of calcium by sensing oxidative stress. Because hemorrhagic shockleads to profound redox stress and is characterized by alterations incellular calcium concentrations, we hypothesized that CaMK medi-ate inflammation and end-organ injury after hemorrhagic shock.Methods: C57/Bl6 mice were treated with either the broad CaMKinhibitor KN93 (16 mg/kg, IP, n�7) or, as a control, its inactiveanalog KN92 (16 mg/kg, IP, n�6) at both 12 hours and 1 hourprior to hemorrhagic shock (H/S). H/S was induced to a meanarterial pressure of 25 mm Hg, as determined by femoral arterialcannulation, maintained for 2.5 hours, and then followed by 4hours of resuscitation with shed blood and twice that volume ofLactated Ringer’s (LR) solution. Sham animals underwent unilat-eral femoral artery cannulation only (n�5/group). After resusci-tation, serum was harvested and assayed for ALT concentrationas a measure of hepatic injury. Systemic inflammation was as-sessed by measuring serum levels of IL-10 and IL-6 by ELISA.Results: Relative to sham, control mice subjected to H/S exhibitedsignificant increases in serum ALT (112 � 13 vs. 520 � 98 IU/L,p � 0.05), IL-6 ( 57.7 � 11.1 vs. 189.6 � 34.7 pg/ml, p � 0.05), andIL-10 (20.4 � 20.1 vs. 149.9 � 26.7 pg/ml, p� 0.05). CaMK inhi-bition with KN93 resulted in significantly lower serum ALT levelsafter hemorrhage when compared to those animals that receivedpretreatment with KN92 (296 � 79 vs. 520 � 98 IU/L, p� 0.05).Similarly, CaMK inhibition with KN93 resulted in significantlylower serum IL-10 levels after hemorrhage when compared tothose animals that received pretreatment with KN92 (64.3 � 23.4vs. 149.9 � 26.7, p� 0.05). Conclusions: These findings indicatethat CaMK play a previously unsuspected role in the initiation ofinflammation and liver injury induced by hemorrhagic shock. Fur-thermore, inhibition of CaMK may prove to be a useful therapeuticstrategy for preventing complications and limiting end-organ injuryafter hemorrhagic shock.

QS388. HEMORRHAGIC SHOCK INDUCES THE RELEASEOF 5-HETE AND THROMBOXANE B2 IN POST-SHOCK MESENTERIC LYMPH. Janeen R. Jordan1,Ernest E. Moore2, Sagar S. Damle1, Simona Zarini1, AnirbanBanerjee1; 1University of Colorado Health Science Center,Denver, CO; 2Denver Health Medical Center, Denver, CO

Background: Mesenteric lymph is the mechanistic link betweensplanchnic hypoperfusion and acute lung injury (ALI), but theculprit mediator(s) remain elusive. Our previous work has shownthat systemic administration of a phospholipase A2 (PLA2) inhib-itor attenuated postshock ALI. We also identified a neutral lipidfraction within the postshock mesenteric lymph (PSML) respon-sible for neutrophil (PMN) priming. Arachidonic acid (AA) is me-tabolized by many key enzymes including 5-Lipoxygenase (5-LO)and COX 1&2. Consequently, we hypothesized that AA is a keymediator in the pathogenesis of postshock inflammation via5-HETE and thromboxane B2 (TXB2). Methods: Hemorrhagicshock was induced in SD rats by controlled hemorrhage to a MAPof 30mmHg and sustained for 40min. Midline laparotomy wasperformed in select animals and the mesenteric duct was cannu-lated for lymph collection and diversion. Resuscitation was per-formed by infusing 2x SB volume in NS over 30min, followed by ½SB volume returned over 30min, then completed with 2x SBvolume in NS over 60min. 5-HETE and TXB2 were measured inthe lymph, plasma and lungs by methanol extraction and MS fromboth diverted and undiverted animals. ANOVA with Tukey wasemployed; significance � p� 0.05. Results: AA is increased in thePSML. 5-HETE was increased in PSML and in the postshockplasma, but did not increase significantly in the lung in undivertedanimals compared to diverted animals (see figure). TXB2 increasedin the lymph and in the undiverted lung samples but not in theplasma (see figure).

Conclusion: This data suggests that splanchnic I/R activates thePLA2 mediated release of AA into the mesenteric lymph where itmay be metabolized by 5-LO; however, it may also be delivered to thelungs and metabolized by COX enzymes. 5-HETE and TXB2 areproduced and are available to affect PMN and platelet infiltration orbe converted to other metabolites to incite inflammation.

