Urokinase-type Plasminogen Activator (uPA) Induces Pulmonary Microvascular Endothelial Permeability through Low Density Lipoprotein Receptor-related Protein (LRP)-dependent Activation of Endothelial Nitric-oxide Synthase*

Urokinase plasminogen activator (uPA) and PA inhibitor type 1 (PAI-1) are elevated in acute lung injury, which is characterized by a loss of endothelial barrier function and the development of pulmonary edema. Two-chain uPA and uPA-PAI-1 complexes (1–20 nm) increased the permeability of monolayers of human pulmonary microvascular endothelial cells (PMVECs) in vitro and lung permeability in vivo. The effects of uPA-PAI-1 were abrogated by the nitric-oxide synthase (NOS) inhibitor l-NAME (ND-nitro-l-arginine methyl ester). Two-chain uPA (1–20 nm) and uPA-PAI-1 induced phosphorylation of endothelial NOS-Ser1177 in PMVECs, which was followed by generation of NO and the nitrosylation and dissociation of β-catenin from VE-cadherin. uPA-induced phosphorylation of eNOS was decreased by anti-low density lipoprotein receptor-related protein-1 (LRP) antibody and an LRP antagonist, receptor-associated protein (RAP), and when binding to the uPA receptor was blocked by the isolated growth factor-like domain of uPA. uPA-induced phosphorylation of eNOS was also inhibited by the protein kinase A (PKA) inhibitor, myristoylated PKI, but was not dependent on PI3K-Akt signaling. LRP blockade and inhibition of PKA prevented uPA- and uPA-PAI-1-induced permeability of PMVEC monolayers in vitro and uPA-induced lung permeability in vivo. These studies identify a novel pathway involved in regulating PMVEC permeability and suggest the utility of uPA-based approaches that attenuate untoward permeability following acute lung injury while preserving its salutary effects on fibrinolysis and airway remodeling.

Lung inflammation is accompanied by increased pulmonary microvascular (PMV) 3 permeability leading to the exudation of plasma proteins into alveolar spaces, interstitial edema, impaired gas exchange, and increased morbidity and mortality (1). Considerable efforts have been made to understand how endothelial barrier function is lost early in the inflammatory process (2), but this knowledge has yet to translate into specific means to prevent excess permeability from developing and thereby to improve the clinical outcome. Transudation of plasma proteins into airways promotes the formation of a provisional fibrin matrix that can lead to pulmonary fibrosis. Plasminogen activators (PA), primarily urokinase PA (uPA) and tissue-type PA (tPA), convert plasminogen to plasmin, which in turn lyses fibrin matrices and thereby exerts anti-fibrotic effects in the lungs (3). PAs exert a protective role in several experimental models of acute lung injury (ALI) and pulmonary fibrosis (3)(4)(5)(6)(7)(8), and in one study, patients with ALI showed significant improvement in oxygenation after treatment with uPA (9).
However, the salutary effects of uPA on lung repair, largely derived from its catalytic activity, are partially offset by deleterious extrafibrinolytic effects on vascular and airway tone (10 -14). Moreover, diverse pulmonary insults involved in ALI decrease PA activity (15)(16)(17)(18)(19) and markedly increase the concentration of its inhibitor PAI-1 (20). Furthermore, uPA at concentrations measured in lung tissue during inflammation (ϳ20 nM) impairs pulmonary artery contractility, increases vascular permeability, and enhances airway hyperresponsiveness (10,11,14). Our data indicate that these effects are mediated through distinct portions of the uPA molecule and engage different receptor pathways than those involved in fibrinolysis (10,14).
