Lipoteichoic Acid from Staphylococcus aureus Induces Lung Endothelial Cell Barrier Dysfunction: Role of Reactive Oxygen and Nitrogen Species

Tunneled central venous catheters (TCVCs) are used for dialysis access in 82% of new hemodialysis patients and are rapidly colonized with Gram-positive organism (e.g. Staphylococcus aureus) biofilm, a source of recurrent infections and chronic inflammation. Lipoteichoic acid (LTA), a cell wall ribitol polymer from Gram-positive organisms, mediates inflammation through the Toll-like receptor 2 (TLR2). The effect of LTA on lung endothelial permeability is not known. We tested the hypothesis that LTA from Staphylococcus aureus induces alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM) that result from activation of TLR2 and are mediated by reactive oxygen/nitrogen species (RONS). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin, the activation of the TLR2 pathway was assessed by Western blot, and the generation of RONS was measured by the fluorescence of oxidized dihydroethidium and a dichlorofluorescein derivative. Treatment with LTA or the TLR2 agonist Pam(3)CSK(4) induced significant increases in albumin permeability, IκBα phosphorylation, IRAK1 degradation, RONS generation, and endothelial nitric oxide synthase (eNOS) activation (as measured by the p-eNOSser1177:p-eNOSthr495 ratio). The effects on permeability and RONS were effectively prevented by co-administration of the superoxide scavenger Tiron, the peroxynitrite scavenger Urate, or the eNOS inhibitor L-NAME and these effects as well as eNOS activation were reduced or prevented by pretreatment with an IRAK1/4 inhibitor. The results indicate that the activation of TLR2 and the generation of ROS/RNS mediates LTA-induced barrier dysfunction in PMEM.


Introduction
Sepsis is the second leading cause of death among patients with end-stage renal disease (ESRD) on hemodialysis (HD) [1]. Grampositive organisms (e.g., Staphylococcus spp.) account for more than 70% of bacteremia/sepsis episodes primarily due to vascular access-related sources (e.g., tunneled central venous catheters) [1]. HD patients have a greater 30-day mortality risk compared to other populations with non-hospital acquired methicillin-resistant Staphylococcus spp. bacteremia [2].
Lipopolysaccharide (LPS) derived from the cell wall of Gramnegative bacteria is a known inducer of sepsis, the systemic inflammatory response syndrome (SIRS) and acute lung injury [3]. However, the cell wall components of pathogenic Gram-positive bacteria (e.g., Staphylococcus aureus) also mediate, at least in part, the vascular injury associated with the SIRS and acute lung injury [4,5]. Pulmonary edema associated with increased vascular permeability is a complication of Gram-positive sepsis [6]. Principal components of the Gram-positive bacteria cell wall are lipoteichoic acid (LTA) [4,5,6] and peptidoglycan. LTA, unique to Gram-positive bacteria, consists of a hydrophilic region of polymers of ribitol phosphate or glycerophosphate substituted in the C2 position of glycerol by sugars in glycosidic linkage and Dalanine in an ester linkage [7]. Peptidoglycan is a transmembrane polymer consisting of glycan strands of N-acetylglucosamine and N-acetylmuramic acid that are, in Gram-positive bacteria, crosslinked by pentaglycine bridges. LTA derived from Staphylococcus aureus induces endothelial dysfunction, circulatory shock and organ injury in different models of Gram-positive sepsis [8,9]. Peptidoglycan may act to amplify LTA-induced induce systemic inflammatory response syndrome [9,10].
Classically, LTA binds to the toll-like receptor 2 (TLR2) which results in the activation of a MyD88-dependent pathway. In response to LTA, two pairs of intracellular adapter proteins-MyD88 and Toll-IL-1 resistance (TIR) domain-containing adapter protein (TIRAP) are recruited to the TLR receptor. Recruitment and activation of the IRAK proteins (1 and 4), follows and TRAF 6 subsequently activates the IKK complex (a,b and NEMO) which is the event immediately preceding NFkB activation and cytokine transcription [11,12]. Peptidoglycan is a co-stimulator of the innate immune system in the presence of LTA, however, studies of highly purified peptidoglycan in the absence of LTA fail to stimulate TLR2 [13,14]. Other pathways involved with LTA-TLR2 mediated alterations in endothelial function are not clear. It is known that reactive oxygen and nitrogen species mediate, in part, endothelial dysfunction during sepsis [15]. The literature indicates oxidant stress (e.g. reactive nitrogen and oxygen species) causes phosphorylation and redistribution of occludin and decreased levels of VE-cadherin associated with loss of peripheral cortical actin and increased actin stress fibers. The loss of actin peripheral bands is associated with disruption of a,b,c catenins and actin binding proteins (e.g.,filamin) with increased cell-cell gap formation [16].
Yet, the ability of LTA to cause lung endothelial barrier dysfunction that is dependent on reactive oxygen/nitrogen species is not known; therefore, the aim of this study is to investigate the mechanism of LTA -induced endothelial barrier dysfunction in a lung microvessel endothelial cell monolayer model.

