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INTRODUCTIONSepsis, septic shock, and multiple

organ failure continue to be the mostcommon causes of death in noncardiacintensive care units (1-3). Despite ad-vances in the management of traumavictims, the incidence of sepsis and sep-tic shock has increased significantly overthe past two decades (3-6). Humanadrenomedullin (AM) is a 52-amino acidpeptide which was first isolated frompheochromocytomas by Kitamura et al.and reported in 1993 (7). Rat AM has 50amino acid residues, with two amino aciddeletions and six substitutions comparedwith human AM (8). Circulating levels ofAM increase significantly in patients withseptic shock (9-11) and systemic inflam-matory response syndrome (12), and after

major surgery (13). Upregulation of AMcan also be observed after administrationof endotoxin in vivo as well as in vitro invascular smooth muscle cells andmacrophages (14-20). Our recent studieshave demonstrated that the small intes-tine is a major source of AM productionand release during sepsis (21). Studies byElsasser et al. (22) have demonstrated thepresence of a specific AM binding protein(120 and/or 140 kDa) in mammalianblood. Pio et al. purified the binding pro-tein, which they named AMBP-1, anddiscovered that AMBP-1 is identical tohuman complement factor H (23). Theprimary site of AMBP-1 (factor H)biosynthesis is the liver (24-26), and theonly extrahepatic source of AMBP-1 inhumans under in vivo conditions is the

small amount produced in the lungs (27).The finding that AMBP-1 potentiatesAM-induced cAMP accumulation in cul-tured Rat-2 fibroblast cells (23) suggeststhat AMBP-1 plays an important role inAM-induced vascular relaxation. Thus,circulating AMBP-1 can positively affectthe bioactivity of AM under normal aswell as disease conditions (23). To thisend, our results have indicated thatAMBP-1 improves vascular responsive-ness to AM stimulation (28). Moreover,we have shown that AMBP-1 productionis decreased in sepsis, and this reductionin AMBP-1 appears to be responsible forthe vascular AM hyporesponsivenessobserved in the hypodynamic phase (28).Moreover, coadministration of AM andAMBP-1 downregulates proinflammatorycytokines (29), maintains cardiovascularstability (30), prevents endothelial cellapoptosis (31) in sepsis and reducessepsis-induced mortality (30).

Studies have indicated that downregu-lation of vascular endothelial constitutive

Adrenomedullin and Adrenomedullin Binding Protein-1 ProtectEndothelium-Dependent Vascular Relaxation in Sepsis

Address correspondence and reprint requests to Ping Wang, Laboratory of Surgical Re-

search, The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, NY

11030. Phone: (516) 562-3411; Fax: (516) 562-2396; E-mail: pwang@nshs.edu.

Submitted December 15, 2006; Accepted for publication June 20, 2007.

Mian Zhou, Subir R Maitra, and Ping WangDepartment of Surgery, North Shore University Hospital and Long Island Jewish Medical Center, and The Feinstein Institute forMedical Research, Manhasset, New York, USA

Downregulation of vascular endothelial constitutive nitric oxide synthase (ecNOS) contributes to the vascular hyporesponsive-ness in sepsis. Although coadministration of the potent vasodilatory peptide adrenomedulin (AM) and the newly discovered AMbinding protein (AMBP-1) maintains cardiovascular stability and reduces mortality in sepsis, it remains unknown whetherAM/AMBP-1 prevents endothelial cell dysfunction. To investigate this possibility, we subjected adult male rats to sepsis by cecalligation and puncture (CLP), with or without subsequent intravenous administration of the combination of AM (12 µg/kg) andAMBP-1 (40 µg/kg). Thoracic aortae were harvested 20 h after CLP (i.e., the late stage of sepsis) and endothelium-dependentvascular relaxation was determined by the addition of acetylcholine (ACh) in an organ bath system. In addition, ecNOS geneand protein expression was assessed by RT-PCR and immunohistochemistry, respectively. The results indicate that ACh-induced(i.e., endothelium-dependent) vascular relaxation was significantly reduced 20 h after CLP. Administration of AM/AMBP-1 pre-vented the reduction of vascular relaxation. In addition, ecNOS gene expression in aortic and pulmonary tissues was downregu-lated 20 h after CLP and AM/AMBP-1 attenuated such a reduction. Moreover, the decreased ecNOS staining in thoracic aortaeof septic animals was prevented by the treatment with AM/AMBP-1. These results, taken together, indicate that AM/AMBP-1 pre-serves ecNOS and prevents reduced endothelium-dependent vascular relaxation (i.e., endothelial cell dysfunction) in sepsis. Inlight of our recent finding that AM/AMBP-1 improves organ function and reduces mortality in sepsis, it is most likely that the pro-tective effect of these compounds on ecNOS is a mechanism responsible for the salutary effect of AM/AMBP-1 in sepsis.Online address: http://www.molmed.orgdoi: 10.2119/2007–00113.Zhou

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nitric oxide synthase (ecNOS) contributesto vascular hyporesponsiveness in sepsis(32-34). Although administration ofAM/AMBP-1 is beneficial in sepsis,whether AM/AMBP-1 prevents endothe-lial cell dysfunction under such condi-tions is not known. We therefore hypoth-esized that AM/AMBP-1 preservesendothelium-dependent vascular relax-ation by preventing ecNOS downregula-tion in sepsis. Endothelium-dependentvascular relaxation was determined bythe addition of acetylcholine (ACh) in anorgan-bath system in aortas from septicand sham-operated animals with orwithout AM/AMBP-1 treatment. Thegene and protein expression of ecNOS inaortic and pulmonary tissues was deter-mined by RT-PCR and immunohisto-chemical analysis, respectively, in thoseanimals with sepsis.