A number of mediators have been identified that can alter PMV permeability within minutes. Among these is a vascular endothelial growth factor (VEGF), which acts through activation of endothelial NOS (eNOS) (NOS-3) (38,39). VEGF-induced permeability of retinal microvascular endothelial cells has been shown to require endogenous uPA (40). eNOS converts L-arginine (L-Arg) to L-citrulline (L-Ctr) to generate NO, which causes vasorelaxation and increases microvascular permeability (38,39,(41)(42)(43). However, the role of NO in the pathogenesis of ALI remains controversial. Low levels of endogenous NO help to prevent pulmonary edema from developing in isolated rabbit lungs following ischemia-reperfusion or vagal stimulation (44 -46), whereas excessive NO appears detrimental in that it increases vascular permeability (43,47).
The cellular and mechanistic basis underlying the loss of pulmonary endothelial barrier function has been studied in detail (reviewed in Refs. 2, 48, and 49), but the role of PAs, although clearly implicated in lung injury and repair (27), has not been studied in detail. Here we report that uPA and uPA-PAI-1 complexes increase PMV endothelial permeability by binding to LRP, which activates cAMP-dependent PKA and eNOS, generates NO, and disrupts intercellular contacts in the lung microvasculature.
Permeability in Vivo-Lung permeability was determined by measuring extravasation of intravenously injected Evans blue dye as described (53) with minor modifications (50). Bronchoalveolar lavage (BAL) was performed using 1.5 ml of warmed (30°C) sterile Hanks' balanced salt solution. BAL fluid was collected and centrifuged at 14,000 ϫ g for 20 min at 4°C, the supernatant was removed, and the optical density at 620 nm was measured.
Nitric-oxide Synthase Activity-eNOS activity was assayed using a NOS assay kit. Briefly, cells were disrupted by 30 strokes in a Dounce homogenizer in cold homogenization buffer. eNOS activity in supernatants was assessed by the conversion of L-[ 14 C]Arg to L-[ 14 C]citrulline. Incubations performed in parallel in the presence of the eNOS inhibitor L-NNA (1 mM) served as the negative control. eNOS activity was expressed as pmol of L-Ctr/mg of protein/min. L-Ctr formation was calculated using the formula pmol L-Ctr ϭ (cpm a -cpm p )/cpm s ϫ 290, where cpm a and cpm p are cpm in the absence and presence, respectively, of L-NNA and cpm s represents cpm in the standard. All standards contained 0.1 Ci of L-[ 14 C]Arg corresponding to 290 pmol.
cAMP-dependent Protein Kinase A Activity-PKA activity was assayed using a PKA assay kit. Briefly, cells were disrupted in cold extraction buffer (25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM ␤-mercaptoethanol, and protease and phosphatase inhibitor mixtures) by 30 strokes in a Dounce homogenizer. Lysates were clarified by centrifugation, and kinase activity was measured according to the manufacturer's protocol. Radiolabeled peptides were separated from the residual [␥-32 P]ATP using P81 phosphocellulose paper, and radioactivity was quantified.
Fluorescent Detection of S-Nitrosylation and Immunofluorescence-To visualize S-nitrosylated proteins in PMVEC incubated with uPA-PAI-1 (as described above) by immunofluorescence, a biotin-switch assay was performed as described (56). In conjunction, cells were immunostained to detect ␤-catenin and fibrillar actin as described above.
Statistical Analysis-Differences between groups were compared using the one-way analysis of variance statistical test. Statistical analyses were performed using the EZAnalyse add-in to Microsoft Excel software. Significance was set at a p value of less than 0.05.

RESULTS
uPA Induces Pulmonary Vascular Permeability in Vivo-In view of findings that uPA Ϫ/Ϫ mice are protected against LPSinduced pulmonary edema (57) and that uPA increases endothelial permeability in vitro (40), we asked whether uPA regulates pulmonary vascular permeability in vivo. Intravenous injection of scuPA (0.1-2 mg/kg) in mice increased lung permeability in a dose-dependent manner as measured by the extravasation of intravenously administered Evans blue dye into the BAL fluid (Fig. 1A). To elucidate the mechanism, we investigated how uPA affects endothelial permeability in vitro.
