S1 Fig: PAI-1 inhibition of HUVEC monolayers with TM5275 leads to loss of transendothelial resistance. (A) and (B) Transendothelial electrical resistance (TER) was measured by electric cell-substrate impedance sensing (ECIS).

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HUVEC were grown to confluence in ECIS arrays and treated with either TM5275 (50 μM, 100 μM, 200 μM) or solvent (DMSO 0.2%). 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 normalized resistance after four hours of PAI-1 inhibition with TM5275 from the same experiment, basal resistance is the normalized resistance just before addition of inhibitor.

(C) and (D) TER was measured as described in (A) and (B). After 4 hours of treatment with TM5275 (100 μM) or solvent (DMSO 0.2%) the old medium was replaced with fresh medium with or without inhibitor (or solvent). The transendothelial resistance 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 TM5275 (n = 3) and recovery for two hours (n = 2) (mean + SEM,. p.

S2 Fig: PAI-1 depletion results in decreased electrical resistance and monolayer gaps. (A) HUVECs were grown to confluency and treated with shRNA as indicated. HUVECs depleted for PAI-1 showed gap formation. Merge shows VE-cadherin in red and F-actin in green. (B) Transendothelial electrical resistance (TER) was measured by electric cell-substrate impedance sensing (ECIS). HUVEC pretreated with shPAI-1 or shCTRL were plated in ECIS arrays and monitored for resistance.

No change in the initial spreading of the cells was detected; however, when forming a stable monolayer, a reduced resistance was measured for HUVECs that were depleted for PAI-1. (C) Quantification of resistance of HUVECs after 20 hours of plating.

Data are mean ± s.e.m. Experiment is carried out three times independently form each other and per experiment in duplicate. (D) Western blot analysis shows efficient depletion of PAI-1. Actin is shown as loading control.(TIF). S3 Fig: PAI-1 inhibition leads to disruption of VE-cadherin-mediated cell junctions. (A) and (B) HUVEC were treated with PAI-039 (25 μM) or solvent (DMSO 0.1%) for four hours and stained for VE-cadherin (green), actin and (A) p120 catenin or (B) α-catenin (red) (scale bar 50 μm).

(C) Illustration of junction width measurements. Five junctional measurements per cells were taken as shown in this picture. (D) TER was measured by ECIS.

HUVEC were grown to confluence in ECIS arrays and treated with either PAI-039 (25 μM) or Control (DMSO 0.1%) and Y-27632 (10 μM) as indicated. Resistance values were normalized to the basal resistance one hour before addition of inhibitors. Treatment with Y-27632 did not prevent decrease in TER after PAI-039 treatment.

Graph is representative of one experiment (mean value of quadruplicates + SD).(TIF). S5 Fig: PAI-1 inhibition with PAI-039 or TM5275 does not alter VE-cadherin and PECAM-1 expression on HUVEC. (A), (B), (C) FACS analysis of HUVEC treated with TM5275 (100 μM, control: DMSO 0.2%) or PAI-039 (25 μM, control: DMSO 0.1%) as indicated.

PAI-1 inhibition did not affect VE-cadherin expression on HUVEC (n = 3, geometric mean fluorescence intensity + SEM, n.s. = non-significant). (D) Densitometric quantification of PAI-1 in conditioned media of HUVEC treated with PAI-039 as described in (n = 3, mean + SEM, n.s.

= non-significant).(TIF). S6 Fig: Immuno-coprecipitation of VE-cadherin from HUVEC cells after PAI-1 inhibition shows no effect on interaction of VE-cadherin with α-catenin or p120 catenin. 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 (A) α-catenin or (B) p120 catenin (ECL) and then for VE-cadherin (Odyssey). The bottom half (.

Methodology/Principal FindingsWe 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.

IntroductionEndothelia line all blood vessels and form a barrier between the circulation and surrounding tissues. The barrier function is maintained and regulated by the adherens junctions that connect neighboring cells.