QS389. ACTIVATED PROTEIN C REDUCES SYSTEMIC IN-FLAMMATION AND PRESERVES HEPATIC PERFU-SION AFTER TRAUMA AND SEPSIS. Steve Keller1,Toan Huynh1, Min Shin2, Jerrod Kraftchick2, Mark Clem-ens2; 1Carolinas Medical Center, Charlotte, NC; 2The Uni-versity of North Carolina at Charlotte, Charlotte, NC

Background: Mild systemic stress induces liver blood flow to becomeheterogeneous (heterogeneity of perfusion - HoP). In sepsis, HoP dimin-ishes, indicating a state of decompensation. Here, we assessed HoP andpro-inflammatory cytokine levels after aPC treatment in a model offemur fracture (FFx) and cecal ligation and puncture (CLP). We hy-pothesized that aPC attenuates pro-inflammatory response andthereby preserves HoP. Methods: Two groups of Sprague Dawley ratsunderwent sham, CLP, or FFx followed 24 hours by CLP. Thereafter,animals in group 1 received saline; group 2 received aPC (1mg/kg),twice daily for four days. Plasma and liver tissue were harvested; IL-6and IL-8 levels were determined by ELISA. In separate experiments,hepatic HoP was determined by in vivo intravital microscopy. Liverinjury was assessed by plasma AST. Results: Animals treated withsaline after CLP and FFx�CLP showed increased levels of plasma andhepatic IL-6 and IL-8, and a decreased HoP. Treatment with aPCattenuated this response (see Table).

Group IL-6 (P) IL-8 (P) IL-6 (L) IL-8 (L)HoP

(�m/sec)AST (IU/

L)

Sham � saline 0 � 0† 21 � 17† 824 � 437† 767 � 124† 244 � 7† 51 � 7†Sham � aPC 0 � 0† 15 � 6† 124 � 118† 696 � 113† 237 � 7† 34 � 5†CLP � saline 5806 � 3389 492 � 22 2133 � 641 5005 � 687 167 � 19 227 � 14CLP � aPC 0 � 0* 52 � 39* 379 � 127* 1403 � 302* 269 � 8* 85 � 7*FFx � CLP �

saline9975 � 4104 481 � 16 3479 � 152 4193 � 578 166 � 20 206 � 50

FFx � CLP �aPC

0 � 0* 24 � 13* 464 � 160* 766 � 71* 298 � 8* 131 � 40

* p�0.05 vs. saline treated counterpart.

† p�0.05 vs. CLP�Saline and FFx�CLP�Saline. P�plasma (pg/mL); L�liver (pg/mL); n � 5

animals/group.

422 ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS

Conclusion: There is significant increase in temperature gradientin exposed bowel segments during mesenteric ischemia that does notimmediately improve during reperfusion. Further experiments willbe needed to clarify the pattern of thermal homeostasis in reperfusedsegments of bowel.

QS393. TREM1 MEDIATES ACTIVATION OF HUMAN ENDO-THELIAL CELLS. Fernando A. Rivera, Ming-Mei Liu, SueVanek, Agnes Burris, Joseph P. Minei; University of TexasSouthwestern Medical Center, Dallas, TX

Introduction: The triggering receptor expressed on myeloid cells(TREM)-1 is a recently identified molecule involved in the inflam-matory response. Activation of the human endothelium plays animportant role in the pathogenesis of sepsis. We examine the effectsof TREM-1 on human lung and vascular endothelial cell function.Methods: Primary human microvascular and lung endothelial cellswere incubated with recombinant human TREM-1. IL-8, solubleICAM-1 (sICAM-1), and soluble VCAM-1 (sVCAM-1), were measuredsupernatants by ELISA. Results: rhTREM-1 increased ICAM-1,VCAM-1, and IL-8 when compared with control, (P � .0001). AntiTREM-1 antibodies significantly decrease the adhesion molecule andIl-8 responses (P �.002). Conclusions: rhTREM-1 elicits proinflam-matory responses on endothelial cells and may contribute to alter-ations in endothelial cell function in human inflammation.