uPA and uPA-PAI-1 Complexes Induce Permeability of PMVEC Monolayers-We examined the effect of uPA on the permeability of PMVEC monolayers using FITC-dextran. The addition of enzymatically active tcuPA (20 nM) induced a more than 2-fold increase in transendothelial permeability, and the monolayer maintained increased permeability for more than 2 h (Fig. 1B). scuPA increased permeability to ϳ30% of that seen in the complete absence of cells and was comparable in magnitude with the effect of VEGF (5 nM) (supplemental Fig. S1A). Both scuPA and tcuPA increased endothelial permeability in vitro in a dose-dependent manner beginning at concentrations as low as 1 nM (supplemental Fig. S1B).
PAI-1 is dramatically elevated in the lungs during ALI to levels well above uPA (5,16,19). PAI-1 and uPA are also synthesized and secreted by PMVECs (supplemental Fig. S2). We hypothesized that exogenous tcuPA (or scuPA converted on the cell surface to tcuPA) binds to cell-associated PAI-1, forming stable enzymatically inactive complexes that promote endothelial permeability. To test this hypothesis, we compared the effects of tcuPA (20 nM), PAI-1 (40 nM), and preformed uPA-PAI-1 complexes on the permeability of PMVEC monolayers. PAI-1 was added in molar excess to ensure that all available uPA was complexed, as some recombinant PAI-1 might have undergone conversion to the latent form before exposure to tcuPA. tcuPA (20 nM) and PAI-1 (40 nM) added alone significantly (p Ͻ 0.05) increased the permeability of PMVEC to a similar extent (Fig. 1B), suggesting engagement of each with their respective endogenous counterparts. In support of this conclusion, preformed uPA-PAI-1 complex significantly (p Ͻ 0.01) increased the permeability of PMVEC monolayers to a greater extent than the same concentration of tcuPA or PAI-1 added individually (Fig. 1B). Therefore, the finding that PAI-1 increased the potency of uPA to induce permeability might be of significance in the setting of lung disease.
uPA-PAI-1 Induces Redistribution of ␤-Catenin-Endothelial permeability and intercellular contacts are regulated by ␤-catenin (58). Dissociation of ␤-catenin from VE-cadherin and redistribution from adherens junctions to the cytoplasm in response to stimuli such as VEGF increases endothelial permeability (40,56,58). Likewise, the addition of uPA-PAI-1 caused ␤-catenin to dissociate from VE-cadherin as evidenced by loss of co-immunoprecipitation (Fig. 1C). This was accompanied by an expansion of the intercellular contact zones that stained for ␤-catenin within 10 min of exposure to uPA-PAI-1, an effect that was sustained for at least 60 min (Fig. 1D).
uPA-PAI-1-induced PMVEC Permeability Is Mediated through eNOS-eNOS-dependent nitrosylation of ␤-catenin leads to its redistribution and increased endothelial permeability (56). eNOS is partially localized to intercellular contacts/ tight junctions (59). Therefore, we asked whether inhibition of eNOS activity would prevent uPA-PAI-1-induced redistribution of ␤-catenin. The eNOS inhibitor L-NAME (0.1 mM) attenuated uPA-PAI-1-induced dissociation of ␤-catenin from VEcadherin as evidenced by co-immunoprecipitation (Fig. 1C) and by more restricted ␤-catenin staining at intercellular contacts (Fig. 1D, L-NAME panels). L-NAME also reduced PMVEC permeability evoked by uPA-PAI-1 in parallel (Fig. 1E). Phosphorylation of eNOS-Ser 1177 in response to 2 nM tcuPA was seen at 15 min, peaked at 30 min, and decreased by 60 min (Fig. 3A), whereas the more profound effect of uPA-PAI-1 was evident at 15 min and persisted for at least 60 min, the latest data point studied (supplemental Fig. S3B). In contrast, the phosphorylation induced by 2 nM scuPA was first seen at 30 min, and the signal declined by 60 min (supplemental Fig. S3B). These data suggest that preformed uPA-PAI-1 complexes more rapidly initiate and sustain the signaling cascade, whereas tcuPA must first bind to endogenous PAI-1, and scuPA must be converted to tcuPA on the EC surface (51) prior to binding to PAI-1, which delays eNOS phosphorylation.