The most important cell adhesion protein in endothelial adherens junctions is VE-cadherin (Vascular Endothelial-cadherin), which ensures that endothelial cells stay connected and restrains the leakage from blood vessels. VE-cadherin interacts with p120 catenin, which prevents internalization of VE-cadherin , and with β- and α-catenin which anchor VE-cadherin to the actin cytoskeleton ,. Disruption of VE-cadherin-based junctions, e.g. In response to inflammatory mediators like TNFα, leads to a loss of endothelial integrity which is accompanied by increased endothelial permeability ,.PAI-1 is a serine protease, which is expressed and secreted by endothelial cells, hepatocytes, adipocytes, megakaryocytes and neuronal cells. PAI-1 has an important role in keeping thrombus formation and fibrinolysis in balance. As an inhibitor of urokinase (uPA) and tissue plasminogen activator (tPA) PAI-1 prevents the formation of plasmin from plasminogen and therefore inhibits the (excessive) degradation of fibrin. Apart from the role of PAI-1 as an inhibitor of fibrinolytic activity, other PAI-1 functions for cell adhesion to extracellular matrix, tissue remodeling, migration, proliferation and apoptosis have been described –.

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Patients with PAI-1 deficiency have an increased risk for hemorrhaging after surgery, menorrhagia and epistaxis. This phenotype has to date been solely ascribed to the function of PAI-1 as an inhibitor of fibrinolysis.We show here that PAI-1 function is necessary for the maintenance of endothelial monolayer integrity. Further we show that PAI-1 inhibition with the small molecule inhibitors PAI-039 and TM5275 disrupts endothelial cell-cell junctions and causes a loss of transendothelial resistance of HUVEC monolayers and increased vascular permeability in zebrafish. We link PAI-1 function to the maintenance of endothelial cell barriers both in vivo in zebrafish and in vitro with HUVEC cultures. This indicates that therapeutic administration of PAI-1 antagonists like PAI-039 ought to be used with caution to avoid side effects like leakage of the vessel wall. MaterialsHUVEC pools were purchased from Invitrogen (Breda, The Netherlands).

EGM-2 medium and SingleQuots ™ for HUVEC cell culture were purchased from Lonza (Verviers, Belgium). PAI-1 inhibitors PAI-039 (tiplaxtinin) and TM5275 were purchased from Axon Medchem BV (Groningen, The Netherlands) and diluted in DMSO (referred to as solvent). PAI-1 rabbit polyclonal antibody was a kind gift from Dr. Sacha Zeerleder. VE-cadherin monoclonal mouse antibody, clone BV6, was purchased from Millipore (Amsterdam, The Netherlands) and actin AC-40 monoclonal mouse antibody was purchased from Sigma (Zwijndrecht, The Netherlands).

GM130 rabbit monoclonal antibody was from Cell Signaling (Leiden, The Netherlands). Goat polyclonal anti-VE-cadherin C-19 and rabbit polyclonal anti-α-catenin were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Mouse monoclonal antibodies directed against mouse monoclonal PECAM-1 AF488 WM59, VE-cadherin AF 647 55-7H1, IgG1 AF674, β-catenin and p120 catenin 98/pp120 were purchased from BD Biosciences (Amsterdam, The Netherlands). Texas Red-X Phalloidin, chicken anti-goat AF647, chicken anti-rabbit AF488, chicken anti-rabbit AF594, chicken anti-mouse AF488 and IgG1 AF488 were from Invitrogen (Breda, The Netherlands). Electric cell-substrate impedance sensing (ECIS)Transendothelial electrical resistance of HUVEC monolayers was measured using ECIS. HUVEC were added at 100,000 cells per well (0.8 cm 2) of L-cysteine (10 mM, Sigma) and fibronectin (10 μg/mL, Sigma) coated electrode arrays (8W10E PET, Ibidi, Planegg/Martinsried, Germany) in a ECIS Z Theta controller from Applied Biophysics Inc. (Troy, NY, USA).

We used a frequency of 4 kHz to continuously measure transendothelial resistance at 37°C at 5% CO 2. Multiples of samples were used in each experiment. Once the endothelial cells form monolayers the resistance values plateau. We referred to the plateau resistance of endothelial monolayers as “basal resistance”. Inhibitors were added to endothelial monolayers two or more hours after the resistance values reached the plateau. Data were normalized to resistance values one hour before addition of inhibitors. Immunofluorescence imaging of HUVECHUVEC were cultured on fibronectin-coated glass coverslips until they formed confluent monolayers.