MATERIALS AND METHODSExperimental animals. Male adult

Sprague-Dawley rats (275-315 g), pur-chased from Charles River Laboratories(Wilmington, MA, USA), were used inthis study. All surgical procedures wereperformed using aseptic technique withthe exception of induction of sepsis bycecal ligation and puncture (CLP). Theexperiments described below were per-formed in adherence to the National In-stitutes of Health (NIH) guidelines forthe use of experimental animals. Thisproject was approved by the InstitutionalAnimal Care and Use Committee of theFeinstein Institute for Medical Research.

Animal model of sepsis. Polymicro-bial sepsis was induced in rats by CLP asdescribed previously (35). Briefly, ratswere fasted overnight prior to the induc-tion of sepsis, but allowed water ad libi-tum. The animals were anesthetized withisoflurane inhalation, and a 2-cm ventralmidline abdominal incision was made.The cecum was then exposed, ligatedwith 3-0 silk suture just distal to the ileo-cecal valve to avoid intestinal obstruc-tion, punctured twice with an 18-gaugeneedle, and returned to the abdominalcavity. The punctured cecum wassqueezed to expel a small amount of

fecal material. The incision was thenclosed in layers, and fluid resuscitationby subcutaneous administration of 3 mL/100 g body wt. normal saline wasperformed immediately after CLP. Sham-operated animals underwent the samesurgical procedure except that the cecumwas neither ligated nor punctured. Stud-ies were then conducted 20 h after theinduction of sepsis or sham-operation.It should be noted that 20 h after CLPrepresents the late, hypodynamic stageof polymicrobial sepsis (35,36).

Administration of AM/AMBP-1. Thefasted animals were anesthetized withisoflurane inhalation, and a 1.0-cm inci-sion was made in the neck. A 200-µLAlzet mini-osmotic pump (infusion at aconstant rate of 8 µL/h) was prefilledwith synthetic rat AM solution (20 µg/mL sterile normal saline; Pheonix Phar-maceuticals, Belmont, CA, USA) and in-serted into the right jugular vein througha silastic catheter. After closure of theneck incision, CLP was performed afterthe implantation of the pump. HumanAMBP-1 (40 µg/kg body wt; Cortex, SanLeandro, CA, USA) was then infused viathe femoral vein using a Harvard pumpat a rate of 50 µL/min for a period of 20 min. The selected dosages of AM andAMBP-1 were previously found to signif-icantly reduce circulating concentrationsof TNF-α, IL-1β, and IL-6 (29), and de-creased sepsis-induced mortality (30).Vehicle-treated animals received an equalamount of sterile normal saline insteadof AM/AMBP-1. Twenty hours after CLPor sham-operation the thoracic aortaeand lungs were harvested for variousmeasurements, as described below.

Determination of vascular relaxation.Immediately after the death of the ani-mals by an overdose of isoflurane inhala-tion, the thoracic cavity was rapidlyopened and the thoracic aorta was re-moved. The blood vessels were im-mersed in ice-cold Krebs-Ringer bicar-bonate solution (composition in mM:NaCl, 118.3; KCl, 4.7; CaCl2, 2.5; MgSO4,1.2; KH2PO4, 1.2; NaHCO3, 25.0; Ca-EDTA, 0.026; glucose, 11.1, Na pyruvate,1.0) that was aerated with 95% O2: 5%

CO2 (pH 7.4; PO2 580 mmHg). The tho-racic aorta was cut into approximately2-mm rings. The aortic rings were thenmounted on specimen holders andplaced in a glass organ chamber contain-ing 5-mL aerated Krebs-Ringer bicarbon-ate solution at 37 °C. One holder was sta-tionary and the other was connected toan isometric force-displacement trans-ducer coupled to a polygraph. The vas-cular rings were incubated in the aeratedKrebs-Ringer bicarbonate solution about30-60 min. When the basal tension wasstable, a contraction of approximately 1-gwas induced by 2×10-7 M norepineph-rine. An endothelium-dependent va-sodilator, acetylcholine (ACh, concentra-tion range from 10-8 to 5×10-6 M) or anendothelium-independent vasodilator,nitroglycerine (NTG, concentration rangefrom 5×10-9 to 10-6 M) was added to theorgan bath, and the percentage of vascu-lar relaxation was then determined. Atthe end of the experiment, the viabilityof the vascular ring preparations waschecked by the addition of norepineph-rine. There was no significant decreasein vascular contraction induced by thisreagent.

