CORRECTION

eNOS-derived nitric oxide regulates endothelial barrier functionthrough VE-cadherin and Rho GTPases

Annarita Di Lorenzo, Michelle I. Lin, Takahisa Murata, Shira Landskroner-Eiger, Michael Schleicher,Milankumar Kothiya, Yasuko Iwakiri, Jun Yu, Paul L. Huang and William C. Sessa

There was an error published in J. Cell Sci. 126, 5541-5552.

Both Annarita Di Lorenzo and William C. Sessa should have been designated as authors for correspondence. They can be contacted at

and2039@med.cornell.edu and william.sessa@yale.edu, respectively.

We apologise to the readers for any confusion that this error might have caused.

Journal of Cell Science JCS153601.3d 27/3/14 16:13:36The Charlesworth Group, Wakefield +44(0)1924 369598 - Rev 9.0.225/W (Oct 13 2006)

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 000, 000–000 doi:10.1242/jcs.153601

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eNOS-derived nitric oxide regulates endothelial barrierfunction through VE-cadherin and Rho GTPases

Annarita Di Lorenzo1,*, Michelle I. Lin1,*, Takahisa Murata2, Shira Landskroner-Eiger1, Michael Schleicher1,Milankumar Kothiya3, Yasuko Iwakiri4, Jun Yu1, Paul L. Huang5 and William C. Sessa1,`

1Department of Pharmacology and Vascular Biology and Therapeutics Program, Yale University School of Medicine, New Haven, CT 06520, USA2Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku,Tokyo 113-8657, Japan3Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY 10065, USA4Division of Digestive Diseases, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06536, USA5Harvard Medical School, Cardiovascular Research Center, Massachusetts General Hospital-East, 149 East 13th Street, Charlestown, MA 02129,USA

*These authors contributed equally to this work`Author for correspondence (william.sessa@yale.edu)

Accepted 16 August 2013Journal of Cell Science 126, 5541–5552� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.115972

SummaryTransient disruption of endothelial adherens junctions and cytoskeletal remodeling are responsible for increases in vascular permeabilityinduced by inflammatory stimuli and vascular endothelial growth factor (VEGF). Nitric oxide (NO) produced by endothelial NO

synthase (eNOS) is crucial for VEGF-induced changes in permeability in vivo; however, the molecular mechanism by whichendogenous NO modulates endothelial permeability is not clear. Here, we show that the lack of eNOS reduces VEGF-inducedpermeability, an effect mediated by enhanced activation of the Rac GTPase and stabilization of cortical actin. The loss of NO increased

the recruitment of the Rac guanine-nucleotide-exchange factor (GEF) TIAM1 to adherens junctions and VE-cadherin (also known ascadherin 5), and reduced Rho activation and stress fiber formation. In addition, NO deficiency reduced VEGF-induced VE-cadherinphosphorylation and impaired the localization, but not the activation, of c-Src to cell junctions. The physiological role of eNOS

activation is clear given that VEGF-, histamine- and inflammation-induced vascular permeability is reduced in mice bearing a non-phosphorylatable knock-in mutation of the key eNOS phosphorylation site S1176. Thus, NO is crucial for Rho GTPase-dependentregulation of cytoskeletal architecture leading to reversible changes in vascular permeability.

Key words: Nitric oxide, eNOS, VEGF, Cytoskeleton, VE-cadherin, Cadherin 5, Src

IntroductionAlterations in endothelial barrier function play an important

role in the pathogenesis of many disease states, including

inflammation, wound healing, edema, acute lung injury, stroke

and cancer (Wojciak-Stothard and Ridley, 2002). Endothelial

barrier maintenance and the response of the quiescent barrier

to locally produced vasoactive agents, such as histamine,

prostaglandins, thrombin and VEGF is primarily mediated by

dynamic changes in adherens junctions (AJs) and increased

centripetal tension created by actomyosin contractility (Dejana,

2004; Feng et al., 1999; Weis and Nelson, 2006).

During an inflammatory process, the microvascular

permeability is selectively increased in post-capillary venules,

where the AJs are the major type of inter-endothelial junctions.

Vascular endothelial cadherin (VE-cadherin, also known as

cadherin 5), a cell-type-specific cadherin, is highly expressed on

endothelial cells (ECs) and forms a complex with proteins of

the armadillo family including p120-catenin, b-catenin and

plakoglobin, contributing to the stability of the EC barrier. The

disruption of AJs by permeability agents is in part mediated

by tightly regulated protein phosphorylation, endocytosis and

intramembrane cleavage. In particular, many studies have

reported that tyrosine phosphorylation of VE-cadherin and its

binding partners induced by histamine (Andriopoulou et al.,

1999; Shasby et al., 2002), tumor necrosis factor-a (TNF-a)

(Angelini et al., 2006) or VEGF (Esser et al., 1998) correlates

with the destabilization of endothelial AJs. Most recently, the

crucial importance of AJ regulation during inflammation has

been determined in mice expressing a ‘stabilized’ VE-cadherin

(Broermann et al., 2011) where the acute responses to

inflammatory mediators are markedly attenuated.

VEGF is a potent vascular permeability factor that plays an

essential role in both physiological and pathological angiogenesis

(Ferrara et al., 2003). VEGF exerts its effects on vascular

permeability through the activation of the Src family kinases, in

particular c-Src (Eliceiri et al., 1999; Zachary and Gliki, 2001) and

phosphoinositide 3 kinase (PI3K)/Akt-dependent phosphorylation

and, hence, activation, of endothelial nitric oxide synthase (eNOS)

(Ackah et al., 2005; Papapetropoulos et al., 1997). Pharmacological

inhibition of Src family protein tyrosine kinases (PTKs) reduces

VEGF-induced phosphorylation of AJ components and vascular

permeability (Eliceiri et al., 1999; Weis et al., 2004) suggesting that

there is a crucial role for c-Src in regulating EC barrier function in

response to activation of growth factor receptors.

By contrast, both VEGF-induced vascular leak and increases in

blood flow can be blocked pharmacologically by inhibitors of

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NOS or its downstream targets, soluble guanylate cyclase orprotein kinase G (Mayhan, 1999; Murohara et al., 1998;

Papapetropoulos et al., 1997; Ziche et al., 1997). Moreover, acell-permeable peptide derived from the endogenous inhibitor ofeNOS function, caveolin, blocks edema formation and hydraulic

conductivity in post-capillary venules that is induced by mustardoil, carrageenan, platelet-activating factor and VEGF(Bernatchez et al., 2005; Bucci et al., 2000; Hatakeyama et al.,2006). The inhibitor data are complemented by genetic studies

showing that the loss of eNOS, but not other NOS isoforms,abrogates VEGF-induced permeability (Fukumura et al., 2001;Gratton et al., 2003a). The activity of eNOS can be regulated by

phosphorylation by several proteins kinases such as AMP-activated protein kinase (AMPK), protein kinase A and Akt. Theloss of Akt1 also reduces VEGF-stimulated permeability (Ackah

et al., 2005), as well as inflammation and histamine-inducedvascular permeability in vitro and in vivo, which, in the lattercases, are mediated by hypo-phosphorylation of VE-cadherin (DiLorenzo et al., 2009).