For PAI-1 inhibition and to avoid formation of precipitates we removed the conditioned media from the cultured cells and diluted PAI-039 (25 μM, or as indicated) or TM5275 (50 μM, or as indicated) directly in the conditioned media before adding the media back to the cells. As controls we used equivalent amounts of solvent (DMSO, 0.1 or 0.2% as inidicated). We incubated with the inhibitors for up to four hours and then rinsed the cells with phosphate buffered saline (PBS) + 1 mM CaCl 2 + 0.5 mM MgCl 2 (PBS) and fixed with 4% (v/v) paraformaldehyde in PBS for 10 minutes. Unspecific antibody binding was blocked by incubating coverslips in 3% (w/v) bovine serum albumin (BSA) in PBS for 30 minutes. For staining with antibodies or phalloidin were diluted in 1% BSA in PBS and coverslips were incubated with the antibody solutions for 60 minutes. Fluorescent imaging was performed using a confocal laser-scanning microscope (Meta, Carl Zeiss MicroImaging) using a 63x oil lens (NA 1.40).Image analyses were performed using Image J.

To account for the variability of junction width of the cells we took the average junction width at five sites on each cells. We measured the average junction width of five cells from three different fields of view from both solvent-treated and PAI-039-treated cells from four independent experiments. For analysis of VE-cadherin fluorescence intensity at the Golgi apparatus we first outlined the area of Golgi localization manually on the basis of GM130 stainings. Then we measured size and the intensity of VE-cadherin staining within that area. We analyzed data from ten cells per treatment and experiment from three independent experiments. Fluorescence recovery after photobleaching (FRAP)For FRAP endothelial cells were virally transduced with VE-cadherin-GFP (pLV-CMV-Ires-Puro SIN) and cultured for one day. Then cells were trypsinzed and transferred to Nunc ™ Lab-Tek ™ 8-chambered coverglasses (Thermo Scientific, Amsterdam, The Netherlands).

After 24 hours 25 μM PAI-039 or 0.1% DMSO (solvent) were added to the culture media. For live-cell imaging Lab-Tek coverglasses were placed in a humidified CO 2 containing atmosphere (5%) in a heating chamber at 37°C with a a confocal laser-scanning microscope (Meta, Carl Zeiss MicroImaging) and a 63x oil lens (NA 1.40). FRAP imaging was performed after two to three hours of incubation with PAI-039 or DMSO. Photobleaching of 5x5 pixel areas (512x512 resolution) at cell-cell junctions was performed with 100 iterations of 488-nm laser illumination at maximum power (25 mW). Fluorescence recovery was measured by time-lapse imaging. FRAP analysis was performed using Image J, Excel and GraphPad prism. Corrections for cell movements, bleaching and background were made.

The data displayed are of average FRAP from five independent experiments with five to ten FRAPs performed per treatment and experiment. ELISAQuantikine human VE-cadherin immunoassay (R&D, Abingdon, UK) was used to measure the release of cleaved VE-cadherin in conditioned media from HUVEC. We cultured HUVEC in supplemented EGM-2 until they reached confluence and then added fresh medium supplemented with either 0.1% DMSO (solvent) or 25 μM PAI-039. After four hours we collected the conditioned media, centrifuged the media to remove cellular debris and snap froze the samples. For the immunoassay samples were measured in duplicate according to the manufacturer’s protocol.

Fluorescence-activated cell sorting (FACS)For FACS stainings HUVEC were cultured and treated as described above. HUVEC were rinsed with PBS twice and then detached using Accutase (GE Healthcare, Eindhoven, The Netherlands). Detached cells were resuspended in ice-cold PBS supplemented with 0.5% (w/v) BSA, centrifuged and resuspended in PBS + 0.5% BSA. Cells were incubated with fluorescently labelled antibodies for VE-cadherin or PECAM-1 or isotype matched controls for 30 minutes on ice followed by three washes. Fluorescence was measured using an LSR II (BD, Breda, The Netherlands).

