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and Septic Vascular DysfunctionSignaling in the Pathogenesis of Septic Shock

BκA Pivotal Role of Endothelial-Specific NF-

LiuJianqiang Ding, Dongmei Song, Xiaobing Ye and Shu Fang

http://www.jimmunol.org/content/183/6/4031doi: 10.4049/jimmunol.0900105August 2009;

2009; 183:4031-4038; Prepublished online 19J Immunol 

Referenceshttp://www.jimmunol.org/content/183/6/4031.full#ref-list-1

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A Pivotal Role of Endothelial-Specific NF-�B Signaling inthe Pathogenesis of Septic Shock and Septic VascularDysfunction1

Jianqiang Ding,2*† Dongmei Song,2*† Xiaobing Ye,2*† and Shu Fang Liu3*†

Although the role of NF-�B in the pathogenesis of sepsis and septic shock has been extensively studied, little is known aboutthe causative contribution of endothelial-intrinsic NF-�B to these pathological processes. In this study, we used transgenic(TG) mice (on FVB genetic background) that conditionally overexpress the NF-�B inhibitor, mutant I-�B�, selectively onendothelium and their transgene-negative littermates (wild type (WT)) to define the causative role of endothelial-specificNF-�B signaling in septic shock and septic vascular dysfunction. In WT mice, LPS challenge caused systemic hypotension,a significantly blunted vasoconstrictor response to norepinephrine, and an impaired endothelium-dependent vasodilatorresponse to acetylcholine, concomitant with a markedly increased aortic inducible NO synthase expression, significantlyelevated plasma and aortic levels of nitrite/nitrate, increased aortic TNF-� expression, and decreased aortic endothelial NOsynthase (eNOS) expression. In TG mice whose endothelial NF-�B was selectively blocked, LPS caused significantly lesshypotension and no impairments in vasoconstrictor and endothelium-dependent vasodilator responses, associated with sig-nificantly reduced aortic inducible NO synthase expression, decreased plasma and aortic levels of nitrite/nitrate, reducedaortic TNF-� expression, and increased aortic eNOS expression. TNF-� knockout mice prevented LPS-induced eNOS down-regulation. WT mice subjected to cecal ligation and puncture showed significant systemic hypotension, which was preventedin TG mice. Our data show that selective blockade of endothelial-intrinsic NF-�B pathway is sufficient to abrogate thecascades of molecular events that lead to septic shock and septic vascular dysfunction, demonstrating a pivotal role ofendothelial-specific NF-�B signaling in the pathogenesis of septic shock and septic vascular dysfunction. The Journal ofImmunology, 2009, 183: 4031– 4038.

S eptic shock and septic vascular dysfunction are character-ized by systemic hypotension, persistent vasodilatation,hyporesponsiveness to vasoconstrictors, and impairment

of endothelium-dependent vasodilator response. The mecha-nisms of septic shock and septic vascular dysfunction are com-plex and multiple (1–3). One well-established mechanism is theactivation of NF-�B pathway. Bacterial pathogens and theirproducts, such as LPS, activate NF-�B, which causes inducibleNO synthase (iNOS)4 expression (2, 3), leading to an excessiveproduction of NO. NO released subsequently causes vasodila-tation, vascular hyporeactivity, and hypotension by activatingsoluble guanylyl cyclase-dependent mechanism (4 –7). NF-�B

activation mediates the expression of numerous cytokines,which lead to further activation of NF-�B, amplifying, and per-petuating the inflammatory response (2). LPS and cytokines,such as TNF-�, down-regulate endothelial NO synthase (eNOS)expression (8, 9), which is believed to be an important molec-ular event underlying the impaired endothelium-dependent va-sodilator response (10, 11). Animal studies have demonstratedthat inhibition of NF-�B activation inhibits multiple inflamma-tory gene expression (2, 12), reverses systemic hypotension (2,13–15), corrects myocardial dysfunction (16), and prevents theimpairment of endothelium-dependent vasodilatation (2, 14).

The pathogenic role of NF-�B activation in septic shock and septicvascular dysfunction is unquestionable. However, the contribution ofindividual cell-specific NF-�B to these pathological processes is sig-nificantly less clear. The pathophysiology of sepsis and septic shockinvolves complex cell-cell and mediator-mediator interactions (1, 2).Emerging evidence suggests that different cell-intrinsic NF-�B mayplay distinct role in the pathophysiology of sepsis (17–19). Elucida-tion of the contribution of individual cell-specific NF-�B to the com-plicated pathological process of septic shock will help to better un-derstand the pathologic mechanisms of septic shock.

