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2007;27;e35-e137 Arterioscler Thromb Vasc Biol Arteriosclerosis, Thrombosis, and Vascular Biology Annual Conference 2007

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Abstracts

Arteriosclerosis, Thrombosis, andVascular Biology Annual Conference 2007

April 19-21, 2007Palmer House HiltonChicago, IL

Sponsored by the American Heart Association Council on Arteriosclerosis, Thrombosis,and Vascular Biology and the Council on Nutrition, Physical Activity, and Metabolism.Co-sponsored by the National Heart, Lung, and Blood Institute.

Conference Program Committee

Chair: Martha K. Cathcart, PhD, FAHA

William C. Aird, MD; Alan Daugherty, PhD, DSc, FAHA; George E. Davis, MD, PhD; WilliamP. Fay, MD, FAHA; Ronald M. Krauss, MD, FAHA; Mark W. Majesky, PhD; Gwendalyn J.Randolph, PhD; Lawrence L. Rudel, PhD, FAHA; Jonathan Smith, PhD, FAHA; Nancy R.Webb, PhD

Abstracts for the oral and poster presentations are provided in this special on-line supplement.


2007 ATVB Oral Presentations1

Pharmacological Inhibition of PCSK9 in Hyperlipidemic Mice SignificantlyReduces Serum LDL-C While Increasing Hepatic Low-Density LipoproteinReceptor Protein Abundance

Mark Graham, Kristina M M Lemonidis, Charles Whipple, Amuthakannan Subramaniam,Brett P Monia, Rosanne M Crooke; Isis Pharmaceuticals, Carlsbad, CA

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proprotein convertasefamily of proteases that has been previously demonstrated to promote the degradation of thelow density lipoprotein receptor (LDLr) through an undefined mechanism. Mutational analysisin humans has demonstrated that genetic polymorphisms which inactivate PCSK9 producedramatic reductions in serum LDL-C and further, appear to reduce the risk of coronary arterydisease (CAD). These encouraging epidemiological findings have been corroborated usingPCSK9 knockout mice, which exhibit the same phenotypic profile (i.e. decreased LDL-C andincreased hepatic LDLr). For this reason, we have developed second generation antisenseoligonucleotide inhibitors (ASOs) which target both human and mouse forms of PCSK9 mRNAto define their hypolipidemic effects both in vitro and in vivo. Due to their optimalpharmacokinetic/pharmacodynamic properties, 2MOE-modified ASOs have been extensivelyexploited to inhibit a broad range of therapeutically attractive liver gene targets such as apoB,Lp(a) and ACAT2. Administration of the PCSK9 ASO (ISIS 394814) to high fat fed mice for 6weeks (i.p., 100mg/kg/wk) resulted in reductions in both total cholesterol and LDL-C, 53% and38%, respectively. In addition, hepatic RNA and protein analysis revealed that ISIS 394814reduced PCSK9 mRNA expression by 92% while increasing LDLr protein levels greater than2-fold relative to controls. The magnitude of these effects is consistent with results previouslyreported in PCSK9 knockout mice. Based on these data, additional studies are in progress inLDLr deficient and additional mouse models to further demonstrate the specificity andpharmacological efficacy of this drug. These promising in vivo results suggest that inhibitingPCSK9 may indeed represent a novel therapeutic approach for reducing LDL-C in man.

2Intestinal Cholesterol Absorption Is Required for the LXR Agonist toIncrease Plasma HDL Cholesterol in Mice

Weiqing Tang, Yinyan Ma, Lin Jia, Liqing Yu; Wake Forest Univ Sch of Medicine,Winston-Salem, NC

Genetic manipulation has established the liver as the major source of HDL in normal mice.However there is about 30% of plasma HDL contributed to the small intestine. It has beenreported that liver X receptor (LXR) agonist GW3965 raises plasma HDL cholesterol levels inwildtype and liver-specific ABCA1 knockout mice but not in mice lacking intestinal ABCA1,indicating that the intestine plays an important role under circumstance in the biogenesis ofplasma HDL cholesterol. The source of cholesterol for intestinal HDL formation is unclear. Wehypothesize that cholesterol absorption from the gut lumen plays a role in the LXRagonist-stimulated HDL formation. To test our hypothesis, mice lacking Niemann-Pick C1-Like1 (NPC1L1) (L1-KO mice), a gene that is essential for intestinal cholesterol absorption, weretreated with the LXR agonist T0901317 at 25 mg/kg BW/day for 7 days. As expected,T0901317-treated wildtype mice showed a dramatic increase in the plasma HDL cholesterollevel but this effect was almost abolished in the L1-KO mice in which a much greater fecalcholesterol excretion was observed instead. The intestinal ABCA1 mRNA level was about 4-foldlower in the untreated L1-KO versus wildtype mice, and increased 4.4-fold and 7.8-fold in theT0901317-treated wildtype and L1-KO mice, respectively. Hepatic ABCA1 failed to respond toT0901317 in both wildtype and L1-KO mice although hepatic ABCG5/G8 mRNA levels werehigher in the T0901317-treated versus untreated animals. In conclusion, intestinal cholesterolabsorption is required for LXR agonist to increase plasma HDL cholesterol in mice.

3HDL Transport Through Endothelial Cells Is Mediated by ABCG1 and SR-BI

Lucia Rohrer, Madeleine Zemp, Iris Lorenzi, Marc Lehnert, Arnold von Eckardstein; UnivHosp, Zurich, Switzerland

High density lipoproteins (HDL) and their main protein constituent, apolipoprotein A-I (apoA-I),exert potentially anti-atherogenic properties within the arterial wall. However, it is not clear howthey are transported from the blood stream into the vascular wall. Previously we have shownthat ABCA1 mediates apoA-I transcytosis in endothelial cells. Here we investigated theinteraction of HDL with endothelial cells. Endothelial cells bound and cell associate both125I-apoA-I and 125I-HDL with high affinity and in a saturable manner. Binding and cellassociation of HDL was only competed by excess HDL not by apoA-I and albumin. In contrastbinding and cell association of apoA-I is competed by excess apoA-I and HDL and not byalbumin. Biotinylation experiments showed that endothelial cells internalize labeled HDL andonly minor amounts of the internalized HDL was degraded. Cultivated in a Transwell system,the cells transported a fraction of 125I-HDL from the apical to the basolateral compartment asan intact protein in a competable and temperature sensitive manner. Moreover, RNAinterference to modulate ATP binding cassette G1 (ABCG1) and SR-BI resulted in reduced HDLcell surface binding, internalization and transport. We conclude that endothelial cellstranscytose HDL in an ABCG1 and/or SR-BI dependent process.

4Impact of Apolipoprotein M Expression on Nascent Pre- HDL Formationby ATP Binding Cassette Transporter A1 (ABCA1)

Anny Mulya, Amanda Wibley, Soon-Kyu Chung, Abraham K Gebre, Gregory S Shelness,John S Parks; Wake Forest Univ Sch of Med, Winston-Salem, NC

ApoM is a novel apolipoprotein that mainly associates with high density lipoproteins (HDLs) inplasma and has been reported to play a role in pre-beta (preb) HDL formation. We investigatedthe role of apoM expression on the initial steps of nascent preb HDL particle assembly byABCA1 using HEK293 cells. Neither control nor ABCA1 expressing HEK293 cells expresseddetectable apoM mRNA or protein based on real time PCR and Western blot analysis,respectively. Control and ABCA1-expressing cells were transfected with apoM C-FLAG andradiolabeled with 35S-Met/Cys prior to immunoprecipitation of apoM from medium and celllysates. Results showed that only 1% of the total cellular apoM was secreted both in theabsence or presence of ABCA1 expression. Immunofluorescence with anti-FLAG antibodydemonstrated that apoM protein was located primarily at intracellular sites and not at theplasma membrane of transfected cells. To investigate the role of apoM in nascent preb HDLformation, ABCA1-expressing or control cells, transfected with empty vector, apoM WT or apoMC-FLAG, were incubated with 125I-lipid-free apoA-I for 24 hours. Conditioned media wereharvested and fractionated by FPLC to observe HDL particle size distribution as monitored byelution of 125I apoAI. Preb HDL particles were formed in the absence of apoM expression (WTor C-FLAG); however, apoM expression promoted formation of larger-sized nascent preb HDL.Immunoprecipitation of the different sized FPLC-isolated preb HDL with anti-apoA-I antibodyfollowed by apoM Western blot analysis of pellet and supernatant showed that very littlesecreted apoM associated with preb HDL particles. Our results suggest that apoM is poorlysecreted by HEK293 cells, is not required for preb HDL assembly by ABCA1, and interactspoorly with secreted nascent preb HDL. However, apoM expression results in the appearanceof larger preb HDL particles in media. We propose that apoM may function catalytically at anintracellular site to transfer lipid onto preb HDL during or after their formation by ABCA1.

5Docosahexaenoic Acid Impairs the Formation of Fully Lipidated VLDL byOxidative Modification, Aggregation, and Degradation of the PrecursorParticles

Vatsala Maitin, NYU Sch of Medicine, New York, NY; Kevin J Williams, Thomas JeffersonUniv, Philadelphia, PA; Carly St. Germain, Zemin Yao, Univ of Ottawa, Ottawa, Canada;Edward A Fisher; NYU Sch of Medicine, New York, NY

Background: One mechanism of the lipid-lowering effects of n-3 fatty acids includingdocosahexaenoic acid (DHA) is reduced hepatic VLDL secretion by promoting intracellularapolipoprotein B (apoB) degradation. We reported that this is a non-proteasomal post-ERprocess; activated by intracellular conversion of DHA to lipid peroxides. DHA-treatment ofhepatocytes also resulted in the intracellular formation of malondialdehyde-modified highmolecular weight apoB aggregates. Objective: To determine the stage in the VLDL secretorypathway where oxidative modification and degradation of apoB occurs. Results: In pulse-chasestudies, initial stages (up to 30 min of chase, C30) of lipoprotein assembly were not significantlydifferent between oleic acid (OA) and DHA treated cells, with similar luminal quantities of totalapoB, pre-VLDL and VLDL. At C45, compared to OA, a decrease in luminal apoB was observedin DHA-treated cells, but in contrast to OA, this apoB was not recovered in the medium.Interestingly, the C45 time point in case of DHA coincided with accumulation of apoB-aggregates, partially accounting for the loss of intracellular apoB and reduced formation ofluminal VLDL. In experiments carried out at 20oC to block protein exit from the Golgi,comparable amounts of pre-VLDL particles accumulated in OA vs. DHA-treated cells, but withDHA, only 50% of these were able to form fully lipidated VLDL, consistent with the amountrecovered in the medium. The same trend was observed for lipoproteins extracted fromGolgi-specific microsomes. Co-administration of desferrioxamine (DFX), an iron-chelator thatprevents lipid peroxidation, caused DHA-treated cells to assume an OA-like profile, accumu-lating luminal VLDL-apoB that was secreted. Using an antibody for light-chain 3 (LC3), aspecific autophagosomal marker, apoB aggregates were shown to be enriched in cells treatedwith DHA compared to OA or DHADFX. Conclusions: (1) DHA allows step I (pre-VLDL) particleformation and their transport to the Golgi (2) Oxidative damage of the pre-VLDL apoB and/orits association with peroxidized lipids in the Golgi results in reduced luminal formation ofsecretion-competent VLDL particles (3) Aggregated apoB is co-localized with autophagosomes.

