Co-Culture and Crosstalk between Endothelial Cells and Vascular Smooth Muscle Cells Mediated by Intracellular Calcium

This chapter aims to describe the basic procedure needed to establish primary cultures of vascular smooth muscle cells (VSMCs) (1 ), and vascular endothelial cells (ECs) (2 ). Direct visualization of crosstalk between two types of cell was first accomplished by means of 2-dimensional Ca²⁺ image analysis of co-cultured smooth muscle cells and ECs (3 ). In order to carry out image analysis of intracellular calcium ions, these two cell types need to be grown in co-culture, and the methods for that are also described. Since Tsien’s group developed calcium-sensitive fluorescent dyes, outstanding progress has been made in the field of intracellular calcium dynamics and cellular functions. These dyes are suitable for longer and repetitive experiments and much improve signalto-noise ratios without foregoing calcium-sensitive photoprotein (aequorin) or metallochromic dyes for measurement of intracellulcar calcium concentration (4 ). Progress made includes excitation-contraction coupling of several muscle cells, excitation-secretion coupling in endocrine cells, and intra- and intercellular signaling mechanisms (5 ). More recently, progress has been made in elucidating the behavior of nitric oxide, a multifunctional effector molecule, which not only conveys signals for vasorelaxation, neurotransmission, and cytotoxicity, but plays a critical role in atherosclerosis, apoptosis, and cellular redox states (6 ,7 ,8 ). All of this progress has been greatly aided by the image analysis system first described by Fay and coworkers (9 ). Therefore, this chapter also describes the basic principle of intracellular calcium image analysis. We have used the term isoculture to refer to one type of cells that were grown in isolation and the term co-culture to designate that two types of cells coexisted in the same dish.