TRAUMA/CRITICAL CARE VIII: SIGNALING II

QS394. THE GENOMIC ANALYSIS OF THE HETEROGENE-ITY HYPOXIA RESPONSES IN RENAL PROXIMALTUBULE EPITHELIAL CELLS AFTER KNOCK-DOWN OF HIF-1� AND HIF-2�. Zhen Wang1, Edwin H.Rodriguez2, Michael T. Longaker1, Patrick O. Brown1, Jen-Tsan A. Chi3, George P. Yang1; 1Stanford University, Stan-ford, CA; 2UCSF, San Francisco, CA; 3Duke University,Durham, NC

Introduction: The cellular response to hypoxia is critical for isch-emic acute kidney failure and kidney carcinoma. HIF-1� is an im-portant transcription factor in the response to hypoxia and is ex-pressed ubiquitously throughout the body. HIF-2� responds to thesame physiologic cues but has a much more restricted pattern ofexpression and is expressed most highly in the kidney. The questionarises as to whether HIF-1� and HIF-2� have the same or differentfunctions in the kidney. To determine this, we chose to use the power

of cDNA microarray to analyze global expression profiles by knock-ing down HIF-1� and HIF-2� using siRNA in human renal proximaltubule epithelial cells (RPTECs). Methods: Dicer-generated humanHIF-1� and HIF-2� siRNA were transfected into RPTECs by elec-troporation with non-specific siRNA as controls. After 24h, RPTECswere subject to 21% or 2% O2 for 12h. Global gene expression profileswere analyzed with cDNA microarrays. The genetic changes associ-ated with hypoxia were derived from zero-transformation betweenthe samples of 2% O2 and corresponding 21% O2 control at the sametime point. After selecting for genes with significant changes bysignificance analysis of microarray (SAM), the zero-transformedchanges of each gene were grouped by hierarchical clustering andvisualized with the Treeview program. The siRNA efficiency ofHIF-1� and HIF-2� was proven by real-time PCR. Results: Real-time PCR demonstrated the siRNA was 90% efficient. Based uponSAM analysis, 447 genes were down regulated and 94 genes were upregulated by HIF-1� activation. HIF-2� appeared to regulate moregenes with 321 genes down regulated, and 954 genes up regulated.When comparing genes that share regulation by HIF-1� and HIF-2�siRNA, we found that only 178 showed similar regulation of the 970genes demonstrating more than 2.8 fold expression difference com-pared to controls. The vast majority, 792 genes, was not regulated byHIF-1� and HIF-2� in a coordinated manner, including importanthypoxia response genes such as CXCR4. Conclusion: Global anal-ysis of cellular responses to hypoxia after HIF-1� or HIF-2� siRNAhas revealed that while they regulate some genes in a similar man-ner, the majority of their down stream responses are unique to eachtranscription factor. This suggests that their functions are not re-dundant, but rather that each transcription factor plays a uniquerole in the response to hypoxia in kidneys. Detailed analysis of thesegenetic changes can lead to greater understanding of cellularchanges associated with different human diseases in the kidney.

QS395. WITHDRAWN

QS396. NITRATED OLEIC ACID IS AN ANTI-INFLAMMATORYSIGNALING MEDIATIOR IN RAT HEPATOCYTES. JonCardinal, Geetha Jeyabalan, Allan Tsung, Rajeev Dhupar,Bruce Freeman, David Geller; Univeristy of Pittsburgh,Pittsburgh, PA

Introduction: Nitrated fatty acids are emerging as a class of en-dogenous anti-inflammatory signaling molecules. We tested the hy-pothesis that nitrated oleic acid (OA-NO2) could inhibit known me-diators of inflammation in cultured rat hepatocytes. Methods: Rathepatocytes were treated with escalating doses of OA-NO2 versusoleic acid (OA) alone. Gamma-interferon (IFN-g) stimulated inter-feron regulatory factor-1 (IRF-1) protein expression was measuredby western blot analysis. Cytokine stimulated inducible nitric oxidesynthase (iNOS) messenger RNA levels, protein expression and ni-trite production were measured by reverse transcriptase polymerasechain reaction (RT-PCR), western blot and nitrite assay respectively.Cytokine stimulated nuclear factor kappa-beta (NF-KB) activationwas measured by electromobility shift assay (EMSA). Cell viabilitywas measured by lactate dehydrogenase (LDH) assay. Results:IFN-g stimulated IRF-1 protein expression was inhibited by OA-NO2in a dose-dependent manner. Cytokine stimulated NF-KB activationwas inhibited by OA-NO2 in a dose dependent manner. Cytokinestimulated iNOS mRNA, protein expression and nitrite productionwere inhibited by OA-NO2 in a dose dependent manner. None ofthese effects were seen with OA alone (Table 1). Cell viability wasunaffected by OA-NO2 or OA alone (data not shown). Conclu-sion(s): Our results indicate that nitrated oleic acid (OA-NO2)serves as an anti-inflammatory mediator in cultured rat hepatocytes.Future investigations will focus on in vivo delivery of this agent toelucidate its role as a possible therapeutic agent during inflamma-tory disease states.

424 ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS