Free Radical Biology & Medicine 48 (2010) 128–135

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Free Radical Biology & Medicine

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Original Contribution

Ascorbate protects endothelial barrier function during septic insult:Role of protein phosphatase type 2A

Min Han a,b, Suresh Pendem a, Suet Ling Teh a, Dinesh K. Sukumaran c, Feng Wu a, John X. Wilson a,⁎a Department of Exercise and Nutrition Sciences, State University of New York at Buffalo, 3435 Main Street, Buffalo, NY 14214-8028, USAb Division of Nephrology, Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan,Hubei 430030, People's Republic of Chinac Department of Chemistry, State University of New York at Buffalo, Buffalo, NY 14260, USA

Abbreviations: BSA, bovine serum albumin; DHAA,2′,7′-dichlorodihydrofluorescein; IFN-γ, interferon-γ;phosphate-buffered saline; p-NPP, p-nitrophenyl phphosphatase type 2A; PP2Ac, PP2A catalytic subunit.⁎ Corresponding author. Fax: +1 716 829 2428.

E-mail address: jxwilson@buffalo.edu (J.X. Wilson).

0891-5849/$ – see front matter © 2009 Elsevier Inc. Adoi:10.1016/j.freeradbiomed.2009.10.034

a b s t r a c t

a r t i c l e i n f o

Article history:Received 13 July 2009Revised 7 October 2009Accepted 13 October 2009Available online 17 October 2009

Keywords:AscorbateEndothelial cellsInterferon-γLipopolysaccharideNADPH oxidaseOccludinPermeabilityProtein phosphatase type 2ASepsisFree radicals

Endothelial barrier dysfunction contributes to morbidity in sepsis. We tested the hypothesis that raising theintracellular ascorbate concentration protects the endothelial barrier from septic insult by inhibiting proteinphosphatase type 2A. Monolayer cultures of microvascular endothelial cells were incubated with ascorbate,dehydroascorbic acid (DHAA), the NADPH oxidase inhibitors apocynin and diphenyliodonium, or the PP2Ainhibitor okadaic acid and then were exposed to septic insult (lipopolysaccharide and interferon-γ). Understandard culture conditions that depleted intracellular ascorbate, septic insult stimulated oxidant productionand PP2A activity, dephosphorylated phosphoserine and phosphothreonine residues in the tight junction-associated protein occludin, decreased the abundance of occludin at cell borders, and increased monolayerpermeability to albumin. NADPH oxidase inhibitors prevented PP2A activation and monolayer leak, showingthat these changes required reactive oxygen species. Okadaic acid, at a concentration that inhibited PP2Aactivity and monolayer leak, prevented occludin dephosphorylation and redistribution, implicating PP2A inthe response of occludin to septic insult. Incubation with ascorbate or DHAA raised intracellular ascorbateconcentrations and mitigated the effects of septic insult. In conclusion, ascorbate acts within microvascularendothelial cells to inhibit septic stimulation of oxidant production by NADPH oxidase and thereby preventsPP2A activation, PP2A-dependent dephosphorylation and redistribution of occludin, and disruption of theendothelial barrier.

© 2009 Elsevier Inc. All rights reserved.

Rising rates of hospitalization and death due to sepsis show thatthis disease is a worsening health care problem [1]. Endothelial barrierdysfunction occurs in systemic capillaries and venules of patients withsepsis. This dysfunction increases the microvessels' permeability tomacromolecules, such as albumin, and frequently leads to plasmaextravasation, edema, organ failure, and septic shock [2–5]. Becauseno effective pharmacological therapy is available to prevent theincrease in permeability, there is a pressing need to identify themolecular mechanisms of this pathology and potential treatments.

Endothelial barrier dysfunction is part of the systemic inflamma-tory response commonly initiated by pathogenic bacteria, theircomponents (e.g., lipopolysaccharide; LPS), and inflammatory cyto-kines (e.g., interferon-γ; IFN-γ). Studies of barrier function inendothelial and epithelial cell cultures have shown that these septic

dehydroascorbic acid; H2DCF,LPS, lipopolysaccharide; PBS,osphate eiwo; PP2A, protein

ll rights reserved.

insults decrease phosphorylation of threonine residues in occludin,stimulate redistribution of occludin and other tight junction proteinsaway from cell borders, and increase paracellular permeability tomacromolecules [6–8]. It has also been observed, in epithelial cellcultures, that serine/threonine protein phosphatase 2A (PP2A)dephosphorylates threonine residues in occludin and this change isassociated with disassembly of tight junctions and increased para-cellular permeability [9–11]. Septic insult (LPS + IFN-γ) in micro-vascular endothelial cell cultures stimulates production of NADPHoxidase-derived superoxide, which forms peroxynitrite that nitratestyrosine residues in the PP2A catalytic subunit (PP2Ac). This nitrationin PP2Ac is associated with PP2A activation and endothelial barrierdysfunction [12].