The vascular endothelium is considered an important mechanismregulating vascular tone and vascular homeostasis (4, 20). In responseto physiological stimuli and hemodynamic forces, endothelial cells(ECs) release contracting and relaxing factors, including endothelin,NO, and prostacyclin. These factors alter vascular tone by directlycausing vasoconstriction or vasodilatation, and by synergistically orcounteractively interacting with neurotransmitters, enhancing or di-minishing the neurally induced vascular contraction or relaxation (4,20). Endothelium-derived NO inhibits adrenergic neural contraction

*Centers for Heart and Lung Research, and Immunology and Inflammation, FeinsteinInstitute for Medical Research, and †Division of Pulmonary and Critical Care Med-icine, Long Island Jewish Medical Center, New Hyde Park, NY 11040

Received for publication January 12, 2009. Accepted for publication July 14, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grant GM063907 and theFaculty Award Program of the Feinstein Institute for Medical Research.2 J.D., D.S., and X.Y. contributed equally to this work.3 Address correspondence and reprint requests to Dr. Shu Fang Liu, Long IslandJewish Medical Center, 270-05 76th Avenue, Research Building, RM B371, NewHyde Park, NY 11040. E-mail address: Sliu@lij.edu4 Abbreviations used in this paper: iNOS, inducible NO synthase; Ach, acetylcholine;CLP, cecal ligation and puncture; Con, control; EC, endothelial cell; eNOS, endo-thelial NO synthase; I-�B�mt, mutant I-�B�; KO, knockout; MBP, mean arterialblood pressure; NE, norepinephrine; SNP, sodium nitroprusside; VSMC, vascularsmooth muscle cell; WT, wild type.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

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(4), and mediates the vasodilator response to acetylcholine, the cho-linergic neural transmitter (4), as well as the vasodilator responses tovasoactive humoral substances (4).

The central role of NF-�B in septic pathologies and the pre-dominant influence of endothelium on vascular homeostasis sug-gest that activation of endothelial-specific NF-�B signaling mayplay an essential role in the development of septic shock and septicvascular dysfunction. However, the causative role of endothelial-intrinsic NF-�B in septic shock has not been studied. A number ofstudies have examined endothelial NF-�B activation and its role inendothelial inflammation (21–24). However, those studies wereperformed with cultured ECs, and have not addressed the patho-genic role of endothelial NF-�B activation in septic shock andseptic vascular dysfunction. Several previous studies, includingour own, have shown that inhibition of NF-�B activation usingchemical NF-�B inhibitors alleviates septic shock and septic vas-cular dysfunction (13–15). However, those inhibitors inhibitNF-�B activation in all cell types, and may have effects that are notrelated to NF-�B inhibition. The causal contribution of endothe-lial-intrinsic NF-�B to the pathogenesis of septic shock and septicvascular dysfunction has never been examined, due to lack of aninvestigative tool.

Recently, we have created and characterized transgenic (TG) micedesignated as EC-TG mice, in which a mutant I-�B� (I-�B�mt), aspecific inhibitor of NF-�B, is expressed in ECs under the control ofthe tetracycline gene regulatory system (17). The EC-TG mice displayendothelial-restricted blockade of NF-�B pathway (17) and enable usto selectively inhibit endothelial NF-�B activation in vivo under phys-iological setting. A preliminary study using those mice showed thatendothelial NF-�B blockade partially reversed endotoxemic hypoten-sion (17). The current study extends our preliminary study by exam-ining the effects of selective blockade of endothelial NF-�B pathwayon the cascades of molecular events that lead to septic shock andseptic vascular dysfunction in LPS and cecal ligation and puncture(CLP) models of sepsis. We demonstrated that blockade of endothe-lial-specific NF-�B signaling is sufficient to abrogate the molecularcascades leading to septic vascular dysfunction. Our data define themechanistic role of endothelial-intrinsic NF-�B in the pathogenesis ofseptic shock and septic vascular dysfunction, and provide new in-sights into the molecular mechanisms of sepsis and septic shock.

Materials and MethodsAnimal groups

The generation and characterization of the EC-TG mice that conditionallyoverexpress I-�B�mt selectively on endothelium have been previously de-scribed (17). In this study, we used this mouse strain to define the causativecontribution of endothelial-intrinsic NF-�B to septic shock and septic vas-cular dysfunction. We studied eight groups of mice (8–10 wk, on FVBgenetic background), as follows: transgene-negative control or sham (wildtype (WT)-Con, WT-sham), transgene-negative LPS or CLP (WT-LPS,WT-CLP), TG control or sham (TG-Con, TG-sham), and TG LPS or CLP(TG-LPS, TG-CLP). We also studied four groups of mice on B6129S ge-netic background (from The Jackson Laboratory; stock numbers, WT mice,101045, TNF-� knockout (KO), 003008), as follows: WT-Con, WT-LPS,TNF-� KO control (TNF-�-KO-Con), and TNF-�-KO-LPS. All animalexperiments were approved by the institutional animal care and use com-mittee and complied with National Institutes of Health Guide.

Measurement of systemic blood pressure

Mice were anesthetized with tribromoethanol (300 mg/kg, i.p.), intubated,and ventilated with a mouse ventilator, as we have previously described(13). We chose to use tribromoethanol as anesthetics because it causes lesscardiovascular depression (25). A microcannula was inserted into carotidartery for continuously monitoring systemic blood pressure. Mouse bodytemperature was kept constant with a servo-controlled electronic blanketand intraanal thermal probe. After a 30-min equilibration period and mea-surement of basal blood pressure, mice were injected with saline or LPS

(Escherichia coli 0111:B4, 2.5 mg/kg, i.p.). Systemic blood pressure wasrecorded for 4 h, and mean arterial blood pressure (MBP) was calculated.In a separate set of experiments, mice were injected with saline or LPS (10mg/kg, i.p.). At 24 h after saline or LPS injection, systemic blood pressurewas recorded, as described above.

For the CLP model, mice were anesthetized and cannulated at 18 h afteroperation, and systemic blood pressure was recorded, as described above.

Assessment of vascular reactivity in vivo

Mice were anesthetized and cannulated at 5.5 h after saline or LPS (10mg/kg, i.p.) injection. Because basal blood pressure influences vascularreactivity, mice that had low initial MBP were resuscitated with 6% dex-tran in 7.5% NaCl during the equilibration period to ensure a comparablebaseline MBP among all groups. Following the measurement of baselineMBP, dose-response relationship to �-adrenergic receptor agonist, norepi-nephrine (NE; 30, 100, and 300 ng/kg, i.v. bolus injection), to the endo-thelium-dependent vasodilator, acetylcholine (Ach; 60, 200, and 600 ng/kg, i.v. bolus injection), or to the endothelium-independent vasodilator,sodium nitroprusside (SNP; 60, 200, and 600 ng/kg, i.v. bolus injection),was recorded in three separate sets of experiments. The maximum increaseor decrease in MBP elicited by each dose of NE, Ach, or SNP was calcu-lated and compared.

Assessment of vasoreactivity in isolated mesenteric vascular bed

At 6 h after saline or LPS (10 mg/kg, i.p.) injection, the mesenteric vascularbed was isolated, as previously described (26), and perfused with oxygen-ated physiological salt solution at a constant flow rate of 200 �l/min. Be-cause perfusion flow rate is constant, changes in perfusion pressure repre-sent changes in vascular resistance. Following a 40-min equilibrationperiod, dose response to NE (30, 100, and 300 ng) was recorded. To studyvasodilator response, perfusion pressure was elevated by �60 mmHg byperfusing the mesenteric vascular beds with 100 �M NE. After a sustainedelevation in perfusion pressure was achieved, dose response to Ach or SNP(1, 10, and 100 ng) was recorded in two separate sets of experiments. Achor SNP was injected into the perfusion circuit immediately proximal to themesenteric artery. The maximal increase or decrease in perfusion pressurecaused by each dose of NE, Ach, or SNP was calculated and compared.

CLP model of sepsis

Mice in sham groups were subjected to sham, and in CLP groups subjectedto CLP operation, as we have previously described (17), using an 18-gaugeneedle. At 18 h postoperation, mice were cannulated for systemic bloodpressure measurement, as described above.

Western blot

Aortic levels of iNOS and eNOS proteins were determined by Westernblot, as we previously described (17), using Abs against iNOS, eNOS, andactin (all from Santa Cruz Biotechnology).

Assays for plasma and aortic levels of nitrite/nitrate, and aorticlevel of TNF-�

Plasma and aortic levels of nitrite/nitrate, the stable metabolic product ofNO, were measured using nitrite/nitrate assay kit (Cayman Chemical).Aortic level of TNF-� was determined using ELISA kit (eBioscience).

Immunohistochemical staining

Cryosections (6 �m) were prepared from aorta of each group of mice at 6 hafter saline or LPS injection, fixed with paraformaldehyde, permeabilized,blocked with blocking solution, and incubated with rabbit anti-TNF-� Ab(Abcam) or rabbit anti-iNOS Ab (Santa Cruz Biotechnology) overnight at4°C. Specific binding was detected with biotinylated secondary Ab-HRPcomplexes using VECTASTAIN Elite ABC kits (Vector Laboratories).Ag-Ab complexes were visualized using 3�,3�-diaminobenzidine (VectorLaboratories). Sections were counterstained with hematoxylin, mounted,and viewed under light microscope.

Statistical analysis

Data were expressed as mean � SEM, and analyzed using ANOVA orKruskal-Wallis rank test, followed by Holm-Sidak method or Dunnett’stest for post hoc analysis. The null hypothesis was rejected at 5% level.

