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

Microvascular endothelial cells (MECs) grown in Matrigel form capillary-lik e structures. We hypothesized that these "capillaries" better mimic communi cation properties of microvessels than conventional cell monolayers. MECs w ere isolated from the rat hindlimb skeletal muscle. Functional communicatio n was tested by visualizing the spread of microinjected 6-carboxyfluorescei n (CF) dye and by measuring a conducted change of membrane potential after micropipette application of 500 mM KC1 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 sig nificantly greater than that of cells grown in monolayers (-24 +/- 2 mV). K CI and ATP caused local depolarization (18 +/- 3 mV) and hyperpolarization (21 +/- 3 mV) in capillaries that yielded conducted 13 +/- 3 mV depolarizat ion and 15 +/- 5 mV hyperpolarization at a 300-mum distal site, respectivel y. 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 c apillaries. Cells grown under both conditions expressed connexin 43, as dem onstrated by immunohistochemistry and Western blotting. We conclude that ce lls grown in capillaries yield substantially larger local and communicated responses than cells in monolayers and thus offer a more sensitive model fo r mechanistic studies of MEC communication. (C) 2000 Academic Press.