Effect of cyclic AMP on barrier function of human lymphatic microvascular tubes - PubMed (original) (raw)
Effect of cyclic AMP on barrier function of human lymphatic microvascular tubes
Gavrielle M Price et al. Microvasc Res. 2008 May.
Abstract
This work examines the effect of cyclic AMP (cAMP) on the in vitro barrier function of tubes of human dermal lymphatic microvascular endothelial cells (LECs). Under baseline conditions, the barrier function of LEC tubes was weak, with diffusional permeability coefficients to bovine serum albumin and 10 kDa dextran of 1.4(-0.6)(+0.9)x10(-6) cm/s and 1.7(-0.5)(+0.8)x10(-6) cm/s (geometric mean+/-95% CI), respectively, and 1.2+/-0.5 (mean+/-95% CI) focal leaks per mm. Exposure to low concentrations (3 microM) of a cell-permeant analog of cAMP did not alter the barrier function. Exposure to higher concentrations (80 and 400 microM) and/or the phosphodiesterase inhibitor Ro-20-1724 (20 microM) lowered permeabilities and the number of focal leaks, and increased the selectivity of the barrier. Decreased permeabilities were accompanied by an increase in continuous VE-cadherin staining at cell-cell borders. Exposure to 1 mM 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, did not increase permeabilities. LECs expressed the lymphatic-specific master transcription factor Prox-1, regardless of whether barrier function was weak or strong. Our results indicate that the permeability of LEC tubes in vitro responds to cAMP in a manner similar to that well-described for the permeability of blood microvessels.
Figures
Figure 1
LEC cultures and tubes. (A) Phase-contrast image of LECs, and fluorescence image of a culture stained for the lymphatic markers Prox-1 (red) and podoplanin (green) and DNA (blue). (B) Phase-contrast image of LEC tubes cultured under 3 μM db-cAMP, 80 μM db-cAMP, and 400 μM db-cAMP + 20 μM Ro-20−1724 (Ro), and fluorescence images after 26 minutes of perfusion with Alexa 488-conjugated 10 kDa dextran. A focal leak (indicated by asterisk) is present in the 3 μM db-cAMP tube. LEC tubes cultured under 0 μM db-cAMP appeared similar to those under 3 μM db-cAMP.
Figure 2
Permeabilities of LEC tubes to BSA and 10 kDa dextran at day 3 post-seeding. (A) BSA permeability coefficients for LEC tubes treated with 0 μM db-cAMP (n = 14), 3 μM db-cAMP (n = 22), 80 μM db-cAMP (n = 22), 400 μM db-cAMP + 20 μM Ro-20−1724 (n = 21), and 0 μM db-cAMP + 1 mM ddA (n = 13). (B) 10 kD dextran permeability coefficients for LEC tubes. (C) Size selectivity of LEC tubes, defined as the ratio of permeability to 10 kD dextran over the permeability to BSA. (D) The occurrence of focal leaks per millimeter in LEC tubes.
Figure 3
Expression of Prox-1 and junctional markers in LEC tubes. (A) Prox-1 (false color red). Inset, negative Prox-1 staining in a BEC tube. (B) VE-cadherin. Insets, VE-cadherin staining magnified 1.8X. (C) PECAM. (D) ZO-1. Images are representative of three separate experiments.
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