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ResultsSelective blockade of endothelial NF-�B reduced systemichypotension

To define the role of endothelial intrinsic NF-�B signaling in sep-tic shock, WT-Con and TG-Con were injected with saline, andWT-LPS and TG-LPS mice were injected with LPS. MBP wasmonitored for 4 h. Baseline MBP was identical in the four groupsof mice and decreased slightly over time, most likely due to loss ofblood or body fluids (Fig. 1A). At 3 and 4 h post-LPS, WT-LPSmice showed a marked drop in MBP, which was significantly lessin TG-LPS (Fig. 1A). The effect of endothelial NF-�B blockade onthe development of septic shock was further examined at late timepoints in both LPS and CLP models of sepsis. Compared withcontrol or sham group of mice, WT-LPS or WT-CLP mice showeda significant drop in MBP at 24 h after LPS injection (Fig. 1B) orat 18 h after CLP operation (Fig. 1C), which was significantlyattenuated or prevented in TG-LPS or TG-CLP mice (Fig. 1, B andC). These results unveil an important role for endothelial-specificNF-�B signaling in the development of septic shock.

Selective blockade of endothelial NF-�B restoredvasoconstrictor response to NE

A major feature of septic vascular dysfunction is the repressedvasoconstrictor response to catecholamine. We have thereforeexamined whether endothelial-selective NF-�B blockade altersthe pressor response to NE in control and endotoxemic mice.Because basal vascular tone affects vascular reactivity, micewith low initial MBP were resuscitated to ensure comparablelevels of MBP among all groups of mice before starting the NEtrial. Baseline MBP was 94 � 1, 91 � 2, 93 � 1, and 93 � 1mmHg for WT-Con, WT-LPS, TG-Con, and TG-LPS group,respectively. As expected, NE caused a dose-dependent eleva-tion in MBP. Compared with that in WT-Con and TG-Conmice, the NE-elicited elevation in MBP decreased significantlyin WT-LPS at all three doses (Fig. 2A). In contrast, the NE-elicited elevation in MBP in TG-LPS mice was comparable tothat in WT-Con and TG-Con mice, and was significantly higherthan that in WT-LPS mice (Fig. 2A).

In vivo vasoreactivity is affected by systemic factors such ascardiac output and reflex. To avoid these effects, we further as-

sessed the NE response in isolated perfused mesenteric vascularbeds. Because the vascular bed was perfused at constant flow rate,changes in perfusion pressure represent alteration in vascular re-sistance. Mesenteric perfusion pressure was comparable among thefour groups of mice at baseline, but was elevated by NE injectionin a dose-dependent manner (Fig. 2B). Compared with WT-Conand TG-Con mice, the NE-elicited elevation in mesenteric perfu-sion pressure was greatly attenuated in WT-LPS mice (Fig. 2B),but was not affected in TG-LPS mice (Fig. 2B). These results in-dicate that blockade of endothelial-intrinsic NF-�B abrogates LPS-induced repression of the vasoconstrictor response to NE, suggest-ing that activation of endothelial-intrinsic NF-�B pathway plays animportant role in the development of vascular hyporesponsivenessto NE in endotoxemic mice.

FIGURE 1. Selective blockade of endothelial NF-�B reversed systemic hypotension. A, WT and TG mice (EC-TG) that conditionally overexpressI-�B�mt, a specific inhibitor of NF-�B, selectively on endothelium were injected with saline or E. coli LPS (2.5 mg/kg, i.p.). MBP was monitored.At 3 and 4 h post-LPS, MBP dropped significantly in WT-LPS mice, but not in TG-LPS mice. �, p � 0.05, compared with the other three groups.Mean � SEM of seven mice in each group. B, WT and EC-TG mice were injected with saline or E. coli LPS (10 mg/kg, i.p.). MBP was recordedat 24 h after LPS injection. Compared with WT-Con (W-C) and TG-Con (T-C) mice, LPS caused a significant drop in MBP in WT-LPS (W-L), butnot in TG-LPS (T-L) mice. �, p � 0.02, compared with the other three groups. Mean � SEM of eight mice in each group. C, WT and EC-TG micewere subjected to sham or CLP operation. At 18 h postoperation, MBP was measured. Compared with WT-sham (W-S) and TG-sham (T-S) mice,CLP caused a significant drop in MBP in WT-CLP (W-P), but not in TG-CLP (T-P) mice. �, p � 0.05, compared with the other three groups. Mean �SEM of eight mice in each group.

FIGURE 2. Selective blockade of endothelial NF-�B restored vasocon-strictor response to NE. WT-Con and TG-Con mice were injected withsaline, and WT-LPS and TG-LPS mice were injected with LPS (10 mg/kg,i.p.). At 6 h following LPS injection, vasoconstrictor response to NE wasassessed. A, Vasoconstrictor response to NE (i.v. bolus injection) in anes-thetized mice in vivo. Mean � SEM of six to seven animals in each group.B, Vasoconstrictor response to NE in isolated perfused mesenteric vascularbed. Mean � SEM of seven to eight animals in each group. �, p � 0.05,compared with the other three groups. LPS blunted the NE response inWT-LPS mice, but not TG-LPS mice.