Pretreatment with the antioxidant ascorbate attenuates theincrease in monolayer permeability caused by LPS in aortic endothe-lial cell cultures [13]. Parenteral administration of ascorbate alsodecreases edema formation in LPS-injected or burn-injured animals,as well as in patients with severe burn injury [14–19]. This study wasdesigned to identify the mechanism of action of ascorbate. We testedthe hypothesis that intracellular ascorbate stabilizes the endothelialbarrier during septic insult by inhibiting PP2A.

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Materials and methods

Cell cultures

The State University of New York at Buffalo Institutional AnimalCare and Use Committee approved the procedures. Microvascularendothelial cells were isolated from murine skeletal muscles andgrown in DMEM/F12 medium containing 10% fetal bovine serum,2 mM L-glutamine, 5 U/ml heparin, 100 U/ml penicillin, 100 μg/mlstreptomycin, and 25 μg/ml endothelial cell growth supplement, asdescribed previously [20].

Chemicals

L-Ascorbic acid was purchased from Sigma–Aldrich. This moleculebecomes ascorbate when put into solution. Dehydro-L-(+)-ascorbicacid dimer (DHAA)waspurchased as a dry powder fromFluka Chemical(a division of Sigma–Aldrich) and stored at −20°C. The certificate ofanalysis indicated that the purity of the DHAAwas 98.3%. The identity ofthe product was confirmed to be DHAA by spectroscopy in the vendor'slab, as well as by HPLC-based electrochemical assay and quantitativeNMR in our labs. Fetal bovine serum was purchased from Hyclone.Catalase and p-nitrophenyl phosphate eiwo (p-NPP) were fromCalbiochem. Anti-occludin antibody (Cat. No. 71-1500), L-glutamine,penicillin, phosphate-buffered saline (PBS), and streptomycin werefrom Invitrogen. Anti-phosphoserine and anti-threonine antibodies(Cat. Nos. sc-81514 and sc-5267, respectively) were from Santa CruzBiotechnology. Anti-PP2Ac antibody (Cat. No. 610556), endothelial cellgrowth supplement, and protein A–agarose beads were from BDBiosciences. Anti-β-actin antibody (Lot 21107) was from Rockland.CellTiter-Fluor cell viability assay kit was from Promega. CompetitiveELISA kit for murine type IV collagen (1014 Strip Plate) was purchasedfrom Exocell. Protease inhibitor cocktail was from Roche. Radio-immunoprecipitation assay buffer was from Cell Signaling Technology.Bicinchoninic acid protein assay kit was from Pierce. All other chemicalswere obtained from Sigma–Aldrich.

Experimental procedures

Microvascular endothelial cells were maintained without endo-thelial cell growth supplement for 7 days, and the serumconcentrationwas decreased to 2% for the final 2 days before the cells were used forexperiments. The cells were incubated with ascorbate, DHAA, or otherdrugs for the indicated periods before exposure to either septic insult[25 ng/ml LPS (Escherichia coli 055:B5) and 100 U/ml IFN-γ dissolvedin bovine serum albumin (BSA) solution] or control (BSA only).

Intracellular ascorbate concentrations were determined by HPLCwith electrochemical detection using a previously described method[20]. Cells in 35-mm dishes were washed twice with 2.5 ml of ice-coldPBS and then scrape-harvested into 500 μl of cold water. Aliquotswere combinedwith metaphosphoric acid (final concentration 0.85%)for subsequent ascorbate assay and the remainder of the cell harvestwas analyzed for total cell protein content.

Oxidant production was measured using 2′,7′-dichlorodihydro-fluorescein diacetate (H2DCF diacetate). This molecule diffusespassively into cells, is deesterified of diacetate by intracellularesterases, and then is oxidized to fluorescent dichlorofluorescein byoxidants such as peroxynitrite and hydroxyl radical [21,22]. Confluentmicrovascular endothelial cells, in 96-well plates, were washed withPBS and incubated 30 min with H2DCF diacetate (10 μM) in the dark.Subsequently the cells were washed twice with PBS and theirfluorescence was measured at excitation and emission wavelengthsof 485/20 and 528/20 nm, respectively.

