Long-term objectives in this area include understanding the dynamic changes in gene expression during dysregulation of homeostasis in cancer and chronic wounds. We have developed a new model of endothelial differentiation, in which endothelial cells from a monolayer migrate into a collagen layer, followed by a second layer containing fibroblasts. Once the cells reach the fibroblasts in the second collagen layer, they form capillary networks with apparent lumen. In the absence of fibroblasts, there is greatly reduced migration and no tube formation, and those cells that migrate show high levels of apoptosis. The induction of mature capillary-like networks by fibroblasts was inhibited by separating the fibroblast-containing layer from the endothelial cell monolayer with membranes that restricted cellular migration by the pore size (0.4 m) while allowing exchange of soluble factors. When 0.4 m membranes prevented close or direct fibroblast-endothelial cell interactions, endothelial cells migrated into the acellular collagen layer but formed only a few scattered immature networks, which were likely supported by fibroblast-derived soluble factors. Membranes with 12 m pore size, on the other hand, allowed transmigration and cell-cell contact, and stimulated formation of mature capillary networks, suggesting that direct cell-cell contact between fibroblasts and endothelial cells is essential for capillary network formation.

Fibroblasts induce differentiation of human microvascular endothelial cells from monolayers into capillary-like, three-dimensional networks, through close cell-cell interactions. Anti-human PECAM immunohistochemistry of reconstruct cross sections and anti-human vWF VIII immunofluorescence of whole-mounted gels. Gels were prepared with or without fibroblasts within the upper layer of the collagen reconstructs that were laid over an HMVEC monolayer. (A)-Mature, capillary-like morphology resembling an in vivo microvascular network was observed by PECAM immunohistochemistry of gel cross sections only with the addition of fibroblasts to the overlaid collagen gels, at 5 days of incubation (A) and 11 days of incubation (B-C). Findings were confirmed by vWF VIII immunofluorescence and confocal microscopy of whole-mounted gels (D-E). PECAM-negative or vWF F VIII-negative cells (fibroblasts), seen by the nuclear counterstain, were noted in close proximity around the endothelial structures that appear annular on cross sections (black arrows, A) or tubular on whole-mount projection (red arrows, D-E). Fibroblasts within the gel (D insert) are seen as many vWF VIII-negative blue-stained nuclei surrounding the green-stained endothelial tubule. In E, the insert shows a tubular structure seen on oblique cross section. Controls not containing fibroblasts within the upper layer of the reconstructs did not show capillary-like differentiation by either PECAM (F) or vWF F VIII (G) staining. Experiments in which endothelial cells and fibroblasts were separated by semiporous membranes demonstrated that direct cell-cell contact is required for endothelial cell tube formation. Magnification 10X A-G, insert in A 20X.