612-Lipoxygenase Deficiency Inhibits Angiotensin II-induced AbdominalAortic Aneurysm Rupture and Incidence in Apolipoprotein E-deficient Mice

Nicholas Hatch, Fjoralba Babamusta, Victoria L King; Univ of Kentucky, Lexington, KY

Objective: Arachidonic acid is metabolized through the cyclooxygenase-1 and -2 (COX-1 and-2), 5-lipoxygenase (5-LO) and 12/15-lipoxygenase (12-LO) pathways which produce a host ofbiologically active fatty acid derivates that affect cardiovascular disease. We have recentlydemonstrated that selective pharmacological inhibition of the COX-2 pathway markedlyattenuated angiotensin II (AngII)-induced AAA formation. Interestingly, 5-LO deficiency alsoattenuated AAA formation in an experimental animal model of AAAs. Genetic or pharmacolog-ical inhibition of one of these pathways may result in the shunting of arachidonic or linoleic aciddown the12-LO pathway. Therefore, we sought to determine the effect of 12-LO deficiency onAngII-induced AAA formation. Methods: Age matched male C57BL/6 apolipoprotein E (apoE)deficient (12-LO/) and C57BL/6 12-LO deficient apoE-/- (12-LO-/-) mice were infused with

e-36 Arterioscler Thromb Vasc Biol June 2007


AngII (1,000 ng/kg/min) for 28 days. Results: 12-LO deficiency in apoE-/- mice did not alterAngII induced increases in systolic blood pressure ( Saline: 12-LO/: 117 6 vs 12-LO-/-:125 6 mmHg; AngII: 12-LO/: 160 5 vs 12-LO-/-: 154 6 mmHg) or plasma totalcholesterol concentrations. Interestingly, 12-LO deficiency markedly decreased AngII-inducedaneurysmal mortality (12-LO/: 41% vs 12-LO-/-: 3%; P 0.001). The decrease inmortality was related to a significant reduction in abdominal aortic rupture (12-LO/: 23 %vs 12-LO-/-: 3%; P 0.016) and an ablation of aortic arch rupture (12-LO/: 18% vs12-LO-/-: 0.0%; P 0.013). 12-LO deficiency also modestly decreased AngII-inducedabdominal aortic expansion (12-LO/: 1.82 0.15 vs 12-LO-/-:1.44 0.11 mm; P 0.045) resulting in an reduction in the incidence (12-LO/: 81.25% vs 12-LO-/-: 50% ; P 0.018) and severity (P 0.015) of AngII-induced AAAs. Plasma 11-dehydro thromboxane B2concentrations were increased in 12-LO-/- mice in response to 28 days of AngII infusion(12-LO/: 192 29 vs 12-LO-/-: 406 73 pg/ml, P 0.011). Conclusion: These findingssuggest that 12-LO generated eicosanoids play a fundamental role in AngII-induced aneurys-mal rupture leading to an increase in mortality, as well as, contribute to the incidence andseverity of AngII-induced AAA formation.

7Tetrahydrobiopterin Reverses Preexisting Hypertension-induced VentricularRemodeling by Recoupling eNOS

An L Moens, Carlo Tocchetti, Elizabeth Ketner, Azeb Haile, Champion Hunter, Robert Weiss,Johns Hopkins Med Insts, Baltimore, MD; Pawel Kaminski, Michael S Wolin, New York MedCollege, Valhalla, NY; David A Kass, Jeffrey Rade; Johns Hopkins Med Insts, Baltimore, MD

Background: Pressure overload triggers eNOS as a prominent source of myocardial ROS thatcontribute to dilatory remodelling and cardiac dysfunction. Administration of tetrahydrobiopterin(BH4) can prevent pressure-load remodelling. The aim of this study was to investigate that BH4can reverse established non-decompensated heart failure by interacting with uncoupled eNOS,and on this way prevent the evolution to end-stage heart failure. Methods: Compensatedcardiac remodelling was induced in 60 mice by transverse aortic constriction (TAC). After 4wks,mice were randomized to receive BH4 (200mg/kg/d, n30) or placebo (n30) for the following5wks. Echocardiography, MRI and PV-loop analysis were performed. Cold SDS-PAGE wasperformed to evaluate eNOS dimer/monomer. ROS generation was evaluated with dihydro-ethidium (DHE) confocal staining and chemiluminescence. NOS activity and downstream NOproducts (PKG and P-VASP) were determined. Isolated myocyte studies were performed.Myocyte dimensions and fibrosis (score 0: abscent - 3 pronounced) were histologicallyevaluated. Overexpression of endothelial GTPCH, the rate limiting enzyme of BH4 synthesis,was evaluated in this TAC model (n15 mice). Results: BH4 significantly reversed cardiachypertrophy (heart weight 24017 mg at 4wks, 32418 mg at 9wks and 212 11mg at9wks with BH4 p0.001, idem for myocyte dimensions, wall thickness and calculated LVmass) and diminished fibrosis (score 2.10.4 vs. score 0.60.4 with BH4, p0.05). BH4prevented the evolution towards cardiac decompensation (ejection fraction: 45.71.6% at4wks, 34.72% at 9wks and 53.44.5% at 9wks with BH4, p0.001 and confirmed by MRIand PV loop analysis). BH4 recoupled the already uncoupled eNOS and increased its activityback to the normal level. Superoxide generation (total and NOS-dependent) was markedlyreduced by BH4. BH4 improved fractional shortening and calcium-kinetics in isolated myocytes.Endothelial upregulation of BH4 by GTPCH-Tg had no beneficial effect on remodeling.Conclusion: BH4 can reverse established cardiac remodelling by re-coupling uncoupled eNOSand as a consequence less NOS dependent ROS is generated, leading to less hypertrophy andfibrosis and an amelioration of cardiac function.

8New Pathway to Gene Expression in Human Endothelial Cells: Filamin BTranslation Is Preserved by Internal Ribosome Entry in Virally Infected EC

Huimiao Jiang, Brandt B Jones, John Kriesel, Douglas I Schmid, Andrew S Weyrich, Guy AZimmerman, Larry W Kraiss; Univ of Utah, Salt Lake City, UT

Introduction: Eukaryotic translation initiation occurs either at the 5 cap of the untranslatedregion (UTR) or at an internal ribosome entry site (IRES). Messenger RNA containing IRES maystill be translated despite cellular stress that impairs cap-dependent translation. WhetherIRES-mediated translation occurs in stressed EC is unknown. Hypothesis: IRES-containingmRNAs are selectively enriched in polyribosomes from human EC when cap-dependenttranslation is impaired by viral infection. Methods: Cap-dependent translation in primary humanumbilical vein EC was disabled by Poliovirus Type 1 infection. Polysome-associated RNA fromvirus- or mock-treated EC was analyzed by whole genome microarray with later verification byreal-time RT-PCR. The presence of an IRES in the 5UTR of candidate mRNA was confirmedusing a dicistronic luciferase reporter assay. Results: Infection of human EC was verified byimmunochemistry (poliovirus I antigen). Four hours post-infection, loss of polysome peaks inribosomal profiles and detection of eIF4G cleavage products on western blots indicatedrepression of cap-dependent translation. On microarray analysis, 277 mRNAs were enriched2-fold in polysome fractions from infected v control EC including several known to containan IRES (Cyr 61, Pim1). Sequences enriched in polysomes from infected EC with 5UTR 100bp are more likely to contain an IRES; 20 were selected for further analysis, including filaminB (not previously known to have an IRES). Real-time PCR confirmed the microarray findings for18/20 mRNAs. The filamin B 5UTR (165 bp) was cloned into a dicistronic luciferase reporterplasmid. Translation of the downstream luciferase product after transfection of the reporterconstruct into EAHY surrogate EC confirmed the presence of an IRES in the filamin B 5UTR.Conclusions: (1) Translation of filamin B, an actin-binding protein implicated in mechanotrans-duction and cell signaling, is preserved in virally infected EC via an IRES-mediated mechanism,identifying another mechanism by which EC regulate the pattern of expressed protein inresponse to microbial challenge and stress; (2) Microarray analysis of poliovirus-infected EC isan effective large-scale method to screen for IRES-containing mRNAs in EC.

9Reoxygenation Leads to Dissociation of Histone Deacetylase 7 fromHypoxia-inducible Factor-1a

Hiroyuki Kato, Shiori Tamamizu-Kato, Vasanthy Narayanaswami, Bruce N Ames; ChildrensHosp, Oakland Rsch Institute, Oakland, CA

Interruption of cerebral blood flow causes hypoxia leading to the death of neurons and glia.Hypoxia-inducible factor-1a (HIF-1a) plays an important role in cell survival under oxygendeprivation by regulating the transcription of genes involved in glucose metabolism, cellproliferation, and angiogenesis. Products of genes targeted by HIF-1a include vascularendothelial growth factor (VEGF), erythropoietin, and glucose transporter-1, which play a rolein improved cell survival under hypoxia. When oxygenation is normal, HIF-1a is rapidlydegraded through the ubiquitin-proteasome pathway, which is triggered by theoxygenadependent hydroxylation of proline residues (Pro402 and Pro564) in HIF-1a. Thesehydroxylated proline residues are recognized by the von-Hippel-Lindau tumor suppressorprotein (pVHL), a component of an E3 ubiquitin ligase complex (pVHL, Elongin B, and ElonginC). The E3 ubiquitin ligase complex promotes ubiquitination of HIF-1 leading to degradation ofHIF-1a in 26S proteasome in mammalian cells in normal oxygen concentrations. Under hypoxia,this oxygen dependent-degradation system is repressed. Previously, we identified histonedeacetylase (HDAC)7 as a binding protein of HIF-1a using yeast two-hybrid system. HDAC7 wasalso shown to increase HIF-1a transcriptional activity under hypoxia. In this study, weinvestigated the roles of HDAC7 in degradation of HIF-1a under re-oxygenation. We found thatdegradation of HIF-1a was correlated with translocation of HDAC7 from the nucleus to thecytoplasm upon re-oxygenation. Using a mutant of HIF-1a (Pro402A/P564A), we also found thatthe stabilized HIF-1a mutant localized in the nucleus under re-oxygenation whereas HDAC7was exported to the cytoplasm. The amino acids substitution mutations of nuclear exportsequences (NES) in HDAC7 (NES mut.) blocked translocation of HDAC7 to the cytoplasm andstabilized HIF-1a in the nucleus upon re-oxygenation. Moreover NES mut HDAC7 bound HIF-1aand suppressed ubqutination of HIF-1a upon re-oxygenation. Taken together, these resultssuggest that the dissociation of HDAC7 from HIF-1a and translocation of HDAC7 to thecytoplasm may lead to degradation of HIF-1a upon re-oxygenation.