FACS analyses were performed with BD FACSDiva (BD, Breda, The Netherlands) and FlowJo (Oregon, USA) software. Immunoprecipitation and western blottingFor immunoprecipitation cells were rinsed twice with ice-cold PBS and then lysed with ice-cold NP-40 lysis buffer (25 mM Tris, 100 mM NaCl, 10 mM MgCl 2, 10% (v/v) glycerol and 1% (v/v) Nonidet P-40, pH 7.4) supplemented with protease-inhibitor cocktail tablets (Roche Applied Science).

After 10 minutes cells were scraped off dishes and centrifuged at 20,000 xg for 10 minutes at 4°C. The supernatant was incubated with 2 μg of mouse monoclonal VE-cadherin antibody and 50 μl protein G-sepharose beads (GE Healthcare, Dassel, Germany) at 4°C under continuous mixing. The beads were then washed five times with NP-40 lysis buffer (5000 xg, 30 s, 4°C) and boiled (5 min) in sample buffer containing SDS and 4% β-mercapto-ethanol. Samples from immunoprecipitation and total cell lysates were subjected to SDS-PAGE and transferred to 0.2 μm Protran ™ nitrocellulose membranes (Whatman/GE Healthcare, Dassel, Germany). Membranes were blocked with 5% (w/v) milk powder dissolved in Tris-buffered saline with Tween (TBST, 150 mM NaCl, Tris 10 mM, 0.1% (v/v) Tween20, pH 8.0). The nitrocellulose membranes were probed with primary antibodies over night at 4°C and subsequently incubated with secondary antibodies conjugated with HRP.

Secondary HRP-conjugated antibodies were purchased from Dako (Heverlee, Belgium). Between probing blots were rinsed with TBST. Bands were visualized with an enhanced chemiluminescence detection system (Thermo Scientific, Amsterdam, The Netherlands). Alternatively or subsequent to probing with HRP-conjugated antibodies, secondary antibodies conjugated with IR680 or IR800 dyes were used and visualized with the Odyssey infrared detection system (Licor Westburg, Leusden, The Netherlands). For probing with Odyssey secondary antibodies we removed residual signal from HRP by washing membranes with 1% (w/v) azide in TBST for 30 minutes and subsequent probing with different primary and secondary antibodies. Statistical analysisThe data were analyzed using SPSS, Excel, and Graph Pad/Prism (v6.04). The zebrafish fluorescence intensity measurements were analyzed using Mann-Whitney U test.

The effect of different concentrations of PAI-039 was statistically evaluated using one-way ANOVA with a Tukey post hoc test for multiple comparisons. All other analyses were performed using unpaired t-test. Inhibition of PAI-1 function in zebrafish leads to vascular leakageTo date the increased bleeding diathesis of patients without PAI-1 has been ascribed to the importance of PAI-1 in preventing fibrinolysis. PAI-1 is a serine protease and an inhibitor of uPA and tPA and thus prevents the activation of plasminogen to plasmin.

Plasmin in turn degrades fibrin clots and prevents thrombosis from overshooting fibrin formation. Thus lack of PAI-1 can lead to excessive fibrinolysis and as a consequence bleeding due to lack of wound closure. Nonetheless, there are more causes to bleeding disposition. And notably, PAI-1 is directly produced and secreted by endothelial cells and we hypothesized that defects of the vessel barrier could add to the phenotype seen in patients lacking PAI-1.

To investigate this hypothesis we used PAI-039 (tiplaxtinin), a small molecule inhibitor of PAI-1, which binds to PAI-1 close to the vitronectin binding site and inhibits PAI-1 protease activity. PAI-1 antagonists like PAI-039 have been successfully used to prevent thrombosis in disease models –.

We first tested if PAI-1 inhibition could cause a phenotype resembling the one seen in patients, particularly hemorrhage and vascular leakage. To investigate the function of PAI-1 in vivo, we examined the effects of PAI-1 inhibiton in the zebrafish ( D. Rerio) vasculature. Zebrafish PAI-1 shares 42.0% sequence identity with the human homologue ,.