Reagents
All reagents were obtained from Sigma Chemical Company (St. Louis, MO) unless otherwise noted.

Cells
Pulmonary microvessel endothelial cell culture. Rat lung microvessel endothelial cells (RLMVEC) were studied using our previously published methods [17]. In brief RLMVEC were obtained at 4th passage (Vec Technologies, Rensselaer, NY). The preparations were identified by Vec Technologies as pure populations by 1) the characteristic ''cobblestone'' appearance as assessed by phase contrast microscopy, 2) the presence of factor VIII-related antigen (indirect immunofluorescence), 3) the uptake of acylated low-density lipoproteins, and 4) the absence of smooth muscle actin (indirect immunofluorescence). For all studies, RLMVEC were cultured from 4 to 10 passages in culture medium, MCDB-131 complete media (VEC Technologies) supplemented with 20% fetal bovine serum (FBS) (Hyclone; Hyclone Laboratories, Logan, UT). The cells were maintained in 5% CO 2 plus humidified air at 37uC. A confluent pulmonary microvessel endothelial cell monolayer (PMEM) was reached within two to three population doublings, which took 3-4 days.
Treatment medium. For all studies, incubation of PMEM with LTA, PAM, Urate, Tiron, L-NAME, IRI and all corresponding controls were performed with phenol red-free DMEM (pf-DMEM) (Life Technologies, Grand Island, NY) supplemented with 10% FBS (20% FBS for 24 hour studies) to avoid a potential antioxidant effect of phenol.

Endothelial Permeability Assay
The assay of endothelial cell monolayer permeability is adapted from our previously described technique [17]. Transwells (6.5 mm diameter, 8 mm pore size; Corning Costar, Corning, NY) were coated with Rat Tail Collagen Type 1 (BD Biosciences, Bedford, MA). RLMVEC (0.1610 6 ) in MCDB-131 were plated in the Transwells according to manufacturer's instructions and allowed to reach confluence within 3-5 days (37uC, 5% CO 2 ). The experimental apparatus for the study of transendothelial transport in the absence of hydrostatic and oncotic pressure gradients have been described [17]. In brief, the system consists of two compartments separated by a microporous polycarbonate membrane lined with the endothelial cell monolayer as described above. The luminal (upper) compartment (0.1 ml) was suspended in the abluminal (lower) compartment (0.6 ml). The entire system was kept in a CO2 incubator at a constant temperature of 37uC. The fluid height in both compartments was the same to eliminate convective flux.
Endothelial permeability was characterized by the clearance rate of Evans Blue-labeled albumin [24]. A buffer solution containing Hanks' balanced salt solution (Life Technologies) containing 20 mM HEPES buffer and 0.5% bovine serum albumin (BSA) was used on both sides of the monolayer (HHB buffer). The luminal compartment buffer was labeled with a final concentration of 0.057% Evans blue dye in a volume of 100 ml. The absorbance of free Evans blue in the luminal and abluminal compartments was always ,1% of the total absorbance of Evans blue in the buffer. At the beginning of each study a luminal compartment sample was diluted 1:100 to determine the initial absorbance of that compartment. Abluminal compartment samples (100 ml) were taken every 10 min for 60 min. To maintain a constant hydrostatic pressure, the sample removed from the abluminal compartment was replaced with an equal volume (100 ml) of HHB buffer. The absorbance of the samples was measured in a BioTek Synergy2 plate reader (BioTek Instruments, Winooski, VT) at 620 nm. The clearance rate of Evans bluelabeled albumin was determined by least-squares linear regression between 10 and 60 min for the control and experimental groups. Mean 6 SEM albumin permeability for control experiments was 0.03560.004 mL/min.

