genesis, transcription regulation, cell survival, and apoptosis. PKC-zis responsible for the serine phosphorylation and activation of Sp1,an important transcription factor for many genes, including matrixmetalloproteinases. The aim of this study was to determine PKC-zinvolvement in endothelial cells (EC) exposed to CS and SS.
METHODS: Microvascular EC were exposed to up to 8 hours ofeither SS at 14 dynes/cm2, or CS at 60 cycles per minute 24%maximal strain. Lysates were studied for the expression and activity ofPKC-z through immunoblotting analysis and by performing activityassays in which lysates were immunoprecipitated, labeled with theisotope gamma-32P, and then analyzed using liquid scintillationspectroscopy.
RESULTS: PKC-z phosphorylation in EC exposed to SS peaked atone hour with an increase in protein expression of 1.73 � 0.34 fold(n�5; p�0.05) and in activity of 2.92 � 0.34 fold (n�3; p�0.05).EC exposed to CS had no significant variation in PKC-z expressionor activation. In addition, Sp1 affinity increased in EC exposed to SSbut not to CS.
CONCLUSIONS: Different hemodynamic forces affect EC differ-ently. SS but not CS activates PKC-z and increases the affinity of Sp1to its nuclear binding site. Differential activation of PKC may explainthe varied response of EC to these physical forces.
Delivery of exogenous ATP maintains sodium-potassium pump activity in endothelial cells duringchemical hypoxiaClaudio Maldonado PhD, Federico V Grossi MD, Mashiur Khan BS,Chirag Soni BS, Mansim C Okafor PhD, Dalibor Vasilic MD,William C Ehringer PhD, Gustavo Perez-Abadia MD,Nicholas A Delamere PhD, John H Barker MDUniversity of Louisville, Louisville, KY
INTRODUCTION: Our group has developed a tissue preservationsolution that contains a novel fusogenic lipid vesicle (FLV) that de-livers ATP directly into the cytosol of cells. In preservation studies inour laboratory, amputated ischemic limbs briefly perfused with ATP-loaded FLVs (ATP-FLVs) remained viable for up to 21h at roomtemperature. These limbs were successfully transplanted to twin-likerecipients, and histologic analysis of limb muscles showed an intactvascular tree surrounded by significant myocyte regeneration sug-gesting a patent microcirculation. The purpose of this study was toexamine mechanisms by which ATP-FLVs maintained cells viableduring hypoxia. We hypothesized that delivery of exogenous ATPduring hypoxia reserved endothelial cell viability by maintainingactivity of the ouabain sensitive sodium-potassium ATPase (Na,KATPase) pump.
METHODS: Human umbilical vein endothelial cells (HUVECs)cultured in separate wells were incubated in Krebs solution;Krebs�ATP; Krebs�FLVs; and Krebs�ATP-FLVs. Chemical hyp-oxia was induced for 4h with potassium cyanide (KCN). Cell viabil-ity was assessed using staining techniques, and activity of the Na,KATPase pump was quantified using rubidium (86Rb) uptake.
RESULTS: HUVECs subjected to chemical hypoxia and treatedwith ATP-FLVs were 97�/-2% viable compared to 11�/-8% ofHUVECs receiving no treatment (p�0.05). ATP-FLVs preservedHUVEC Na,K ATPase pump activity compared to cells treated withKrebs only or with Krebs�ATP (5.9�/-1.7 vs. 0.6�/-0.2 or 1.1�/-0.4 nanomoles potassium/mg protein/min, p�0.05).
CONCLUSIONS: Exogenous delivery of ATP with ATP-FLVs dur-ing chemical hypoxia is effective in sustaining activity of the Na,KATPase pump, which is important in maintaining cellular ionic ho-meostasis, and ultimately, HUVEC viability.
Novel role of toll-like receptor 4 in limb ischemia-reperfusionHassan Albadawi MD, Fateh Entabi MD, Mark Conrad MD,David Stone MD, Michael Watkins MDMassachusetts General Hospital, Harvard Medical School, Boston,MA
INTRODUCTION: Pattern recognition molecules including Toll-like 4 receptor (TLR4), play a major role in the innate immuneresponse (i.e. inflammation) to sepsis. These experiments were de-signed to study the role of TLR4 in skeletal muscle injury in a murinemodel of hindlimb ischemia-reperfusion.
METHODS: TLR4 mutant mice C3H/HeJ and their wild typeC3H/OuJ (WT) were subjected to 3 hours unilateral hindlimb isch-emia (tourniquet) followed by 48 hours of reperfusion. At the end ofreperfusion, hindlimb tissue viability was assessed using an assay ofmitochondrial activity (MTT). ELISA was used to evaluate locallevels of pro-inflammatory cytokines KC, MIP2, IL-1beta, the acutephase reactant IL-6 and the angiogenic factor VEGF in protein ex-tracts from the limbs. Data was analyzed using an unpaired t test andexpressed as mean � SE.
RESULTS: Tissue viability (% contralateral limb) was significantlyhigher in the TLR4 mutated mice vs. WT after 48 hours reperfusion.Tissue KC, MIP2 and IL-1beta were higher in the TLR4 mutatedmice, whereas IL-6 and VEGF proteins were similar to WT mice.
CONCLUSIONS: TLR4 deficient mice manifest reduced reperfu-sion injury evident by higher mitochondrial activity compared toWT. Paradoxically, decreased injury was associated with higher tissuelevels of pro-inflammatory cytokines. These data suggest that TLR4pathway plays a significant role in modulating the inflammatoryresponse to skeletal muscle ischemia-reperfusion.
TLR4 mutated(n�6)
WT(n�7)
Viability (%) (MTT) 87.4 � 3* 74 � 4KC pg/mg 82 � 8* 53 � 10MIP2 pg/mg 55 � 4** 31 � 5IL-1� pg/mg 29 � 7* 14 � 2IL-6 pg/mg 11 � 1 8 � 2VEGF pg/mg 52 � 2 49 � 4
*p � 0.05 vs. WT; **p � 0.01 vs. WT.
S101Vol. 201, No. 3S, September 2005 Vascular Surgery I