The permeability of endothelial monolayers to Evans blue-coupledBSA was determined as described previously [12]. In brief, themicrovascular endothelial cells were grown on gelatin-coated inserts

(3 μm pore size) in 12-well plates (BD Biosciences). Evans blue-coupled BSA and uncoupled BSA were added to the upper chamberand lower chamber, respectively, and incubated 1 h with the cells.Finally, the Evans blue-coupled BSA in the lower chamber wasmeasured at 595 nm.

Cell viability was measured using the Promega CellTiter-Fluor cellviability assay according to the manufacturer's protocol. Briefly,endothelial cells in 96-well plates were incubated with 100 μl ofCellTiter-Fluor reagent for 30 min at 37°C and then fluorescence wasdetermined at 400 nm/505 nm. Type IV collagen was measured byExocell 1014 Strip Plate competitive ELISA according to the manufac-turer's instructions.

PP2A activity was measured as okadaic acid-inhibitable phospha-tase activity by themethod described previously [12]. A 100-μl aliquotof cell harvest (containing a protein concentration of 500 μg/ml) wasmixed with 100 μl of assay buffer (5 mM p-NPP, 3 mMMnCl2, 0.1 mMEDTA, 50mMTris–Cl, pH 7.0) with orwithout 50 nM okadaic acid, andthen it was incubated 10 min at 30°C. The hydrolysis of p-NPP wasdetermined at 405 nm and the PP2A activity was calculated as thedifference between total phosphatase activity and okadaic acid-insensitive phosphatase activity.

Western blot analysis of proteins was performed as follows. Cellswere rinsed twice with PBS and scrape-harvested in radioimmuno-precipitation assay buffer containing protease inhibitor cocktail. Thecell harvests were sonicated on ice and then centrifuged for 10 min at14,000 g at 4°C. Next, the supernatants were collected and the proteinconcentration was determined by bicinchoninic acid protein assay.Cell proteins were separated by 10% SDS–polyacrylamide gelelectrophoresis and transferred to polyvinylidene difluoride mem-brane. Then the blocked membranes were incubated with anti-PP2Acantibody or anti-β-actin antibody for 2 h, followed by incubation withhorseradish peroxidase-conjugated secondary antibodies for 1 h atroom temperature. Protein bands were detected by ECL chemilumi-nescence and quantified with Quantity One (Bio-Rad) software.

Immunoprecipitation of proteins was performed using aliquots ofcell harvests containing 800 μg of total protein each. The cell harvestswere incubated overnight at 4°C with anti-occludin antibody and thenincubated for 3 h at 4°C with protein A–agarose beads. Theimmunoprecipitates were washed four times with ice-cold radio-immunoprecipitation assay buffer and then boiled in Laemmli'ssample buffer and separated by electrophoresis. Subsequently theproteins were transferred to polyvinylidene difluoride membrane.Finally, specific antibodies were used to detect phosphoserine,phosphothreonine, and occludin.

Immunofluorescence microscopy was performed using cellsgrown on glass coverslips. The cells were fixed by incubation with3% paraformaldehyde for 20 min, permeabilized by incubation with0.2% Triton X-100 for 20 min, and blocked by incubation with 5% BSAfor 30 min at room temperature. Next, the cells were incubatedsequentially with anti-occludin antibody for 2 h and goat AlexaFluor488-conjugated anti-rabbit IgG antibody for 1 h. Finally, the cells'labeled occludin was visualized by fluorescence microscopy.

Statistics

Data are presented as means±SD and were analyzed with thePrism statistical program. Comparisons between mean values wereperformed with one-way ANOVA followed by the Tukey multiplecomparison test. Pb0.05 was considered significant.

Results

Intracellular ascorbate and oxidant production

Microvascular endothelial cells were incubated for 12 h withascorbate, DHAA, or DMEM vehicle and then were incubated for 24 h

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with LPS+ IFN-γ (septic insult) or BSA (control). The concentration ofascorbate and DHAA added to the cell cultures was 500 μM, becausethis was the peak ascorbate concentration achieved in plasma by anascorbate infusion protocol that inhibited edema formation inpatients [19]. Ascorbate was not detectable in the cells or mediumof cell cultures that were treated with vehicle instead of ascorbate orDHAA (Fig. 1A). Intracellular ascorbate concentrations were elevatedto similar levels by incubation with either ascorbate or DHAA (Fig.1A). Delayed exposure to LPS + IFN-γ did not alter the intracellularascorbate concentration derived from exogenous ascorbate or DHAA(Fig. 1A).

