Cyclic AMP potentiates vascular endothelial cadherin-mediated cell-cell contact to enhance endothelial barrier function through an Epac-Rap1 signaling pathway

Abstract

Cyclic AMP (cAMP) is a well-known intracellular signaling molecule improving barrier function in vascular endothelial cells. Here, we delineate a novel cAMP-triggered signal that regulates the barrier function. We found that cAMP-elevating reagents, prostacyclin and forskolin, decreased cell permeability and enhanced vascular endothelial (VE) cadherin-dependent cell adhesion. Although the decreased permeability and the increased VE-cadherin-mediated adhesion by prostacyclin and forskolin were insensitive to a specific inhibitor for cAMP-dependent protein kinase, these effects were mimicked by 8-(4-chlorophenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate, a specific activator for Epac, which is a novel cAMP-dependent guanine nucleotide exchange factor for Rap1. Thus, we investigated the effect of Rap1 on permeability and the VE-cadherin-mediated cell adhesion by expressing either constitutive active Rap1 or Rap1GAPII. Activation of Rap1 resulted in a decrease in permeability and enhancement of VE-cadherin-dependent cell adhesion, whereas inactivation of Rap1 had the counter effect. Furthermore, prostacyclin and forskolin induced cortical actin rearrangement in a Rap1-dependent manner. In conclusion, cAMP-Epac-Rap1 signaling promotes decreased cell permeability by enhancing VE-cadherin-mediated adhesion lined by the rearranged cortical actin.

FIG. 1.

cAMP enhances barrier function of monolayer VE cells. (A) Vascular permeability, reflecting barrier function, was analyzed by measuring the fluorescence of FITC-labeled dextran across the monolayer-cultured HUVECs as described in Materials and Methods. HUVECs grown on transwell filters were incubated with control (Cont), 0.1 μM AM, 200 μM Iso, 200-ng/ml PGE2, 10-μg/ml PGI2, 1 mM IBMX, 1 mM dbcAMP, and 10 μM FSK for 30 min. Average permeability ± standard deviation is expressed as a percentage compared to the control. (B) The effects of PGI2 and FSK on vascular permeability were quantified in the presence (+) or absence (−) (Vehicle) of 2 U of thrombin (Thr)/ml. Average permeability ± standard deviation is expressed as the increase relative to that observed in unstimulated HUVECs in the vehicle. Data shown are the results from at least three independent experiments. Significant differences from the control (A) or between two groups (B) determined by Student's t test are indicated by a single asterisk (P < 0.05) or double asterisks (P < 0.01).

FIG. 2.

cAMP induces AJ formation. (A) HUVECs cultured on a glass base dish were stimulated with 10 μg of PGI2/ml (upper panels) or with 10 μM FSK (lower panels) for 20 min and shown as phase-contrast images. Left and right panels show the cells before and after stimulation, respectively. The arrows indicate the sites of cell-cell contacts induced by PGI2 and FSK. The area boxed by the white broken line is enlarged in the right top of the panels. Bars, 50 μm. (B) Subconfluent HUVECs stimulated with vehicle (Cont), 10-μg/ml PGI2, and 10 μM FSK for 45 min were fixed, stained with anti-VE-cadherin antibody, and visualized with Alexa 488-conjugated secondary antibody through a confocal microscope (BX50WI; Olympus). Note that VE-cadherin (green) was accumulated at the cell-cell contact upon PGI2 and FSK stimulation. Bars, 50 μm. (C) Translocation of VE-cadherin was assessed by Triton X-100 solubility. HUVECs were stimulated with vehicle (top), 10-μg/ml PGI2 (middle), and 10 μM FSK (bottom) for the time indicated at the top and fractionated with cytoskeleton-stabilizing buffer as described in Materials and Methods. The Triton X-100-insoluble fraction was subjected to SDS-PAGE followed by Western blot analysis (WB) with anti-VE-cadherin.

FIG. 3.