To examine if PAI-1 inhibition with PAI-039 can mimick the effects of PAI-1 deficiency in patients, we injected fluorescent TMR-dextran into zebrafish embryos to visualize any leakage from blood vessels. We observed leakage of 2000 kDa TMR-dextran from intersegmental vessels of 3dpf zebrafish embryos after six hours of treatment with 50 μM PAI-039. Further, we observed increased vascular permeability of 70 kDa dextran in the hindbrain vasculature after three to four hours of treatment with PAI-039 (25 μM) compared to controls. These results indicate that, similar to the observations in patients, inhibition of PAI-1 function leads to loss of blood vessel integrity in zebrafish embryos. PAI-1 inhibition in zebrafish leads to vascular leakage.(A) Zebrafish embryos three-days post-fertilization (3dpf) were incubated with PAI-039 inhibitor (50 μM) or solvent (DMSO 1%) for 6 hours.

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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. Inhibition of PAI-1 function leads to decreased transendothelial resistanceNext we tested if PAI-1 inhibition with PAI-039 could directly affect the integrity of endothelial monolayers. The disruption of vascular integrity and increased vascular permeability are accompanied by loss of transendothelial electrical resistance.

We investigated the effect of PAI-1 inhibition on HUVEC monolayers in real time with ECIS. We observed that PAI-1 inhibition with PAI-039 leads to a concentration-dependent decrease in transendothelial resistance. We determined concentrations of 25 μM PAI-039 as effective in reducing transendothelial resistance of HUVEC monolayers while lower concentrations had no significant effect on transendothelial resistance.

Interestingly, within less than an hour the loss in transendothelial resistance was maximal. However, when we washed out the inhibitor we observed a fast recovery of the transendothelial resistance. We also tested another PAI-1 inhibitor, TM5275. TM5275 binds to different epitope of PAI-1 than PAI-039. Similar to inhibition with PAI-039 we observe a significant decrease in transendothelial resistance and a recovery after washing out of the inhibitor.

In line with the inhibitor data, silencing of PAI-1 using shRNA induced gaps in between the endothelial cells and reduced the basal resistance of the endothelial monolayer. Note that the initial spreading of the endothelial cells was not altered in the absence of PAI-1. These experiments indicate that PAI-1 inhibition directly affects the integrity of endothelial monolayers and causes a marked decrease in endothelial resistance. The PAI-1 induced loss of barrier function, as well as its recovery are fast, indicating that PAI-1 inhibition acts very close or directly on the endothelial cell-cell junctions. PAI-1 inhibition of HUVEC monolayers leads to loss of transendothelial resistance.(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.

Inhibition of PAI-1 function leads to disrupted endothelial junctionsThe main regulator of endothelial integrity are adherens junctions and especially VE-cadherin which mediates homophilic binding of endothelial cells and contributes to the electrolyte barrier. Blocking VE-cadherin interactions with VE-cadherin antibodies leads to a dramatic decrease in endothelial resistance. To investigate if the observed decrease of endothelial transendothelial resistance was accompanied by visible disruptions to endothelial monolayers, we cultured endothelial cells to confluence and immunostained for VE-cadherin as well as α-, β- and p120 catenin and the actin cytoskeleton. We observed a marked disruption of endothelial adherens junctions and gap formation between cells after PAI-1 inhibition. In controls the adherens junctions of confluent HUVEC form wide zones of overlap between endothelial cells where VE-cadherin connects adjacent cells. These junctional networks of cell-cell adhesion have been described previously as reticular junctions. After PAI-1 inhibition the reticular junctions are replaced by thinner junctions.

After PAI-1 inhibition with PAI-039 the average junction width decreased from 2.7 μm to 1.25 μm. Within two hours of removing the inhibitor the junction width recovered.

We further observed accumulation of VE-cadherin around the nucleus ( arrowhead). PAI-1 inhibition did not affect the colocalisation of VE-cadherin with α-catenin, β-catenin and p120 catenin ( and ). Finally, we detected stress fibers in cells treated with PAI-039 (, arrowhead + asterisk, ). Stress fibers can cause gap formation by mediating contractility. They are formed downstream of RhoA and Rho kinase activity. We inhibited Rho kinase activation with the pharmacological inhibitor Y-27632 while at the same time inhibiting PAI-1 activity with PAI-039 and measured transendothelial resistance with ECIS. However Rho kinase inhibition with Y-27632 did not prevent the loss of transendothelial resistance.