LPS + IFN-γ increased oxidant production in cells that had notreceived ascorbate or DHAA (Fig. 1B). This effect of LPS + IFN-γ wasabolished in cells previously incubated with ascorbate or DHAA (Fig.1B). Although donation of reducing equivalents from ascorbate totransition metals may increase the concentrations of ascorbate radicaland hydrogen peroxide [23], this mechanism did not account for theeffect of ascorbate on the oxidant production we measured, becauseDHAA had an equally large effect (Fig. 1B). Taken together, the resultsof Figs. 1A and 1B indicate that intracellular ascorbate inhibits theoxidant production induced by septic insult.

Endothelial barrier dysfunction, collagen production, and PP2A activity

LPS + IFN-γ increased microvascular endothelial cell monolayerpermeability to Evans blue-coupled BSA markedly (Fig. 2A). Thiseffect was prevented by pretreating the cells with ascorbate or DHAA

Fig. 1. Incubations with ascorbate and DHAA increase intracellular ascorbateconcentration and prevent LPS + IFN-γ-induced oxidant production in microvascularendothelial cells. The cells were incubated with ascorbate (AA; 500 μM), DHAA(500 μM), or DMEM vehicle for 12 h. Subsequently they were incubated with LPS(25 ng/ml) + IFN-γ (100 U/ml) or vehicle (BSA; Control) for 24 h. (A) Intracellularascorbate concentration was determined by HPLC-based electrochemical assay andexpressed as nmol ascorbate/mg cell protein. (B) Oxidant production was assessedby dichlorodihydrofluorescein oxidation assay and expressed as a percentage of thevalue for vehicle control. Plotted are means±SD for three experiments. ⁎Pb0.05compared to vehicle control. #Pb0.05 compared to the combination of vehicle(DMEM) and LPS + IFN-γ.

Fig. 2. Ascorbate and DHAA prevent LPS + IFN-γ-induced barrier dysfunction.Microvascular endothelial cells were treated as described for Fig. 1. (A) Permeabilityof endothelial monolayers was measured with Evans blue-coupled albumin andexpressed as a percentage of the value for vehicle control. (B) Cell viability wasmeasured by CellTiter-Fluor cell viability assay and expressed as a percentage of thevalue for vehicle control. Plotted are means±SD for four experiments. ⁎Pb0.05compared to vehicle control. #Pb0.05 compared to the combination of vehicle (DMEM)and LPS + IFN-γ.

(Fig. 2A). Although reactions of ascorbate with transition metals mayincrease hydrogen peroxide in cell culture media, addition of ahydrogen peroxide scavenger (catalase, 500 U/ml) to the medium didnot alter the protective effects of ascorbate and DHAA on monolayerpermeability (data not shown). Cell viability was not altered by thesetreatments (Fig. 2B). The similar viabilities of control and LPS + IFN-γ-stimulated cells suggest that cell death was not the cause of thechange in monolayer permeability induced by septic insult.

To elucidate the mechanism by which intracellular ascorbatestabilizes the endothelial barrier, type IV collagen production, PP2Acprotein expression, and PP2A activity were measured. Incubations ofcell cultures for 36 hwith ascorbate or DHAA increased the productionof type IV collagen and these effects were not altered by addition ofLPS + IFN-γ during the final 24 h (Fig. 3). LPS + IFN-γ did not changePP2A protein expression (Fig. 4A) but did increase PP2A activity (Fig.4B). Furthermore, both ascorbate and DHAA inhibited the PP2Aactivation induced by LPS + IFN-γ (Fig. 4B).

