Plasminogen Activator Inhibitor-1 Controls Vascular Integrity by Regulating VE-Cadherin Trafficking

Background Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is expressed and secreted by endothelial cells. Patients with PAI-1 deficiency show a mild to moderate bleeding diathesis, which has been exclusively ascribed to the function of PAI-1 in down-regulating fibrinolysis. We tested the hypothesis that PAI-1 function plays a direct role in controlling vascular integrity and permeability by keeping endothelial cell-cell junctions intact. Methodology/Principal Findings We utilized PAI-039, a specific small molecule inhibitor of PAI-1, to investigate the role of PAI-1 in protecting endothelial integrity. In vivo inhibition of PAI-1 resulted in vascular leakage from intersegmental vessels and in the hindbrain of zebrafish embryos. In addition PAI-1 inhibition in human umbilical vein endothelial cell (HUVEC) monolayers leads to a marked decrease of transendothelial resistance and disrupted endothelial junctions. The total level of the endothelial junction regulator VE-cadherin was reduced, whereas surface VE-cadherin expression was unaltered. Moreover, PAI-1 inhibition reduced the shedding of VE-cadherin. Finally, we detected an accumulation of VE-cadherin at the Golgi apparatus. Conclusions/Significance Our findings indicate that PAI-1 function is important for the maintenance of endothelial monolayer and vascular integrity by controlling VE-cadherin trafficking to and from the plasma membrane. Our data further suggest that therapies using PAI-1 antagonists like PAI-039 ought to be used with caution to avoid disruption of the vessel wal

PAI-1 inhibition reduces cellular PAI-1 and VE-cadherin levels while surface levels remain unaltered.

<p>(A) HUVEC were treated with PAI-039 (25 μM) or solvent (DMSO 0.1%) for four hours and total cell lysates (TCL) and conditioned media (CM) were collected. VE-cadherin and PAI-1 levels in total cell lysates are reduced whereas secreted PAI-1 is unchanged. (B) Densitometric quantification of Western blots showed reduced (non–significant) VE-cadherin and PAI-1 after inhibitor treatment. VE-cadherin and PAI-1 levels were normalized to actin and relative density is displayed (n = 10, mean + SEM, n.s. = non-significant). (C) Levels of soluble VE-cadherin (sVE-cadherin) in conditioned media of HUVEC treated with PAI-039 or solvent (as described at (A)) were determined by ELISA. Levels of sVE-cadherin shed into conditioned media were reduced after PAI-039 treatment (n = 4, mean + SEM, n.s. = non-significant). (D) FACS analysis of HUVEC treated as described before. PAI-039 treatment had no effect on VE-cadherin surface levels (n = 3, geometric mean fluorescence intensity + SEM, n.s. = non-significant).</p

Fluorescence recovery after photobleaching (FRAP) of VE-cadherin at the plasma membrane is unaltered after PAI-1 inhibition.

<p>(A) HUVEC were transduced with lentivirus to express VE-cadherin-GFP. HUVEC were treated with PAI-039 (25 μM) or solvent (DMSO 0.1%) for two hours before FRAP imaging was performed. Fluorescence recovery was monitored for five minutes. (B) The average FRAP from five independent experiments are displayed (solvent: mean + SEM, PAI-039 mean–SEM). (C) The average FRAP from five experiments 240 seconds after bleaching is not different from solvent treated cells (mean + SEM, n.s. = non-significant).</p

PAI-1 inhibition leads to disruption of VE-cadherin-mediated cell-cell junctions.

<p>(A) HUVEC were treated with PAI-039 (25 μM) or solvent (DMSO 0.1%) for four hours and stained for VE-cadherin (green), β-catenin (red) and actin (scale bar 50 μm). PAI-039 treatment causes disruption of junctions, gap formation (asterix) and stress fiber formation. (B) Zooms of images in (A). Arrowhead indicates accumulation of VE-cadherin in endothelial cells, arrowhead plus asterix indicates stress fibres. (C) Average junction width of cells treated with PAI-039 (25 μM) or solvent (DMSO 0.1%) for four hours (n = 4) and after junction recovery in response to washout of inhibitor (n = 2) (mean + SEM, *** p < 0.001, n.s. = non-significant).</p

PAI-1 inhibition in zebrafish leads to vascular leakage.

<p>(A) Zebrafish embryos three-days post-fertilization (3dpf) were incubated with PAI-039 inhibitor (50 μM) or solvent (DMSO 1%) for 6 hours. Vascular leakage was visualized by injecting 2000 kDa tetramethylrhodamine(TMR)-dextran into the duct of cuvier. Intersegmental vessels are depicted in green, vascular lumen and leakage from vessels in red (scale bar 100 μm). (B) Quantification of extracellular fluorescence data from at least 15 fish were analysed for PAI-039 or solvent treatments respectively (mean + SEM, * p < 0.05). (C) 3dpf zebrafish were injected with 70 kDa TMR-dextran and incubated with PAI-039 inhibitor (25 μM) or solvent (DMSO 1%) for 3–4 hours. Vascular leakage occurs in the hindbrain of zebrafish (arrow heads) (scale bar 100 μm). (D) Quantification of extracellular fluorescence data from at least 23 fish were analysed for PAI-039 or solvent treatments respectively (mean + SEM, ** p < 0.01).</p

PAI-1 inhibition leads to the accumulation of VE-cadherin at the Golgi apparatus.

<p>(A) HUVEC were treated with PAI-039 (25 μM) or solvent (DMSO 0.1%) for four hours and stained for VE-cadherin (green) and GM130 (red) (scale bar 50 μm). Inset are magnified on the right side and the Golgi apparatus is outlined as for the intensity measurements. (B) PAI-039 treatment leads to the accumulation of VE-cadherin at the Golgi apparatus, (C) whereas the size of the Golgi apparatus is decreased (n = 3, mean + SEM, ** p < 0.001).</p

PAI-1 inhibition of HUVEC monolayers leads to loss of transendothelial resistance.

<p>(A) and (B) Transendothelial resistance (TER) was measured by electric cell-substrate impedance sensing (ECIS). HUVEC were grown to confluence in ECIS arrays and treated with either PAI-039 (6.25 μM, 12.5 μM, 25 μM) or solvent (DMSO 0.1%). Resistance values were normalized to the basal resistance one hour before addition of inhibitor. (A) is representative of one experiment (mean value of quadruplicates). (B) is representative of the normalized resistance after four hours of PAI-039 of three independent experiments, basal resistance is the normalized resistance before addition of inhibitor (mean + SEM, * p < 0.05). (C) and (D) Transendothelial resistance was measured as described in (A) and (B). After 4 hours of treatment with PAI-039 (25 μM) or solvent (DMSO 0.1%) medium was replaced with fresh medium with or without inhibitor (or solvent). TER returned to basal values within two to four hours. (C) is representative of one experiment (mean value of quadruplicates). (D) is the summary of four hour inhibition with PAI-039 (n = 10) and recovery for two hours (n = 4) (mean + SEM, ** p < 0.01).</p

Immuno-coprecipitation of VE-cadherin from HUVEC cells after PAI-1 inhibition shows no effect on interaction of VE-cadherin with β-catenin.

<p>HUVEC were treated with PAI-039 (25 μM) or solvent (DMSO 0.1%) for four hours and total cell lysates (TCL) were subjected to immuno-coprecipitation (IP) of VE-cadherin. After SDS-PAGE proteins were blotted onto nitrocellulose membranes and the top half (> 70 kDa) first probed for β-catenin (ECL) and then for VE-cadherin (Odyssey). The bottom half (< 70 kDa) was probed for β-actin. Short and long exposures are depicted here.</p