To ensure that eNOS-Ser 1177 phosphorylation in response to tcuPA activates the enzyme, we examined whether NO is generated (61). The phosphorylation of eNOS (Fig. 3A) and the time dependence of conversion of L-Arg to L-Ctr (Fig. 3B), indicating the generation of NO, followed a similar time course (42). The generation of NO was abolished by pretreating the cells with the NOS inhibitor L-NNA (1 mM; data not shown), affirming the specificity of the reaction.
uPA-PAI-1 Induces Nitrosylation of ␤-Catenin and Redistribution of Fibrillar Actin in PMVEC-NO generated by eNOS in response to VEGF promotes S-nitrosylation of ␤-catenin (56). Therefore, we investigated whether activated p-eNOS-Ser 1177 co-localizes with ␤-catenin after exposure of PMVEC to uPA-PAI-1. uPA-PAI-1 increased the appearance of p-eNOS-Ser 1177 -positive staining in PMVEC monolayers, which partially co-localized with ␤-catenin at intercellular contacts (Fig.  4A). To elucidate whether uPA-PAI-1-induced NO generation causes S-nitrosylation of ␤-catenin and other proteins in PMVEC, we employed a biotin-switch technique (56) to visualize intracellular S-nitrosothiols. The addition of uPA-PAI-1 increased total cellular levels of S-nitrosothiol and its appearance at intercellular contacts where it co-localized with ␤-catenin (Fig. 4B, leftmost panel). Using immunoprecipitation of ␤-catenin and WB detection of S-nitrosothiol, we found that uPA-PAI-1-induced S-nitrosylation of ␤-catenin in PMVEC was inhibited by L-NAME, indicating the involvement of NO (Fig. 4C). This was confirmed by using a biotin-switch technique and fluorescent detection of S-nitrosothiols (supplemental Fig. S4). uPA-PAI-1 also caused a marked coincident redistribution of actin filaments toward intercellular contacts (Fig.  4B, F-actin panels) that co-localized with ␤-catenin (Fig. 4B,  rightmost panels), a conformation reported to contribute to EC retraction and increased permeability (62).
Role of PKA and PI3K-Akt Pathways in Activation of eNOS-Phosphorylation of eNOS-Ser 1177 can be mediated by PKA, phosphatidylinositol-3 kinase (PI3K)-dependent protein kinase B (PKB/Akt), or AMP-dependent kinase (AMPK) (63,64). PKA regulates permeability through phosphorylation of cytoskeletal proteins (36,37), and PKB/Akt is implicated in vascular leakage during acute inflammation (65). Therefore, we asked whether PKA or PI3K-dependent PKB/Akt was responsible for uPAinduced eNOS activation by using selective inhibitors of each pathway. As exposure to uPA or uPA-PAI-1 has not been shown to cause metabolic stress in ECs, we did not study AMP-  dependent kinase. PMVEC were preincubated with the PI3K inhibitor LY294002, the PKB/Akt inhibitor AktI, or the PKA inhibitor mPKI for 1 h prior to adding tcuPA or uPA-PAI-1 for 30 min. uPA induced phosphorylation of eNOS-Ser 1177 at all concentrations tested (1-20 nM). Phosphorylation was com-pletely inhibited by the PKA inhibitor mPKI (Fig. 5A). In contrast, LY294002 and AktI had no effect, suggesting that the PI3K/Akt pathway is not essential for uPA-induced phosphorylation of eNOS. The same pattern of inhibition was seen when uPA-PAI-1 was studied (data not shown). Although uPA-in- FIGURE 4. A, uPA-PAI-1 treatment reveals co-localization of ␤-catenin and p-eNOS-Ser 1177 at intercellular contacts in PMVEC. Cells were incubated with uPA-PAI-1 (20:40 nM) for 10 min, fixed, and stained for ␤-catenin (red) and p-eNOS-Ser 1177 (green) as described under "Experimental Procedures." Nuclei were counterstained with