NADPH oxidase is the principal source of superoxide in LPS +IFN-γ-treated microvascular endothelial cells, and inhibition ofNADPH oxidase by apocynin or diphenyliodonium (DPI) preventsstimulation by LPS + IFN-γ of superoxide production [12,24].Apocynin and DPI had no significant effects on the basal level ofendothelial cell monolayer permeability or PP2A activity (i.e.,absent LPS + IFN-γ; Figs. 5 and 6). The increase in monolayerpermeability induced by LPS + IFN-γ was prevented by apocyninand DPI (Fig. 5). The induction of PP2A activity by LPS + IFN-γwas inhibited by apocynin and DPI, whereas PP2Ac proteinexpression was not changed (Fig. 6). These results implicated

Fig. 3. Incubation with ascorbate and DHAA increases type IV collagen production in thepresence or absence of delayed LPS + IFN-γ. Microvascular endothelial cells weretreated as described for Fig. 1 and then aliquots of medium were collected forcompetitive ELISA of type IV collagen. ⁎Pb0.05 compared to vehicle control. #Pb0.05compared to the combination of vehicle (DMEM) and LPS + IFN-γ.

Fig. 5. NADPH oxidase inhibitors prevent LPS + IFN-γ-induced barrier dysfunction inmicrovascular endothelial cell monolayers. The cells were incubated with apocynin(250 μM), DPI (5 μM), or DMEM vehicle for 1 h. Subsequently they were incubated withLPS (25 ng/ml) + IFN-γ (100 U/ml) or vehicle (BSA; Control) for 24 h and thenmonolayer permeability was measured. Plotted are means±SD for three experiments.⁎Pb0.05 compared to vehicle control. #Pb0.05 compared to the combination of vehicle(DMEM) and LPS + IFN-γ.

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NADPH oxidase in the endothelial barrier dysfunction and PP2Aactivation after exposure to LPS + IFN-γ.

Occludin phosphorylation and localization

LPS + IFN-γ treatment of cells decreased the abundance ofphosphorylated serine and threonine residues in occludin immuno-precipitates (Fig. 7). The decreased phosphorylation of occludin wasassociated with a loss of the protein from regions of cell–cell contact(Fig. 7). Pretreatment of the cells with ascorbate or DHAA preventedthese effects of LPS + IFN-γ (Fig. 7). To explore whether PP2Amediated the dephosphorylation of occludin, okadaic acid was used toinhibit PP2A specifically. The concentration of okadaic acid we usedhas been shown to inhibit PP2A activity and septic induction ofendothelial monolayer leak [12]. Okadaic acid prevented the LPS +IFN-γ-induced decrease in phosphoserine and phosphothreonine

Fig. 4. Ascorbate and DHAA prevent LPS + IFN-γ-induced PP2A activation.Microvascular endothelial cells were treated as described for Fig. 1. (A) PP2Ac and β-actin (loading control) protein expression was measured by Western blot analysis, andthe cell treatments (LPS + IFN-γ, ascorbate, and DHAA) had no detectable effect on thePP2Ac:β-actin ratio. A representative Western blot is shown. (B) PP2A activity wasmeasured as okadaic acid-inhibitable phosphatase activity and expressed as apercentage of the value for vehicle control. Plotted are means±SD for nineexperiments. ⁎Pb0.05 compared to vehicle control. #Pb0.05 compared to thecombination of vehicle (DMEM) and LPS + IFN-γ.

levels and maintained the normal localization of occludin at cellborders (Fig. 8). These results suggested that intracellular ascorbateprotects the endothelial barrier from septic insult by preventingPP2A-mediated dephosphorylation and redistribution of occludin.

Discussion

Septic insults induce leaks in endothelial and epithelial barriers bydisrupting intercellular tight junctions [6–8]. This disruption ismediated by activation of PP2A [12]. Because endothelial barrierdysfunction contributes tomorbidity in sepsis [2–5], there is an urgentneed to identify the molecular mechanisms of this pathology andpotential treatments. This study discovered that intracellular ascor-bate protects endothelial barrier function during septic insult and thatthis protection is associated with inhibition of oxidant production,PP2A activation, and occludin dephosphorylation and redistribution.

Fig. 6. NADPH oxidase inhibitors prevent LPS + IFN-γ-induced PP2A activation.Microvascular endothelial cells were treated as described for Fig. 4. (A) PP2Ac and β-actin protein expressionwasmeasured byWestern blot analysis and the cell treatments(LPS + IFN-γ, apocynin, and DPI) had no detectable effect on the PP2Ac:β-actin ratio. Arepresentative Western blot is shown. (B) PP2A activity. Plotted are means±SD for sixexperiments. ⁎Pb0.05 compared to vehicle control. #Pb0.05 compared to thecombination of vehicle (DMEM) and LPS + IFN-γ.

