Cerebral pericytes constitute an essential component of the blood-brain barrier (BBB) and are involved in blood vessel assembly. Recently, we reported on the induction of a BBB-specific enzyme expressed by cerebral pericytes (pericytic aminopeptidase N/pAPN) in coculture with cerebral endothelial cells. We completed this in vitro BBB system by adding astrocytes to these mixed cultures of endothelial cells and pericytes. Under these triculture conditions, endothelial cells and pericytes reorganize into capillary-like structures (CLSs). Capillary formation can also be achieved by the application of transforming growth factor beta 1 (TGF-b1) in the culture medium of endothelial-pericyte cultures lacking astrocytes. In contrast to the effect achieved by astrocytes, pericytes did not assemble with endothelial cells. In both cases (application of astrocytes or TGF-b1), endothelial cells underwent apoptosis. However, endothelial cells that form CLSs in the presence of pericytes appeared to be resistant to induction of apoptosis. On the basis of these observations, we concluded that astrocytes have a profound influence on the morphogenetic events underlying the organization of the vessel wall; that the effect of TGF-b1 is different from the astrocytic effect because it lacks induction of endothelial-pericyte association; and that pericytes stabilize CLSs formed by endothelial cells in coculture with astrocytes.

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We used a 3D in-vitro model of angiogenesis to investigate the effects of different growth factors on vessel formation and stabilization in vitro. Vascular endothelial growth factor (VEGF) was the only factor that induced the formation, elongation and sprouting of capillary-like structures (CLS) by bovine retinal capillary endothelial cells (BREC), an effect that was dose-dependent and saturable. Basic fibroblast growth factor 2 (FGF2) enhanced capillary formation in the presence of VEGF, leading to a more complex network of CLS and a higher rate of BrdU incorporation than VEGF alone, indicating that whereas VEGF acts as a morphogen, FGF2 is primarily a mitogen. Addition of transforming growth factor β1 (TGFβ1) to the 3D assay along with VEGF and FGF2, reduced tube formation in a dose-dependent manner. When added at the time of cell plating TGFβ1 completely suppressed formation of VEGF/FGF2-stimulated CLS. Angiopoietin 1 (Ang1) prevented regression of the TGFβ1-induced CLS, an effect that was blocked by angiopoietin 2 (Ang2), but required the continuous presence of VEGF.