DAPI. Arrowheads indicate p-eNOS-Ser 1177 -positive staining co-localized with ␤-catenin at intercellular contacts. B, uPA-PAI-1 induces S-nitrosylation at intercellular contacts in PMVEC. PMVEC were incubated with uPA-PAI-1 (20:40 nM) for the indicated times. Fixed cells were subjected to a biotin-switch assay, as described under supplemental "Methods." S-Nitrosylation was visualized using Alexa 488-conjugated avidin (green). ␤-Catenin immunostaining was performed in parallel (red). Fibrillar actin was detected using Alexa 647-conjugated phalloidin, which is pseudocolored in cyan. Nonspecific staining was determined in control cells incubated with Alexa 488-streptavidin. Arrowheads indicate the presence of S-nitrosylation at intercellular junctions (merge with ␤-catenin) on the left. C, uPA-PAI-1 induces S-nitrosylation of ␤-catenin. PMVEC were starved and incubated with uPA-PAI-1 (20:40 nM) for 60 min or preincubated for 30 min with L-NAME (200 M) prior to uPA-PAI-1 treatment. Cells were lysed, and ␤-catenin was immunoprecipitated using mouse anti-␤-catenin Abs as described under "Experimental Procedures." S-Nitrosylated ␤-catenin and total ␤-catenin were detected by WB using rabbit anti-Snitroso-cysteine mAbs and anti-␤-catenin mAbs, respectively, and the corresponding HRP-conjugated secondary Abs. duced phosphorylation of PKB/Akt at Ser 473 was inhibited in the presence of LY294002, this seemed to be unrelated to uPAinduced eNOS-Ser 1177 phosphorylation because neither LY294002 nor AktI inhibited uPA-induced permeability (data not shown).
LRP Mediates uPA-PAI-1-induced Activation of eNOS-uPA and uPA-PAI-1 can bind to uPAR and LRP simultaneously (24,30). However, the affinity of uPA-PAI-1 for LRP is almost 100fold higher than the affinity of either its constituents (60). tcuPA has also been shown to alter cell signaling through the activation of NMDAR-1 in lung airways (14). To elucidate which receptors contribute to uPA and uPA-PAI-1-induced eNOS phosphorylation, we used the following receptor-specific antagonists: isolated GFD, which competes with uPA for uPAR binding (66); RAP (67) and anti-LRP mAb, which block LRP; and an antagonist of NMDAR-1, MK-801 (68). Phosphorylation of eNOS induced by tcuPA was blocked by the anti-LRP Ab and RAP but not by MK-801 as determined by WB analysis (Fig.   5B). GFD alone (2-50 nM) did not induce any response. However, 50 nM GFD blocked the effect of 2 nM tcuPA, but inhibition was overridden at 20 nM tcuPA (supplemental Fig. S5). The same effect was seen when uPA-PAI-1 was studied (data not shown). These data suggest that the binding of uPA or uPA-PAI-1 to LRP is sufficient to induce PKA-mediated eNOS activation. However, high affinity binding to uPAR and the presence of PAI-1 lowers the concentration required for LRPmediated engagement and activation.
LRP and PKA Mediate uPA-PAI-1-induced Permeability of PMVEC-The binding of uPA to LRP initiates activation of PKA in some cell types (31)(32)(33)69), and phosphorylation of eNOS is inhibited by mPKI (Fig. 5A). Therefore, we next asked whether tcuPA and/or uPA-PAI-1 induce activation of PKA. The addition of tcuPA or uPA-PAI-1 led to the activation of PKA in a time-dependent manner as detected by 32 P phosphorylation of a PKA-specific peptide (Fig. 6A). The activation of PKA by tcuPA (20 nM) was inhibited by RAP (Fig. 6B). RAP and mPKI also significantly suppressed uPA-PAI-1-induced PMVEC permeability, suggesting that binding of the complexes to LRP precedes the activation of PKA (Fig. 7A).