Fig. 7. Ascorbate and DHAA prevent LPS + IFN-γ-induced dephosphorylation andredistribution of occludin. Microvascular endothelial cells were treated as described forFig. 1. (A) p-Serine and p-threonine levels were measured in occludin immunopreci-pitates by Western blot analysis. A representative Western blot is shown. (B) Ratios ofphosphoserine:occludin and phosphothreonine:occludin in occludin immunoprecipi-tates. Plotted are means±SD for three experiments. ⁎Pb0.05 compared to vehiclecontrol. #Pb0.05 compared to the combination of vehicle (DMEM) and LPS+ IFN-γ. (C)Cellular distribution of immunoreactive occludin was visualized by immunofluores-cence microscopy. Representative micrographs are shown.

Fig. 8. LPS + IFN-γ-induced dephosphorylation and redistribution of occludin areprevented by the PP2A inhibitor okadaic acid. Microvascular endothelial cells wereincubated with okadaic acid (OA; 0.5 nM) or DMEM vehicle for 1 h and then incubatedwith LPS+ IFN-γ or vehicle (BSA; Control) for 24 h. (A) p-Serine and p-threonine levelswere measured in occludin immunoprecipitates by Western blot analysis. Arepresentative Western blot is shown. (B) Ratios of phosphoserine:occludin andphosphothreonine:occludin in occludin immunoprecipitates. Plotted are means±SDfor three experiments. ⁎Pb0.05 compared to vehicle control. #Pb0.05 compared to thecombination of vehicle (DMEM) and LPS + IFN-γ. (C) Immunofluorescencemicrographs of occludin in microvascular endothelial cells.

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There is abundant evidence that ascorbate influences endothelialbarrier function under certain conditions. Observations in patientswith scurvy and in scorbutic animals show that severe ascorbatedeficiency can increase capillary permeability and edema formation,even in the absence of sepsis [25–27]. In contrast, administration ofascorbate does not affect acutely the microvascular permeability toprotein in normal, nonseptic animals. For example, bolus intravenousinjection of ascorbate (100 mg/kg) does not change microvascularpermeability in normal rats, even though this dose is large enough toprevent the microvascular leak induced by intravenous injection ofarachidonate [28].

Why endothelial barrier dysfunction occurs under nonseptic,scorbutic conditions in situ is not known. In vitro approaches to

solving this problem have yielded conflicting results. Addition ofascorbate to the medium of nonseptic endothelial cell cultures hasbeen reported to decreasemonolayer permeability tomacromolecules

Fig. 9. Diagram of the mechanism by which intracellular ascorbate and okadaic acid(OA) prevent the induction of endothelial barrier dysfunction by LPS + IFN-γ. The solidarrows indicate stimulation and the dotted arrows indicate inhibition.

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in some studies [29,30]. For instance, incubation of EA.hy926endothelial cells with extracellular ascorbate increases intracellularascorbate concentration and inhibits the diffusion of inulin (molecularweight range 5000–5500) across the endothelial monolayer [30]. Also,daily addition of ascorbate for 5 days to artery endothelial cell culturesinhibits the trans-monolayer diffusion of small and large fluoresceindextrans (molecular weights 4000 and 70,000, respectively) [29]. Inthe present experiments, microvascular endothelial cell monolayercultures that were grown under standard conditions did not containdetectable levels of ascorbate in either the cells or the medium.Compared to these ascorbate-deficient cultures, elevation of intracel-lular ascorbate concentration by incubation with ascorbate or DHAAdid not change monolayer permeability to Evans blue-coupled BSA(molecular weight 70,000) in the absence of septic insult. This failureof ascorbate to alter monolayer permeability under nonsepticconditions may be clinically relevant because it was observed inexperiments with endothelial cells derived from exchange vessels(capillaries and venules) and with a macromolecule (albumin) that isabundant in plasma.

