Permeability of human endothelial monolayers: effect of vasoactive agonists and cAMP

Abstract

Permeability coefficients of human umbilical vein endothelial cell monolayers cultured on polycarbonate filters were determined by monitoring transendothelial albumin transport. Permeability was determined as a function of time in culture and in the presence of vasoactive agonists. Permeability decreased with increasing time in culture. All agonist experiments were performed with 15-day cultures because this time point best modeled the in vivo permeability barrier function. Permeability of endothelial monolayers decreased significantly in the presence of the stable prostacyclin analogue iloprost (6 nM), dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP, 0.5 mM)-3-isobutyl-1-methylxanthine (IBMX, 0.1 mM), 8-bromo cAMP (0.5 mM)-IBMX, dibutyryl cAMP-theophylline (0.5 mM), or IBMX. A 9.6-fold increase in permeability resulting from thrombin [0.15 U/ml (1 nM)] treatment was inhibited by pretreating the monolayers with dibutyryl cAMP-IBMX, 8-bromo cAMP-IBMX, dibutyryl cAMP-theophylline, dibutyryl cAMP, IBMX, iloprost, or D-Phe-Pro-Arg-CH2-alpha-thrombin (1 nM). The thrombin-induced permeability increase was not significantly altered by pretreating monolayers with aspirin (5 microM) or indomethacin (50 microM). Inactivated forms of thrombin, diisopropylflurophosphate-alpha-thrombin (1 nM) and D-Phe-Pro-Arg-CH2-alpha-thrombin, did not significantly affect permeability. Monolayer permeability was not altered in response to bradykinin (1 microM). These results suggest a mediating role for intracellular cAMP in the permeability barrier function of endothelial monolayers.

Pseudomonas aeruginosa exotoxin A induces pulmonary endothelial cytotoxicity: protection by dibutyryl-cAMP

“…Db-cAMP has been demonstrated to attenuate lung injury from a number of injurious stimuli [17][18][19][20][21]. However, the mechanism of protection in these instances is not known, although there is evidence that in some in vivo models it may exert a beneficial response by reducing pulmonary vascular pressures [19,20].…”

“…However, the mechanism of protection in these instances is not known, although there is evidence that in some in vivo models it may exert a beneficial response by reducing pulmonary vascular pressures [19,20]. However, there is also evidence that this protection may be related to decreasing endothelial permeability [20,21].…”

“…However, the mechanism of protection in these instances is not known, although there is evidence that in some in vivo models it may exert a beneficial response by reducing pulmonary vascular pressures [19,20]. However, there is also evidence that this protection may be related to decreasing endothelial permeability [20,21]. A further hypothesis to explain the protective influence of Db-cAMP on pulmonary endothelial cell injury is that Db-cAMP may exert its in vitro protective influence by activating protein kinase A and promoting endothelial cell membrane stability [22].…”

PKA inhibits RhoA activation: a protection mechanism against endothelial barrier dysfunction

“…The cAMP-dependent protein kinase (PKA) has significant and profound protective actions against increases in endothelial permeability. We and others (4,10,32,42,43) have shown that the ubiquitous cellular messenger cAMP prevents increases in endothelial permeability in response to a wide range of inflammatory mediators, including oxidants. Although growing evidence indicates that cAMP activates both PKA-dependent and -independent actions (8,11,26), our recent observations show that protective actions of cAMP against barrier dysfunction are likely mediated predominantly through PKA-dependent signaling mechanisms (32,43).…”

Methods for Forming Human Microvascular Tubes In Vitro and Measuring Their Macromolecular Permeability

“…Traditionally, the permeability of human endothelium has been examined in planar cell cultures in vitro (12). These cultures lack physiologically relevant shear stress, and the permeability coefficients are averaged over large areas and long times.…”