Endothelial barrier function is also regulated by the tensileforces within the endothelial monolayer. Rho, Rac and Cdc42,

members of the Rho GTPase family, are key regulators of theactin cytoskeleton; in particular Rho governs stress fiberformation, actomyosin bundles and focal adhesions, whereasRac and Cdc42 have been shown to promote formation of

lamellipodia and filopodia, respectively, in cultured cells whenstimulated with a vasoactive compound (Ridley, 2001; Ridleyand Hall, 1992; Ridley et al., 1992). Rho GTPases can also

regulate endothelial cell contractility by controlling thephosphorylation state of myosin light chain (MLC), through theactivation of MLC kinases, a Ca2+/CaM-dependent MLC kinase

(MLCK) and the Rho-associated kinase (Rho-kinase) (Amanoet al., 1996). However, how eNOS-derived NO regulates themechanisms responsible for cytoskeletal alterations, EC barrier

breakdown and vascular permeability remains elusive.

In the present study, we show that eNOS-derived NO regulatesthe rearrangement of the actin cytoskeleton and the integrity of

endothelial barrier by affecting the adhesive properties of AJs.The loss of NO in ECs stabilizes cortical actin structures byincreasing the interaction of VE-cadherin with TIAM1 at the cell

junctions, leading to an augmentation of Rac activity, therebypromoting cortical actin assembly. Conversely, Rho activation,as well as Src-dependent VE-cadherin phosphorylation, wasmarkedly attenuated in eNOS-deficient cells. The primacy

of eNOS during permeability changes in vivo is illustratedby impaired VEGF-, histamine- and inflammation-drivenpermeability in mice harboring a non-phosphorylatable knock-

in mutation of eNOS at S1176, a key residue necessary for eNOSactivation. These data show that NO is a key molecule promotingendothelial cell permeability, linking the cytoskeleton to VE-

cadherin phosphorylation through regulation of the endothelial-junction-associated Rac guanine-nucleotide-exchange factor(GEF) TIAM1.

ResultseNOS is required for VEGF-induced permeability inendothelial cells

Several studies have demonstrated that eNOS is essential for

VEGF-mediated changes in microvascular permeability(Aramoto et al., 2004; Fukumura et al., 1997; Gratton et al.,2003a; Hatakeyama et al., 2006; Murohara et al., 1998).

However, the precise mechanism by which eNOS modulatesvascular leakage is not known. To address potential mechanisms,

VEGF-induced changes in barrier function was assessed inhuman microvascular endothelial cells (HDEMCs) using avariety of approaches to interfere with eNOS function.Treatment of HDEMCs with eNOS small interfering RNA

(siRNA), but not control siRNA resulted in a .80% reduction ineNOS protein levels (Fig. 1A,B). Treatment of ECs with VEGFresulted in the time-dependent activation of VEGFR2, MAPK42

and MAPK44 (hereafter MAPK42/44; also known as MAPK3and MAPK1, and ERK1 and ERK2, respectively) andphosphorylation of eNOS on S1177 in control cells (Fig. 1A,

left panel), and a reduction in the latter in eNOS knockdown cells(Fig. 1A, right panel). Next, VEGF-induced changes in trans-endothelial electrical resistance (TEER) were measured in eNOS-deficient cells. As expected, VEGF induced a drop in TEER

followed by a recovery to baseline, consistent with transientchanges in barrier function. Silencing of eNOS significantlyattenuated VEGF-induced changes in barrier function compared

to siRNA control ECs (Fig. 1C). Furthermore, inhibition ofeNOS using a conventional NOS inhibitor, L-nitroargininemethyl ester (L-NAME), or the cell-permeable peptide

cavtratin, which corresponds to the inhibitory domain ofcaveolin-1 (Bernatchez et al., 2005; Bucci et al., 2000), alsosignificantly reduced the changes in TEER after VEGF

administration (Fig. 1D,E). These data show that eNOS-derivedNO promotes physiological changes in EC barrier function in

vitro.

VEGF-induced stress fiber formation is markedly reducedin the absence of NO

Following VEGF stimulation, changes in TEER are associated

with the reorganization of actin filaments into dense stress fiberscausing cell contraction and endothelial permeability (Soga et al.,2001; van Nieuw Amerongen et al., 2003). Thus, we examined

whether the loss of eNOS would have any effect on VEGF-mediated actin cytoskeletal reorganization. As expected, F-actin(visualized with phalloidin staining) in contact-inhibitedmonolayers was predominantly localized at the cell periphery

(Fig. 2A, panel a), and upon stimulation with VEGF(30 minutes), F-actin reorganized into stress fibers.Interestingly, F-actin reorganization was largely abrogated in

HDMECs transfected with siRNA eNOS, which exhibited adistinct pattern of junction-associated belt of cortical actin(Fig. 2A, panel d compared to panel b). The persistence of

cortical actin due to the loss of eNOS was confirmed in mouselung endothelial cells (MLECs) isolated from wild-type (WT)and eNOS2/2 mice. As seen in Fig. 2B, WT ECs responded toVEGF with the formation of stress fibers, and this effect was

reduced in eNOS2/2 ECs. Moreover, the necessity of eNOS forVEGF-induced changes in cytoskeletal reorganization was shownby the rescuing the eNOS knockout phenotype by adenoviral re-

expression of eNOS (Ad-eNOS; Gratton et al., 2003b; Scotlandet al., 2002) but not a control adenovirus encoding b-galactosidase (Ad-b-Gal; Fig. 2C). Finally, to rule out potential

artifacts due to culturing ECs or interactions between ECs and theextracellular matrix, the formation of stress fibers was examinedby ‘en face’ imaging of F-actin in endothelial layers of intact

blood vessels isolated from WT or eNOS2/2 mice. As shown inFig. 2D, cortical F-actin was clearly delineated betweenindividual ECs from intrapulmonary arteries isolated from WT

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and eNOS2/2 mice. Consistent with cultured EC data, VEGF

failed to induce a stress fiber re-organization in ECs of eNOS2/2

blood vessels compared to WT endothelium. Collectively, these

results demonstrate the importance of eNOS in mediating

cytoskeletal rearrangements in ECs upon VEGF stimulation.

The loss of eNOS affects VEGF-mediated Rac and Rho

activation

The Rho family of small GTPases, including RhoA, Rac1 and

Cdc42, regulate the dynamics and structures of F-actin and

therefore influence cell shape, EC adhesion and endothelial

barrier function (Hall, 1998a; Hall, 1998b; Ridley, 2001;

Wojciak-Stothard and Ridley, 2002). Because the loss of eNOS

increased cortical actin and impaired VEGF-induced stress fiber

formation, the activation of Rac was assessed using p21 activated

kinase (PAK)-conjugated agarose beads (Benard et al., 1999).