Mechanism of uPA-induced Pulmonary Vascular Permeability in Vivo-To assess the relevance of our in vitro findings that uPA-and uPA-PAI-1-induced lung microvascular permeability involves LRP-, PKA-, and eNOS-dependent mechanisms, we examined the effect of specific inhibitors of each pathway on uPA-induced lung permeability in vivo. Mice were given an intravenous injection of mPKI, Fc-RAP, or L-NAME. One hour later, scuPA (1 mg/kg) and Evans blue dye were injected intravenously. Permeability was accessed 1 h later by measuring the transudation of the dye from its circulation into the BAL fluid. Each of the three inhibitors significantly (p Ͻ 0.05) protected the endothelial barrier function in uPA-treated mice (Fig. 7B) consistent with our in vitro findings.

DISCUSSION
The loss of PMV endothelial barrier function as a result of lung injury leads to acute transudation of fluid and plasma proteins into the alveolar space, impairing oxygenation. uPA and PAI-1 are elevated in several lung disorders associated with increased vascular permeability, including ALI, acute respiratory distress syndrome, infectious pneumonia, pulmonary fibrosis, and asthma (3-8, 15-19, 70). uPA plays a salutary role in some forms of ALI through its ability to lyse fibrin deposits in a timely manner and thereby helps prevent fibrosis (8,19). However, uPA also inhibits pulmonary arterial contraction, which impairs the physiological shunting of blood from underventilated to well ventilated airways (14) and may increase pulmonary vascular permeability (10,11,14,40,71). These observations led us to investigate the effect of uPA on the PMV endothelium.
VEGF induces vascular permeability in cultured bovine retinal EC through a process that requires uPA and uPAR, and exogenously added uPA increases the permeability of retinal microvascular endothelial monolayers in vitro (40,56). However, little is known about the mechanisms underlying the effect of uPA on PMV permeability or the involvement of PAI-1.

uPA Induces Endothelial Permeability via eNOS Activation
We focused on PMVEC because the loss of their contact integrity compromises lung function. Our data indicate that uPA-PAI-1 increases the permeability of PMVEC in culture and lung permeability in vivo. In pathological settings where uPA and PAI-1 are elevated (15,16,17,19,72) disruption of the endothelial barrier by uPA-PAI-1 complexes may permit uPA and other proteins ready access to the underlying vascular smooth muscle cells, which in turn may exacerbate hypoxemia by impairing pulmonary vascular contractility. 4 The induction of pulmonary vascular permeability requires the binding of uPA or uPA-PAI-1 to LRP. There is a precedent for the involvement of LRP in regulating tissue permeability (73), e.g. permeabilization of the blood-brain barrier induced by tissue-type PA (74), although the mechanism remains uncertain. Some investigations indicate that LRP signal transduction is dependent on the enzymatic activity of the PA, which cleaves the extracellular domain of the receptor (75), whereas other studies implicate uPA-PAI-1-mediated association of domain 3 of uPAR with LRP (76). Whether direct binding of agonists to LRP induces intracellular signaling or whether binding to receptors other than uPAR may be involved in LRP signaling is unknown but both are suggested by our findings.
Several lines of evidence indicate that uPA induces LRP-mediated PMVEC permeability through a non-catalytic mechanism. First, exogenous tcuPA and PAI-1 had comparable effects on permeability, which we posit results from binding to its cellular counterpart, leading to the formation of enzymatically inactive complexes. Second, preformed uPA-PAI-1 complexes, which have much greater affinity for LRP than single-or twochain uPA (60), mediate LRP-dependent intracellular signaling and induce permeability at lower concentrations and earlier times, and the effect is more sustained than seen with either tcuPA or PAI-1 alone. Our data are also in line with previous findings that PAI-1 sustains a uPA signaling response by engaging an LDL receptor homologue, the very low density lipoprotein receptor VLDLR (77). Third, displacement of tcuPA from uPAR by its GFD fragment raised the concentration required to mediate permeability, likely by impairing the formation of uPA-PAI-1 complexes on the cell surface. Lastly, a catalytically inactive uPA variant induced permeability but only at 20-fold higher concentrations than required for uPA-PAI-1 complexes to do so.