Endothelial cells adhere to a VEC-Fc-coated dish through homophilic ligation of VE-cadherin. (A) HUVECs were plated onto the VEC-Fc-coated dish and time-lapse imaged at the time points (in minutes) indicated on the panels. Bar, 20 μM. (B) HUVECs were plated on the Fc-coated dish (top panel) or the VEC-Fc-coated dish (bottom panel) for 1 h and phase-contrast imaged after removal of nonadherent cells by washing with PBS-Ca/Mg. (C) HUVECs were plated onto either an Fc- or VEC-Fc-coated dish in the absence (−) or presence (+) of 5 mM EGTA and 10 μM FSK for 7 min. Cell adhesion was quantified as described in Materials and Methods. (D) Adhesion of HUVECs to a collagen-coated dish in the presence or absence of 5 mM EGTA was analyzed by a method similar to that described for panel C. (E) Adhesion of HUVECs, HAECs, and HeLa and HEK293 cells to the VEC-Fc-coated dish was examined as described in the legend for panel C. Cells adhering to the dishes of total input cells (percentage) is expressed as the mean ± standard deviation by measuring alkaline phosphatase activity of adherent cells divided by that of total input cells. Representative results from three independent experiments were shown in all panels.

FIG. 4.

cAMP potentiates VE-cadherin-dependent cell adhesion. (A) HUVECs were plated onto a VEC-Fc-coated dish in the presence of PGI2 at the concentrations indicated at the bottom for 7 min. Cell adhesion was quantified as described in Materials and Methods. Mean adhesion activity ± standard deviation is expressed as the increase compared with that observed in unstimulated cells. (B) HUVECs were plated onto the VEC-Fc-coated dish in the absence (circle) or presence (square) of 10-μg/ml PGI2 for the time indicated at the bottom. The percent adhesion was calculated by measuring the alkaline phosphatase activity of adherent cells divided by that of total input cells. (C) HUVECs stimulated with cAMP-elevating ligands similar to that described in the legend to panel A were assessed for adhesion activity. The concentration of stimulants was the same as described in the legend to Fig. 1A. (D) The effect of FSK on cell adhesion was analyzed by a method similar to that described for panel A, except that cells were preincubated for 10 min before plating. (E) The effect of 10 μM FSK on time-dependent adhesion was analyzed as described in the legend to panel B, except that cells were preincubated for 10 min before plating. (F) HUVECs stimulated with the reagent indicated at the same concentration used as described in the legend to Fig. 1A were analyzed for cell adhesion by a method similar to that described for panel D. Data are expressed as means ± standard deviations of the results from three independent experiments in panels A, C, D, and F. Representative results from three independent experiments wereshown in panels B and E. A significant difference from the control determined by Student's t test is indicated with a single asterisk (P < 0.05) or double asterisks (P < 0.01).

FIG. 5.

cAMP-enhanced VE-cadherin-dependent cell adhesion and endothelial barrier function does not depend upon PKA. (A) Permeability across monolayer HUVECs grown on transwell filters were assessed by measuring FITC-labeled dextran as described in the legend to Fig. 1A. The effect of 10-μg/ml PGI2 on cell permeability without pretreatment (Vehicle) or with pretreatment with 5 μM H89, a specific PKA inhibitor, for 10 min is indicated as the percent permeability compared to that observed in untreated cells. +, present; −, absent. (B) The effect of 10 μM FSK on cell permeability without pretreatment (Vehicle) and with pretreatment with H89 was assessed similar to that described for panel A. (C) The effect of pretreatment of HUVECs with 5 μM H89 on FSK-induced reduction of 2-U/ml thrombin-induced permeability was analyzed. Permeability indicates the increase relative to that observed in untreated cells. (D) HUVECs untreated or pretreated with H89 for 10 min prior to stimulation with 10-μg/ml PGI2 were analyzed for cell adhesion as described in the legend to Fig. 4A. (E) HUVECs untreated or pretreated with H89 for 10 min prior to stimulation with 10 μM FSK were analyzed for cell adhesion as described in the legend to Fig. 4D. For panels A to E, data are expressed as means ± standard deviations of the results from triplicate samples. Similar results were obtained in at least three independent experiments. Significant differences between two groups determinedby Student's t test are indicated by a single asterisk (P < 0.05) or double asterisks (P < 0.01). (F) HUVECs serum starved in 1% BSA-containing medium 199 for 6 h, followed by pretreatment with (+) or without (−) 5 μM H89 for 10 min, were stimulated with vehicle and 10 μM FSK for 10 min. Phosphorylation of CREB was assessed by Western blot analysis with anti-CREB (CREB) and anti-phospho-CREB-specific (pCREB) antibodies.