Following VEGF stimulation, maximal Rac activation in WT

ECs was achieved within 2 minutes and dropped back down to

basal levels at 10 minutes, whereas eNOS-deficient ECs

exhibited increased basal as well as sustained Rac activation

throughout the VEGF timecourse (Fig. 3A). Moreover, treatment

of eNOS-depleted HDEMCs with a Rac inhibitor (NSC 23766)

was able to disrupt the ring of cortical actin and partially restore

stress fiber formation upon VEGF stimulation (Fig. 3C),

suggesting that the loss of eNOS results in a hyperactive GTP-

bound state of Rac, which cannot be further activated by VEGF.

In addition to Rac, VEGF is known to activate RhoA in ECs,

leading to the formation of stress fibers (Burridge and

Wennerberg, 2004). Thus, we examined GTP-Rho levels in

eNOS-deficient cells using GST–Rhotekin-conjugated agarose

beads (Reid et al., 1996). Basal and VEGF-stimulated Rho

activation was largely abrogated in eNOS2/2 ECs compared to

WT cells (Fig. 3B), suggesting that the defective stress fiber

formation observed in eNOS2/2 cells was due to inactivation of

Rho. The infection of eNOS-depleted HDMECs with an

adenovirus encoding the constitutively active form of Rho (Ad

RhoL63) readily led to the formation of stress fibers in these cells

compared to HDMECs treated with eNOS siRNA and infected

with a control virus encoding Lac Z (Ad b-Gal) (supplementary

material Fig. S1). This suggests that eNOS is upstream of Rho

activation (Siddiqui et al., 2011) and that the loss of stress fiber

formation due to eNOS deficiency can be restored by a

downstream constitutively active Rho. Many studies have

indicated that Rho, through its direct downstream effectors,

Rho kinases, can phosphorylate myosin light chain (MLC) kinase

(Amano et al., 1996) or inhibit MLC phosphatase, which results

in increased MLC phosphorylation (Kimura et al., 1996). In

addition, Rac-activated PAK can also phosphorylate MLC or

myosin II (Kiosses et al., 1999; Zeng et al., 2000). Thus, we

investigated whether the loss of eNOS, either through the

decreased activation of Rho and/or hyperactivation of Rac, could

affect MLC phosphorylation. As shown in the western blots in

Fig. 3D, VEGF-induced phosphorylation of MLC was reduced in

eNOS-depleted HDMECs compared to the control, suggesting a

role of eNOS upstream of Rho and MLC activation in the

VEGF-activated signaling cascade. The reduction in MLC

Fig. 1. eNOS activity is crucial for VEGF-induced permeability in HDEMCs. (A) HDMECs with control or eNOS siRNA were stimulated with vehicle (control,

0 minutes) or VEGF (100 ng/ml) for 5, 10 and 30 minutes and the cell lysates were analyzed by western blotting for eNOS, P-eNOS1177, P-1175-VEGFR-2,

VEGFR-2, P-MAPK42/44, MAPK42/44 (where P indicates the phosphorylated form). (B) Quantification of eNOS levels in HDEMCs by densitometry from four

independent experiments and expressed relative to the amount of b-actin. The data shown represent the mean6s.e.m. from five individual experiments.

*P,0.05 compared to HDEMCs with siRNA control. (C) TEER measurements of post-confluent HDEMCs transfected with control (black line) or eNOS (red line)

siRNA expressed as fractional resistance (%) of the TEER basal values. HDEMCs were stimulated with VEGF (100 ng/ml; addition indicated by the arrow)

and TEER measured overtime. *P,0.05 compared to HDEMCs with control siRNA. (D) TEER measurements of HDEMCs treated with L-NAME (1 mM) or vehicle

followed by VEGF (100 ng/ml) stimulation. *P,0.05 compared to vehicle-treated HDEMCs. (E) TEER measurements of HDEMCs treated with the eNOS

inhibitor cavtratin peptide (10 mM) or the peptide control (antennapedia internalization peptide, AP) for 1 hour, measured after VEGF stimulation for 5 minutes.

*P,0.05 compared to AP-treated cells (n53). The data shown represent the mean6s.e.m. from three individual experiments.

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Fig. 2. The loss of eNOS prevents VEGF-induced stress fiber formation in ECs in vitro and ECs of intrapulmonary arteries in situ. (A) HDEMCs

with control or eNOS siRNA were stimulated with vehicle (basal) or VEGF (100 ng/ml) for 30 minutes, then fixed and stained with phalloidin to label

F-actin (green) and nuclei (blue). (B) WT and eNOS2/2 MLECs were stimulated with vehicle or VEGF (100 ng/ml) for 30 minutes and labeled as in A.

(C) eNOS2/2 MLECs were transduced with adenovirus encoding either control lac Z (Ad b-gal) or eNOS (Ad eNOS) at 100 MOI for 3 days before stimulation

with vehicle or VEGF (100 ng/ml) and immunolabeled similarly to in A. These figures are representative of three separate experiments. (D) Intrapulmonary

arteries isolated from WT or eNOS2/2 mice were stimulated with vehicle or VEGF (100 ng/ml) for 15 or 30 minutes. Blood vessels were then fixed, cut open

longitudinally and immunolabeled with phalloidin as described in A. These figures are representative of five animals from each group.

Fig. 3. eNOS deficiency enhances Rac activity while

reducing Rho activity. (A) WT and eNOS2/2 MLECs were

stimulated with VEGF (100 ng/ml) for the indicated times and

Rac activity was measured as described in the Materials and

Methods. Total Rac and eNOS levels were determined in the

whole cell lysates before GST–PAK incubation. The graph

shows the densitometric ratio of active Rac to total Rac. The

data are representative of three independent experiments.

(B) WT and eNOS2/2 MLEC were stimulated with VEGF

(100 ng/ml) for the indicated times and cell lysates processed

for Rho activity. Total Rho and eNOS levels were determined in

the whole cell lysates before GST–Rhotekin incubation. The

graph shows the densitometric of active Rho to total Rho. The

data are representative of three independent experiments.

(C) HDEMCs with control or eNOS siRNA were pre-incubated

with Rac inhibitor (25 mM, 6 hours) and stimulated with

VEGF (100 ng/ml) following immunolabeling of F-actin

(green) and nuclei (blue). (D) Western blotting for P-T18/19

MLC2 and MLC2 in whole cell lysates from HDEMCs with

eNOS or control siRNA stimulated with VEGF (100 ng/ml) for

the indicated times. The data represent three independent

experiments. (E) HDEMCs with eNOS or control siRNA were

stimulated with vehicle or SNAP, an NO donor (100 mM,

1 hour) and immunolabeled for F-actin (green) and nuclei

(blue). Images are representative of three experiments.