10Role of Autotaxin, a Plasma Lysophospholipase D, in Hemostasis andThrombosis

Zehra Pamuklar, Univ of Kentucky, Lexington, KY; Shuying Liu, The Univ of Texas M DAnderson Cancer Cntr, Houston, TX; Anping Dong, Univ of Kentucky, Lexington, KY; GordonB Mills, The Univ of Texas M D Anderson Cancer Cntr, Houston, TX; Andrew J Morris,Susan S Smyth; Univ of Kentucky, Lexington, KY

Lysophosphatidic acid (LPA) is a bioactive lipid mediator produced by platelets and found inplasma and atherosclerotic plaques. LPA stimulates platelets, leukocytes, endothelial cells, andsmooth muscle cells to regulate cell growth, differentiation, survival, motility, and contractility.LPA is thus poised to serve as a key regulator of vascular cell function. LPA is generated inlarge part by the secreted lysophospholipase D autotoxin (ATX; enpp2). Mice with only one ATXgene (enpp2/-) have plasma LPA levels that are 50% of wild type mice. ATX deficiency inmice (enpp2-/-) results in embryonic lethality due to vascular defects, implicating ATX andpotentially LPA in vascular development. We observed excessive bleeding in three founder linesof transgenic FVB mice globally overexpressing ATX (ATX-TG). To characterize the bleedingdefect further, tail vein bleeding times were performed. The mean bleeding time in control FVBmice was 3 2.7 min (n 6), whereas none of the ATX-TG mice (n 10) stopped bleedingwithin 10 min (p 0.001). Platelet counts were similar in control (928 184 x 103/mm3) andATX-TG (808 156 x 103/mm3), and platelets from control and ATX-TG mice displayed similarlevels of platelet membrane glycoproteins (GPIIb/IIIa, GPIb, GPVI, GPIa/IIa) as measured by flowcytometry. Upon stimulation by different agonists (ADP, collagen, thrombin), platelets fromATX-TG mice also exposed P-selectin and bound fibrinogen as did control platelets. Nodifferences in shear-induced platelet aggregation in whole blood from control and ATX-TG micewere observed (surface coverage 10.25 1.73% and 9.21 1.6%, respectively, p 0.595).Thus, ex vivo studies were not able to recapitulate a platelet function defect. Additionally,clotting times were normal in the ATX-TG mice. To determine if thrombus formation was alteredin the ATX-TG mice, mice were studied in the ferric chloride-induced carotid artery thrombosismodel. In control mice, thrombus formation occluded the vessel in 10 1.4 min (n 3);whereas none of the ATX-TG mice (n 3) formed occlusive thrombus within 30 min(p0.001). In summary, our results suggest that ATX, and potentially LPA, regulate hemostasisand thrombosis through effects on vascular function.

11Variable Growth Rates and Diameter-Dependent Expression of VascularEndothelial Growth Factor Receptors in Experimental Abdominal AorticAneurysms

Maureen M Tedesco, Masahiro Terashima, Francis G Blankenberg, Zoia Levashova, StanfordUniv Med Cntr, Stanford, CA; Marina Backer, Joseph Backer, SibTech, Inc, Newington, CT;Mien Sho, Eiketsu Sho, Michael V McConnell, Ronald L Dalman; Stanford Univ Med Cntr,Stanford, CA

Purpose: Transmural inflammation and adventitial neovascularization are important patho-physiologic correlates of both human and experimental abdominal aortic aneurysm diseaseprogression. We hypothesized that adventitial VEGF receptor expression would be increased indiseased aortic segments of the Apoprotein E deficient angiotensin II AAA murine model.Methods: Apoprotein E deficient mice with a C57/Bl6 background were infused withAngiotensin II (1000 ng/kg/min) via a subcutaneous osmotic pump. The mice were maintainedon a high fat diet. Luminal diameter was determined in vivo via serial transabdominalultrasound imaging examinations (n 22). Greatest lumen diameter was recorded. Near-infrared fluorescent imaging of VEGF receptors with single chain VEGF-Cy5.5 conjugate injected

2007 ATVB Oral Presentations e-37


intravenously (10g/mouse) was performed on select mice (n 3) once AAA diameter reachedat least 175% of baseline aortic diameter. Results: All AAAs identified were suprarenal andexhibited a variable growth rate. By postoperative day 12, 45% of the mice demonstrated alarge AAA (defined as 175% or greater lumen diameter dilation compared to normal) withtransabdominal ultrasound. We visualized large AAAs in 82% of the mice by postoperative day24, and 95% of the mice by postoperative day 30 with ultrasound. In vivo and ex vivofluorescence imaging of VEGF receptors with scVEGF-Cy5.5 in select mice demonstratedincreased signal in the larger AAA (245% dilated) compared to the smaller AAA (183% dilated).Conclusion: We have demonstrated a variable growth pattern and size dependent enhance-ment in VEGFR-2 expression in a mouse model of AAA disease. VEGF receptors may proveuseful as a clinical marker of AAA progression.

12Sustained Angiogenic Activity of Thrombin-Resistant Mutants of FGF-1 onHuman Endothelial Cells in Fibrin Hydrogel

Luke P Brewster, Loyola Univ Med Cntr, Oak Park, IL; Eric M Brey, Illinois Institute ofTechnology, Chicago, IL; Dominick Buffalino, Howard P Greisler; Loyola Univ Med Cntr,Maywood, IL

Objective: To design a thrombin resistant mutant of FGF-1 (R136K), ligate it to a collagenbinding domain (CBD) to create R136K-CBD, and then to test their angiogenic activity on humanendothelial cells (EC) in vitro using a 3-D fibrin hydrogel matrix. Methods: Thrombin resistanceof growth factors were quantified by SDS-PAGE analysis after incubating the growth factors inthrombin (2U/mL) at 37 degrees Celsius. Angiogenic activity was tested in our 3-Dangiogenesis assay, which incorporates growth factors into a fibrin hydrogel matrix whereinECs are clustered as a multicellular aggregate; the pellets are incubated in media and thelength of tubule invasion is quantified over time; gels without growth factors served as control.Results: R136K and R136K-CBD exhibited thrombin resistance that was superior to that ofFGF-1 (Figure A,B). By day 2, R136K-CBD and R136K, but not FGF-1, stimulated significantlygreater lengths of EC sprouting than the untreated control group (R136K-CBD: 28744,P.007; R136K: 29547, P.005; FGF-1: 24768, P.14; control: 18857). At3 days, all treatment groups reached statistical significance over the untreated control(P.0001), but after day 3, FGF-1 treated ECs sprouts regressed, while both R136K andR136K-CBD continued to demonstrate sprout lengthening which was statistically significant(P.0002) compared to control (Figure C,D). Conclusion: R136K-CBD is resistant to thrombinproteolysis and demonstrates angiogenic activity superior to that of FGF-1. This combinationgrowth factor/matrix delivery vehicle may provide sustained angiogenic activity for applicationin vascular tissue engineering.

13Critical Role of Endothelial Notch1 Signaling in Postnatal Angiogenesis

Kyosuke Takeshita, Nagoya Univ, Nagoya, Japan; James K Liao; Brigham and WomensHosp, Harvard Med Sch, Cambridge, MA

Notch receptors are important mediators of cell fate during embryogenesis, but their role inadult physiology, particularly in postnatal angiogenesis, remains unknown. Of the Notchreceptors, only Notch1 and Notch4 are expressed in vascular endothelial cells. Here we showthat blood flow recovery and postnatal neovascularization in response to hindlimb ischemia inhaploinsufficient global or endothelial-specific Notch1/- mice, but not Notch4-/- mice, wereimpaired compared with wild-type mice. The expression of vascular endothelial growth factor(VEGF) in response to ischemia was comparable between wild-type and Notch mutant mice,suggesting that Notch1 is downstream of VEGF signaling. Treatment of endothelial cells withVEGF increases presenilin proteolytic processing, gamma-secretase activity, Notch1 cleavage,and Hes-1 (hairy enhancer of split homolog-1) expression, all of which were blocked by treatingendothelial cells with inhibitors of phosphatidylinositol 3-kinase/protein kinase Akt or infectingendothelial cells with a dominant-negative Akt mutant. Indeed, inhibition of gamma-secretaseactivity leads to decreased angiogenesis and inhibits VEGF-induced endothelial cell prolifera-tion, migration, and survival. Overexpression of the active Notch1 intercellular domain rescuedthe inhibitory effects of gamma-secretase inhibitors on VEGF-induced angiogenesis. Thesefindings indicate that the phosphatidylinositol 3-kinase/Akt pathway mediates gamma-secretase and Notch1 activation by VEGF and that Notch1 is critical for VEGF-induced postnatalangiogenesis. These results suggest that Notch1 may be a novel therapeutic target forimproving angiogenic response and blood flow recovery in ischemic limbs.

14A Novel Translational Pathway Represses Vascular Endothelial GrowthFactor Expression and Angiogenic Activity by Activated Monocytes

Partho Sarothi Ray, Paul L Fox; Lerner Rsch Institute, Cleveland Clinic, Cleveland, OH

Post-transcriptional regulation of gene expression in macrophages is a principal mechanismlimiting inflammation. Early pro-inflammatory actions of the cytokine interferon- (IFN-) laterswitch to anti-inflammatory, contributing to inflammation resolution. In activated monocytes,IFN- elicits formation of the IFN--activated inhibitor of translation (GAIT) complex that inhibitstranslation of ceruloplasmin, an acute phase inflammatory protein. Bioinformatic andriboimmunoprecipitation-microarray analyses reveal multiple transcripts as potential targets ofGAIT-mediated translational silencing. Our identification of the angiogenic factor VEGF as acandidate target was particularly significant because it is a potent angiogenic factor mediatinginflammatory and tumor angiogenesis. VEGF synthesis by macrophages is induced in tumorsand in chronic inflammatory conditions such as atherosclerosis; however, no negativeregulatory mechanisms are known. We show that translation of VEGF mRNA in activatedmonocytes is silenced by GAIT complex-binding to a defined 3UTR RNA element. IFN-increases VEGF mRNA transcription but causes delayed silencing of VEGF protein synthesis andsuppression of macrophage angiogenic activity as shown by endothelial cell proliferation andtube-formation assays. Our results are the first to show negative regulation of VEGF expressionunder inflammatory conditions. GAIT-mediated translation repression of VEGF, and otherinflammatory genes, might constitute a post-transcriptional operon that limits the response toinflammatory stimuli and contributes to the resolution of chronic inflammation.

15Atox1 as a Novel Copper-dependent Transcription Factor for CellProliferation: Role in Neovascularization

Ha Won Kim, Masuko Ushio-Fukai, Tohru Fukai; Univ of Illinois at Chicago, Chicago, IL

Copper plays a fundamental role in regulating cell proliferation involved in angiogenesis.Recently, we found that antioxidant-1 (Atox1), previously appreciated as a copper chaperone,functions as a novel copper dependent transcription factor that mediates copper-induced cellproliferation. We performed present study to test the hypothesis that Atox1 play an importantrole in postnatal neovascularization. Using mouse ischemia hindlimb model, we found thatneovascular formation in the ischemic hindlimb was significantly impaired in Atox1-/- mice ascompared with WT (35.96.7% decrease) as evaluated by laser Doppler blood flow. Capillarydensity in ischemic hindlimb at 7 days after ischemia was markedly reduced in Atox1-/- miceas compared to WT mice (27.84.7% decrease). In addition, superoxide production whichplays a critical role in angiogenesis was significantly decreased in ischemic hindlimb ofAtox1-/- mice as examined by lucigenin assay (33.55.2% decrease). Interestingly, thenumbers of CD34/CD31- and c-kit/CD31- double positive endothelial progenitor cells (EPCs)derived from bone marrow and peripheral blood were markedly decreased in Atox1-/- miceafter ischemia as compare to WT (37.5- and 22.4% decrease, respectively) as analyzed byFACS. To gain insight into the molecular mechanism, we performed EMSA and CHIP assays andfound that Atox1 binds to the promotor region of cyclin D1 in a copper dependent manner.Moreover, copper-induced increase in cell proliferation and protein levels of cyclin D1 ismarkedly inhibited in Atox1 deficient mouse fiblobrasts and by knockdown of Atox1 usingsiRNA. Taken together, we provide the first evidence that Atox1 functions as a copperdependent transcription factor for cyclin D1 and thus stimulates cell proliferatioin, therebypromoting neovascularization induced by tissue ischemia.

16Candidate Susceptibility Loci for Vascular Remodeling Identified Through aGenome-wide Association Study of In-stent Restenosis

Santhi K Ganesh, NHGRI/NIH, Bethesda, MD; Kimberly Skelding, Mayo Clinic, Rochester,MN; Jungnam Joo, Gang Zheng, NHLBI, Bethesda, MD; Adong Yu, NHGRI/NIH, Bethesda,MD; Nancy Geller, NHLBI, Bethesda, MD; Stephen Pan, NHGRI/NIH, Bethesda, MD; EricBillings, NHLBI, Bethesda, MD; Laxmi Mehta, William Beaumont Hosp, Royal Oak, MI;Kathleen ONeill, NHLBI, Bethesda, MD; Robert Simari, David Holmes Jr, Mayo Clinic,Rochester, MN; William ONeill, William Beaumont Hosp, Royal Oak, MI; Giulia Kennedy,Affymetrix, Inc, Santa Clara, CA; Elizabeth G Nabel; NHLBI/NHGRI/NIH, Bethesda, MD

Introduction: Risk-conferring genes responsible for complex diseases are characterized byvariable expressivity, low penetrance, epistasis and locus heterogeneity, making the analysisof complex genetic traits challenging. For complex cardiovascular diseases, candidate geneapproaches have achieved limited success, making genome-wide association study (GWAS)designs appealing. Molecular and genetic studies suggest that in-stent restenosis (ISR) isprimarily an inflammatory and proliferative disease, with distinct roles for cell cycle proteins,growth factors, and inflammatory cytokines. Methods: To investigate the genetic basis of ISR,we designed a case control GWAS on a dataset of 116,000 single nucleotide polymorphisms(SNPs) assayed in 407 patients (150 cases, 257 controls). We undertook a haplotype analysisof regions highlighted by the presence of two or more SNPs within 250 kb of one another andwith p0.001 (unadjusted) in univariate tests of allelic association. Haplotypes were definedin the regions and tested for association with ISR, using a Bonferroni correction for allhaplotypes tested. Results: We identified eight candidate susceptibility loci containing fivegenes: NOV, ARNTL, TAF4B, PKP4 and a hypothetical protein FLJ21986. We observedexpression of these genes by RNA and protein analysis in normal, atherosclerotic and restenotichuman coronary arteries, supporting the hypothesis that these genes may mediate vascularhomeostasis and adverse vascular remodeling. We estimated each subjects haplotypes andexamined the time to development of ISR as a quantitative trait in a Cox regression model anddemonstrated a significant effect of allele dose for each of the eight regions identified(p0.00038 to 0.035, after Bonferroni correction), suggesting that allele copy numbercontributes to ISR. We additionally analyzed the data using the updated BRLMM algorithm tocall genotypes and identified some of the same genes and EPHB1 and ST18. The additional



regions demonstrate significant allele dose effect as well (p0.003). Conclusions: Haplotypeanalysis of GWAS using SNP markers is a useful approach to identify candidate genes forcomplex vascular diseases. These areas of association with ISR warrant further investigation.

17Ambient Particulate Pollutants in the Ultrafine Range PromoteAtherosclerosis and Systemic Oxidative Stress

Jesus A Araujo, Berenice Barajas, UCLA Sch of Medicine, Los Angeles, CA; MichaelKleinman, Univ of California, Irvine, Irvine, CA; Xuping Wang, Brian J Bennett, Ke Wei Gong,Mohamad Navab, UCLA Sch of Medicine, Los Angeles, CA; Jack Harkema, Michigan StateUniv, Lansing, MI; Constantinos Sioutas, Univ of Southern California, Los Angeles, CA;Aldons J Lusis, Andre Nel; UCLA Sch of Medicine, Los Angeles, CA

Background - Air pollution has been associated with significant adverse health effects leadingto increased cardiovascular morbidity and mortality. Cumulative evidence supports thepathogenic role of particulate matter (PM) in atherosclerosis. While EPA is currently regulatingparticles with an aerodynamic diameter of 2.5 m (PM2.5), there is increasing evidence thatthe smaller particulate components shed into polluted urban air from vehicular emissions mayeven be more dangerous due to their high content of pro-oxidative chemicals. Methods andResults - To test our hypothesis that the smaller the particles, the larger the cardiovasculareffect, we used particle concentrator technology to compare the atherogenic effects of ambientparticles with aerodynamic diameter 0.18 m (ultrafines) vs. fine particles 2.5 m (PM2.5)in downtown Los Angeles. Two experimental protocols were used. In the first, 6-week-old maleC57BL/6J apoE null mice were placed on a chow diet and exposed to concentrated ambientPM (CAPs) for a combined total of 75 hours over a period of 40 days; while in the second,2-month-old male apoE null mice were fed a high fat diet (HFD) and exposed to CAPs for acombined total of 120 hours over a period of 58 days. Control groups included mice exposedto filtered air (FA) or left non-exposed (NE). Chow-fed mice exposed to concentrated ultrafinesdeveloped 25%, 55% and 91% greater aortic atherosclerotic lesions than animals exposed toPM2.5, FA or NE mice, respectively (n1417/group, p0.05). Exposure to ultrafine particlesresulted in an inhibition of the antiinflammatory capacity of plasma high density lipoproteinsand increased systemic oxidative stress markers as evidenced by a significant (i) increase inliver malondialdehyde levels, (ii) upregulation of Nrf2 and Nrf2-related phase-2 responseantioxidant genes (e.g, catalase, superoxide dismutase, NQO-1). HFD-fed apoE null miceexposed to concentrated ultrafines also exhibited evidence of increased systemic oxidativestress but not of greater atherosclerosis. Conclusions - Ultrafine particles concentrate theproatherogenic effects of ambient PM and constitute a significant cardiovascular risk factor thatmay need to be considered in air quality regulation, similar to PM2.5.

18Induction of Apoptosis in Established Atherosclerotic Lesions PromotesInflammation and Monocyte Recruitment in Apoe-/- Mice

Emmanuel Gautier, Thierry Huby, Betty Ouzilleau, John Chapman, Phillipe Lesnik; INSERMU551, Paris, France

Introduction: The impact of apoptosis on atherosclerosis progression may be deleterious asapoptotic cell clearance appears to be defective in atherosclerotic plaques. We evaluated theimpact of the induction of lesional apoptosis on intraplaque inflammation and monocyterecruitment in a new genetically-modified mouse model. Methods: Mice expressing theDiphteria Toxin Receptor (DTR) under the control of the CD11c promoter were crossed withApoe-/- mice. Induction of apoptosis in CD11c lesional cells was achieved by injection ofDiphteria Toxin (DT, 4ng/g). Apoe-/- and DTR Apoe-/- mice were fed a Western diet for 8 weeksand divided into 3 groups: Apoe -/- DT (control), DTR Apoe-/- PBS (control) and DTRApoe-/- DT. Two days after injection, chemokines and monocyte markers expression wasevaluated in the aorta, while apoptosis and newly recruited macrophage detection wereassessed in aortic root lesion. Results: Increased TUNEL staining in the aortic tissue of DTRApoe-/- DT mice as compared to control mice (p0.05) was associated with increasedmRNA expression for the small inducible chemokines MCP-1, MIP-1, MIP-1 and MIP-2(p0.05 versus controls). Concomitantly, mRNA expression of monocyte markers (CD11b,CCR2, CX3CR1) was significantly increased in the aorta of DTR Apoe-/- DT mice ascompared to controls. Moreover, analysis of aortic root lesions revealed the presence of newlyrecruited macrophages in areas of apoptotic cell accumulation, which was indicative ofmonocyte recruitment. Monocyte tracing using fluorescent beads confirmed that apoptotic celldeposition promoted recruitment of circulating monocytes in the aorta of DTR Apoe-/- DTmice. Conclusions: These data suggest that apoptosis associated with impaired apoptotic cellclearance may promote inflammation and recruitment of monocytes in established atheroscle-rotic lesions.

19Distinctive Expression of Chemokines and Transforming Growth Factor-Signaling in Human Arterial Endothelium During Atherosclerosis

Oscar L Volger, Joost O Fledderus, Academic Med Cntr, Univ of Amsterdam, Amsterdam,The Netherlands; Natasja Kisters, Cardiovascular Rsch Institute Maastricht, Univ ofMaastricht, Maastricht, The Netherlands; Ruud D Fontijn, Perry D Moerland, Academic MedCntr, Univ of Amsterdam, Amsterdam, The Netherlands; Johan Kuiper, Theo J van Berkel,Gorlaeus Laboratories, Leiden Univ, Leiden, The Netherlands; Ann-Pascale J Bijnens, Mat JDaemen, Cardiovascular Rsch Institute Maastricht, Univ of Maastricht, Maastricht, TheNetherlands; Hans Pannekoek, Anton J Horrevoets; Academic Med Cntr, Univ of Amsterdam,Amsterdam, The Netherlands

Objectives: Knowledge about the in vivo role of endothelium in chronic human atherosclerosishas mostly been derived by insights from mouse models. Therefore, we set out to establish bymicroarray analyses the gene expression profiles of endothelium from human large arteries, asisolated by Laser Microbeam Microdissection, having focal atherosclerosis of the early or the

advanced stage. Within individual arteries the endothelial transcriptomes of initial (N7) andadvanced (N6) lesions were compared pairwize to the unaffected sides, thus limiting geneticand environmental confounders. Results: Specific endothelial signature gene sets withchanged expression levels in either early (N718) or advanced atherosclerosis (N403),relative to their paired plaque-free controls, were identified (paired Cyber-T test: FDR-correctedp0.05). Gene Set Enrichment Analysis identified distinct sets of chemokines and differentialenrichments (p0.05) of NF-kappa B-, p53- and TGF--related genes in advanced plaques,compared with initial plaques. Immunohistochemistry on a separate set of human arteries withfocal atherosclerosis of the early- (N6) or the advanced-stage (N5) validated thediscriminative value of corresponding endothelial protein expression between early (fractalkine/CX3CL1, IP10/CCL10, TBX18) or advanced (BAX, NFKB2, phosphorylated SMAD2) stages ofatherosclerosis and versus their plaque-free controls. Conclusions: The functional involvementof TGFbeta-signaling in directing its downstream gene repertoire was substantiated by aconsistent detection of activated endothelial SMAD2 in advanced lesions. Thus, we identifiedtruly common, local molecular denominators of pathological changes to vascular endothelium,with a marked distinction of endothelial phenotype between early and advanced plaques.

20Inactivation of Macrophage Fatty Acid Synthase Decreases Atherosclerosis

Jochen G Schneider, Manu V Chakravarthy, Kara N Standley, Clay F Semenkovich;Washington Univ, St. Louis, MO

Fatty acid metabolism is disturbed in atherosclerotic lesions but whether it affects lesionformation is unknown. To test the hypothesis that fatty acid synthesis affects atherosclerosis,we inactivated macrophage fatty acid synthase (FAS) in apoE-deficient Cre-Lox mice. FASKOM(FAS KnockOut in Macrophages) animals showed essentially no FAS mRNA or enzyme activityin macrophages. FASKOM mice and their wild type littermates on the apoE-null backgroundwere fed a Western diet for 8 weeks. There was no effect of genotype on body weight, glucosemetabolism or serum lipids in either sex. FASKOM mice compared to controls had significantlylower systolic and diastolic blood pressures both at baseline on chow diet (928/737 vs1027/8310, p0.05) and with high fat feeding (1008/756 vs 10812/829,p0.05). Compared to littermate controls (n17), FASKOM mice (n21) had 40% lessatherosclerosis in the abdominal aorta (p0.0001), 20% less in the aortic arch (p0.05), and23% less in the thoracic aorta (p0.05). In other tissues, FAS is necessary for endogenousactivation of the nuclear receptor PPARalpha and expression of its target genes. In elicitedmacrophages from FASKOM mice, there was no effect on PPARalpha mRNA (as expected), butexpression of the PPARalpha target genes CPT1 and ACO was decreased compared tomacrophages from control mice, consistent with decreased activation of PPARalpha in theabsence of FAS. There was also a robust induction of mRNA for the antiatherosclerotic oxysterolreceptor LXRalpha as well as its targets ABCA1 and SREBP1c in FASKOM as compared tocontrol macrophages. Treatment of FASKOM macrophages with a PPARalpha agonist normal-ized the expression of PPARalpha target genes as well as LXRalpha. These results suggest thatmacrophage FAS promotes atherosclerosis in apoE-deficient mice by decreasing LXRalphaexpression through endogenous activation of PPARalpha.

21A Cluster of Basic Residues Within the Factor V B-domain Contributes toPreserving the Procofactor State

Mettine H A Bos, Michael Boltz, Rodney M Camire; The Childrens Hosp of Philadelphia,Philadelphia, PA

Blood coagulation factor V (FV) circulates as an inactive procofactor protein and must beproteolytically processed to express procoagulant activity. Thrombin is the principal activator ofFV and cleaves three peptide bonds, thereby removing a large central B-domain in order togenerate active FV (FVa). Mechanistic explanations as to how B-domain release facilitates FVactivation are not adequately developed. Recent studies from our laboratory using a panel ofB-domain truncated FV variants suggest that B-domain sequences within Asp902-Glu1033contribute to preserving the FV procofactor state. Interestingly, the Lys963-Lys1008 portion ofthis sequence is very basic (18 out of 46 residues are Arg or Lys) and is highly conserved, eventhough most of the B-domain is poorly conserved within the vertebrate lineage. The aim of thecurrent study was to examine whether this basic region is sufficient to maintain the FVprocofactor state. Using the previously characterized and constitutively active FV-810 derivativeas scaffold (B-domain sequence Pro811-Gly1491 deleted), we expressed and purified three FVvariants with portions of the basic region inserted: FV-1007BR with Lys963-Glu1007 inserted,FV-1015BR with Lys963-Leu1015 inserted, and FV-1033BR with Lys963-Glu1033 inserted.Each of the proteins migrated as a single band on SDS-PAGE and was processed by thrombinto yield the expected heavy and light chains. Using both a one-stage PT-based clotting assayand a purified prothrombinase assay, we were able to demonstrate that insertion of the basicregion into FV-810 converted this cofactor-like species back to the procofactor state.Furthermore, additional experiments showed that all derivatives exhibited full cofactor activityfollowing treatment with thrombin. These observations indicate that the highly conserved basicregion (Lys963-Lys1008) is able to maintain the procofactor state, even when assembled intoa B-domain that lacks 75% of the full length B-domain sequence and structure. Based onour data, we speculate that the basic sequence might serve an inhibitory function, possibly bydirectly concealing binding interactions that govern the function of the active cofactor species.

22The Utility of Quantitative Calf Muscle Near-infrared Spectroscopy in theFollow-up of Acute Deep Vein Thrombosis

Takashi Yamaki, Motohiro Nozaki, Hiroyuki Sakurai, Masaki Takeuchi, Kazutaka Soejima,Taro Kono; Tokyo Womens Med Univ, Tokyo, Japan

BACKGROUND: To investigate patterns of venous insufficiency and changes in calf muscledeoxygenated hemoglobin (HHb) levels after an acute deep vein thrombosis. METHODS: A total



of 78 limbs with an acute deep vein thrombosis (DVT) involving 156 anatomic segments wereevaluated with duplex scanning and near-infrared spectroscopy (NIRS) at 1 month, 3 months,6 months, and 1 year. Venous segments were examined whether they were occluded, partiallyrecanalized, and totally recanalized, and the development of venous reflux was noted. The NIRSwas used to measure calf muscle HHb levels. Calf venous blood filling index (HHbFI) wascalculated on standing, then the calf venous ejection index (HHbEI) and the venous retentionindex (HHbRI) were obtained after exercise. RESULTS: The segments investigated were thecommon femoral vein (CFV; 38 segments), femoral vein (FV; 37), popliteal vein (POPV; 44), andcalf veins (CV; 37). At 1 year, thrombi had fully resolved in 67% of the segments, 27% remainedpartially recanalized, 6% were occluded. The venous occlusion was predominant in the FV(24%) at 1 year. On the contrary, rapid recanalization was obtained in CV than proximal veinsat each examination (p0.01). Venous reflux was predominant in POPV (55%), followed by FV(19%), and no reflux was found in CV. At 1 year, the HHbFI in POPV reflux patients wassignificantly higher than these with resolution (0.190.14, 0.110.05mol/lsec, p0.009,respectively). Similarly, there was a significant difference in the HHbRI between the two groups(3.081.91, 1.421.56, p0.002, respectively). In patients with FV occlusion, the value ofHHbRI was significantly higher than these with complete resolution (2.591.50, 1.421.56,p0.011, respectively). CONCLUSIONS: The lower extremity venous segments show differentproportions of occlusion, partial recanalization, and total recanalization. The CV shows morerapid recanalization than proximal veins. The NIRS-derived HHbFI and HHbRI could bepromising parameters as the overall venous function in the follow-up of acute DVT. Thesefindings might be very helpful for physician in detecting patients who require much longerfollow-up studies.

23A Unique Function for Low-density ReceptorRelated Protein-1: AComponent of a 2-Receptor System Mediating Specific Endocytosis ofPlasma-derived Factor V by Megakaryocytes

Beth A Bouchard, Natalie T Meisler, Univ of Vermont, Burlington, VT; Michael E Nesheim,Queens Univ, Kingston, Canada; Dudley K Strickland, Univ of Maryland Sch of Medicine,Baltimore, MD; Paula B Tracy; Univ of Vermont, Burlington, VT

Factor V is endocytosed by megakaryocytes from plasma via a specific, receptor-mediated,clathrin-dependent mechanism to form the functionally and physically unique platelet-derivedfactor V pool. Because of its ability to endocytose proteins involved in hemostasis, the role oflow density lipoprotein receptor-related protein-1 (LRP-1) in factor V endocytosis by themegakaryocyte-like cell line, CMK, was examined. Equilibrium binding of 125I-factor V to CMKcells is defined by a sigmoidal binding isotherm, suggesting that factor V binding is cooperativeand mediated by a two-receptor system. Furthermore, 125I-factor V binding is reversible andpartially sensitive to receptor associated protein (RAP), a known LRP-1 ligand. Based on theseobservations a two-receptor model for factor V binding to megakaryocytes was hypothesized.In this model, factor V binds to a specific receptor facilitating binding of another factor Vmolecule to LRP-1 or a like molecule, which subsequently endocytoses factor V. The purposeof the current study was to identify which member of the LRP-1 receptor family is involved.Using RT-PCR, expression of an LRP-1 transcript in CMK cells was demonstrated. In contrast,transcripts representing other LRP family members could not be identified. Cell surfaceexpression of LRP-1 antigen by CMK cells was confirmed by flow cytometry using polyclonalanti-LRP-1 antibodies. Greater than 70% of the CMK cells expressed LRP-1 on their cellsurface. Co-localization of endocytosed AlexaFluor488-factor V and LRP-1 demonstrated thatall of the factor V positive cells expressed LRP-1. These same anti-LRP-1 antibodies were usedto displace 40% of the bound 125I-factor V from the surface of the cells. Furthermore, factorVIII, a known ligand of LRP-1, inhibited 125I-factor V endocytosis equally as well as factor Vwhen present at a 25-fold molar excess. These combined observations confirm our model ofthe cell surface events regulating factor V binding to megakaryocytes, and represent a novelparadigm whereby an essential coagulation protein is endocytosed from plasma and modifiedintracellularly to yield a functionally distinct molecule. Furthermore, a role for LRP-1 inendocytosis of a protein not destined for lysosomal degradation is unique.

24Induction of Tissue Factor and Loss of Thrombomodulin Activities uponInflammatory Stimulation in Vivo

Hugo ten Cate, Kim Frederix, Ingeborg Kooter, Rene van Oerle, Karly Hamulyak, Henri MSpronk; Academic Hosp Maastricht, Maastricht, The Netherlands

Introduction: Inflammation induces organ specific changes in expression of tissue factor (TF)and thrombomodulin (TM) antigen, but it is unknown whether this translates to a netprocoagulant phenotype in vivo. Hypothesis: induction of TF and suppression of TM activitiescontribute to organ-associated thrombogenicity in response to specific inflammatory stimuliincluding endotoxin (LPS) and particulate matter (PM). Design and results: in C57BlJ/6 miceTFactivity in the lungs was induced more by intraperitoneal LPS (25.8 5.3 pM) than by saline(12.7 1.7 pM, p0.05); TF activities in other organs (brain, heart, spleen, liver, kidney) werecomparable for LPS and saline. Addition of lung homogenate from control mice to plasmamarkedly attenuated the plasma endogenous thrombin potential (ETP) (175 61 vs. 1437 112 nM. min; p0.01). This inhibitory effect was due to TM in the lungs, because I) it wasabsent in protein C deficient plasma; II) lungs from TMpro/pro mice with a 100-fold reducedpotential to activate protein C did not inhibit thrombin generation in plasma (ETP: 1686 209nM.min). LPS challenge abolished the inhibitory activity of the lungs, indicated by a significantincrease in ETP (941 523 vs.194 159 nM.min); LPS did not affect other organs in theireffect on thrombin generation. We applied these assays to analyze the consequences ofintratracheal instillation of increasing concentrations of PM, an environmental determinant ofchronic lung- and cardiovascular disease, to spontaneous hypertensive rats PM(road tunneldust: 0.31310 mg/kg bodyweight) dose-dependently induced an increase in TF activitiesafter 4 and 48 hours of exposure compared to controls (1045 472, 1021 62 for 10 mg/kgdose vs. 397 231 pM for control, p0.02). At the highest dose of PM the ETP increasedsignificantly after 4 hours (390 148 vs. 175 61 for saline, p0.05) and after 48 hours(1441 289 nMmin vs. saline: 164 64 nMmin, p0.0001), while TF activity remained

stably elevated in this period, suggesting progressive loss of TM activity. Conclusion:inflammation-induced prothrombotic activity is established by induction of TF in an organspecific manner and the loss of TM activity is a major determinent of this effect in vivo.

25Modulation of LPS-induced Inflammation and Coagulation by the PI3K-AktPathway

James P Luyendyk, Michael Tencati, Todd Holscher, Nigel Mackman; Scripps Rsch Institute,La Jolla, CA

Lipopolysaccharide (LPS) stimulation of monocytes/macrophages activates various intracellularsignaling pathways, including the mitogen-activated protein kinases (MAPKs) and thephosphatidylinositol-3-kinase (PI3K)-Akt pathway. LPS activation of the MAPK pathways isrequired for the expression of inflammatory cytokines and tissue factor (TF), the principalactivator of blood coagulation. We have shown that pharmacologic inhibition of PI3K enhancesLPS signaling and the induction of inflammation and coagulation, both in monocytic cells andin endotoxemic mice. To extend these findings, we determined the effect of geneticallyreducing or enhancing activation of the PI3K-Akt pathway on LPS induction of inflammatorymediators and TF in peritoneal macrophages (PMs) and in mice. We utilized PMs lacking p85,which have decreased PI3K-Akt activity and PMs lacking the phosphatase PTEN, which haveincreased Akt activity. LPS activation of the MAPKs and expression of TF, IL-6 and TNF wasenhanced in p85-/- PMs. Furthermore, inflammation and coagulation were enhanced inendotoxemic wild type mice lacking p85-/- in hematopoietic cells. LPS activation of the MAPKsand the expression of TF, IL-6 and TNF were reduced in PTEN-/- PMs. Our results indicate thatLPS activation of the PI3K-Akt pathway in macrophages inhibits the MAPK signaling pathwaysand reduces inflammation and coagulation. Activation of PI3K or inhibition of PTEN mayrepresent novel strategies to reduce inflammation and coagulation in endotoxemia and sepsis.

26Angiotensin II-induced Hypertrophy and Fibrosis Is Prevented byAngiotensin 17 Overexpression in the Heart

Alvaro Yogi, Univ of Ottawa, Ottawa, Canada; Chantel Mercure, Clinical Rsch Institute ofMontreal, Montreal, Canada; Augusto C Montezano, Glaucia E Callera, Univ of Ottawa,Ottawa, Canada; Rita C Tostes, Univ of Sao Paulo, Sao Paulo, Brazil; Rhian M Touyz, Univof Ottawa, Ottawa, Canada; Timothy L Reudelhuber; Clinical Rsch Institute of Montreal,Montreal, Canada

Background: In vitro studies demonstrate that Ang-17 opposes many actions of Ang II.Whether similar effects occur in vivo remains unclear. Objectives: We tested the hypothesisthat Ang17 protects the heart from damage induced by Ang II and assessed some signalingpathways whereby this occurs. Methods and results: Transgenic mice over-producingAng17 (TG/Ang17) exclusively in the heart (8-fold) were generated. Basal blood pressure andcardiac contractility were similar between groups. To evaluate whether Ang17 exerts directcardioprotective effects, TG/Ang17 and control mice were infused with Ang II (350ng/kg/min,19 days). Ang II-infused TG/Ang17 mice developed less ventricular hypertrophy and fibrosisversus controls, in spite of developing similar levels of hypertension (p0.05). Increasedmembrane translocation of the NAD(P)H oxidase subunit p47phox, an index of activity, wasobserved in Ang II-infused controls. This was blunted in TG/Ang17 mice. Phosphorylation ofcardiac c-Src was increased by Ang II in controls (2.5-fold) but not in TG/Ang17 mice.Activation of redox-sensitive growth signaling molecules, Akt and p38MAPK, was increased byAng II in controls (23-fold, p0.001) without effect in TG/Ang17 mice. Ang II increasedcardiac ERK 1/2 phosphorylation in control and TG/Ang17 mice. Effects of Ang17overproduction on cardiac cell cycle regulatory proteins were also assessed. Expression ofCdk2 /cyclin E and Cyclin D/Cdk4 was increased in Ang II-infused controls (12-fold) andabrogated in TG/Ang17. Increased expression of cell cycle inhibitors p21 (1-fold) and p53(1.5-fold) by Ang II was observed in controls but not in TG/Ang17 mice. p27 expression wasnot different between groups. Conclusion: Our data indicate that Ang17 protects the heartfrom an Ang II-dependent hypertensive challenge. This is associated with downregulation ofNAD(P)H oxidase, c-Src, Akt and p38MAPK, but not with ERK1/2. Moreover Ang(17) influencescell cycle regulatory proteins. Our findings suggest that the negative modulatory effects ofAng-(17) may represent a protective mechanism whereby potentially deleterious actions ofAng II are counterbalanced.

27Requirement of RhoA Serine 188 Phosphorylation for cGMPKinase-mediated Vascular Protection

Naoki Sawada, Hiroshi Itoh, Kazutoshi Miyashita, Hirokazu Tsujimoto, Kazuwa Nakao; KyotoUniv Graduate Sch of Medicine, Kyoto, Japan

[Background] cGMP agonists such as natriuretic peptides dilate blood vessels and inhibitvascular remodeling. Small GTPase RhoA and its effector ROCK, crucial mediators of



vasocontraction and vascualr growth, antagonize cGMP effects. We previously demonstratedthat cGMP-dependent protein kinase type I (cGK I), the direct cGMP effecter, phosphorylatesRhoA at Ser188 and inhibits its downstream signaling. However, the in vivo relevance of thisinteraction remains elusive. This study assessed our hypothesis that cGK I phosphorylation ofRhoA mediates vascular protection by cGMP agonists. [Results] Ser188 phosphorylation ofRhoA, as assessed by phospho-specific antibodies, was diminished in the aortas of cGK Ihaploinsufficient mice (cGK I/-), whereas RhoA/ROCK activity was enhanced. Brain natriureticpeptide transgenic mice (BNP-Tg), with 3- to 4-fold increase in plasma cGMP levels, showedaugmented phosphorylation of RhoA with less ROCK activity as compared to control. cGK I/-mice treated with angiotensin II (Ang II) developed exaggerated BP elevation, cardiachypertrophy, coronary artery medial thickening (MT) and perivascular fibrosis (PVF), andexpression of fibrogenic cytokines and extracellular matrix (ECM) proteins. ROCK inhibitorY-27632 markedly normalized these changes except elevated BP. To assess the role of Ser188phosphorylation of RhoA in cGK I-mediated vascular protection, we generated transgenic micethat express cGK I-unphosphorylatable mutant RhoA (A188RhoA) or wild-type RhoA (wtRhoA)in arterial smooth muscle using SM22alpha promoter. A188RhoA-Tg exhibited 2.7-foldaugmented ROCK activity and 2-fold increase in MT and PVF compared to non-Tg, whereasthese changes were milder in wtRhoA-Tg mice. While wtRhoA/BNP double Tg (DTg) displayednormalized MT and PVF, those of A188RhoA/BNP DTg remained worse (1.5-fold) than non-Tg.Ang II treatment induced comparable increase (1.5- to 4-fold) in MT, PVF and ECM geneexpression in A188RhoA/BNP DTg and non-Tg mice. By contrast, these changes induced byAng II were blunted in wtRhoA/BNP DTg and BNP-Tg mice. [Conclusion] Our data suggest thatSer188 phosphorylation and inhibition of RhoA by cGK I is required for cGMP/cGK I-mediatedprotection against vascular remodeling.

28Cyclooxygenase-2 Expression Increases Vascular Inflammation andAbdominal Aortic Aneurysm Formation in Mice

Jonathan M Gitlin, Darshini B Trivedi, Charles D Loftin; Univ of Kentucky, Lexington, KY

Abdominal aortic aneurysms (AAAs) are associated with a profound inflammatory responsewithin the vessel wall throughout propagation of the disease. Increased expression ofcyclooxygenase-2 (COX-2) is suggested to contribute to the disease in humans. We examinedthe hypothesis that COX-2-dependent proliferation or inflammation in the abdominal aortacontributes to AAA formation. AAAs were induced by angiotensin II infusion and comparedbetween COX-2-deficient mice and their wild-type littermate controls. COX-2-deficient miceshowed significantly reduced AAA incidence at multiple time-points following angiotensin IIinfusion (day 3, 31% 516 for COX-2/ versus 6% 116 for COX-2-/-, P 0.07; day 7, 43% 1535for COX-2/ versus 12% 326 for COX-2-/-, P 0.01; day 21, 37% 1130 for COX-2/ versus4% 128 for COX-2-/-, P 0.01; day 28, 54% 1324 for COX-2/ versus 0% 023 for COX-2-/-, P0.01). COX-2 has previously been shown to be required for angiotensin II-induced smoothmuscle cell proliferation. To examine the role of proliferation in COX-2-dependent AAAformation, we determined the effect of COX-2 expression on activation of AKT and ERK1/2. Asdetermined by western blot, the levels of phosphorylated AKT or ERK1/2 were not significantlydifferent between angiotensin II-infused COX-2/ and COX-2-/- mice, suggesting that COX-2does not contribute to altered proliferation during AAA formation. Angiotensin II-induced AAAformation is initially characterized by macrophage infiltration into the wall of the abdominalaorta. Therefore, we examined macrophage infiltration as a potential mechanism by whichCOX-2 contributes to AAA formation. At time-points where angiotensin II was shown to induceCOX-2, mRNA expression of the macrophage marker CD68 was significantly attenuated in theabdominal aorta of COX-2-/- mice, as compared to wild-type controls (for COX-2/ versusCOX-2-/-: day 3 was 0.6 0.05 versus 0.3 0.03, n 9, P 0.01; day 7 was 0.7 0.1versus 0.2 0.05, n 10, P 0.01). Furthermore, abdominal aortas of COX-2-/- miceshowed attenuated expression of the chemokines MCP-1 and MIP-1alpha. In conclusion,increased COX-2 expression in the abdominal aorta may contribute to AAA formation byenhancing macrophage recruitment.

29Integrin Signaling Is Critical for Pathological Angiogenesis

Ganapati H Mahabeleshwar, Weiyi Feng, David Phillips, Tatiana Byzova; Lerner RschInstitute, Cleveland, OH

The process of postnatal angiogenesis plays a crucial role in pathogenesis of numerousdiseases, including but not limited to tumor growth/metastasis, diabetic retinopathy, and intissue remodeling upon injury. However, the molecular events underlying this complex processare not well understood and numerous issues remain controversial, including the regulatoryfunction of integrin receptors. To analyze the role of integrin phosphorylation and signaling inangiogenesis, we generated knock-in mice that express a mutant beta3 integrin unable toundergo tyrosine phosphorylation. Two distinct models of pathological angiogenesis revealedthat neovascularization is impaired in mutant beta3 knock-in mice. In an ex vivo angiogenesisassay, mutant beta3 knock-in endothelial cells did not form complete capillaries in responseto vascular endothelial growth factor (VEGF) stimulation. At the cellular level, defective tyrosinephosphorylation in mutant beta3 knock-in cells resulted in impaired adhesion, spreading, andmigration of endothelial cells. At the molecular level, VEGF stimulated complex formationbetween VEGF receptor-2 and beta3 integrin in wild-type but not in mutant beta3 knock-inendothelial cells. Moreover, phosphorylation of VEGF receptor-2 was significantly reduced incells expressing mutant beta3 compared to wild type, leading to impaired integrin activation inthese cells. These findings provide novel mechanistic insights into the role of integrin-VEGF axisin pathological angiogenesis.

30Matrix Metalloproteinase-1: Role in Aneurysm Formation in Vivo andRegulation by Nicotine in Vascular Smooth Muscle Cells

Vincent Lemaitre, Jeanine DArmiento; Columbia Univ, New York, NY

The role of matrix metalloproteinase-1 (MMP-1) and the molecular mechanisms regulating itsexpression in aneurysm remain unknown. Transgenic mice expressing human MMP-1 inmacrophages were crossed into the ApoE-null / Timp-1 (Tissue inhibitor ofmetalloproteinases-1) knockout background, which develops a high number of micro-aneurysms associated with atherosclerosis. Transgenic mice (n11) and their littermatecontrols (n10) were given a high-fat diet for ten weeks and sacrificed. Transgenic mice hadlarger aneurysms (45,21628,111 m2, n33) compared to the controls (26,18522,026m2, n28; P0.005). These aneurysms were also characterized by the bulging of thedestroyed media into the adventitia and an increased deposition of fibrillar collagen (transgenic: 3214% of the aneurysm area, controls : 125%, n11 in each group; P0.008), a markerof severity in the human disease. Since smoking is a major risk factor for aneurysmdevelopment and rupture, we tested the hypothesis that nicotine could modulate the expressionof MMP-1 in vascular cells. Human aortic vascular smooth muscle cells were treated withnicotine, at concentrations found in the circulation of moderate smokers (10 nM to 1000 nM).After 24 hours, nicotine increased the expression of MMP-1, both at the mRNA and proteinlevels (P0.05). This up-regulation was abrogated by specific inhibitors of p38 and ERK,suggesting that nicotine induces MMP-1 through the MAP kinases signaling pathway.Western-blot analysis of cell lysates showed that nicotine activates the Jak/STAT kinasepathway, leading to increased phosphorylation of Jak2, ERK, p38, Jnk, and Stat3, andsubsequent induction of MMP-1 expression. Our data demonstrates that MMP-1 enhances theseverity of mouse aneurysm in vivo, and that nicotine induces MMP-1 expression in smoothmuscle cells through the Jak/Stat kinase pathway. This study suggests that increasedsusceptibility to aneurysm formation and rupture in smokers might result from augmentedcollagenase activity in the vessel wall, due to a chronic exposure to circulating nicotine

31Notch Signaling Negatively Regulates Endothelial Lineage Specification

Rhiannon Penkert, UNC-Chapel Hill, Chapel Hill, NC; Kevin Vogeli, Univ of SouthernCalifornia, Los Angeles, CA; Jessica Alsio, UNC-Chapel Hill, Chapel Hill, NC; Didier Stainier,UCSF, San Francisco, CA; Suk-Won Jin; UNC-Chapel Hill, Chapel Hill, NC

Endothelial cells and hematopoietic cells are thought to arise from a common progenitor, thehemangioblast. This notion was initially suggested as a result of the observation that these twocell types develop in close proximity during embryonic development. Although the dataacquired from cell culture experiments suggest the existence of the hemangioblast, thepresence of bipotential progenitors has yet to be demonstrated in vivo until our recent study.We have reported the first in vivo evidence for the existence of hemangioblast during embryonicdevelopment using zebrafish gastrula as a model system. As the initial step to delineate themolecular signaling pathways that regulate hemangioblast lineage specification, we analyzedthe function of Notch signaling pathway in this process. Previous study indicated that Notchcould function as a bi-modal switch in the hemangioblast equivalent cell population inDrosophila. We find that Notch signaling appears to promote hematopoietic fate overendothelial fate. Embryos with reduced Notch activity show a significant increased number ofangioblasts at the expense of hematopoietic progenitors. This early requirement of Notchactivity can be separated from the previously described function of Notch in promoting arterialendothelial fate. Furthermore, by using lineage tracing we show that a certain population ofcells which would give rise to primitive erythrocytes transfate into endothelial precursors inembryos with compromised Notch signaling. Although further analyses need to be done to testwhether the increased number of angioblasts is due to the fate changes within thehemangioblast lineage or the direct transformation of hematopoietic progenitors, our datasuggest that Notch signaling might functions as a cell fate switch by negatively regulatingendothelial fate.

32The Atheroprotective Effect of Flk-1-based DNA Vaccination in LDLReceptorDeficient Mice Is Enhanced by Coexpression of CCL21

Ramona J Petrovan, Audrey S Black, Joshua Bulgrien, David J Bonnet, Linda K Curtiss; TheScripps Rsch Institute, La Jolla, CA

Suppressors of angiogenesis can reduce plaque neovascularization and inhibit atheroscleroticlesion growth. Vaccination of hyperlipidemic LDL receptor-deficient (LDLr-/-) mice with an oralDNA vaccine specific for the murine VEGF receptor 2 (Flk-1) evoked a CD8 T cell-mediatedresponse that suppressed atherosclerosis-related angiogenesis and attenuated lesion progres-sion. Appropriate adjuvants can improve effector T cell function and increase the efficacy ofDNA vaccines. We therefore tested the hypothesis that co-expression of the secretorychemokine CCL21 with Flk-1 can enhance the atheroprotective effect of the Flk-1 vaccination.Empty expression vector, Flk-1 or Flk-1/CCL21 vaccinated LDLr-/- mice (n 8/group) were feda high fat diet for 16 weeks. Consistent with previous findings, there was no difference in totalcholesterol levels between all groups of mice, despite the expected increase in plasmacholesterol after high fat diet consumption. Compared to both Flk-1 and control immunizedanimals, immunization with the vector encoding Flk-1/CCL21 led to increased in vitro lysis ofmurine endothelial target cells expressing Flk-1 by T cell-enriched splenocytes. Co-expressionof CCL21 led to increased protection against diet-induced atherosclerosis in both male andfemale mice, as demonstrated in both cross sections of the aortic sinus and in lipid stained enface preparations of the aorta. A time course study of lesion development revealed that theextent of aortic sinus lesions was substantially decreased in LDLr-/- mice vaccinated with theFlk-1/CCL21 construct after only 4 weeks of consuming the high fat diet. Both foam-cell lipidaccumulation and macrophage infiltration in aortic segments within areas of disturbed bloodflow were altered by the Flk-1-based DNA vaccination at early stages of the disease. In



conclusion, these studies demonstrated that co-expression of CCL21 with Flk-1 improved T cellmediated immune responses against Flk-1 and reduced lesion progression in hyperlipidemicmice.