Immunoblot Analysis
Preparation of PMEM lysates. RLMVEC were seeded into 6 or 12-well plastic culture plates and incubated for 3-4 days until confluent. After interventions, the PMEM were washed on ice two times with ice-cold PBS, then ice-cold extraction buffer (Tris HCl: 10 mM-pH 7.5; SDS: 0.1%; Triton X-100:0.5%; Sodium Deoxycholate: 0.5%; DTT: 0.1 mM) supplemented with 1x mammalian protease inhibitor cocktail and 1x phosphatase inhibitor cocktails 1 and 2 was added to the cells. Lysates were cleared by centrifugation at 18,5006g for 45 minutes at 4uC. Cell lysate protein concentrations were determined by BCA assay (Thermo Scientific, Rockford, IL) against BSA protein standards. All samples were normalized for protein content, diluted 4:1 in 5x Laemmli buffer, heated 10 min at 95uC, and stored at 280uC.
Blots were stripped prior to reprobing with Restore PLUS Western Blot Stripping Buffer (Thermo) for 15 minutes at RT.
Detection and analysis. Blots were imaged with a Chemidoc XRS (Bio-Rad) and net band intensity units were measured with Image Lab image analysis software (Bio-Rad). All blots contained equal numbers of samples of each experimental treatment. The mean band intensity of all samples on an individual blot was normalized across all blots to an arbitrarily chosen value to compensate for interblot variability and individual sample band values were then adjusted proportionally to reflect that normalized mean.

Fluorescence Detection of RONS in PMEM
Ethidium. RLMVEC (0.3610 6 /2.0 ml of culture medium) were plated in six-well culture dishes and allowed to grow to confluence. After treatment, PMEM were washed 1X with PBSand incubated with 1 mM dihydroethidium (DHE) (Life Technologies) in phenol free DMEM with 10% FBS (1 ml/well) at 37uC for 0.5 h. PMEM were then washed on ice 2x with ice-cold DPBSto remove free probe and scraped with 1 ml of ice-cold DPBSinto microtubes. The cells were centrifuged at 8,000 g for 10 min at 4uC, and the pellet was resuspended in 0.4 ml of ice-cold DPBS. The cell suspensions were sonicated on ice for 15

Statistics
All data points are mean values of treatment samples. All samples were generated over 2 to 4 experiments within which there were 2 to 4 replicates for each treatment, except for 96-well microplate format studies, which contained 8-12 replicates per treatment. Each PMEM well represents a single sample. There are at least 4-10 samples per group in all studies. A one-way analysis of variance was used to compare values among the treatments. If significance among treatments was noted, a post hoc multiplecomparison test was done with a Bonferroni (parametric-equal variance) or a Duncan (nonparametric-unequal variance) test to determine significant differences among the groups. All data are reported as means 6 SE. Significance was at P,0.05.