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phosphorylation in eNOS-deficient cells was confirmed by

immunofluorescence labeling of phosphorylated MLC in WT

and eNOS2/2 cells treated or not with VEGF (supplementary

material Fig. S2). The NO dependency of stress fiber formation

was tested in eNOS-depleted HDMECs treated with the NO

donor, S-nitroso-N-acetylpenicilamine (SNAP). As seen in

Fig. 3E, SNAP induced actin filament formation in both

control and eNOS-depleted HDMECs (Fig. 3E), suggesting that

NO levels per se are able to induce the formation of stress fibers.

eNOS is required for VEGF-driven c-Src-dependent

VE-cadherin phosphorylation

To assess the mechanisms underlying eNOS regulation of

barrier function, we examined the relationship between eNOS

and VE-cadherin, a well-documented pathway that is important

for VEGF-induced EC barrier breakdown, (Esser et al., 1998).

As shown in Fig. 4A, and quantified in Fig. 4B, treatment of

HDMECs with VEGF induced the time-dependent change in

VE-cadherin tyrosine phosphorylation, and the co-association of

c-Src and eNOS in the VE-cadherin immunocomplex.

Interestingly, VE-cadherin tyrosine phosphorylation and

recruitment of c-Src was diminished in eNOS-depleted ECs,

as well as in cells treated with L-NAME, using either an

antibody against VE-cadherin or the anti-phosphorylated-

tyrosine antibody PY20 to immunoprecipitate the complex

(supplementary material Fig. S3A,B). However, the activation

of c-Src (measured as P-418), its downstream target focal

adhesion kinase (FAK P-861), VEGFR2 and MAPK

phosphorylation in whole cell lysates were not affected by the

lack of eNOS (Fig. 4C, with quantitative phosphoprotein:total

protein ratios below and supplementary material Fig. S3C).

Finally, the localization of c-Src was examined by

immunofluorescence microscopy. As seen in Fig. 4D, VEGF-

induced c-Src translocation to VE-cadherin-positive junctions

was reduced HDMECs treated with eNOS siRNA (Fig. 4D, and

quantified in Fig. 4E). Collectively, these results support the

importance of eNOS-derived NO in VEGF-induced c-Src

recruitment and phosphorylation of VE-cadherin.

Fig. 4. eNOS is required for VEGF-driven c-Src-translocation to cellular junctions and VE-cadherin phosphorylation. (A) HDEMCs transfected with

eNOS or control siRNA were serum starved and stimulated with VEGF for the indicated times. VE-cadherin immunoprecipitates were analyzed by

immunoblotting against phosphorylated tyrosine (4G10, PY20 clone), VE-cadherin and also for co-immunoprecipitation of c-Src and eNOS proteins. (B) The

amount of tyrosine-phosphorylated VE-cadherin was quantified by densitometry from four independent experiments and expressed as fold increase compared with

untreated controls. (C) Western blot analysis on whole cell lysates (WCL) from HDEMCs treated as described above. The activation of additional pathways (P-

419-Src, Src, P-861-FAK, FAK, P-p38, p38; where P indicates the phosphorylated form) was examined after VEGF (100 ng/ml) stimulation. Densitometric

quantification of phosphorylated:total for each protein is shown below the blot. (D) Immunofluorescent co-staining of VE-cadherin (green) and c-Src (red) in

HDEMCs transfected with eNOS or control siRNA, then serum starved and stimulated with VEGF (100 ng/ml) for 15 minutes. Scale bar: 20 mm. The images

have been captured at a 0.3 mm slice thickness (z-stack) by using a Zeiss Axiovert epifluorescence microscope and a 636 oil immersion objective, following

deconvolution by Openlab software (Improvision, Lexington, MA). Data are representative of at least three experiments. Scale bar: 30 mm. (E) Pearson’s

correlation analysis of VE-cadherin and Src colocalization to the plasma membrane.

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NO regulates VEGF-induced F-actin assembly through

localization of TIAM1 to cell junctions

The inter-relationship between Rho GTPases and VE-cadherin

plays an important role in the regulation of endothelial barrier

integrity. In confluent ECs, the VE-cadherin complex can initiate

the activities of many Rho GTPases by engaging cytosolic Rho

GEFs including TIAM1 and VAV2 (activators of Rac) as well as

Rho GTPase-activating proteins (GAPs) such as p190RhoGAP

(an inactivator of RhoA). In confluent ECs, TIAM1-induced Rac

activation leads to the formation of F-actin belts at the cell

periphery, promoting cadherin-mediated cell-to-cell adhesion

(Lampugnani et al., 2002), as well as the partitioning of VE-

cadherin molecules into detergent-resistant basolateral domains.

Because the loss of eNOS enhanced the levels of Rac-dependent

cortical actin assembly, we investigated whether NO influenced

cell adhesion strength by determining the partitioning of VE-

cadherin into detergent-resistant domains. WT and eNOS2/2 ECs

were grown to confluence on glass coverslips, and treated with

0.5% Triton X-100 and the detergent-resistant proteins stained

with antibodies for VE-cadherin and TIAM1. Immunofluorescent

staining showed an increase of VE-cadherin and TIAM1

colocalized in the insoluble junctional domains of eNOS2/2

cells compared to that in WT ECs (Fig. 5A). Similar results of

enhanced TIAM1 labeling at junctions were obtained in

HDMECs treated with L-NAME (Fig. 5B), suggesting that the

lack of NO strengthened the cell-to-cell adhesion in a TIAM1 and

Rac-dependent fashion. To directly test whether the loss of NO

leads to augmented TIAM1 junctional localization, we evaluated

the fraction of TIAM1 that co-immunoprecipitated with VE-

cadherin in HDMECs treated with L-NAME. The antibody for

TIAM1 detects multiple proteins in western blots, therefore

siRNA against TIAM1 was used to identify the specificity of

the antibody. As shown in supplementary material Fig. S4,

siRNA against TIAM1 reduced the levels of TIAM1 (molecular

mass band of ,220 kDa) but did not reduce the other bands.

Inhibition of eNOS with L-NAME increased the formation of a

Fig. 5. NO regulates VEGF-induced F-actin formation through TIAM1 localization to cellular junctions. (A) WT and eNOS2/2 MLECs were plated

onto glass coverslips, and were treated with 0.5% Triton X-100-containing buffer for 5 minutes on ice. The soluble fraction was removed and the ECs were

gently washed with PBS before fixation with paraformaldehyde 4% and immunofluorescent staining of VE-cadherin (red) and TIAM1 (green).