FIG. 6.

cAMP induces Rap1 activation. (A) Serum-starved HUVECs kept in medium 199 containing 1% BSA overnight were stimulated with cAMP-elevating agonists for 2.5 min as indicated at the top and at the concentrations described in the legend to Fig. 1A. GTP-bound Rap1 was detected by pull-down assay as described in Materials and Methods. Activation indicates the ratio of the poststimulation GTP-Rap1 intensity of total Rap1 intensity to the prestimulation GTP-Rap1 intensity of total Rap1 intensity. (B) Rap1 activation was analyzed by detecting GTP-bound Rap1 with lysates from HUVECs stimulated with PGI2 for 2.5 min at the different concentrations indicated at the top. (C) Rap1 activation was analyzed by detecting GTP-bound Rap1 with lysates from cells stimulated with 10-μg/ml PGI2 for the time period indicated at the top. (D) Serum-starved HUVECs similar to those described in the legend to panel A were stimulated with the reagents indicated at the top for 10 min at the same concentrations described in the legend to Fig. 1A. Rap1 activation was assessed by a method similar to that described for panel A. (E) The effect of 10 μM FSK on time-dependent Rap1 activity was examined as described for panel C. Representative results from at least three independent experiments are shown for all panels.

FIG. 7.

Activation of Epac is sufficient to enhance VE-cadherin-dependent cell adhesion and endothelial barrier function. (A) Serum-starved HUVECs in medium 199 containing 1% BSA were stimulated with 0.2 mM 8-CPT-2′-O-Me-cAMP (8CPT) for the indicated time. Rap1 activity was determined as described in the legend to Fig. 6A. The result is a representative from three independent experiments. (B) Permeability of cells treated with the reagents as indicated on the bottom for 30 min was analyzed as described in the legend to Fig. 1A. (C) The effect of 0.2 mM 8CPT-2′-O-Me-cAMP on 2-U/ml thrombin-induced permeability was analyzed as described in the legend to Fig. 1B. (D) Effect of 8CPT-2′-O-Me-cAMP on VEGF-induced permeability was assessed by intradermal Miles assay as described in Materials and Methods. Amounts of extravasation of Evan blue in mouse dermal skin were measured 60 min after intradermal injection of vehicle and VEGF together with (+) or without (−) 8CPT. Mean leakage ± standard deviation of the results from 6 mice per group is expressed as nanograms of weight of extravasated Evans blue per milligram of weight of dermal skin. A photograph on the bottom shows leakage of Evans blue in dermal skin. (E) HUVEC adhesion to the VEC-Fc-coated dish in the presence of 0.2 mM 8CPT and 1 mM dbcAMP for 7 min was analyzed as described in the legend to Fig. 4F. In panels B, C, and E, data are expressed as means ± standard deviations of the results from triplicate samples. A significant difference from the control in panels B and E or between two groups in panels C and D was determined by Student's t test and indicated by a single asterisk (P < 0.05) or double asterisks (P < 0.01).

FIG. 8.

Rap1 plays a critical role in VE-cadherin-dependent cell adhesion and endothelial barrier function. (A) Rap1 inactivation was assessed by detecting GTP-Rap1 in HUVECs infected with different MOI of adenovirus-expressing Rap1GAPII (RapGAP) as indicated at the top. An adenovirus-expressing LacZ at an MOI of 50 was used as a control. GTP-bound Rap1 (GTP-Rap) was detected by pull-down assay as described in Materials and Methods. Rap1 (Rap) and Rap1GAPII (RapGAP) expression were examined by Western blot analysis. (B) The permeability of FITC-dextran across HUVECs infected with adenovirus as indicated at the bottom was analyzed as described in Materials and Methods. Data are the means ± standard deviations of the results from three independent experiments and are expressed as increases relative to those of LacZ-infected cells. (C) Monolayer HUVECs infected with either an adenovirus-expressing LacZ or that expressing Rap1V12 at an MOI of 50 for 24 h were medium changed and cultured for another 24 h. The permeability of cells upon 2-U/ml thrombin stimulation (Thr) after starvation for 1 h was analyzed as described in the legend to Fig. 1B. Data are the means ± standard deviations of the results from five independent experiments and are expressed as inductions relative to those of untreated HUVECs infected with the LacZ-expressing virus. (D) HUVECs were transfected with either empty vector (Mock), plasmids expressing Rap1GAPII (RapGAP), EpacΔcAMP, or Rap1V12 together with the luciferase reporter construct. Transfected cells were plated on the VEC-Fc-coated dish and allowed to adhere for 15 min. Cell adhesion was analyzed as described in Materials and Methods. Data are expressed as increases compared to those of mock-transfected cells. The results indicate the means ± standard deviations of the results from triplicate samples. Similar results were obtained in three independent experiments. Significant differences between two groups in panel C or from the control in panel D are determined by Student's t test and are indicated by a single asterisk (P < 0.05) or double asterisks (P < 0.01).