LTA Induces Activation of the TLR-2 Pathway in Pulmonary Endothelium
The presence of TLR2 in RLMVEC was confirmed by detection on Western blots with anti-TLR2. TLR2 was identified on the pulmonary endothelial cells independent of 1 hour challenge with vehicle, TNFa, LTA, or PAM. A representative Western blot is shown in Figure 1a. Known consequences of TLR pathway activation, primarily shown in leukocytes, include IRAK1 activation, phosphorylation, and depletion as well as phosphorylation and depletion of IkBa during NFkB activation [25]. Representative Western blots of IRAK1, IRAK4, IkBa and phospho-IkBa-Ser 32/36 (p-IkBa) from PMEM treated with either LTA or PAM for 0.5, 4 and 24 hours compared to vehicle are shown in Figure 1b and the band density data for all similar blots is given in Figure 1c-f. Both LTA and PAM caused a late (4-24 hr.) sustained decrease in IRAK1 with no changes in IRAK4, and a rapid (0.5 hr.) and sustained (4-24 hr.) increase in p-IkBa with a concomitant decrease in the total IkBa cellular pool. These results suggest activation of the Toll-like receptor pathway; NFkB activation and IRAK1 degradation subsequent to its activation through TLR signaling.
To verify these results, an IRAK1/4 inhibitor (IRI) was utilized to block the downstream effects of TLR2 activation. An initial PMEM dose response to IRI was performed that indicated a 2 hour pretreatment with 10 mM IRI was optimal for curtailing IkBa phosphorylation following LTA treatment while still maintaining monolayer integrity. Higher doses of IRI (25-50 mM) resulted in large gaps in the cell monolayer visible under low resolution phase microscopy (data not shown). Representative Western blots of cell lysates from PMEM treated with either LTA or PAM in the presence of IRI (10 mM) for 0.5, 4 and 24 hours (Figure 2a) reveal a marked decrease in the generation of p-IkBa. The p-IkBa band density data from all blots of this treatment group (Figure 2b-d) show that this decrease is significant and sustained throughout the study period (,28%-62% less than agonist alone, p#0.02, for all treatments). The data of Figures 1 and 2 support the idea that both LTA and PAM activate TLR2 in rat lung microvessel endothelial cell monolayers and that this activation can be at least partially inhibited with an IRAK1/4 inhibitor.

Activation of the TLR-2 Pathway Induces Endothelial Barrier Dysfunction Mediated by Reactive Oxygen/ nitrogen Species
Endothelial cell monolayer permeability changes were assayed 24 hours after treatment with either LTA or PAM. Both LTA and PAM significantly increase permeability to albumin compared to control PMEM (mean 6 SEM: 81611% and 262670% increase versus untreated controls, p.0.01, respectively). This increase in permeability is ameliorated or prevented by co-treatment with the superoxide scavenger tiron, the reactive nitrogen species scavenger, urate, the NOS inhibitor L-NAME, or by treatment with the IRAK1/4 inhibitor IRI (Figure 3).
There is a marked increase in DHE oxidation after a 30 minute treatment of PMEM with either LTA or PAM,(mean 6 SEM: 69617% and 89620% increase versus untreated controls, p.0.01, respectively). Co-treatment of LTA-or PAM-treated monolayers with tiron, urate or L-NAME prevents the oxidation of DHE (Figure 4a). Treatment of PMEM grown in 96-well plates with LTA or PAM for 2 hours resulted in a significant increase in DCFDA oxidation whereas the degree of DCFDA oxidation in LTA-or PAM-treated PMEM was significantly reduced in the presence of IRI (mean 6 SEM: 25.265.2% vs 59.3613%, LTA+IRI vs LTA alone, p,0.04 and 33.466.7% vs 84.269.6%, PAM+IRI vs PAM alone, p,0.01) (Figure 4b).