(B) Immunofluorescent co-staining of VE-cadherin (green) and TIAM1 (red) in HDEMCs plated on glass-bottomed dishes (MatTek) transfected with eNOS

siRNA or control siRNA and treated with 0.5% Triton X-100-containing buffer for 5 minutes on ice. The arrows show TIAM1 at the junctions. (C) HDEMCs were

treated with L-NAME or vehicle, and VE-cadherin was immunoprecipitated from the cell lysates. Western blotting for TIAM1 and VE-cadherin was carried

out on the whole cell lysates (WCL) and on the immunoprecipitates (IP). (D) Basal and VEGF-stimulated primary WT and eNOS2/2 MLEC lysates were

subjected to immunoprecipitation of VE-cadherin and immunoprecipitates were analyzed by immunoblotting against TIAM1. The amount of TIAM1 co-

immunoprecipitated was quantified and is reported as a ratio to VE-cadherin (VEC). (E) Immunofluorescent staining of F-actin (green) in HDEMCs transfected

with eNOS siRNA or control siRNA, or co-transfected with eNOS siRNA and TIAM1 siRNA, serum starved and stimulated with VEGF (100 ng/ml) for

30 minutes. Arrows indicate cortical actin; arrowheads indicate stress fibers formed upon VEGF stimulation. The images have been captured with Zeiss Axiovert

epifluorescence microscope and a 636 oil immersion objective, using Openlab software (Improvision, Lexington, MA). Data are representative of three

independent experiments.

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VE-cadherin–TIAM1 immunocomplex (Fig. 5C). Similar

experiments were performed in WT and eNOS2/2 ECs in the

absence of VEGF stimulation. As shown in Fig. 5D, the loss of

eNOS increased the basal interaction with VE-cadherin, and

VEGF-induced increases in VE-cadherin–TIAM1 immunocomplexes

were not observed in eNOS2/2 cells. Because the fine-tuning of

Rho GTPase function regulates the balance of cortical actin versus

stress fibers, and enhanced junctional VE-cadherin–TIAM1 could

in principle drive Rac activation, we partially knocked down levels

of TIAM1 in control and eNOS-depleted HDEMCs. Notably,

reducing TIAM1 levels augmented VEGF-driven stress fiber

formation in eNOS-depleted cells (Fig. 5E). These findings

suggest that eNOS-derived NO regulates TIAM1-dependent

activation of Rac.

The eNOS–TIAM1 pathway regulates VE-cadherin

phosphorylation

TIAM1-induced Rac activation can promote cadherin-mediated

cell-to-cell adhesion in epithelial cells (Hordijk et al., 1997; Sander

et al., 1999; Sander et al., 1998), fibroblasts (Sander et al., 1999)

and ECs (Lampugnani et al., 2002). The stabilization of mature

junctions in ECs also correlates with reduced VE-cadherin

phosphorylation (Lampugnani et al., 1997) and the loss of eNOS

abrogated VEGF-induced changes in barrier function, stress fiber

formation and VE-cadherin phosphorylation. To study whether

TIAM1 contributes to NO-regulated VE-cadherin phosphorylation,

HDEMCs were treated with control, eNOS siRNA or eNOS and

TIAM1 siRNAs. As shown in Fig. 6A, the loss of eNOS reduced

basal and VEGF-stimulated VE-cadherin tyrosine phosphorylation

after 5 minutes of stimulation (lanes 3 and 4), whereas partial

knockdown of TIAM1 in an eNOS-depleted background, enhanced

VE-cadherin tyrosine phosphorylation (lane 6), as well as c-

Src recruitment to cell junctions (Fig. 6B). Collectively these

findings support the concept that eNOS regulates the dynamics of

TIAM1–VE-cadherin interactions and the balance of cortical actin

versus stress fibers required for reversible changes in EC barrier

function. Mechanistically, our data are consistent with the model

that NO upregulates Rho activation via p190RhoGEF tyrosine

nitration, thereby increasing Rho activity and c-Src recruitment and

VE-cadherin phosphorylation. Owing to competition between c-

Src and TIAM1 for cadherins, in the absence of NO (and low Rho

activity), TIAM1 is recruited to the VE-cadherin complex driving

cortical actin localization. An additional possibility is that NO

could directly nitrosylate TIAM1 reducing its interaction with VE-

cadherin. However, as seen in supplementary material Fig. S4, we

could not detect tyrosine nitration of TIAM1 in COS cells

transfected with TIAM1 treated with the peroxynitrite-generating

compound, SIN-1 (supplementary material Fig. S5).

eNOS phosphorylation at S1176 is essential for agonist-

and inflammation-induced changes in vascular

permeability

Prior work has demonstrated that eNOS-derived NO is necessary

for vascular leakage induced by VEGF and inflammatory irritants

(Bucci et al., 2000; Fukumura et al., 2001; Gratton et al., 2003a;

Murohara et al., 1998). The molecular mechanisms by which

VEGF activates eNOS is through Ca2+-calmodulin and

phosphorylation of eNOS on S1177 (equivalent to S1176 in the

mouse), both mechanisms increasing the rate of electron transfer

from the eNOS reductase to the oxygenase domain resulting in

enhanced NO release (Sessa, 2004). The phosphorylation of

eNOS at S1176 can be mediated by multiple kinases; however, in

the context of angiogenesis, it is known that the serine/threonine

kinase Akt1/PKB is the principal kinase because expression of

eNOS S1176D, but not eNOS S1176A, knocked into the

endogenous eNOS locus in mice, rescues the angiogenic

defects in Akt1-deficient mice (Schleicher et al., 2009). To

directly test whether this site of eNOS phosphorylation is

Fig. 6. Partial knockdown of TIAM1, in eNOS-deficient cells, restores the phosphorylation of VE-cadherin and the recruitment of c-Src to cellular

junctions upon VEGF stimulation. (A) HDEMCs transfected with siRNA control, or siRNA against eNOS or eNOS and TIAM1 were serum starved and

stimulated with VEGF (100 ng/ml) for 10 minutes. VE-cadherin was immunoprecipitated and the associated proteins were analyzed by immunoblotting against

phosphorylated tyrosine (pY; 4G10, PY20 clone) and VE-cadherin. The whole cell lysates (WCL) were analyzed by western blotting for VE-cadherin, eNOS,

TIAM1 and b-actin expression before the incubation with anti-VE-cadherin antibody. (B) HDEMCs transfected with eNOS siRNA or control siRNA, or

with eNOS siRNA and TIAM1 siRNA, were co-labeled for VE-cadherin (green) and c-Src (red) after before and incubation with VEGF (100 ng/ml) for

15 minutes. The images have been captured with a Zeiss Axiovert epifluorescence microscope and a 636 oil immersion objective, using Openlab software

(Improvision, Lexington, MA). Arrows depict c-Src recruitment to the plasma membrane, which colocalizes with VE-cadherin. Data are representative of three

independent experiments.

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necessary for vascular leakage, VEGF- and histamine-induced

vascular permeability was assessed in vivo using a modified

Miles assay. Intradermal injection of VEGF (Fig. 7A and images

in Fig. 7B) and histamine (Fig. 7C), promoted the extravasation

of Evan’s blue (a tracer that binds albumin as index of protein

leakage into tissue) in WT and eNOS S1176D mice suggesting

that both agonists maximally phosphorylate this residue. In

contrast, agonist-induced permeability was reduced in eNOS

S1176A mice. Moreover, to assess whether this phosphosite in

eNOS is crucial for edema and leukocyte infiltration to sites of

inflammation, carrageenan was instilled into air pouches on the

backs of mice. Carrageenan-induced edema and leukocyte

infiltration was reduced in eNOS S1176A mice compared to

WT and S1176D eNOS mice (Fig. 7D,E), demonstrating that

eNOS phosphorylation at the S1176 plays a crucial role in

vascular permeability and leukocyte infiltration.