In summary these results show that PAI-1 inhibition directly affects endothelial cell morphology and function by disrupting VE-cadherin-based junctions. PAI-1 inhibition leads to disruption of VE-cadherin-mediated cell-cell junctions.(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. PAI-1 inhibition affects VE-cadherin traffickingWith immunofluorescence we observed that the colocalisation of VE-cadherin with the β-catenin, α-catenin and p120 catenin after inhibiting PAI-1 is unaltered.

To corroborate this finding we performed co-immunoprecipitations for VE-cadherin. We observed that VE-cadherin levels are reduced in total cell lysates and co-immunoprecipitations after PAI-039 treatment. Along with this, co-immunoprecipitation of β-catenin, α-catenin and p120 catenin are also less efficient (, ). Further to the reduction of VE-cadherin in total cell lysates we found a reduction of PAI-1 in total cell lysates but not in conditioned media.

Immuno-coprecipitation of VE-cadherin from HUVEC cells after PAI-1 inhibition shows no effect on interaction of VE-cadherin with β-catenin.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 (. PAI-1 inhibition reduces cellular PAI-1 and VE-cadherin levels while surface levels remain unaltered.(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).Since PAI-1 is a serine protease inhibitor we explored the possibility that inhibition of PAI-1 could lead to uncontrolled proteolysis of endothelial surface proteins including VE-cadherin by endothelial serine proteases.

VE-cadherin can be a target for proteolytic cleavage by serine proteases and the extracellular domain of VE-cadherin (“soluble” VE-cadherin, sVE-cadherin) can be found in patients with inflammatory diseases like rheumatoid arthritis. Therefore, we next investigated if PAI-1 inhibition leads to the release of sVE-cadherin. For this purpose we collected conditioned media from endothelial cells treated with PAI-039 and performed an ELISA specific for sVE-cadherin. To our surprise we measured less sVE-cadherin, indicating reduced shedding from cells treated with the PAI-1 inhibitor.

In addition we measured surface VE-cadherin expression on endothelial cells by FACS. Interestingly, PAI-1 inhibition with PAI-039 or TM5275 had no effect on the surface levels of VE-cadherin (, ). Also the levels of PECAM-1, another endothelial cell-cell junction protein, were not altered.It was shown that reduced mobility of VE-cadherin increased the resistance of endothelial monolayers ,. Therefore, we next tested if PAI-1 inhibition could affect the lateral mobility of VE-cadherin at the membrane and thus disturb VE-cadherin function. We measured VE-cadherin-GFP mobility at cell-cell junctions by FRAP. For this purpose we transfected VE-cadherin-GFP into endothelial cells and inhibited PAI-1 activity with PAI-039 for two hours before FRAP measurements were made.

Within four minutes VE-cadherin fluorescence recovered for both solvent- and PAI-039-treated HUVEC. There were no significant differences in the fluorescence recovery rates. Fluorescence recovery after photobleaching (FRAP) of VE-cadherin at the plasma membrane is unaltered after PAI-1 inhibition.(A) HUVEC were transduced with lentivirus to express VE-cadherin-GFP.

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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).As mentioned before we noticed an accumulation of VE-cadherin in an intracellular compartment when PAI-039 was used to inhibit PAI-1 function (, arrowhead). Immunofluorescence co-stainings with a Golgi marker showed that VE-cadherin and β-catenin accumulation was localized at the Golgi apparatus. To quantify these effects we measured the size of the Golgi apparatus and the fluorescence intensity for VE-cadherin in that compartment.

Interestingly, the size and fluorescence intensity at the Golgi are inversely related, with increased VE-cadherin fluorescence intensity after PAI-039 inhibition and a decrease in the size of the Golgi. Altogether the data presented here show that PAI-1 inhibition with PAI-039 leads to a reduction of the endothelial monolayer integrity by reducing total VE-cadherin levels while its surface levels and mobility remain unaltered. PAI-1 inhibition leads to the accumulation of VE-cadherin at the Golgi apparatus.(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.