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Selective blockade of endothelial NF-�B prevented theimpairment of endothelium-dependent vasodilator response

We next examined whether endothelial-selective NF-�B blockadeprevents the impairment of endothelium-dependent vasodilator re-sponse, another major feature of septic vascular dysfunction. Micewere injected with saline or LPS, cannulated, and resuscitated, asdescribed above. Baseline MBP was comparable among the fourgroups of mice before initiation of Ach or SNP trial. Both Ach(endothelium-dependent vasodilator) and SNP (endothelium-inde-pendent vasodilator) caused dose-dependent drop in MBP (Fig. 3).Compared with WT-Con and TG-Con mice, WT-LPS mice dis-played a significantly blunted vasodilator response to Ach at allthree doses (Fig. 3A). In contrast, TG-LPS mice showed an Achresponse that was identical with that of WT-Con and TG-Conmice, and was significantly bigger than that of WT-LP mice (Fig.3A). The four groups of mice showed a similar vasodilator re-sponse to SNP (Fig. 3B).

Likewise, Ach and SNP caused dose-dependent drop in perfu-sion pressure in the isolated perfused mesenteric vascular bed (Fig.4). The drops in perfusion pressure evoked by the three doses ofAch, but not by the three doses of SNP, were significantly lessin WT-LPS mice as compared with that of WT-Con and TG-Conmice (Fig. 4). In TG-LPS mice, Ach or SNP caused a drop inmesenteric perfusion pressure that was similar to that in WT-Conand TG-Con mice, and the Ach-mediated response was signifi-cantly bigger than that of WT-LPS mice (Fig. 4). Overall, theseresults illustrate that selective blockade of endothelial NF-�B path-way prevents the LPS-induced impairment of endothelium-depen-dent vasodilator response to Ach, implying that activation of en-dothelial-intrinsic NF-�B plays an important role in theimpairment of endothelium-dependent vasodilator response duringendotoxemia.

Selective blockade of endothelial NF-�B reduced iNOSexpression

LPS causes systemic hypotension and vascular hyporeactivityby inducing iNOS expression, resulting in overproduction ofNO, which causes hypotension and blunts vasoconstrictor re-sponse (5–7). To investigate the mechanism through which en-

dothelial-selective NF-�B blockade restores systemic MBP andvascular reactivity, we determined aortic iNOS protein expres-sion in WT and TG mice. Fig. 5A consists of Western blotphotographs showing that endothelial-selective NF-�B block-ade inhibited LPS-induced iNOS protein expression in aortae.The iNOS bands were quantified using densitometry and sum-marized in Fig. 5B. Aortic level of iNOS protein was negligiblein WT-Con and TG-Con mice, increased markedly in WT-LPSmice, but reduced by �73% in TG-LPS mice, as compared withthat in WT-LPS mice (Fig. 5).

Endothelial-selective NF-�B blockade also inhibited iNOSactivity, as indicated by the reduced plasma and aortic levels ofnitrite/nitrate in TG-LPS mice (Fig. 6). Compared with WT-Con and TG-Con mice, WT-LPS mice showed a �5-fold in-crease in plasma level and 6-fold increase in aortic level ofnitrite/nitrate, which were reduced by �64 and 52% in TG-LPSmice (Fig. 6). Thus, blockade of endothelial-intrinsic NF-�B

FIGURE 3. Selective blockade of endothelial NF-�B abrogated the im-pairment of endothelium-dependent vasodilator response in vivo. WT-Conand TG-Con mice were injected with saline, and WT-LPS and TG-LPSmice were injected with LPS (10 mg/kg, i.p.). At 6 h post-LPS, vasodilatorresponses to three i.v. injected doses of endothelium-dependent vasodila-tor, Ach (A), or endothelium-independent vasodilator, SNP (B), were re-corded in anesthetized mice in two separate sets of experiments. �, p �0.05, compared with the other three groups. Mean � SEM of six to sevenanimals in each group.

FIGURE 4. Selective blockade of endothelial NF-�B abrogated the im-pairment of endothelium-dependent vasodilator response in isolated per-fused mesenteric vascular bed. The isolated, perfused mesenteric vascularbeds were prepared. Perfusion pressure was elevated by perfusing NE.Vasodilator responses to three doses of endothelium-dependent vasodila-tor, Ach (A), or endothelium-independent vasodilator, SNP (B), were re-corded at 6 h after saline or LPS (10 mg/kg, i.p.) injection in two separatesets of experiments. #, p � 0.05 compared with WT-Con group. �, p �0.05, compared with the other three groups. Mean � SEM of 8–10 animalsin each group.