Role of RNS: eNOS Activation
Treatment of RLMVEC monolayers with either LTA or PAM is associated with DHE oxidation that is inhibited by urate or L-NAME, suggesting a role for reactive nitrogen species; thus, eNOS was examined. Phosphorylation of eNOS at ser 1177 activates eNOS; conversely, phosphorylation of eNOS at thr 495 is associated with a decrease in eNOS activity [26]. A measure of eNOS activity was therefore determined by the ratio of phospho-eNOS-ser 1177 / phospho-eNOS-thr 495 (pSer 1177 /pThr 495 ). Both LTA and PAM increased the ratio of pSer 1177 /pThr 495 , suggesting eNOS activation in response to LTA or PAM. This effect was early (0.5 hours) and sustained over 24 hours ( Figure 5). In the presence of IRI, the activation of eNOS in response to LTA or PAM treatment was virtually eliminated. Representative Western blots of p-eNOS-ser 1177 and the corresponding p-eNOS-thr 495 bands from PMEM treated with LTA or PAM in the absence or presence of IRI for 0.5, 4, and 24 hours are shown in Figure 6a. The cumulative eNOS pSer 1177 /pThr 495 band density ratios for these treatments reveal that eNOS activity in PMEM, as defined herein, remains at control levels after exposure to LTA or PAM when an IRAK1/4 inhibitor (IRI) is present. Figure 7 further illustrates the dynamics of eNOS activation and inhibitory site phosphorylation in PMEM exposed to LTA for 4 hours in the presence of increasing amounts of IRI. In the absence of inhibitor, LTA induces both an increase in pSer 1177 and decrease in pThr 495 resulting in a large eNOS pSer 1177 /pThr 495 ratio increase relative to control. The level of pSer 1177 after LTA exposure rapidly falls as the concentration of IRI is increased whereas the level of pSer 1177 in the absence of LTA remains relatively unaffected (Figure 7a). Conversely, both control and LTA-exposed PMEM levels of pThr 495 rise with an increase in IRI concentration (Figure 7b). The LTA-induced decrease in pThr 495 is not eliminated by IRI but contributes less to the overall eNOS pSer 1177 /pThr 495 ratio  change relative to the magnitude of the IRI-induced increase in pThr 495 and decrease in pSer 1177 . This results in a dramatic fall in the pSer 1177 /pThr 495 ratio (eNOS activity) of LTA-exposed PMEM with increasing IRI concentration (Figure 7c). Representative Western blots are shown in Figure 7d which illustrate this effect.