Determination of ecNOS gene expres-sion. The thoracic aortae and lungs (rep-resenting blood vessel–rich tissues) wereharvested from the sham and septic ani-mals with or without AM/AMBP-1 treat-ment. Total RNA was extracted and 4 µgRNA was reverse-transcribed. The result-ing cDNAs were amplified by PCR reac-tion using specific primers for rat ecNOS(forward: GGG CCA GGG TGA TGAGCT CTG; reverse: CCC TCC TGG CTTCCA GTG TCC; NM_021838). The PCRreaction was conducted at 35 cycles.Each cycle consisted of 1 min at 94 °C, 1min at 65 °C, and 2 min at 72 °C. Ratglyceraldehyde 3-phosphate dehydroge-nase (G3PDH) served as a housekeepinggene (Clontech). After the RT-PCR proce-dure, the reaction products were elec-trophoresed in 1.6% TBE-agarose gelcontaining 0.22 μg/mL ethidium bro-mide. The gel was then photographed onPolaroid film and a digital image systemwas used to determine the band density.

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The ratio of ecNOS/G3PDH was thencalculated.

Determination of ecNOS gene expres-sion in pulmonary tissues using real-time PCR. Total RNA extracted frompulmonary tissues was reverse-tran-scribed to cDNA as described above.ecNOS gene expression was determinedwith a real-time PCR technique. Theprimers specific for rat ecNOS were se-lected by a real-time PCR primer designsoftware (Primer Express Software Ver-sion 3; Applied Biosystems, Foster City,CA, USA) from mRNA sequences re-trieved from GenBank (accession #:NM_021838; forward primer: CCG GCG CTACGA AGA ATG; reverse primer: AGTGCC ACG GAT GGA AAT TG). G3PDHwas used as a reference gene (accession#M17701; forward primer: ATG ACTCTA CCC ACG GCA AG; reverse primer:CTG GAA GAT GGT GAT GGG TT).Real-time PCR was performed using the7300 Real-Time PCR system (AppliedBiosystems) with SYBR Green as the de-tection dye. The reaction was carried ina 24 µL final reaction volume containing0.08 µmol concentrations of each forwardand reverse primer, 2 µL cDNA, 9.2 µLH2O and 12 µL SYBR Green PCR MastMix (Applied Biosystems). The thermalprofile for the real-time PCR was 50 °Cfor 2 min and 95 °C for 10 min, followedby 40 cycles of 95 °C for 15 s and 60 °Cfor 1 min. The gene expression was ex-pressed as fold change according to theformula for real-time PCR data analysis.In addition, melting curve analysis wasperformed to confirm the specificity ofthe PCR product in this experiment.

Immunohistochemistry. The aortictissues were collected 20 h after CLP orsham-operation (n = 4 rats/group) andimmediately fixed in the neutralized 4%paraformaldehyde solution overnight.The tissues were then dehydrated andembedded into the paraffin through thestandard histology procedure. The paraf-fin sections were dewaxed and rehy-drated, then microwave antigen retrievalwas performed. To retrieve the antigen,slides were soaked in 20% citric acidbuffer, pH 6.0 (Vector Labs, Burlingame,

CA, USA) and kept at 95 °C for 15 min.The slides were cooled at room tempera-ture for 5 min, rinsed with TBS, then in-cubated in 3% bovine serum albumin for30 min to block nonspecific binding fol-lowed by incubation in 1:50 rabbit anti-ecNOS polyclonal antibodies (BD Bio-sciences, San Diego, CA, USA) for twohours at room temperature. After wash-ing, the sections were reacted with 1:200biotinylated anti-rabbit IgG secondaryantibodies (Vector Labs, Burlingame,CA, USA). Vectastain ABC and DAB sub-strate kits (Vector Labs) were used to re-veal the immunohistochemical reaction.Normal rabbit IgG was substituted toprimary antibody as a negative control.

Statistical analyses. Data are pre-sented as means ± SE. One-way analysisof variance (ANOVA) and Tukey’s testwere used for the comparison amongdifferent groups. The difference wasconsidered significant at P ≤ 0.05.

RESULTSEffects of AM/AMBP-1 on ACh-induced

vascular relaxation. As indicated in Figure 1,vascular relaxation increased to 76-82% atACh concentrations of 5×10-7 to 5×10-6 Min sham-operated animals. However,ACh-induced vascular relaxation de-creased to 38%-48% at the above-men-tioned concentrations of ACh in septicanimals (P < 0.05). Administration ofAM/AMBP-1, however, maintained ACh-induced vascular relaxation to the levelsimilar to sham-operated animals (seeFigure 1A). In contrast to ACh, vascularrelaxation induced by the endothelium-independent vasodilating agent NTG didnot change significantly in septic nor inAM/AMBP-1–treated septic animals com-pared with sham-operated animals (seeFigure 1B). Thus, endothelium-dependentbut not endothelium-independent vascu-lar relaxation is reduced in sepsis, andthis reduction can be prevented by ad-ministration of AM/AMBP-1.