FIGURE 5. Selective blockade of endothelial NF-�B reduced aorticiNOS protein expression. WT-Con (W-C) and TG-Con (T-C) mice wereinjected with saline, and WT-LPS (W-L) and TG-LPS (T-L) mice wereinjected with LPS (10 mg/kg, i.p.). Aortic iNOS protein expression wasdetermined using Western blot at 6 h after LPS injection. A, Western blotphotographs showing inhibition of LPS-induced iNOS protein expressionby selective blockade of endothelial NF-�B. Actin: membrane of iNOSblotting was reblotted to anti-actin Ab. B, The iNOS bands were quantifiedusing densitometry and expressed as arbitrary OD units. �, p � 0.05, com-pared with the other three groups. Mean � SEM of six mice in each group.

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suppresses aortic iNOS expression and activity, suggesting thatendothelial-selective NF-�B blockade prevents systemic hypo-tension and vascular hyporeactivity by inhibiting iNOS expres-sion in vascular tissues.

Selective blockade of endothelial NF-�B prevented LPSdown-regulation of eNOS expression

Ach causes vasodilatation by activating eNOS, resulting in therelease of endothelium-derived NO (10, 11). We have thereforecompared aortic levels of eNOS protein expression betweenWT-LPS and TG-LPS mice. Fig. 7A consists of Western blotphotographs showing that endothelial-selective NF-�B block-ade abrogated LPS-induced eNOS protein down-regulation.Fig. 7B summarized the densitometry quantification of theeNOS bands. Consistent with impaired vasodilator response toAch, WT-LPS mice showed a significantly reduced aortic ex-pression of eNOS protein, as compared with WT-Con and TG-Con mice (Fig. 7). Aortic level of eNOS protein in TG-LPS micewas identical with that of WT-Con and TG-Con mice (Fig. 7).These results suggest that activation of endothelial-specific NF-�Bcontributes to the LPS-induced down-regulation of eNOS expres-

sion, which accounts for the impairment of endothelium-dependentvasodilator response to Ach in endotoxemic mice.

TNF-� is required for LPS-induced eNOS down-regulation

To establish a link between activation of endothelial NF-�Bsignaling and eNOS down-regulation, we examined the effect ofendothelial NF-�B blockade on aortic TNF-� expression, anddetermined the role of TNF-� in LPS-induced eNOS down-regulation in our mouse model. Compared with WT-Con andTG-Con mice, WT-LPS mice displayed a 10-fold increase inaortic level of TNF-� protein, which was reduced by �51% inTG-LPS mice (Fig. 8). To define the role of TNF-� in LPS-induced eNOS down-regulation, we compared aortic levels ofeNOS protein expression between TNF-�-KO mice and theirgenetic background-matched WT mice at 6 h after LPS injec-tion. Compared with that in WT-Con and TNF-�-KO-Con mice,aortic eNOS expression was significantly down-regulated inWT-LPS mice, which was prevented in TNF-�-KO-LPS mice(Fig. 9). This result indicates that TNF-� plays an obligatory rolein LPS-induced eNOS down-regulation. Collectively, these resultssuggest that activation of endothelial NF-�B signaling leads toeNOS down-regulation via TNF-�-dependent mechanisms.

FIGURE 6. Selective blockade of endothelial NF-�B-reduced plasmaand aortic levels of nitrite/nitrate. Plasma (A) and aortic (B) levels of nitrite/nitrate in WT-Con (W-C), TG-Con (T-C), WT-LPS (W-L), and TG-LPS(T-L) groups of mice were determined at 6 h after saline or LPS injectionusing commercial assay kits. �, p � 0.05, compared with the other threegroups. Mean � SEM of six to seven animals in each group.

FIGURE 7. Selective blockade of endothelial NF-�B prevented aorticeNOS down-regulation. Aortic level of eNOS protein expression in WT-Con (W-C), TG-Con (T-C), WT-LPS (W-L), and TG-LPS (T-L) mice wasdetermined at 6 h after LPS injection (10 mg/kg, i.p.) using Western blot.A, Western blot photographs showing endothelial NF-�B blockade pre-vented LPS-induced down-regulation of eNOS protein. Actin: membraneof eNOS blotting was reblotted to anti-actin Ab. B, The eNOS bands werequantified using densitometry, and expressed as arbitrary OD units. �, p �0.05, compared with the other three groups. Mean � SEM of eight mice ineach group.

FIGURE 8. Selective blockade of endothelial NF-�B suppressed aorticTNF-� protein expression. Aortic level of TNF-� protein in WT-Con (W-C), TG-Con (T-C), WT-LPS (W-L), and TG-LPS (T-L) groups of micewas determined at 6 h after LPS injection (10 mg/kg, i.p.) using ELISA kit.�, p � 0.05, compared with the other three groups. Mean � SEM of sixmice in each group.

FIGURE 9. LPS-induced eNOS down-regulation was prevented inTNF-�-KO mice. Aortic level of eNOS protein expression in WT-Con(W-C), TNF-�-KO-Con (K-C), WT-LPS (W-L), and TNF-�-KO-LPS(K-L) mice was determined at 6 h after LPS injection (10 mg/kg, i.p.) usingWestern blot. A, Western blot photographs showing TNF-�-KO preventedLPS-induced down-regulation of eNOS protein. Actin: membrane of eNOSblotting was reblotted to anti-actin Ab. B, The eNOS bands were quantifiedusing densitometry, and expressed as arbitrary OD units. �, p � 0.02,compared with the other three groups. Mean � SEM of six mice in eachgroup.