Discussion
In the present study, LTA activated TLR2 dependent pathways because: (1) TLR2 is detected in the RLMVEC, (2) LTA caused specific depletion of IRAK1, and (3) LTA caused increased p-IkBa-ser 32/36 associated with decreased IkBa. The synthetic, TLR1/2-specific agonist PAM also caused a depletion of IRAK1 and increased p-IkBa-ser 32/36 similar to LTA which further supports the idea that TLR2 mediates the effect of LTA. Recognition of PAM is mediated by TLR2 cooperatively with TLR1 through their cytoplasmic domains to induce the signaling cascade leading to the activation of NFkB. While TLR1 cooperates in initiation of signaling by PAM, signaling is mediated via the TLR 2 pathway and is thus the receptor of significance in mediating the deleterious effects of both LTA and PAM. Importantly, the agonists have no TLR4 activity [27,28]. Signaling activated by LTA is documented to be mediated via TLR2. PAM is a widely proven agonist of TLR2 and, although it causes dimerization of TLR2 with TLR1, in TLR2 knock-out studies, neither PAM nor LTA activate the Toll-receptor cascade [29,30]. Finally, the activation of the TLR2 pathway was not a non-specific effect of endothelial cell activation because TNF did not affect IRAK1 despite the TNF induced increase in p-IkBa-ser 32/36 (data not shown). Importantly, both LTA and PAM caused similar increased albumin clearance rates which supports the idea that TLR2 receptor activation mediates a permeability effect in the rat pulmonary microvessel endothelial cells. The ultrapure LTA from S. aureus used in our experiments contains minimal endotoxin; thus, these observed effects can confidently be attributed to LTA and TLR2 activation. However, it should be noted that there are other Gram-positive cell wall components that are TLR2 ligands that may contribute to endothelial activation such as a principal lipoprotein peptidoglycan. Peptidoglycan has been reported to be a more potent inducer of inflammation than LTA and to act synergistically with LTA [8,29]. A recent study examined the effects of TLR2 agonists (e.g., peptidoglycan-associated lipoprotein and PAM) on human endothelial cell function [28]. The TLR2 agonists activated human umbilical vein, lung, and coronary artery endothelial cells, indicated by increased IL-6 and E-selectin production. In addition, increased FITC-labeled albumin permeability was demonstrated in human umbilical vein endothelial cell monolayers treated with 5 to 15 mg/mL of PAM for 24 hours. Xing et al. recently demonstrated that LTA doses ranging from 50 to 300 ng/mL induced significant barrier dysfunction in human pulmonary artery endothelial cells measured by transendothelial electrical resistance [9]. The authors also showed that intratracheal administration of LTA alone as well as in combination with peptidoglycan induced lung injury in vivo in a murine model that measured bronchial alveolar lavage cell count and protein concentration. LTA also induced activation of key intracellular signaling proteins, evidenced by increased phosphorylation of p38 MAP kinase, HSP 27 and at later time points ERK 1/2 MAP kinase; and similar to our findings, the authors observed degradation of IkBa.
Yet, to the best of our knowledge our study represents the first to show that activation of the TLR2 pathway by ultrapure LTA or PAM induces increased permeability by a mechanism mediated by ROS/RNS generation in lung endothelial cells. LTA and PAM induced significant increases in endothelial monolayer permeability and DHE oxidation which were prevented by the superoxide radical (NO 2 2 ) scavenger tiron, the peroxynitrite (ONOO2) scavenger urate, and the eNOS inhibitor L-NAME. This indicates LTA induces the generation of reactive oxygen species (i.e., scavenging by tiron) and reactive nitrogen species (i.e., scavenging by urate and eNOS inhibition with L-NAME) that increase endothelial permeability. The mechanism for the LTA induced oxidant dependent increase in permeability is not clear from the present study. However, the literature indicates oxidant stress causes phosphorylation and redistribution of occludin and decreased levels of VE-cadherin associated with loss of peripheral cortical actin and increased actin stress fibers. The loss of actin peripheral bands is associated with disruption of a,b,c catenins and actin binding proteins (e.g.,filamin) with increased cell-cell gap formation. Moreover PLC, PLD and PLA2 are activated during oxidant stress resulting in increased endothelial kinase (e.g., PKC) activity and generation of lipid mediator (thromboxane A2) [16,17]. Previous work by our group and others have shown that ROS/RNS mediate endothelial dysfunction [16]. TLR2 activation induces AKT activation and AKT activates eNOS via phosporylation of eNOS-ser-1177 [34]. Also, previous studies indicate that the TLR2 receptor agonist, LTA, induces endothelial dysfunction: therefore, we studied the relationship between TLR2 activation and ROS/RNS production. As an example of this relationship, in the present study, the TLR2 receptor agonists LTA and PAM both cause an increase in the activation of eNOS evidenced by the increase in phospho-eNOS-ser 1177 /phospho-eNOS-thr 495 which results in increased production of ROS and RNS.