Effects of AM/AMBP-1 on ecNOSgene expression. Because ACh-inducedvascular relaxation is mediated by NOproduced by the activated ecNOS, wedecided to determine ecNOS gene

expression. In the aortic tissues, ecNOSgene expression decreased by 27% afterthe onset of sepsis (Figures 2A and 3).However, administration of AM/AMBP-1 prevented the downregulation ofecNOS gene expression in the aortic tissues (Figures 2A and 3). Similarly,

Figure 1. (A) Effects of AM/AMBP-1 onacetylcholine (ACh)-induced vascular re-laxation in aortic rings 20 h after cecal lig-ation and puncture (CLP). The data areexpressed as percentage (%) of the vascu-lar relaxation from the initial tension in-duced by 2×10-7 M norepinephrine. Values(n = 6/group) are presented as means ± SEand compared by one-way ANOVA andTukey’s test: *P < 0.05 versus sham-oper-ated animals; #P < 0.05 versus septic ani-mals. (B) Effects of AM/AMBP-1 on nitro-glycerine (NTG)-induced vascularrelaxation in the aortic ring 20 h aftercecal ligation and puncture (CLP). Thedata are expressed as percentage (%) ofthe vascular relaxation from the initial ten-sion induced by 2 ×10-7 M norepinephrine.Values (n = 6/group) are presented asmeans ± SE. There was no significant differ-ence between sham and septic animalswith or without AM/AMBP-1 treatment.

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pulmonary ecNOS gene expression de-creased by 36% in 20 h after CLP, andAM/AMBP-1 treatment maintained itsexpression to a level similar to that ofsham-operated animals (Figures 2B and 4).In addition, real-time PCR results indi-cate a decrease of 42% in pulmonaryecNOS gene expression at 20 h after CLP,and an increase of 67% in ecNOS geneexpression following AM/AMBP-1 treat-ment (Figure 5).

Effects of AM/AMBP-1 on ecNOSprotein levels. Immunohistochemistrywas used to determine the effect of AM/AMBP-1 on ecNOS protein levels in theaorta. As indicated in Figure 6, ecNOS-

positive staining is limited on the toplayer of the aortic tissues, indicating aor-tic endothelial cells (see Figure 6A). At 20h after the onset of sepsis, ecNOS stain-ing decreased (see Figure 6B), and ad-ministration of AM/AMBP-1 preventedsuch a decrease in ecNOS protein (seeFigure 6C).

DISCUSSIONWe have previously demonstrated that

AMBP-1 improves AM-induced vascularrelaxation in aortic tissues from septicanimals and that vascular levels ofAMBP-1 decreases significantly duringthe late stage of sepsis (28). Our resultssuggested that AMBP-1 plays an impor-tant role in modulating vascular respon-siveness to AM during sepsis and circu-lating AM-AMBP-1 complexes should beconsidered as an important factor in thetransition from hyperdynamic to hypo-dynamic circulation during the progres-sion of polymicrobial sepsis (28). In thepresent study, we investigated whetherAM/AMBP-1 preserves endothelium-dependent vascular relaxation by pre-venting ecNOS downregulation in sepsis.

Plasma levels of AM begin to rise asearly as two hours after CLP and pro-gressively increases to 30 h after theonset of sepsis (37). Elevated levels ofAM play a major role in producing the

hyperdynamic phase of sepsis, but tran-sition to the late, hypodynamic phaseoccurs despite continued high circulatinglevels of AM. Decreased vascular respon-siveness to AM appears to explain thisparadox and may be responsible for in-ducing the phase change. Studies of AM-induced vascular relaxation in thoracicaortic rings showed no change at either5 or 10 h after CLP, but a significant de-crease occurred at 20 h after the onset ofsepsis (38). These results therefore sug-gest that vascular AM hyporesponsive-ness contributes to the transition fromthe early, hyperdynamic phase to thelate, hypodynamic phase during the

Figure 2. Representative gels of the gene expression of ecNOS (324 bp) and housekeep-ing gene G3PDH (983 bp) in the aortic (A) and pulmonary tissues (B) 20 h after sham-op-eration or cecal ligation and puncture (CLP) with or without AM/AMBP-1 treatment.

Figure 3. The ratio of ecNOS versus G3PDHPCR products in the aortic tissues is pre-sented as means ± SE (n = 7/group) andcompared by one-way ANOVA and Tukey’stest: *P < 0.05 versus sham-operated ani-mals; #P < 0.05 versus septic animals.

Figure 4. The ratio of ecNOS versus G3PDHPCR products in the pulmonary tissues ispresented as means ± SE (n = 5/group) andcompared by one-way ANOVA andTukey’s test: *P < 0.05 versus sham-operatedanimals; #P < 0.05 versus septic animals.

Figure 5. Alteration in pulmonary ecNOSgene expression at 20 h after cecal liga-tion and puncture (CLP) or sham-opera-tion, measured by real-time PCR tech-nique. The data (fold change) arepresented as means ± SE (n = 5) and com-pared by one-way ANOVA and Tukey’stest. *P < 0.05 versus sham-operated ani-mals; #P < 0.05 versus septic animals.

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progression of sepsis (39). Further studyshows that pentoxifylline (PTX) adminis-tered after CLP preserved the vascularresponse to AM in late sepsis (40). PTXtreatment did not alter the increase inblood AM concentration following CLP,but it did decrease the elevated levels ofTNF-α, IL-1β and IL-6 seen in the latephase (40). In addition to its well knownvasodilatory effects, AM also modulatesproduction of inflammatory cytokines.Kaomi et al. (41) reported that AM inhib-ited secretion of cytokine-induced neu-trophil chemoattractant, belonging toIL-8 superfamily in rat alveolarmacrophages stimulated with endotoxin.AM also can suppress IL-1β-inducedTNF-α production in Swiss 3T3 cells (42).

The novel specific AM binding proteinAMBP-1 was first reported by Elsasser etal. (22). AMBP-1 is present in plasma, andthe liver is considered to be the mainsource (29). Our recent studies have

demonstrated that coadministration ofAM and AMBP-1 prevents the transitionfrom the hyperdynamic phase to the hy-podynamic phase, attenuates tissue in-jury and hemoconcentration during thelate hypodynamic stage of sepsis, anddecreases sepsis-induced mortality (30).Neither AM nor AMBP-1 alone was suffi-cient to achieve these beneficial effects(30). Although our recent studies haveindicated that increased levels of plasmaTNF-α in septic animals were signifi-cantly attenuated after the administra-tion of AM/AMBP-1 (29), it remainsunknown whether AM/AMBP-1 directlydownregulates the proinflammatory cy-tokines. Kupffer cells are the mainsource of proinflammatory cytokines insepsis (43), and we have shown that AM/AMBP-1 have a direct downregulatoryeffect on TNF-α from isolated Kupffercells, as well as from macrophage cellline RAW 264.7 cells (44).

The potential roles of vasoactive medi-ators other than AM have been evaluatedin the pathophysiology of sepsis. Nitricoxide (NO) is a potent vasodilator pro-duced by several isoforms of the enzymeNO synthase (NOS), including endothe-lial constitutive NOS (ecNOS) and in-ducible NOS (iNOS). NO derived fromecNOS has been shown to decrease inthe early, hyperdynamic phase of sepsis(33,45). Downregulation of ecNOS con-tributes to vascular hyporesponsivenessin sepsis (32-34). In the present study,ecNOS gene expression decreased by27%-36% after the onset of sepsis. Twospecific mechanisms are responsible forthe vasodilatory effect of AM, a direct effect on vascular smooth muscle cells toincrease intracellular cAMP levels bystimulating AM receptors and adenylatecyclase activity (39,46,47) and an indirecteffect on vascular endothelial cells bystimulating Ca2+ mobilization to increaseendothelium-derived NO release via theactivation of ecNOS (39,48). Activation ofecNOS was observed to lead to the acti-vation of the NO-cGMP pathway (49,50).In the present study, we observed thatthe decreased ACh-induced vascular re-laxation (reflecting ecNOS-derived NO)at 20 h after CLP was prevented by intra-venous administration of AM/AMBP-1.Moreover, the reduced gene expressionof endothelial ecNOS observed at 20 hafter CLP was prevented after adminis-tration of AM/AMBP-1 in aortic (seeFigure 3A) and pulmonary (see Figure 3B)tissues. Furthermore, the results alsoshow that the ecNOS-immunoreactivepositive area in endothelial cells wassignificantly reduced in CLP animals,but was restored to normal levels inAM/AMBP-1-treated animals (see Figure 6).It is interesting to note that the endothe-lium-independent vasodilating agentNTG was not effective in reducing vas-cular relaxation in septic animals (seeFigure 2). This finding suggests that theNO-cGMP pathway is indeed importantin mediating the beneficial effects ofAM/AMBP-1 in sepsis.

It should be pointed out that the drug-control group (sham-operated animals

Figure 6. The immunohistochemical stainings of ecNOS in the thoracic aortae 20 h aftercecal ligation puncture (CLP) with or without AM/AMBP-1 treatment. ecNOS immunoreac-tion products were observed in aortic endothelial cells (A–C). No immunostaining was seenin vascular smooth muscle or connective tissues. ecNOS immunohistochemical staining inthe vascular endothelial cells was reduced in septic animals (B) compared with sham-operated animals (A). Administration of AM/AMBP-1 attenuated the decreased ecNOSstaining (C). There were no immunoreaction products in the negative control section (D).(A), sham 20 h; (B), CLP 20 h; (C), CLP 20 h plus AM/AMBP-1 treatment; (D), negative control.

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with AM/AMBP-1 administration) wasnot included in this study. In this regard,we have previously demonstrated that ad-ministration of AM/AMBP-1 in sham-op-erated rats did not alter circulating levelsof liver enzymes (AST, ALT), lactate, creati-nine, or inflammatory cytokines (TNF-α,IL-10, HMGB-1) (51). Because it is unlikelythat administration of AM/AMBP-1 inshome rats would change ACh-inducedvascular relaxation and ecNOS expression,to save animals resources we did not in-clude the drug-control group in the currentstudy. Our previous study clearly showedthat AMBP-1 increases AM-induced vascu-lar relaxation in sham-operated animals ina dose-response manner under in vitroconditions (28). Moreover, in vitro additionof AMBP-1 can completely restore AM hy-poresponsiveness in the isolated blood ves-sels in a dose-response fashion (28). Al-though plasma levels of AM increase insepsis (37), the levels of AMBP-1 decreasedsignificantly in the blood and tissues undersuch conditions (28,52). The AM hypore-sponsiveness observed in sepsis may becaused by the reduced AMBP-1 produc-tion. In addition, the effect of AM/AMBP-1in sepsis is physiological becauseAM/AMBP-1 has no adverse effect onsham-operated rats (51). Regarding the po-tential effects of AM/AMBP-1 on iNOS ex-pression, it is well recognized that iNOSexpression and iNOS-derived NO produc-tion increase in severe sepsis. While wehave clearly shown that administration ofAM/AMBP-1 in sepsis improves ecNOSexpression and ACh-induced vascular re-laxation (ecNOS-mediated), one would ex-pect that AM/AMBP-1 may have suppres-sive effects on iNOS under suchconditions. However, the precise effect ofAM/AMBP-1 on iNOS expression and ac-tivity in sepsis remains to be determined.

