A new in vitro model for agonist-induced communication between microvascular endothelial cells

Microvascular endothelial cells (MECs) grown in Matrigel form capillary-like structures. We hypothesized that these "capillaries" better mimic communication properties of microvessels than conventional cell monolayers. MECs were isolated from the rat hindlimb skeletal muscle. Functional communication was tested by visualizing the spread of microinjected 6-carboxyfluorescein (CF) dye and by measuring a conducted change of membrane potential after micropipette application of 500 mM KCl or 10 mM adenosine triphosphate (ATP) on the capillary and monolayer. MECs grown under both conditions were dye-coupled, as demonstrated by the spread of CF injected into a single cell. The membrane potential of cells grown in capillaries (-59 +/- 5 mV) was significantly greater than that of cells grown in monolayers (-24 +/- 2 mV). KCl and ATP caused local depolarization (18 +/- 3 mV) and hyperpolarization (21 +/- 3 mV) in capillaries that yielded conducted 13 +/- 3 mV depolarization and 15 +/- 5 mV hyperpolarization at a 300-microm distal site, respectively. In monolayers, local and distal responses to agonists were 3- to 6-fold and 9- to 10-fold less, respectively, than the corresponding responses in capillaries. Cells grown under both conditions expressed connexin 43, as demonstrated by immunohistochemistry and Western blotting. We conclude that cells grown in capillaries yield substantially larger local and communicated responses than cells in monolayers and thus offer a more sensitive model for mechanistic studies of MEC communication.