DiscussionWe here present data that show a role for PAI-1 in the maintenance of vascular integrity. Our results are in agreement with the phenotype of patients that lack PAI-1 activity. While PAI-1 effects on uPA and tPA are undoubtedly involved in causing the bleeding diathesis in patients with a quantitative or qualitative PAI-1 deficiency, our data suggest a more direct role of PAI-1 for maintaining the integrity of blood vessels.The integrity of blood vessels greatly depends on the stability of cell-cell contacts between endothelial cells ,.

Endothelial adherens junctions and especially VE-cadherin are crucial for endothelial integrity and permeability. Whereas a null mutation in VE-cadherin causes severe defects in junctional morphology and vascular morphogenesis, a partial knock-down of VE-cadherin has been shown to increased vascular permeability. In addition, also merely blocking VE-cadherin with antibodies is sufficient to disrupt vascular integrity, increase permeability and cause hemorrhaging in mice. Our experiments show that the inhibition of PAI-1 function with small molecule inhibitors of PAI-1 leads to disrupted endothelial junctions, which is accompanied by a marked decrease of endothelial resistance. To our knowledge this is the first time that PAI-1 function was shown to have a direct effect on endothelial junction integrity.Quiescent endothelia are characterized by overlapping networks of reticular VE-cadherin-based adherens junctions. The permeability of these types of endothelial junctions is low. We noticed that in the absence of PAI-1 activity, VE-cadherin distribution at cell-cell junctions was altered.

The reticular network of VE-cadherin-based junctions was replaced by non-reticular and more “jagged” junctions. These types junctions have been described as junctions undergoing remodeling and correlate with an increase in endothelial permeability –. VE-cadherin on endothelial cells is associated with intracellular molecules like p120 catenin, β-catenin, α-catenin and actin. Particularly p120 catenin has been described as a factor in keeping VE-cadherin at the membrane by masking an endocytic signal ,. However, the interaction of VE-cadherin with its intracellular binding partners was unchanged after PAI-1 inhibition.Only few studies imply a possible connection between PAI-1 and VE-cadherin. VE-cadherin is involved in the regulation of PAI-1 transcription in endothelial cells and VE-cadherin deficient endothelial cells express less PAI-1 ,.

Interestingly, the inverse is also true: we show that the inhibition of PAI-1 function leads to decreased VE-cadherin levels in endothelial cells, albeit the differences we observed did not reach statistical significance. The loss of VE-cadherin expression and the disruption of the endothelial architecture occur concomitantly with the drop of endothelial resistance.

It is interesting to note that both the disruption (within less than an hour) and the recovery (within two hours) of endothelial junctions occur rapidly after addition or removal, respectively, of PAI-1 inhibitors pointing at a fast regulatory mechanism. One possible mechanism for fast regulation of proteins expressed on the cell surface is ectodomain shedding by proteolytic cleavage. The surface levels of VE-cadherin can be regulated in this way ,. To our surprise we did not detect increased ectodomain shedding.

On the contrary, we saw less shedding while the overall surface expression of VE-cadherin remained unaltered. Together with our other results this suggests that the trafficking of VE-cadherin may be impaired when PAI-1 activity is inhibited.The internalization of VE-cadherin in response to VEGF has been associated with increased permeability of endothelial monolayers. Upon closer examination we observed an accumulation of VE-cadherin at the Golgi apparatus which suggests that the transport of newly synthesized VE-cadherin from the Golgi to the plasma membrane may be inhibited. Interestingly, PAI-1 can be found in the Golgi of endothelial cells stimulated with LPS. In our experiments we could not visualize PAI-1 by immunofluorescence, possibly due to low intracellular concentrations of PAI-1, however it would be interesting to know if PAI-1 and VE-cadherin colocalise in the Golgi apparatus. Our data suggests that PAI-1 inhibition leads to less VE-cadherin production in cells and affects the trafficking of VE-cadherin to and conceivably also from the plasma membrane.