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Endothelial NF-�B blockade inhibited TNF-� and iNOSexpression in smooth muscle cells

We next performed immunohistochemical staining of aortic sec-tions from WT and TG mice to identify the cells whose levels ofiNOS and TNF-� expression were inhibited by endothelial-selec-tive NF-�B blockade. TNF-�- and iNOS-positive cells were notdetectable in aortic sections from WT-Con and TG-Con mice (Fig.10, A, B, E, and F), markedly increased in aortic sections fromWT-LPS mice (Fig. 10, C and G), and significantly reduced inaortic sections from TG-LPS mice (Fig. 10, D and H). StrongTNF-� and iNOS staining was localized to ECs and vascularsmooth muscle cells (VSMCs) in aorta of WT-LPS mice (Fig. 10,C and G). In TG-LPS mice, TNF-�- and iNOS-positive ECs werebarely detected, and TNF-�- and iNOS-positive VSMCs were sig-nificantly reduced (Fig. 10, D and H). This result illustrates thatendothelial-selective NF-�B blockade diminishes EC TNF-� andiNOS expression, and also inhibits VSMC TNF-� and iNOSexpression.

DiscussionThe major finding of this study is that endothelial-specificNF-�B signaling plays a pivotal role in the pathogenesis ofseptic shock and septic vascular dysfunction. Challenge of WTmice with LPS resulted in a marked drop in systemic MBP atboth early and late time points, a significantly blunted vasocon-strictor response to NE, and an impaired endothelium-depen-dent vasodilator response to Ach. WT mice that underwent CLPalso showed a significant systemic hypotension. Consistent withthe changes in physiological parameters, WT-LPS mice showeda markedly increased aortic iNOS protein expression, elevatedplasma and aortic levels of nitrite/nitrate, the stable end prod-ucts of NO, increased aortic TNF-� protein expression, anddecreased aortic eNOS protein expression. In sharp contrast toWT-LPS mice, TG-LPS mice with endothelial-selective NF-�Bblockade displayed no significant hypotension, a normal vaso-constrictor response to NE, no impairment in the endothelium-dependent vasodilator response to Ach, reduced aortic iNOSexpression, decreased plasma and aortic levels of nitrite/nitrate,reduced aortic TNF-� expression, and increased aortic eNOS

expression. TG-CLP mice were also prevented from systemichypotension. Taken together with our previous demonstrationthat the EC-TG mice express the NF-�B inhibitor, I-�B�mt,only on endothelium, and display endothelial-selective block-ade of NF-�B activation (17), these results illustrate that block-ade of endothelial-intrinsic NF-�B pathway mitigates the cas-cade of molecular events that lead to septic shock and septicvascular dysfunction, implying that endothelial-specific NF-�Bsignaling plays a pivotal role in the development of septic shockand septic vascular dysfunction.

Several groups, including our own, have shown that inhibi-tion of NF-�B activation reduces systemic hypotension and re-stores or partially restores vascular reactivity in mice or ratssubjected to endotoxemia or multimicrobial sepsis (13–15).However, those studies used nonspecific chemical NF-�B in-hibitors, which may have nonspecific effects. More importantly,those studies inhibited NF-�B in all cell types, and did notaddress the function of cell-specific NF-�B in the pathologicalprocesses. This study is the first to define the specific contri-bution of endothelial-intrinsic NF-�B signaling to septic shockand septic vascular dysfunction, and thus provides novel in-sights into the molecular mechanisms of septic shock and septicvascular dysfunction.

It is well documented that LPS or sepsis causes systemichypotension and vascular hyporesponsiveness by activatingNF-�B-mediated iNOS expression (2, 3), leading to an exces-sive production of NO. The marked inhibition of LPS-inducediNOS expression and activity by selective blockade of endo-thelial NF-�B pathway with a concomitant abrogation of LPS-induced hypotension and vascular hyporeactivity supports thenotion that endothelial NF-�B blockade alleviates septic hypo-tension and septic vascular dysfunction by inhibiting NF-�B-mediated iNOS expression.

