protocol

Original research article

A brief version of this protocol appeared in:
The Journal of Clinical Investigation
Mar 2016

Abstract

Vascular smooth muscle cells (SMC) in the ascending thoracic aorta arise from neural crest cells, whereas SMCs in the descending aorta are derived from the presomitic mesoderm. SMCs play important roles in cardiovascular development and aortic aneurysm formation. This protocol describes the detailed process for explanting ascending and descending SMCs from mouse aortic tissue. Conditions for maintenance and subculture of isolated SMCs and characterization of the vascular SMC phenotype are also described.

Keywords: Tissue culture, Smooth muscle cells, Cell biology

Background

Vascular smooth muscle cells (SMCs) make up the muscular medial layer of arteries. Larger elastic arteries, such as the aorta, have multiple concentric lamellae consisting of aligned smooth muscle cells sandwiched between elastin fibers. The elastin and collagen present within the medial layer of elastic arteries allow it to distribute the force generated by the heart throughout the vessel wall (Wagenseil and Mecham, 2009). Smaller muscular arteries, by contrast, have only an internal and external elastic lamina bounding the smooth muscle layer. These arteries are downstream in the arterial tree and thus bear less force from blood flow.

Vascular smooth muscle cells, unlike cardiac and skeletal muscle cells, are capable of modulating their phenotype in response to vascular injury or environmental cues. Under normal physiologic conditions, quiescent, contractile SMCs populate the artery wall and contract to regulate vascular tone and keep blood flow continuous in response to pulsatile pressures. Contractile SMCs are characterized by high expression of smooth muscle-specific contractile genes, including smooth muscle specific α-actin and myosin heavy chain (Owens et al., 2004). However, in response to injury or mitogenic stimuli, SMCs downregulate expression of the contractile genes and take on a synthetic phenotype: they proliferate rapidly, migrate into the site of injury, and remodel the extracellular matrix by synthesizing both matrix-digesting enzymes and new matrix proteins. Many vascular diseases are associated with a synthetic SMC phenotype, including atherosclerosis (Owens, 1995).

SMCs located in different areas of the body actually arise from diverse embryonic lineages (Majesky, 2007). For example, the SMCs populating the ascending thoracic aorta and the cerebrovasculature are derived from neural crest cells. However, SMCs in the descending thoracic aorta come from mesodermal origins. These distinctions affect the ultimate properties of the SMCs, so it is important when designing experiments to use SMCs from the same developmental origin where the phenotype of interest arises.

In this protocol, we give detailed instructions for isolating and culturing SMCs from the ascending and descending thoracic aortas in the mouse. We have previously used this technique to isolate SMCs from genetically modified mice and their wild-type littermates to provide an in vitro system for looking at the effect of genetic changes on SMC phenotype (Cao et al., 2010; Kuang et al., 2012; Kuang et al., 2016; Papke et al., 2013; Kwartler et al., 2014).

Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Kwartler, C. S., Zhou, P., Kuang, S., Duan, X., Gong, L. and Milewicz, D. M. (2016). Vascular Smooth Muscle Cell Isolation and Culture from Mouse Aorta. Bio-protocol 6(23): e2045. DOI: 10.21769/BioProtoc.2045.
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