Development of a coculture system and use of confocal laser fluorescent microscopy to study human microvascular endothelial cell and mural cell interaction

In the present study, human myometrial microvascular endothelial cells (HMMEC) were cocultured with human vascular smooth muscle cells (VSMC) labeled with fluorescent dyes to examine their morphological interaction using confocal laser fluorescent microscopy. HMMEC and VSMC labeled with fluorescent green and red dyes, respectively, attached to opposite sides of polyethyleneterephthalate membranes and remained viable for up to 96 h. In defined medium, 5% ± 3% of the VSMC cytoplasmic processes and 71% ± 17% of the HMMEC processes extended completely across the 13 μm thickness of the transmembrane. However, 41% ± 21% of the VSMC projections and 10% ± 3% of the HMMEC processes that traversed the membrane made contact with the opposing cell type. In cocultures incubated with angiopoietin-1 (Ang-1), although the number of VSMC or HMMEC projections was not significantly increased, the number of VSMC extending across the membrane and making contact with HMMEC was increased (P < 0.05) to 88% ± 2%. The results of the current study demonstrate that coculture of fluorescent-labeled HMMEC and VSMC on a semipermeable transmembrane coupled with confocal laser fluorescent microscopy provides an in vitro experimental model to study morphological association of microvascular endothelial cells with mural cells. We propose that this system will greatly facilitate study of remodeling of the microvasculature in various organ systems.