Concluding RemarksPAI-1 is an interesting target for therapeutically affecting the uPA and tPA pathways that lead to plasmin activation and fibrinolysis. The use of PAI-1 antagonists, like PAI-039 and TM5275, has been praised as a promising risk-free treatment for the prevention and treatment of thrombotic disease. We here provide data that PAI-1 inhibition may not be without side-effects. In addition to the previously reported effects of PAI-1 deficiency on fibrinolysis, our data show that PAI-1 inhibition can disrupt vascular integrity. We and others have cautioned that the goal of therapeutic PAI-1 inhibition ought to be restricted to bringing PAI-1 activity levels down to physiological values. This requires both careful monitoring of PAI-1 activity in plasma and a vigilant eye on vascular integrity.

S1 Fig PAI-1 inhibition of HUVEC monolayers with TM5275 leads to loss of transendothelial resistance.(A) and (B) Transendothelial electrical resistance (TER) was measured by electric cell-substrate impedance sensing (ECIS). HUVEC were grown to confluence in ECIS arrays and treated with either TM5275 (50 μM, 100 μM, 200 μM) or solvent (DMSO 0.2%). 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 normalized resistance after four hours of PAI-1 inhibition with TM5275 from the same experiment, basal resistance is the normalized resistance just before addition of inhibitor.

(C) and (D) TER was measured as described in (A) and (B). After 4 hours of treatment with TM5275 (100 μM) or solvent (DMSO 0.2%) the old medium was replaced with fresh medium with or without inhibitor (or solvent). The transendothelial resistance 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 TM5275 (n = 3) and recovery for two hours (n = 2) (mean + SEM,. p. S2 Fig PAI-1 depletion results in decreased electrical resistance and monolayer gaps.(A) HUVECs were grown to confluency and treated with shRNA as indicated. HUVECs depleted for PAI-1 showed gap formation. Merge shows VE-cadherin in red and F-actin in green. (B) Transendothelial electrical resistance (TER) was measured by electric cell-substrate impedance sensing (ECIS).

HUVEC pretreated with shPAI-1 or shCTRL were plated in ECIS arrays and monitored for resistance. No change in the initial spreading of the cells was detected; however, when forming a stable monolayer, a reduced resistance was measured for HUVECs that were depleted for PAI-1. (C) Quantification of resistance of HUVECs after 20 hours of plating. Data are mean ± s.e.m. Experiment is carried out three times independently form each other and per experiment in duplicate. (D) Western blot analysis shows efficient depletion of PAI-1. Actin is shown as loading control.(TIF).

S3 Fig PAI-1 inhibition leads to disruption of VE-cadherin-mediated cell junctions.(A) and (B) HUVEC were treated with PAI-039 (25 μM) or solvent (DMSO 0.1%) for four hours and stained for VE-cadherin (green), actin and (A) p120 catenin or (B) α-catenin (red) (scale bar 50 μm). (C) Illustration of junction width measurements. Five junctional measurements per cells were taken as shown in this picture. (D) TER was measured by ECIS. HUVEC were grown to confluence in ECIS arrays and treated with either PAI-039 (25 μM) or Control (DMSO 0.1%) and Y-27632 (10 μM) as indicated. Resistance values were normalized to the basal resistance one hour before addition of inhibitors.

Treatment with Y-27632 did not prevent decrease in TER after PAI-039 treatment. Graph is representative of one experiment (mean value of quadruplicates + SD).(TIF).

S5 Fig PAI-1 inhibition with PAI-039 or TM5275 does not alter VE-cadherin and PECAM-1 expression on HUVEC.(A), (B), (C) FACS analysis of HUVEC treated with TM5275 (100 μM, control: DMSO 0.2%) or PAI-039 (25 μM, control: DMSO 0.1%) as indicated. PAI-1 inhibition did not affect VE-cadherin expression on HUVEC (n = 3, geometric mean fluorescence intensity + SEM, n.s. = non-significant). (D) Densitometric quantification of PAI-1 in conditioned media of HUVEC treated with PAI-039 as described in (n = 3, mean + SEM, n.s. = non-significant).(TIF). S6 Fig Immuno-coprecipitation of VE-cadherin from HUVEC cells after PAI-1 inhibition shows no effect on interaction of VE-cadherin with α-catenin or p120 catenin.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 (A) α-catenin or (B) p120 catenin (ECL) and then for VE-cadherin (Odyssey).

The bottom half (.

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