We showed that LPS impaired endothelium-dependent vasodi-lator response in WT-LPS, but not in TG-LPS mice. The preser-vation of endothelium-dependent vasodilator response in TG-LPSmice could be explained by the prevention of TNF-�-mediatedeNOS down-regulation. First, TNF-� is a NF-�B-regulated geneproduct (2, 3), and blockade of endothelial NF-�B significantly

FIGURE 10. Endothelial NF-�B blockade inhibited TNF-� and iNOS protein expression in ECs and smooth muscle cells. Representative immunohis-tochemical staining for TNF-� (A–D) and iNOS (E–H) expression in aortic sections from WT-Con (A and E), TG-Con (B and F), WT-LPS (C and G), andTG-LPS (D and H) mice at 6 h after LPS injection. The dark brown HRP-diaminobenzidine reaction product shows TNF-�- or iNOS-expressing cells.TNF-� and iNOS staining was strong and evident in ECs (arrow) and VSMCs in WT-LPS aortic section (C and G), but absent in ECs (arrow) andsignificantly reduced in VSMCs of TG-LPS aortic section (D and H). Representative of three experiments. Original magnification �1000.

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inhibited LPS-induced TNF-� expression in TG-LPS mice. Sec-ond, TNF-� is known to down-regulate eNOS expression (8). Wedemonstrated in the current study that TNF-� mediates LPS-in-duced eNOS down-regulation. Third, it is well documented thatAch elicits vasodilatation by stimulating eNOS-mediated endothe-lial NO release (4, 10, 11). Fourth, we demonstrated in this studythat blockade of endothelial NF-�B concomitantly inhibited theLPS-induced TNF-� up-regulation and eNOS down-regulation invascular tissue, and restored endothelium-dependent vasodilata-tion. Inhibition of endothelial NF-�B activation suppresses localinflammation within ECs, which could reduce the production ofreactive oxygen species and increase NO bioavailability (27). Thismay also contribute to the preserved endothelium-dependent va-sodilator response to Ach in TG-LPS mice.

Physiologically, vascular tone is mainly influenced by thephysical and biochemical properties of VSMCs. NF-�B path-way in smooth muscle cells is not inhibited in our EC-TG mice(17). The effectiveness of endothelial-restricted NF-�B inhibi-tion in correcting septic vascular dysfunction and in inhibitingaortic iNOS expression is somewhat unexpected. Endothelial-selective inhibition of NF-�B activation restored systemic MBPto 80% of control level, completely abrogated LPS-induced re-pression of vasoconstrictor responses to NE, and inhibited LPS-induced aortic iNOS protein expression by �73%. These resultshighlight the importance of endothelium- and endothelial-spe-cific NF-�B signaling in septic shock and septic vascular dys-function. These results suggest that cross-talk between ECs andVSMCs may be an important mechanism regulating inflamma-tory response within vascular wall during sepsis. In support ofthis speculation, we demonstrated in this study that selectiveblockade of endothelial NF-�B pathway not only diminishedLPS-induced TNF-� and iNOS expression in ECs, but also sig-nificantly inhibited TNF-� and iNOS expression in VSMCs.Further studies to elucidate the mechanisms underlying theparacrine interactions between ECs and VSMCs will help tobetter understand the molecular mechanisms of vascular wallinflammation during sepsis and other pathological conditions.

Our current results are in good agreement with our previousstudies demonstrating that endothelial selective NF-�B blockadeameliorated multiple organ injury and improved survival in septicmice (17). Our results are also consistent with two recent reportsshowing that endothelial-specific NF-�B suppression attenuatedhypertension-induced renal damage (28) and high fat diet-inducedatherosclerosis (29). It is reported that blockade of endothelialNF-�B pathway enhanced LPS-induced endothelial permeability(30). This result does not necessarily contradict our findings, be-cause different models (conventional vs conditional TG mice) wereused in the two studies. The two mouse models have different basalendothelial barrier integrity and, therefore, display different re-sponse to LPS in term of alteration in endothelial permeability (17,30). Nevertheless, both studies illustrate a critical role of endothe-lial NF-�B in controlling endothelial barrier integrity and function.

Our findings have therapeutic implications. The central rolesof NF-�B in systemic inflammation and in septic pathologyindicate that NF-�B is an ideal target for therapeutic interven-tion (2, 12–16). However, NF-�B inhibition impairs host de-fense mechanism and causes immune suppression (31, 32).Consequently, the beneficial anti-inflammatory effect can beoffset by the detrimental proinfectious effect of NF-�B inhibi-tion, leading to unaltered or even worse outcome. To overcomethis problem, we need to develop novel approaches that canselectively inhibit NF-�B-mediated inflammatory and injuriousresponses (detrimental) without significantly interfering withthe NF-�B-mediated immune and host defense responses (ben-

eficial). Our current and previous demonstrations that selectiveblockade of NF-�B-driven inflammatory response within endo-thelium ameliorated septic multiple organ injury (17), abro-gated septic vascular dysfunction, and improved survival, buthad no effect on bacterial clearance capacity (17) provide ex-perimental basis for endothelial-selective NF-�B inhibition asan innovative strategy to develop sepsis therapies.

AcknowledgmentsWe thank Dr. J. W. Pollard and the staff at Albert Einstein College ofMedicine Transgenic and Gene Targeting Facility for help in generatingTG mice.

DisclosuresThe authors have no financial conflict of interest.

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