In Vitro Vascular Cell Culture Systems – Vascular Smooth Muscle

The phenotypic modulation of vascular smooth muscle cells (VSMCs) from the differentiated state characteristic of normal mature arteries to a dedifferentiated phenotype is critical to normal vascular physiology, including angiogenesis and wound healing. However, this transition is also central to the pathogenesis of atherosclerosis, restenosis, intimal hyperplasia, hypertension, and tumor angiogenesis. In this chapter, we provide an overview of the signaling pathways and target transcription factors, including serum response factor (SRF), myocardin, GATA-6, nuclear factor of activated T cells (NFAT), and Kruppel-like factor (KLF) family members, that regulate VSMC differentiation. We describe in detail methods for isolation and cultivation of VSMC in culture, and methods used to study phenotypic modulation of VSMC in culture. We discuss methods to study and quantitate VSMC morphology, proliferation, migration, contractile protein expression, and promoter regulation, as all of these processes are altered in differentiated versus dedifferentiated VSMC. In addition to the traditional tissue culture approaches, methods are also presented to study vascular cells in dynamic models to assess the effects of shear stress and cyclic strain. We discuss models of VSMC differentiation employed by our laboratory and others, including endothelial cell (EC)/VSMC coculture, insulin-like growth factor I (IGF-I) and the mammalian target of rapamycin (mTOR) inhibitor rapamycin, prostacyclin (PGI2), and retinoids. These models and techniques provide an experimental system in which to study the effects of drugs and toxicants on VSMC, with an emphasis on effects on VSMC phenotype.