Studies have shown that vascular en-dothelial cells play an important role inregulation of tissue perfusion by the re-lease of various vasoactive mediators,such as NO, under normal as well asvarious adverse circulatory conditions(53,54). Endothelium-derived NO is synthesized from L-arginine by Ca2+-de-pendent, constitutive NO synthase (48).

A number of studies have shown that endothelium-derived NO is depressedfollowing endotoxic shock (54,55) as wellas during sepsis (33,53). However, themechanism responsible for the decreasedrelease of endothelial-derived NO duringsepsis remains unknown. In the presentstudy, we have provided a clue to thefinding that ecNOS gene expression isdepressed during sepsis and the protec-tive effect of AM/AMBP-1 in sepsis is topreserve ecNOS gene expression, therebypreventing endothelial cell dysfunction.

ACKNOWLEDGMENTSThis investigation was supported by

National Institutes of Health (NIH)grants R01 GM057468 and R01 HL076179(P Wang). The authors sincerely thankZheng F Ba, Dale E Fowler, ShaolongYang, and David A Ornan for their excel-lent assistance.

REFERENCES1. Bernard GR, Vincent JL, Laterre PF, LaRosa SP,

Dhainaut JF, Lopez-Rodriguez A, Steingrub JS,Garber GE, Helterbrand JD, Ely EW, Fisher CJ Jr.(2001) Efficacy and safety of recombinant humanactivated protein C for severe sepsis. N. Engl. J.Med. 344:699–709.

2. Martin GS, Mannino DM, Eaton S, Moss M. (2003)The epidemiology of sepsis in the United Statesfrom 1979 through 2000. N. Engl. J. Med. 348:1546–54.

3. Hotchkiss RS, Karl IE. (2003) The pathophysiol-ogy and treatment of sepsis. N. Engl. J. Med. 348:138–50.

4. Tracey KJ. (2002) The inflammatory reflex. Nature420:853–9.

5. Riedemann NC, Guo RF, Ward PA. (2003) Novelstrategies for the treatment of sepsis. Nat. Med. 9:517–24.

6. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. (2001) Epide-miology of severe sepsis in the United States:analysis of incidence, outcome, and associatedcosts of care. Crit. Care Med. 29:1303–10.

7. Kitamura K, Kangawa K, Kawamoto M, Ichiki Y,Nakamura S, Matsuo H, Eto T. (1993)Adrenomedullin: a novel hypotensive peptideisolated from human pheochromocytoma.Biochem. Biophys. Res. Commun. 192:553–60.

8. Sakata J, Shimokubo T, Kitamura K, Nakamura S,Kangawa K, Matsuo H, Eto T. (1993) Molecularcloning and biological activities of ratadrenomedullin, a hypotensive peptide. Biochem.Biophys. Res. Commun. 195:921–7.

9. Hirata Y, Mitaka C, Sato K, Nagura T, Tsunoda Y,

Amaha K, Marumo F. (1996) Increased circulat-ing adrenomedullin, a novel vasodilatory pep-tide, in sepsis. J. Clin. Endocrinol. Metab. 81:1449–53.

10. Nishio K, Akai Y, Murao Y, Doi N, Ueda S,Tabuse H, Miyamoto S, Dohi K, Minamino N,Shoji H, Kitamura K, Kangawa K, Matsuo H.(1997) Increased plasma concentrations ofadrenomedullin correlate with relaxation of vas-cular tone in patients with septic shock. Crit. CareMed. 25:953–7.

11. Ehlenz K, Koch B, Preuss P, Simon B, Koop I,Lang RE. (1997) High levels of circulatingadrenomedullin in severe illness: correlationwith C-reactive protein and evidence against theadrenal medulla as site of origin. Exp. Clin. En-docrinol. Diabetes 105:156–62.

12. Ueda S, Nishio K, Minamino N, Kubo A, Akai Y,Kangawa K, Matsuo H, Fujimura Y, Yoshioka A,Masui K, Doi N, Murao Y, Miyamoto S. (1999)Increased plasma levels of adrenomedullin in pa-tients with systemic inflammatory response syn-drome. Am. J. Respir. Crit. Care Med. 160:132–6.

13. Fujioka S. (2001) Increased plasma concentrationof adrenomedullin during and after major sur-gery. Surg. Today 31:575–9.

14. Sugo S, Minamino N, Shoji H, Kangawa K, Kita-mura K, Eto T, Matsio H. (1995) Interleukin-1,tumor necrosis factor and lipopolysaccharide ad-ditively stimulate production of adrenomedullinin vascular smooth muscle cells. Biochem. Biophys.Res. Commun. 207:25–32.

15. Shoji H, Minamina N, Kangawa K, Matsuo H.(1995) Endotoxin markedly elevates plasma con-centration and gene transcription of adrenomedullinin rat. Biochem. Biophys. Res. Commun. 215:531–7.