DiscussionThe disruption of endothelial barrier function causes a marked

increase in vascular permeability, a critical and initial event in the

pathophysiology of many inflammatory diseases, including

stroke, cancer and atherosclersosis. eNOS-derived NO controls

vascular permeability in vivo through multiple actions, including

modulation of blood flow in post capillary venules, protein kinase

G activation and NO-mediated chemical modifications of

junctional proteins. However, the relationship between NO,

Rho GTPases and junctional permeability remains elusive. Here,

we show that the eNOS-derived NO regulates VEGF-induced

stress fiber formation and VE-cadherin phosphorylation by

controlling the balance of Rho versus Rac activation. This

conclusion is supported by in vitro data in human and murine

ECs lacking eNOS and in ex vivo intact pulmonary arteries fromWT and eNOS2/2 mice, showing that the loss of NO stabilizes

the cortical actin belt in a TIAM1–Rac-dependent manner andabrogates Rho-dependent F-actin formation upon VEGFstimulation. Moreover, a central role for NO in regulatingVEGF-mediated downstream signaling is supported by

experiments showing that the absence of NO reduces VEGF-induced phosphorylation of VE-cadherin by impairing Srcrecruitment to cell junctions, yet stimulates the recruitment of

TIAM1 to the VE-cadherin complex. The partial knockdownof TIAM1 in the absence of NO re-establishes the formationof F-actin-rich stress fibers and Src-dependent VE-cadherin

phosphorylation in response to VEGF, supporting the conceptthat NO controls endothelial barrier function by spatiallyregulating TIAM1 versus p190RhoGAP (see Fig. 8 for model).

The crucial role of NO in modulating vascular permeability

has been extensively corroborated by many studies in vivo

(Aramoto et al., 2004; Fukumura et al., 2001; Fukumura et al.,1997; Gratton et al., 2003a; Mayhan, 1999) and in vitro (Di

Lorenzo et al., 2009; Lal et al., 2001). Recently, it wasdemonstrated that eNOS activation is augmented in ECslacking the caveolae coat protein caveolin-1, and that eNOS

hyperactivation results in nitration and impairment ofp190RhoGAP-A function and increased Rho activation, leadingto increased vascular permeability (Siddiqui et al., 2011). These

findings are consistent with our model demonstrating that eNOSdeficiency activates Rac, and reduces Rho activation and stressformation, in response to VEGF in vitro and in intact vessels. TheeNOS and NO dependency of the response is apparent by

experiments showing that reconstitution of eNOS or NO donorsboth promote cytoskeletal responses.

Of particular interest is the novel observation of cortical actin

persistence at the cell periphery of eNOS2/2 mouse lungendothelial cells (MLECs) and HDEMCs with siRNA againsteNOS. This process is largely determined by Rho GTPases, and

our data document that eNOS-derived NO fine-tunes thisresponse in ECs. The persistence of cortical actin isreminiscent of ECs treated with the bioactive lipid sphingosine1-phosphate (Garcia et al., 2001; Shikata et al., 2003b) or the

microtubule-stabilizing drug paclitaxel (Verin et al., 2001), bothof which enhance endothelial barrier function. Mechanistically,physiological concentrations of S1P result in preferential

activation of Rac over Rho (Shikata et al., 2003a; Shikata et al.,2003b) and enhancement of cortical actin rings. Thus, our dataare consistent with the idea that the loss of eNOS enhances

endothelial barrier function or impedes VEGF-mediated vascularpermeability.

It is widely recognized that there is a complex interactionbetween Rho family members during cytoskeletal remodeling

upon growth factor activation or cell migration. Although somestudies suggest a role for Rac upstream of Rho (Lee et al., 1999;Ridley, 2001; Wojciak-Stothard and Ridley, 2002), many others

have shown that Rac activation can lead to Rho downregulation(Burridge and Wennerberg, 2004; Sander et al., 1999). Here, weshowed that in WT MLECs, the activation of Rac at 2 minutes

preceded the activation of Rho at 10 minutes after VEGFstimulation, when Rac activation had already begun to decrease,suggesting that Rac and Rho act in concert in mediating the stress

fiber formation observable after 15–30 minutes. The loss ofeNOS disrupted this fine balance and probably the lack of stressfiber formation was due in part to an inhibitory action of active

Fig. 7. The phosphorylation of eNOS on S1176 is crucial for vascular

permeability and inflammation in vivo. (A) VEGF-induced dermal vascular

permeability was reduced in S1176A eNOS (loss of function) mice

compared to WT and S1176D eNOS (gain of function) mice. The data are

plotted as the VEGF:PBS ratio of individual mice. *P,0.05; n55 mice per

group. (B) Representative images of Evan’s blue leakage upon PBS or VEGF

injection of the mice described in A. (C) Histamine-induced dermal vascular

permeability was reduced in S1176A eNOS mice compared to WT and

S1176D eNOS mice. (D,E) S1176A eNOS mice displayed a significant

reduction of carrageenan-induced (D) exudate formation and (E) neutrophil

recruitment into the air pouches 4 hours post-instillation compared to WT and

S1176D eNOS mice (n55, per group). The data shown represent the

mean6s.e.m. *P,0.05, **P,0.01 compared to WT group.

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Rac on Rho and in part by a direct effect on p190RhoGAP

(Siddiqui et al., 2011).

The sustained activation of Rac by stable overexpression of the

Rac-GEF TIAM1, results in increased cadherin-mediated cell-to-

cell adhesion, cell spreading and downregulation of Rho activity.

Interestingly, the expression of active Rho in cells overexpressing

TIAM1 was able to restore the cell morphology, strengthening

the concept of Rho being downstream of Rac (Sander et al.,

1999). Our data demonstrate an increase of TIAM1 at the cell

junctions in the absence of eNOS, resulting in stable cortical

actin formation and Rho downregulation in response to VEGF.

The partial knockdown of TIAM1 was able to rescue Rho-

activation in response to VEGF, in agreement with the idea of

Rho being downstream of Rac.

Previous studies have shown that the localization of c-Src at

the cell membrane is necessary for its biological functions

(Catling et al., 1993), and specifically that the translocation of c-

Src from the cytoplasm to the plasma membrane upon PDGF

stimulation was inhibited by cytochalasin D, an inhibitor of actin

polymerization (Fincham et al., 1996). These studies support our

data showing that partial knockdown of TIAM1 was able to

restore the defective VEGF-induced translocation of c-Src at the

AJ and VE-cadherin phosphorylation in the absence of eNOS.