Ascorbate clearly has an important role in sepsis. Critically illpatients have decreased plasma levels of ascorbate [31–37]. Further-more, in septic patients the plasma ascorbate concentration correlatesinversely with multiple organ failure incidence and directly withsurvival [31,33]. Experiments with animal models have shown thatascorbate increases survival after septic insult [38–41]. There is alsoclinical evidence that parenteral administration of ascorbate mayimprove outcome. In a randomized, prospective, double-blind,placebo-controlled trial with 226 critically ill patients, 28-daymortality was decreased in the patients who received combinedascorbate and vitamin E by intravenous infusion compared to thosewho did not [42]. Another randomized, prospective trial found adecreased incidence of organ failure and shortened ICU stay forcritically ill patients who began receiving intravenous injections ofcombined ascorbate (3 g/day for up to 28 days) and vitamin E within24 h of traumatic injury or major surgery [37]. Finally, a study ofpatients who received high-dose intravenous ascorbate (1584 mg/kg/24 h) in the first 24 h after burn injury observed that ascorbatetreatment was associated with lower resuscitation fluid volumerequirements, less weight gain, and less wound edema [19]. It may beinferred that ascorbate prevents endothelial barrier dysfunction,because the latter is a major contributor to fluid requirements andedema formation [2]. Parenteral administration of ascorbate alsodecreases edema formation in LPS-injected or burn-injured animalmodels of critical illness [14–18].

Ascorbate pretreatment attenuates the increase in monolayerpermeability to albumin caused by LPS + IFN-γ (25 ng/ml E. coli LPSand 100 U/ml IFN-γ) in microvascular endothelial cell cultures (thisstudy) and by LPS (1 μg/ml E. coli LPS) in aortic endothelial cellcultures [13]. The septic insults that increased permeability did notchange cell viability in our experiments or those of Dimmeler et al.[13]. Therefore the barrier dysfunction and protection caused byseptic insult and ascorbate, respectively, do not depend on changes inendothelial cell viability.

Reaction of ascorbate with transition metals can producehydrogen peroxide, which is a precursor of the strongly oxidizinghydroxyl radical [23]. However, the similar degree of barrierprotection conferred by ascorbate and DHAA indicates thathydrogen peroxide production in the medium is not required.Instead, the stabilization of the barrier is probably due to theintracellular ascorbate that endothelial cells derive from extracellu-lar ascorbate and DHAA.

Ascorbate and DHAA decreased monolayer permeability in LPS +IFN-γ-stimulated (septic) cells but not in unstimulated (nonseptic)control cells, despite similar increases in collagen production in theseptic and nonseptic groups. We infer that the increases in collagenproduction induced by ascorbate and DHAA were not sufficient to

decrease monolayer permeability. This inference is supported by theobservation of May et al. [30] that incubation for 18 h with 300 μMascorbate did not change the extracellular barrier to macromoleculardiffusion through endothelial cell cultures. These observations mustbe compared to those of Utoguchi et al. [29], who reported thatinhibitors of collagen synthesis prevented the decrease in monolayerpermeability caused by daily additions of ascorbate (10–100 μM) tobovine artery endothelial cells that were growing to confluence onporous membrane filters [29]. However, as remarked by May et al.[30], the ascorbate-induced decrease in permeability of the bovineartery endothelial cells alone was relatively greater than that of thefilters and extracellular matrix [29], indicating that ascorbate had aneffect on the cells beyond that due to collagen deposition.

LPS lowers intracellular cAMP concentration as it increasesmonolayer permeability in endothelial cell cultures [43]. Moreover,themonolayer leak canbe blockedwithdrugs that elevate intracellularcAMP [43]. This mechanism is unlikely to account for the protectiveeffect of intracellular ascorbate that we observed, however, becauseascorbate is a competitive inhibitor of adenylate cyclase and decreasesintracellular cAMP levels [44]. A more probable explanation of howintracellular ascorbate stabilizes the endothelial barrier during septicinsult involves NADPH oxidase, superoxide, peroxynitrite, and PP2A(Fig. 9), and these mediators will be discussed next.

In microvascular endothelial cells, LPS + IFN-γ increases proteinexpression of the NOX1 and p47phox subunits of NADPH oxidase,resulting in increased NADPH oxidase activity that becomes theprincipal source of superoxide in these cells [24]. Superoxide is aprecursor of peroxynitrite and the latter increases paracellularpermeability in endothelial cell monolayers [45,46]. Apocynindecreased the basal level of endothelial cell monolayer permeability(i.e., absent LPS + IFN-γ) in a previous experiment (Fig. 6 of Ref.[12]), but neither apocynin nor DPI had this effect in this study andthe reason for this discrepancy is not known. However, this studyobserved a protective effect of NADPH oxidase inhibitors (apocyninand DPI) on cell monolayer leak, which shows that the products ofthis enzyme's activity contribute to septic endothelial barrierdysfunction. Two previous studies also support this inference[47,48], although it should be noted that knockout of p47phox orgp91phox does not confer protection against LPS-induced death[49]. Intracellular ascorbate prevents the induction of endothelialbarrier dysfunction by inhibiting the septic activation of NADPHoxidase and by scavenging superoxide and peroxynitrite (Fig. 9).Intracellular ascorbate abrogates NADPH oxidase activation by

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preventing septic stimulation of the JAK2/STAT1/IRF1 signalingpathway that leads to p47phox expression [24].