16. Ono Y, Kojima M, Okada K, Kangawa K. (1998)cDNA cloning of canine adrenomedullin and itsgene expression in the heart and blood vessels inendotoxin shock. Shock 10:243–7.

17. Zaks-Zilberman M, Salkowski CA, Elsasser T,Cuttitta F, Vogel SN. (1998) Induction ofadrenomedullin mRNA and protein bylipopolysaccharide and paclitaxel (taxol) inmurine macrophages. Infect. Immun. 66:4669–75.

18. Elsasser TH, Sartin JL, Martinez A, Kahl S,Montuenga L, Pio R, Fayer R, Miller MJ, Cuttitta F.(1999) Underlying disease stress augmentsplasma and tissue adrenomedullin (AM) re-sponses to endotoxin: colocalized increases inAM and inducible nitric oxide synthase withinpancreatic islets. Endocrinol. 140:5402–11.

19. Matsui E, Kitamura K, Yoshida M, Kato J, Asada Y,Sumiyoshi A, Eto T. (2001) Biosynthesis and secre-tion of adrenomedullin and proadrenomedullinN-terminal 20 peptide in a rat model of endotoxinshock. Hypertens. Res. 24:543–9.

20. Shindo T, Kurihara H, Kurihara Y, Morita H,Yazaki Y. (1998) Upregulation of endothelin-1and adrenomedullin gene expression in themouse endotoxin shock model. J. Cardiovasc.Pharmacol. 31 (Suppl 1):S541–4.

21. Zhou M, Chaudry IH, Wang P. (2001) The small

4 9 4 | Z H O U E T A L . | M O L M E D 1 3 ( 9 - 1 0 ) 4 8 8 - 4 9 4 , S E P T E M B E R - O C T O B E R 2 0 0 7

A M / A M B P - 1 A N D e c N O S I N S E P S I S

intestine is an important source ofadrenomedullin release during polymicrobialsepsis. Am. J. Physiol. Regul. Integr. Comp. Physiol.281:R654–60.

22. Elsasser TH, Kahl S, Martinez A, Montuenga LM,Pio R, Cuttitta F. (1999) Adrenomedullin bindingprotein in the plasma of multiple species: charac-terization by radioligand blotting. Endocrinol.140:4908–11.

23. Pio R, Martinez A, Unsworth EJ, Kowalak JA,Bengoechea JA, Zipfel PF, Elsasser TH, Cuttitta F.(2001) Complement factor H is a serum bindingprotein for adrenomedullin. The resulting com-plex modulates the bioactivities of both partners.J. Biol. Chem. 276:12292–300.

24. Friese MA, Hellwage J, Jokiranta TS, Meri S,Peter HH, Eibel H, Zipfel PF. (1999) FHL-1/reconectin and factor H: two human complementregulators which are encoded by the same geneare differently expressed and regulated. Mol. Im-munol. 36:809–18.

25. Schwaeble W, Zwirner J, Schulz TF, Linke RP,Dierich MP, Weiss EH. (1987) Human comple-ment factor H: expression of an additional trun-cated gene product of 43 kDa in human liver.Eur. J. Immunol. 17:1485–9.

26. Luo W, Vik DP. (1999) Regulation of complementfactor H in a human liver cell line by interferon-gamma. Scand. J. Immunol. 49:487–94.

27. Schwaeble W, Schwaiger H, Brooimans RA, Barbieri A, Most J, Hirsch-Kauffmann M, Tiefen-thaler M, Lappin DF, Daha MR, Whaley K. (1991)Human complement factor H. Tissue specificityin the expression of three different mRNAspecies. Eur. J. Biochem. 198:399–404.

28. Zhou M, Ba ZF, Chaudry IH, Wang P. (2002)Adrenomedullin binding protein-1 modulatesvascular responsiveness to adrenomedullin inlate sepsis. Am. J. Physiol. Regul. Integr. Comp.Physiol. 283:R553–60.

29. Yang S, Zhou M, Fowler DE, Wang P. (2002)Mechanisms of the beneficial effect ofadrenomedullin and adrenomedullin-bindingprotein-1 in sepsis: Down-regulation of proin-flammatory cytokines. Crit. Care Med. 30:2729–35.

30. Yang S, Zhou M, Chaudry IH, Wang P. (2002)Novel approach to prevent the transition fromthe hyperdynamic phase to the hypodynamicphase of sepsis: role of adrenomedullin andadrenomedullin binding protein-1. Ann. Surg.236:625–33.

31. Zhou M, Simms HH, Wang P. (2004)Adrenomedullin and adrenomedullin bindingprotein-1 attenuate vascular endothelial cellapoptosis in sepsis. Ann. Surg. 240:321–30.

32. Wang W, Mitra A, Poole B, Falk S, Lucia MS,Tayal S, Schrier R. (2004) Endothelial nitric oxidesynthase-deficient mice exhibit increased suscep-tibility to endotoxin-induced acute renal failure.Am. J. Physiol. Renal Physiol. 287:F1044–8.

33. Zhou M, Wang P, Chaudry IH. (1997) Endothelialnitric oxide synthase is downregulated duringhyperdynamic sepsis. Biochim. Biophys. Acta.

1335:182–90.34. Wang P, Ba ZF, Chaudry IH. (1994) Nitric oxide:

to block or enhance its production during sepsis?Arch. Surg. 129:1137–43.