These findings suggest that NO influences F-actin formation to

regulate VEGF-induced c-Src translocation at the junctional

membrane and, therefore, the signaling cascade, leading to

disruption of the endothelial barrier. Although it has been

reported that NO is able to modulate c-Src activity through

cysteine modifications (Rahman et al., 2010), our data showed

that the lack of NO did not affect the VEGF activation of c-Src,

as confirmed by the phosphorylation of its downstream substrate

focal adhesion kinase (FAK) at Y861. This could be explained by

the usage of different cell types and/or different agonists.

However, future studies on the role of eNOS in the regulation

of Rac GEFs might provide further insight into the modulation of

VEGF-induced Src translocation to the membrane.

We have recently shown that phosphorylation of eNOS on

S1176 appears crucial for the actions of Akt in promoting

angiogenesis because eNOS S1176D, but not eNOS S1176A,

alleles rescue the defects caused by loss of Akt1 in mice

(Schleicher et al., 2009). However, the crucial role of this

phosphorylation site in regulating barrier function was unknown.

Here, we demonstrate that the increase in permeability induced

by VEGF and histamine, as well as the acute inflammatory

response to carrageenan, are markedly reduced in knock-in mice

expressing the less-active eNOS S1176A compared to mice

expressing the active form of the enzyme, eNOS S1176D. This

suggests that this phosphorylation site of eNOS is crucial for

its function during changes in in vivo permeability and

inflammation.

Collectively our data show that endogenous eNOS-derived NO

‘fine tunes’ the levels of Rho GTPases by regulating VE-

cadherin-mediated changes in junctional permeability. We show

that pharmacological blockade of eNOS or the genetic loss of

endogenous eNOS stabilizes cortical actin by sustained

recruitment of TIAM1 to the VE-cadherin complex, thereby

promoting Rac activation and AJ stabilization. This effect is

reversible by NO, TIAM1–Rac inhibition and is physiologically

Fig. 8. Schematic view of VEGF-induced NO regulation of the VE-cadherin complex. In basal conditions, the accumulation of TIAM1 at the VE-

cadherin complex in basal conditions is limited by eNOS-derived NO (left panel), while VEGF-induced Rho activation and Src-dependent VE-cadherin

phosphorylation are promoted by the NO. The lack of NO increases the amount of VE-cadherin-bound TIAM1, leading to the formation of stable cortical actin in

basal and VEGF-stimulated conditions (right panel), reduces stress fiber formation and VE-cadherin phosphorylation, with consequent improvement of

endothelial barrier function.

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relevant as revealed in eNOS knockin mice. The regulation of AJ

dynamics strongly supports the autocrine role of NO inendothelial cells, separate from the canonical, paracrine role of

eNOS in controlling blood flow and hemodynamics. The idea

that eNOS-derived NO regulates junctional dynamics is

supported by recent results showing that NO post-translationally nitrosylates b-catenin and p120-catenin

promoting its dissociation from VE-cadherin (Marın et al.,

2012; Thibeault et al., 2010), and tyrosine nitrates p190RhoGAP

(Siddiqui et al., 2011) effects leading to enhanced permeability.Our data showing enhanced TIAM1–VE-cadherin-mediated

stabilization of cortical actin explains why mice deficient in

eNOS or mice harboring a S1176A allele exhibit reduced

vascular leakage in vivo and sheds light on why anti-VEGFtherapy reduces vascular permeability but promotes increased

blood pressure in patients.

Materials and MethodsAnimals

WT C57BL/6 and eNOS2/2 mice were purchased from Jackson’s laboratory (,8–10 weeks of age). Knock-in mice carrying the S1176A and S1176D mutations inthe endogenous eNOS locus were generated as previously described (Schleicheret al., 2009). Congenic male, 8- to 14-week-old S1176D eNOS, S1176A eNOS andcontrol littermates were used. All experiments were approved by the InstitutionalAnimal Care and Use Committee of Yale University.

Carrageenan air-pouch

Air-pouches were generated as previously described (Di Lorenzo et al., 2009). Onday 6, 0.5 ml of carrageenan suspension (1% w/v) in sterile saline was injectedinto the pouch. Exudates and leukocytes were recovered from the pouches at4 hours post-carrageenan. Migrated leukocytes, mainly neutrophils, were countedby use of Trypan Blue, and the total volume of exudate was measured.

Cell culture

Primary human dermal microvascular endothelial cells (HDEMCs) were culturedin EGM2-MV growth medium (Cambrex) as previously described (Di Lorenzoet al., 2009) and were used between passages four and eight for the experimentsdescribed. Eco-Pack2-mT packaging cells producing retroviruses containing thePolyoma middle T-antigen (Primo et al., 2000), were cultured in high glucoseDMEM with 10% FBS. Mouse lung endothelial cells (MLECs), isolated from WTC57BL/6 or congenic eNOS2/2 mice, were immortalized by the Polyoma middleT-antigen and cultured in EBM-2/EGM-2 MV (Cambrex) supplemented with4 mM L-glutamine, penicillin-streptomycin, 100 mg/ml heparin and 15% heat-inactivated FBS, in 37 C and 5% CO2. Immortalized MLECs were used betweenpassages 3 and 12.

Immunofluorescent staining in cultured cells

HDEMCs treated with eNOS and control siRNA, or WT and eNOS2/2 MLECswere grown to confluence, serum starved with 0.15% FBS/EBM-2 overnight andstimulated with 100 ng/ml VEGF (Genentech) for the indicated time. In someexperiments, ECs were treated with vehicle, Rac inhibitor (50 mM, 6 h,Calbiochem) or SNP (Sigma). Cells were washed with PBS and fixed in 3.7%paraformaldehyde for 10 minutes, blocked with 5% BSA in PBS for 10 minutes,permeabilized with 0.2% Triton X-100 in PBS for 5 minutes, and incubatedovernight with antibodies against VE-cadherin (1:200, Santa Cruz Biotechnologyfor HDEMCs; 1:200, Pharmingen for MLECs) and Src (Cell Signaling).Appropriate secondary antibodies, conjugated to Alexa-Fluor-594 or Alexa-Fluor-488 (1:200, Molecular Probes) were applied and Alexa-Fluor-488-labeledphalloidin (1:50, Molecular Probes) was used to visualize the F-actin. DAPI wasused to visualize nuclei. Images were captured with a Zeiss Axiovertepifluorescence microscope followed by deconvolution using the Openlabsoftware (Improvision, Lexington, MA). Images in Fig. 2E and Fig. 6B havebeen captured at a 0.3 mm slice thickness (z-stack) by using a Zeiss Axiovertepifluorescence microscope and a 636 oil immersion objective, followingdeconvolution by Openlab software (Improvision, Lexington, MA).

In situ phalloidin labeling of F-actin in whole blood vessels

Intrapulmonary arteries from WT or eNOS2/2 mice were dissected, cut openlongitudinally, pinned with surgical needles and incubated in serum-free DMEMfor 16 hours at 37 C to quiesce the blood vessels. Specimens were stimulated with100 ng/ml VEGF (Genentech) for 15 or 30 minutes and immediately fixed with4% paraformaldehyde (15 minutes, 4 C). After permeabilization with 0.3% Triton

X-100 (30 minutes at room temperature), Alexa-Fluor-488-labeled phalloidin(1:100, Molecular Probes) was used for ‘en face’ staining of F-actin (15 minutes atroom temperature). Fluorescence images of actin in the endothelium were capturedusing a Zeiss microscope and analyzed using Openlab software.