LPS + IFN-γ increases the serine/threonine protein phosphataseactivity of PP2A in microvascular endothelial cells. The septic insultdoes not alter PP2Ac protein expression. Instead it stimulatesproduction of NADPH oxidase-derived superoxide, which then reactswith nitric oxide to form peroxynitrite that activates PP2A [12].Recent experiments with enterocytes and myocardial cells haveconfirmed that PP2A activity is increased by peroxynitrite [50,51].Peroxynitrite nitrates the tyrosine residues of PP2Ac, therebypreventing their phosphorylation and the consequent inactivation ofPP2A [12]. Additionally, PP2Ac nitrationmay also restrict the access ofmethylesterase enzymes to PP2Ac and thereby prevent demethyla-tion of the catalytic subunit and consequent inactivation of thephosphatase, because PP2Ac methylation facilitates assembly of thePP2A holoenzyme [52]. Last, nitration of PP2Ac at the cell membranemay prevent translocation of the enzyme to the cytosol and thusaccelerate dephosphorylation of occludin at the membrane [53].

The endothelial barrier dysfunction induced by septic insultevidently depends on PP2A activation, because inhibition of PP2Aactivity by okadaic acid or siRNA knockdown of PP2Ac stabilizes thebarrier [12]. This study found that NADPH oxidase inhibitors(apocynin and DPI) and intracellular ascorbate prevent LPS + IFN-γ-induced increases in PP2A activity. Because ascorbate blocks septicactivation of NADPH oxidase, these results support the hypothesisthat intracellular ascorbate abrogates septic activation of PP2A bydecreasing the production of superoxide and its product peroxyni-trite. An alternative hypothesis is that ascorbate-derived hydrogenperoxide inhibits PP2A in endothelial cells, because hydrogenperoxide has been shown to inhibit phosphatase activity in cell-freepreparations of PP2A [54]. However, the effect on PP2A that weobserved in endothelial cells after incubation with ascorbate was alsoevident after incubation with DHAA, and therefore it is notattributable to generation of hydrogen peroxide by reactions ofascorbate with transition metals in the culture medium.

Inflammatory signals stimulate myosin light chain kinase-depen-dent contractions of the actin–myosin cytoskeleton, which widen theintercellular space for transendothelial diffusion of macromolecules,and these signals dissociate intercellular junctions [55]. Phosphory-lation of occludin on tyrosine, serine, and threonine residues, which iscontrolled by protein kinases and protein phosphatases, normallyregulates tight junction permeability [56]. It has been observed inepithelial cell cultures that PP2A dephosphorylates threonine residuesin occludin, and this change is associated with disassembly of tightjunctions and increased paracellular permeability [9–11]. This studyfound that serine and threonine residues in occludin become depho-sphorylated, and occludin becomes less abundant at cell borders,when endothelial cell monolayer permeability is increased by septicinsult. Ascorbate, DHAA, and okadaic acid prevent septic stimulationof PP2A activity and this effect is associated with preservation ofoccludin phosphorylation, occludin localization at cell borders, andlow permeability of the endothelial monolayer to albumin (Fig. 9).The modulations of PP2A activity and monolayer permeability byintracellular ascorbate are consequences of the latter preventingactivation of NADPH oxidase. Taken together, these results supportthe conclusion that intracellular ascorbate prevents microvascularendothelial barrier dysfunction after septic insult by inhibitingNADPH oxidase-dependent activation of PP2A, PP2A-induced de-phosphorylation of occludin on serine and threonine residues, andloss of occludin from tight junctions.

Acknowledgments

The excellent technical assistance of Dr. David P. Schuster andGeorge Kamenos is gratefully acknowledged. The project describedwas supported by Award No. R01AT003643 from the National Center

for Complementary and Alternative Medicine. The content is solelythe responsibility of the authors and does not necessarily representthe official views of the National Center for Complementary andAlternative Medicine or the National Institutes of Health.

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