In macrophages, LTA exposure is associated with increased iNOS and NO release [35]. Both LTA and PAM induced eNOS activation, the likely source of NO and peroxynitrite via the interaction between NO and NO 2 2 R ONOO 2 . We previously showed during TNF-induced endothelial injury that the protective effect of L-NAME was due to competitive inhibition of the substrate L-arginine resulting in decreased nitric oxide, and that urate is a scavenger of ONOO- [22]. Although the limited specificity of urate as a peroxynitrite scavenger is appreciated, reduction of both endothelial cell monolayer permeability and RO/NS generation after either LTA or PAM co-treatment with urate, or with L-NAME, a specific NOS inhibitor, provides compelling evidence for a RNS mediated injury pathway. IRAK1/4 inhibition also prevented the LTA-induced increase in permeability as well as minimizing the LTA-induced oxidation of DCFDA. Inhibition of IRAK had a profound effect on phosphorylation patterns of the major effector sites of eNOS, essentially reversing the TLR2 agonist-induced changes and eliminating eNOS activation. It is known that eNOS is activated by the Akt dependent phosphorylation of eNOS-ser 1177 whereas PKCd has been shown to be responsible for phosphorylation of the eNOS-thr 495 inhibitory site [36,37]. TLR-dependent Akt activation occurs via a TLR/TIRAP/MyD88/PI3K/Akt pathway [38,39] and Dunne et al found that both IRAK1 and IRAK4 directly phosphorylate TIRAP (Mal) [40]. This suggests a direct role for IRAK in the TLR signaling cascade leading to the activation of Akt. LTA increases PKCd activity in fibroblasts [41] and Tiwari et al have reported direct interaction between IRAK1 and PKCd in monocytes [42]. Additionally, Xiong et al demonstrated that NO inhibits IRAK activity in splenocytes, macrophage, and dendritic cells [43]. These findings are consistent with the noted TLR2 agonist-dependent, and IRAK inhibitor reversible, increase in the p-eNOS-Ser 1177 /p-eNOS-thr 495 ratio of the present study. These findings along with our observations also potentially describe a previously unreported self-regulating relationship between the activities of IRAK and eNOS.
In the present study, the protective effect of Tiron indicates the generation of NO 2 2 . NADPH-oxidase dependent NO 2 2 has been shown to mediate various models of endothelial injury such as TNF-induced increased endothelial permeability [44]. NADPHoxidase consists of a NOX2 (gp91phox), p47, p22 and Rac regulated by multiple phosphorylation and translocation events. In smooth muscle cells, LTA induces MAPK activation mediated by a TLR2/MyD88/PI3K/Rac1/Akt pathway [45]. In bone marrow-derived macrophages, Maitra et al demonstrated that IRAK-1 associated with and activated small GTPase Rac1, a known activator of NOX-1 oxidase enzymatic activity [46]. The NOX 4 homolog is also expressed in endothelial cells in which its activity may be independent of Rac and p47phox. In human aortic endothelial cells exposed to LPS, Park et al. have demonstrated the critical role of NOX 4 derived ROS in LPS-induced monocyte adhesion and migration [47]. Recently Lee et al. have shown in smooth muscle cells that treatment with LTA 50 mg/mL induced ROS generation that was inhibited by the NOX inhibitors DPI and apocynin, suggesting ROS generation is NOX dependent. The authors demonstrated NADPH-oxidase activation via a TLR2/MyD88/TRAF6/c-Src/p47phox complex [48]. Indeed, it has been shown that the Src family of tyrosine kinases activates the NADPH-oxidase complex [49]. Finally, the un-coupling of eNOS because of modification of the protein by reactive oxygen/nitrogen species and lack of the subtrates L-arginine and tetrahydrobiopterin, results in generation of NO 2 2 . Thus, in addition to prevention of NO generation, the protective effect of L-NAME may be due to inhibition of NO 2 2 generated by un-coupled eNOS. There are other potential sources of reactive oxygen species such as xanthine oxidase and the mitochondrial respiratory chain. The role of NOX 2 and NOX 4 as a source of LTA-induced, ROSdependent lung endothelial cell barrier dysfunction warrants further exploration.

Conclusion
In summary, our data demonstrate that the TLR2 agonists LTA and PAM activate the TLR2 pathway which is associated with pulmonary endothelial barrier dysfunction. Moreover, ROS and RNS mediate the LTA mediated increase in lung endothelial permeability.