In vitro permeability assay

The barrier function of HDEMCs was assayed by measuring the resistance of acell-covered electrode by using an ECIS instrument (Applied BioPhysics). An8W10E plate was incubated for 15 minutes with L-cysteine (10 mM) solution,followed by 0.1% gelatin for 30 minutes. ECs were plated on the electrode at46103 cells/well. The day after, ECs were transfected with small interference RNA(siRNA) for eNOS or control siRNA. After 72 hours, HDMECs were starvedovernight in 0.15% FBS/EBM-2 and stimulated with VEGF (100 nM). In anotherset of experiments, HDEMCs grown on an 8W10E plate were starved overnight in0.15% FBS/EBM-2 with L-NAME (1 mM) or vehicle, and stimulated with VEGF(100 ng/ml). Resistance was monitored and expressed as the fractional resistanceof the basal value. In a further set of experiments, HDEMCs were plated infibronectin-coated in vitro permeability transwells (0.45 mm, BD DiscoveryLabware) were treated with 10 mM control peptide (AP) or 10 mM cavtratin (AP-Cav) saline in 0.15% FBS/EBM-2. Transwells were subjected to transmonolayerresistance (TMR) measurement by placing EVOM voltohmmeter (World PrecisionInstruments) electrodes above and below the cell monolayer. Resistance readingswere taken at 0, 5 and 30 minutes after VEGF stimulation for the peptide- or drug-treated wells, corrected for background and total transwell surface area, andexpressed as the percentage decrease in resistance from baseline (time50).

Western blotting

WT and eNOS2/2 MLECs, HDEMCs treated with control or eNOS siRNA, ortreated with L-NAME (1 mM) were cultured for 72–96 hours before quiescing in0.15% FBS/EBM-2 for 6–8 hours before stimulation with 100 ng/ml of VEGF(Genentech) at different time points. Cells were lysed in modified RIPA buffercontaining 50 mM Tris-HCl pH 7.4, 0.1 mM EGTA, 0.1 mM EDTA, 1% NP-40,0.1% SDS, 0.1% DOC, 20 mM NaF, 1 mM Na4P2O7, 1 mM Pefabloc SC, 1 mMsodium orthovanadate and protease inhibitor cocktail (Roche Diagnostics). Solubleproteins were resolved by SDS-PAGE and western blotted for P-1177-eNOS, P-1175-VEGFR2, VEGFR2, P-416-Src, Src, FAK, MLC, P-Thr18/Ser19-MLC2(Cell Signaling), P-861-FAK (Invitrogen), eNOS, HSP90 (BD TransductionLaboratories), b-actin (Sigma), MAPK42/44, P-MAPK42/44, VEGFR2,phosphotyrosine (4G10, Millipore) or VE-cadherin (Santa Cruz Biotechnology).The secondary antibodies used for all western blots were conjugated directly toinfrared fluorescent dyes (Alexa-Fluor-780 from Molecular Probes or IRDye800from Li-Cor) to allow direct detection on the OdysseyH Infrared Imaging System(Li-Cor). For immunoprecipitations, cells were lysed in a buffer containing 10 mMHEPES-NaOH pH 7.4, 100 mM NaCl, 50 mM b-glycerophosphate, 2 mM MgCl2,5 mM EGTA, 5 mM EDTA, 1% Igepal (Sigma), 1 mM orthovanadate, 1 mM NaFand protease inhibitors (Roche). Clarified cell extracts were incubated with 2 mg ofantibody against VE-cadherin (Santa Cruz Biotechnology) or phosphotyrosine(PY20). Antigen–antibody complexes were collected using protein-G–Sepharosebeads (Sigma). SDS-PAGE and immunoblotting was performed as describedabove, and used the antibody against VE-cadherin (Santa Cruz Biotechnology) orphophotyrosine (PY20, 4G10).

Rac and Rho assay

MLECs were grown to confluence, quiesced in serum-free EBM-2 for 36–48 hoursand stimulated with 100 ng/ml VEGF for the indicated amount of time. Afterwashing, cells were lysed with Mg2+ lysis buffer (MLB, Upstate) containing25 mM HEPES pH 7.5, 250 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 1% IgepalCA-630 and 10% glycerol supplemented with 20 mM NaF, 1 mg/ml Leupeptin,1 mg/ml Aprotinin and 1 mM sodium orthovanadate. After centrifugation(14,000 rpm, 10 minutes, 4 C) of cell lysates, 250 mg of protein was pre-cleared with 50 ml of 50% GST–Sepharose slurry for 10 minutes at 4 C.Subsequently, 10 mg of GST–PAK-1 agarose beads were added to all samplelysates and incubated at 4 C for 1 hour. The samples were resolved on SDS-PAGEand western blotted for Rac protein. Lysates before GST–PAK-1 pulldown werealso western blotted for total amount of Rac. In a similar manner, the Rho assayMLECs were grown to confluence, quiesced in serum-free EBM-2 for 36–48 hoursand stimulated with 100 ng/ml VEGF for the indicated amount of time and lysed inRho lysis buffer containing 50 mM Tris-HCl (pH 7.2), 500 mM NaCl, 10 mMMgCl2, 1% Triton X-100, 0.1% SDS, 0.5% DOC, 20 mM NaF, 1 mg/mlLeupeptin, 1 mg/ml Aprotinin and 1 mM sodium orthovanadate. The lysates(500 mg of protein from each sample) were pre-incubated with 100 nM GTPgS(30 minutes, 25 C) to label the maximal amount of GTP bound Rho prior to GST-Rhotekin agarose beads (30 minutes, 4 C), followed by wash with Rho wash buffercontaining 50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 10 mM MgCl2 and 1%Triton X-100 with protease and phosphatase inhibitors mentioned above. Sampleswere separated by SDS-PAGE and western blotting for Rho protein. Lysatesbefore GST-Rho pulldown were also western blotted for total amount of Rho.

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AcknowledgementsWe thank Mr. Roger Babbitt for skilled technical assistance. Theauthors have no competing financial interests.

Author contributionsA.D.L., M.I.L., T.M., S.L.E., M.S., M.K., J.Y. performed criticalexperiments and analyzed data; P.L.H. generated the mice; A.D.L.,M.I.L., P.L.H. and W.C.S. conceptualized the study and wrote themanuscript.

FundingThis work was supported by the American Heart Association (grantto A.D.L.); the National Institutes of Health [grant numbers R01HL64793, R01 HL61371, R01 HL081190, RO1 HL096670, P01HL1070205 to W.C.S., RO1 DK082600 to Y.I.]. Deposited in PMCfor release after 12 months.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.115972/-/DC1

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