FIG. 9.

Inactivation of Rap1 reduces PGI2- and FSK-induced barrier function and VE-cadherin-mediated cell adhesion. (A) Monolayer-cultured HUVECs grown on transwell filters were infected with either LacZ-expressing adenovirus (Ad-LacZ) or Rap1GAPII-expressing virus (Ad-RapGAP) at an MOI of 40 for 24 h. Medium was replaced with fresh medium after infection. Cells were cultured for an additional 24 h and treated with 10 μg of PGI2/ml for 30 min after serum starvation for 1 h. Permeability was analyzed as described in Materials and Methods. (B) The effect of 10 μM FSK on permeability in HUVECs infected with Ad-RapGAP was similarly analyzed. (C) HUVECs were infected with either Ad-LacZ or Ad-RapGAP at an MOI of 40 for 24 h. HUVECs resuspended in medium 199 with 0.5% BSA were plated onto VEC-Fc-coated dishes in the presence (+) or absence (−) of 10 μg of PGI2/ml for 7 min. Cell adhesion activity was quantified as described in the legend to Fig. 4A. (D) The effect of FSK on adhesion of HUVECs infected with Ad-RapGAP was analyzed similarly to that described for panel C. Resuspended HUVECs were preincubated with 10 μM FSK for 10 min before plating. Significant differences between two groups determined by Student's t test are indicated by a single asterisk (P < 0.05) or double asterisks (P < 0.01).

FIG. 10.

cAMP induces cortical actin rearrangement in a Rap1-dependent manner. (A) Monolayer-cultured HUVECs starved in 0.5% BSA-containing medium 199 for 3 h were stimulated with vehicle (top row), 10-μg/ml PGI2 (second row), 10 μM FSK (third row), and 0.2 mM 8-CPT-2′-O-Me-cAMP (8CPT) (bottom row) for 30 min. Fixed and permeabilized cells were stained with rhodamine-phalloidin (left column) and with anti-cortactin (center column). Rhodamine images to detect F-actin (red) and Alexa 488 images for cortactin visualized byAlexa 488-labeled secondary antibody (green) were obtained through a confocal microscope (BX50WI). Right panels show the merged images of rhodamine and Alexa 488 images. Bars, 20 μm. (B) HUVECs transfected with an EGFP-expressing vector (left) and pCXN2-Rap1GAPII-IRES-EGFP (right) were serum starved in 0.5% BSA-containing medium 199 for 3 h and stimulated with vehicle (top panels) and 10 μM FSK (bottom panels). Cells were fixed, permeabilized, and stained with Rhodamine-phalloidin. EGFP images (green) and rhodamine images showing F-actin (red) were obtained similar to those in panel A. Arrows and arrowhead indicate transfected and untransfected cells, respectively. Bars, 20 μm. (C) Cell permeability of HUVECs pretreated with 2 μM cytochalasin D (CytoD) for 30 min followed by 10 μM FSK stimulation for 30 min was analyzed as described in the legend to Fig. 1A. −, absent; +, present. (D) The effect of pretreatment of 2 μM cytochalasin D (CytoD) on adhesion of HUVECs stimulated with FSK was analyzed as described in the legend to Fig. 5E. A significant difference between two groups determined by Student's t test is indicated by double asterisks (P < 0.01).