Modulation of angiogenic endothelial permselectivity by the cAMP pathway

The chorioallantoic membrane (CAM) of the chick embryo provides an accessible model of normal angiogenesis in vivo. Previously, we reported a rapid reduction in CAM microvascular permeability to macromolecules between Days 4.5 and 5.0 of the normal 21-day gestation (V. Rizzo et al., 1995, Microvasc. Res. 49, 49-63). Here, we tested the hypothesis that activation of the cAMP signaling pathway at Day 4.5 would acutely increase permselectivity prior to normal differentiation of CAM endothelial barrier properties at Day 5.0. Changes in interstitial optical intensities due to extravasation of a graded series of FITC-dextrans (20, 40, and 70 kDa) were evaluated by computer-assisted image analysis, and endothelial ultrastructure was monitored by transmission electron microscopy. The cAMP analogue 8-bromo-cAMP (10^-4 and 10^-3 M) and forskolin (10^-5 and 10^-4 M), an adenylyl cyclase activator, acutely decreased permeability of the graded FITC-dextran series in a dose-dependent fashion. In addition, the nonspecific phosphodiesterase inhibitor IBMX (10^-4 M) served to increase basal restriction of the 20- and 40-kDa tracers. Further, Rp-cAMPS (10^-4 M), a cAMP antagonist for cAMP-dependent protein kinase, abolished the effects of both 8-bromo-cAMP (10^-3 M) and forskolin (10^-4 M) on FITC-Dextran 40 restriction. In all cases, ultrastructural presentation of both the endothelial cell junctions and the vesicles remained unchanged. The present results are consistent with the concept that exogenous cAMP activation decreased permeability of the angiogenic CAM endothelium at Day 4.5 without concomitant ultrastructural changes in the transendothelial macromolecular exchange pathways. Whether endogenous activity of cAMP contributes to normal differentiation of CAM endothelial barrier properties between Days 4.5 and 5.0 remains to be tested.