35. Wang P, Chaudry IH. (1998) A single hit model ofpolymicrobial sepsis: cecal ligation and puncture.Sepsis 2:227–33.

36. Yang S, Cioffi WG, Bland KI, Chaudry IH, Wang P.(1999) Differential alterations in systemic and re-gional oxygen delivery and consumption duringthe early and late stages of sepsis. J. Trauma47:706–12.

37. Wang P, Zhou M, Ba Z., Cioffi WG, Chaudry IH(1998) Upregulation of a novel potent vasodila-tory peptide adrenomedullin during polymicro-bial sepsis. Shock 10:118–23.

38. Wang P, Yoo P, Zhou M, Cioffi, WG, Ba ZF,Chaudry IH. (1999) Reduction in vascular re-sponsiveness to adrenomedullin during sepsis. J. Surg. Res. 85:59–66.

39. Koo DJ, Zhou M, Chaudry IH, Wang P. (2001)The role of adrenomedullin in producing differ-ential hemodynamic responses during sepsis. J. Surg. Res. 95:207–18.

40. Koo DJ, Yoo P, Cioffi WG, Bland KI, Chaudry IH,Wang P. (2000) Mechanism of beneficial effects ofpentoxifylline during sepsis: maintenance ofadrenomedullin responsiveness and downregu-lation of proinflammatory cytokines. J. Surg. Res.91:70–5.

41. Kaomi H, Kanazawa H, Hirata K, Kurihara N,Yano Y, Otano S. (1995) Adrenomedullin inhibitsthe secretion of cytokine-induced neutrophilchemoattractant, a member of the interleukin-8family, from rat alveolar macrophages. Biochem.Biophys. Res. Commun. 211:1031–5.

42. Isumi Y, Kubo A, Katafuchi T, Kangawa K, Minamir N. (1999) Adrenomedullin suppressesinterleukin-1_-induced tumor necrosis factor-αproduction in Swiss 3T3 cells. FEBS Lett. 463:110–6.

43. Koo DJ, Chaudry IH, Wang P. (1999) Kupffercells are responsible for producing inflammatorycytokines and hepatocellular dysfunction duringearly sepsis. J. Surg. Res. 83:151–7.

44. Wu R, Zhou M, Wang P. (2003) Adrenomedullinand adrenomedullin binding protein-1 downreg-ulate TNF-alpha in macrophage cell line and ratKupffer cells. Regulatory Pept. 112:19–26.

45. Wang P, Ba ZF, Chaudry IH. (1995) Endotheliumdependent relaxation is depressed at the macro-and microcirculatory levels during sepsis. Am. J. Physiol. 269:R988–93.

46. Equchi S, Hirata Y, Iwanski H, Sato K, Watanabe TX,Inui P, Nakajima K, Sakakibara S, Marumo F.(1994) Structure activity relationship ofadrenomedullin, a novel vasodilatory peptide,in cultured rat vascular smooth muscle cells. Endocrinology 135:2454–8.

47. Montuenga LM, Martinez A, Miller MA,Unsworth EJ, Cuttitta F. (1997) Expression ofadrenomedullin and its receptor during embryo-genesis suggests autocrine or paracrine modes of

action. Endocrinology 138:440–51.48. Shimekake Y, Nagata K, Ohta S, Kambayashi Y,

Teraoka H, Kitamuoa K, Eto P, Kangawa K, Matsuo H. (1995) Adrenomedullin stimulatedtwo signal transduction pathways, cAMP accu-mulation and Ca2+ mobilization in bovine aorticendothelial cells. J. Biol. Chem. 270:P4412–7.

49. Hinson JP, Kapas S, Smith DM. (2000)Adrenomedullin, a multifunctional regulatorypeptide. Endocr. Rev. 21:138–67.

50. Nishimatsu H, Suzuki E, Nagata D, Mooiyama N,Satonaka H, Walsh K, Sata M, Kangawa K, Matsuo H, Gotto A, Kitamura T, Hirata Y. (2001)Adrenomedullin induces endothelium depen-dent vasorelaxation via the phosphatidylinositol3-Kinase/Akt-dependent pathway in rat aorta.Circ. Res. 89:63–70.

51. Cui X, Wu R, Zhou M, Dong W, Ulloa L, Yang H,Wang H, Tracey KJ, Simms HH, Wang P. (2005)Adrenomedullin and its binding protein attenu-ate the proinflammatory response after hemor-rhage. Crit. Care Med. 33:391–8.

52. Cui Y, Ji Y, Wu R, Zhou M, Wang P. (2006)Adrenomedullin binding protein-1 is downregu-lated during polymicrobial sepsis in the rat. Int.J. Mol. Med. 17:925–9.

53. Vallet B. (2003) Bench-to-bedside review: En-dothelial cell dysfunction in sepsis: a role inorgan dysfunction? Crit. Care 7:130–8.

54. Yamashita T, Kawashina S, Ohashi Y. (2000) Re-sistance to endotoxin shock in transgenic miceoverexpressing nitric oxide synthase. Circulation101:931–7.

55. Parker JL, Adams HR. (1993) Selective inhibitionof endothelium-dependent vasodilator capacityby Escherichia coli endotoxemia. Circ. Res.72:539–51.