uPA Induces Endothelial Permeability via eNOS Activation
The binding of uPA and uPA-PAI-1 to LRP activates cAMPdependent PKA, which is required to induce PMVEC permeability. Increased cAMP levels have been reported to stabilize or destabilize endothelial barrier function depending on the origin of the ECs (78). The situation in pulmonary endothelium is complex and might involve competing mechanisms that depend upon the subcellular localization of cAMP due to selective anchoring of PKA and that differ in their effect on permeability (36, 37, 79 -81). cAMP, which is generated by plasma membrane-associated adenylate cyclase, activates membraneassociated PKA. This exerts a protective effect on barrier function by phosphorylating/activating filamin and Rac1, thereby stabilizing the cortical actin rim (82). Membrane-associated cAMP-specific phosphodiesterase 4D4 (PDE4D4) hydrolyzes cAMP, which limits its diffusion into the cytosol (83), potentially providing a negative feedback mechanism. In addition, membrane-associated cAMP protects endothelial barrier function by activating the exchange protein directly activated by cAMP (EPAC), which forms a ternary complex with PKA and PDE4D4 and subsequently stabilizes VE-cadherin-based adherens junctions (84). However, if PDE4D4 is inhibited, cAMP diffuses into the cytosol and activates cytosolyic PKA, which initiates a reorganization of the microtubules through phosphorylation of tau-Ser 214 , generating intercellular gaps that increase endothelial permeability (85). Additional studies will be needed to determine whether these or alternative mechanisms are responsible for the uPA-PAI-1-induced PKA-dependent increase in PMV permeability that we observed.
Activation of PKA in response to uPA and uPA-PAI-1 leads to phosphorylation of eNOS-Ser 1177 , activation of the enzyme, and generation of NO, which is known to increase vascular permeability (38,41). The NOS inhibitor L-NAME reduced PMVEC permeability induced by uPA and uPA-PA-1. This is consistent with the known effects of L-NAME in preventing hyperpermeability during the late phase of allergic responses, smoke-induced lung injury, and endotoxin-induced pulmonary edema; inhalation of L-NAME may be beneficial in the treatment of ALI (46, 47, 86 -89).
Recent studies indicate that NO nitrosylates ␤-catenin, which leads to its dissociation from VE-cadherin and redistribution from tight junctions (56). Destabilization of these junctions contributes to the increased permeability of ECs in response to VEGF (40,56). Our studies show that uPA and uPA-PAI-1 have a similar effect, i.e. rapidly inducing NO, which leads to nitrosylation of ␤-catenin concomitant with its dissociation from VE-cadherin. eNOS also has been reported to associate with ␣-actin (90,91), which can undergo S-nitrosylation by NO (92) leading to its dissociation from the integrins, which might result in the redistribution of the actin filaments and the impairment of the endothelial barrier function in response to uPA-PAI-1 that we observed. Whether uPA or uPA-PAI-1 promotes nitrosylation of ␣-actin and how these activities might modulate adherens junctions and alter PMVEC permeability will require additional study.
In summary, the studies presented here show that uPA-PAI-1 at the levels found in ALI induce PMVEC permeability in vitro and increase lung permeability in vivo through uPAR/ LRP-mediated activation of PKA, which is followed by activa-tion of eNOS and NO-dependent disruption of adherens junctions. These findings thereby identify several potential sites at which loss of endothelial barrier function in ALI can be interrupted. Understanding the mechanisms that regulate PMV permeability may assist in controlling host defense and repair in diverse common respiratory disorders.