33Diet-induced Dyslipidemia Is Associated with Accleration of Disease andMortality in Lupus-susceptible LDLr-/- Mice

Amy S Major, Nicole A Braun, Adam C Morgan; Vanderbilt Univ Med Cntr, Nashville, TN

Individuals suffering from systemic lupus erythematosus (SLE) are predisposed to acceleratedatherosclerosis. Unfortunately, the underlying mechanisms for increased vascular disease inlupus are not well understood. Our laboratory has recently developed an animal model ofSLE-accelerated atherosclerosis. We have shown that radiation chimeras consisting ofSLE-derived hematopoietic cells transferred to LDLr-/- mice have increased atherosclerosis.Surprisingly, feeding mice high-fat diet for 8 weeks resulted in significant mortality inSLE-susceptible mice compared to controls. Based on these data, we hypothesized thatincreased dyslipidemia, characterized by an accumulation of non-HDL lipoproteins, isassociated with increased autoimmunity. To test this hypothesis, we created radiation chimerasof LDLr-/- mice that were either SLE-susceptible (LDLr.Sle) or resistant (LDLr.B6). Eight weeksfollowing bone marrow reconstitution, mice were placed on a normal chow or high fat (21%fat, 0.15% cholesterol) diet for eight weeks. All animals fed a high-fat diet had significantlyincreased total cholesterol and triglycerides compared to chow fed mice, however there wereno significant differences within groups between LDLr.B6 control and LDLr.Sle mice. Comparedto all chow-fed animals and high-fat fed LDLr.B6 controls, high-fat fed LDLr.Sle mice exhibitedincreased mortality (37%) and were mildly, but significantly hypertensive. In addition, ECHOanalyses showed that 60% (3 of 5 mice) of the LDLr.Sle mice fed high fat diet had increasedleft ventricular mass compared their LDLr.B6 counterparts. Increased blood pressure did notovertly appear to be due to advanced renal disease as serum creatinine and urea levelsbetween LDLr.Sle mice on chow or high-fat diet did not differ significantly. These datademonstrate that increased dyslipidemia resulting from feeding a high-fat diet can exacerbateautoimmunity and associated vascular complications.

34Chemical Genetic Analysis Reveals the Central Role ofPhosphatidylinositol-3 Kinase and MAP Kinase/ERK Signaling Pathways inArtery/Vein Specification

Charles C Hong, Vanderbilt Univ Sch of Medicine, Nashville, TN; Quinn P Peterson, Ji-YoungHong, Randall T Peterson; Massachusetts General Hosp, Boston, MA

How the endothelial progenitor cells are specified to either the arterial or venous fates is afundamental biological question with significant clinical implications. We reasoned that theartery/vein specification during development is amenable to chemical genetic analysis, in amanner analogous to the classical genetic analyses, which have been instrumental inelucidation of numerous biological pathways in prokaryotes and invertebrates. Specifically, wehypothesized that small molecules found to suppress zebrafish model of arterial deficiency willfunction by augmenting the signaling for arterial specification. In a high-throughput chemicalscreen, we discovered two classes of small molecules that rescued the defective arteriogenesiscaused by a genetic mutation. Using these compounds as chemical tools to dissect thesignaling pathways involved in artery/vein specification, we made the following important andsurprising observations: 1) p42/44 MAP kinase (ERK) activation is a specific and early markerof arterial progenitors, 2) ERK activation is a key determinant of arterial specification, and 3)the phosphatidylinositol-3 kinase (PI3K) has the opposite effect on artery-vein specification. Insummary, our chemical genetic approach has revealed opposing roles of PI3K and ERK inartery/vein specification, a better understanding of which will lead to novel therapies fornumerous conditions, such as ischemic heart/limb diseases and vascular malformations.

35ER Stressors Promote TLR4/SRA-dependent Macrophage ApoptosisThrough Ca2-mediated CaMKII Activation

Tracie A Seimon, Wahseng Lim, Janelle Timmins, Columbia Univ, New York, NY; Kathryn JMoore, Harvard Med Sch, Boston, MA; Douglas T Golenbock, Univ of Massachusetts MedSch, Worcester, MA; Ira A Tabas; Columbia Univ, New York, NY

The macrophage (M) scavenger receptor-A (SRA) and toll-like receptor 4 (TLR4) are patternrecognition receptors (PRRs) of the innate immune system. In vivo data suggest that the SRAand TLR4 contribute to the development of atherosclerosis, although the mechanistic linksbetween these PRRs and atherosclerosis have not been elucidated. Our laboratory has focusedon a critical event in the formation of necrotic atherosclerotic plaques, namely, M death. Werecently discovered that SRA ligands trigger M apoptosis in an SRA- and TLR4-dependentmanner. SRA ligands activate the pro-apoptotic TLR4-JNK pathway but, unlike LPS, silence thepro-survival TLR4-IFN pathway. We also found that ER stressors that induce Ca2mobilization enhance TLR4-dependent signaling and that chelating Ca2 inhibits TLR4-dependent JNK activation and apoptosis. In the current study we show that LPS, whichnormally leads to cell survival, induces apoptosis in the presence of various SRA ligands suchas fucoidan, -amyloid, and advanced glycation endproducts (AGEs). These SRA ligands do notinduce apoptosis when added in the absence of LPS. Moreover, M apoptosis induced by LPSand SRA ligands was markedly amplified in the setting of Ca2-releasing ER stressors likethapsigargin. To probe mechanism, we investigated the role of Ca2-regulated calmodulin-dependent protein kinase II (CaMKII). We found that apoptosis-enhancing Ca2-releasing ERstressors are potent activators of CaMKII. Most importantly, TLR4-dependent JNK activationand apoptosis were blocked by the CaMKII inhibitor KN93. These data and our previous studiessuggest that Ca2-releasing ER stressors contribute to two events necessary for Mapoptosis: enhancement of TLR4-dependent JNK activation through activation of CaMKII; and

induction of the pro-apoptotic CHOP branch of the ER stress pathway through depletion of ERCa2 stores. These findings reveal a novel link between ER stress, Ca2 mobilization, andPRR signaling and have potential implications for M death and plaque necrosis in advancedatherosclerosis and perhaps other diseases where PRRs, ER stress, and cell death are knownto play a role.

36Signal-Dependent Splicing of Tissue Factor Pre-mRNA Modulates theThrombogenicity of Human Platelets: A New Mechanism Linked toDisordered Coagulation in Sepsis

Hansjorg Schwertz, Neal D Tolley, Jason M Foulks, Univ of Utah, Salt Lake City, UT; RachelE Tilley, The Scripps Rsch Institute, La Jolla, CA; Estelle S Harris, Matthew T Rondina, GuyA Zimmerman, Univ of Utah, Salt Lake City, UT; Nigel Mackman, The Scripps Rsch Institute,La Jolla, CA; Andrew S Weyrich; Univ of Utah, Salt Lake City, UT

Introduction: We recently demonstrated that platelets use a previously-unrecognized pre-mRNA splicing pathway to generate tissue factor (TF)-dependent procoagulant activity. Here,we hypothesize that TF pre-mRNA splicing events are activated in patients with sepsis, asyndrome of dysregulated coagulation. Methods: Patients meeting consensus criteria forsepsis were prospectively enrolled. Platelets were freshly-isolated from whole blood within thefirst 24 hours of admission to the ICU. Platelets from non-medicated healthy controls wereassayed in parallel to eliminate differences due to inter-assay variability. TF mRNA expressionpatterns and procoagulant activity was measured in both groups. Platelets were also incubatedwith bacteria from septic patients or bacterial-derived products. Results: Without exception,platelets from healthy controls (n54) expressed TF pre-mRNA. In contrast, platelets from 26of 46 septic patients expressed spliced TF mRNA. The incidence of TF pre-mRNA splicingincreased with increasing severity of illness as measured by APACHE II scores. Consistent withTF mRNA expression patterns, procoagulant activity in platelets from septic patients wassignificantly (p0.05) higher than in healthy controls (45.913.3 vs. 11.12.8 pM). In asubgroup of patients (n16), we also assessed TF pre-mRNA splicing in serial samples. Wefound progressive splicing in the platelets over time. Altogether, 81% of these patientsexpressed spliced TF mRNA at some point during their ICU stay. Gram- (E. coli) or gram (S.aureus) bacteria isolated from septic patients also induced TF pre-mRNA splicing. Similarly,products of E. coli (lipopolysaccharide) or S. aureus (-toxin) induced TF pre-mRNA splicing inplatelets, resulting in accelerated clot formation. Conclusions: These data demonstrate thatcirculating platelets from septic patients spontaneously splice TF pre-mRNA and generateprocoagulant activity. TF pre-mRNA splicing by platelets may contribute to abnormalcoagulation in sepsis and may be a target of future therapeutics.

37Targeting Coagulation Factor XII Provides Protection from PathologicalThrombosis in Myocardial Infarction and Cerebral Ischemia WithoutInterfering with Hemostasis

Thomas Renne, Ulrich Walter, Christoph Kleinschnitz, Guido Stoll, Stefan Frantz, JohannBauersachs, Univ of Wurzburg, Wurzburg, Germany; Hans-Ulrich Pauer, Univ of Gottingen,Gottingen, Germany; David Gailani; Vanderbilt Univ Sch of Medicine, Nashville, TN

Formation of fibrin is critical for limiting blood loss at a site of blood vessel injury (hemostasis),but may also contribute to vascular thrombosis. Hereditary deficiency of factor XII (FXII), theprotease that triggers the intrinsic pathway of coagulation in vitro, is not associated withspontaneous or excessive injury-related bleeding, indicating FXII is not required for hemostasis.We demonstrate that deficiency or inhibition of FXII protects mice from ischemic brain injuryand cardiac ischemia/reperfusion damage. Following transient middle cerebral artery occlu-sion, the volume of infarcted brain in FXII deficient and FXII inhibitor-treated mice wassignificantly less than in wild type controls, without an increase in infarct-associatedhemorrhage. FXII-null and inhibitor treated mice were largely protected from ischemia/reperfusion injury and survival rate was significantly higher in myocardial infarction models.Targeting FXII reduced fibrin formation in ischemic vessels, and reconstitution of FXII deficientmice with human FXII restored fibrin deposition. Mice deficient in the FXII substrate factor XIwere similarly protected from vessel-occluding fibrin formation in brain and heart, suggestingthat FXII contributes to pathologic clotting through the intrinsic pathway. These datademonstrate that some processes involved in pathologic thrombus formation are distinct fromthose required for normal hemostasis. As FXII appears to be instrumental in pathologic fibrinformation, but dispensable for hemostasis, FXII inhibition may offer a selective and safestrategy for preventing stroke, myocardial infarction and other thromboembolic diseases.

38The Antithrombotic Potential of Selective Blockade of Talin-dependentIntegrin IIb3 (Platelet GPIIb-IIIa) Activation

Brian G Petrich, Univ of California, San Diego, La Jolla, CA; Susan J Monkley, Univ ofLeicester, Leicester, United Kingdom; Anthony W Partridge, Nima Yousefi, Sanford J Shattil,Univ of California, San Diego, La Jolla, CA; David R Critchley, Univ of Leicester, Leicester,United Kingdom; Mark H Ginsberg; Univ of California, San Diego, La Jolla, CA

Studies in vitro and with cultured cells indicate that talin binding to the 3 cytoplasmic domainis a final step in platelet integrin IIb3 (GPIIb-IIIa) activation. We tested the significance oftalin-mediated integrin activation by generating platelet-specific talin1 knock-out mice (PKO).Platelets from PKO mice showed a dramatic reduction in agonist-induced llb3 activation asdetermined by soluble fibrinogen binding. In addition, more than 90% of PKO mice showedpathological bleeding that was associated with reduced survival. To determine whether thephenotype of PKO mice was due to lack of talin binding to integrin 3, we generated 3integrin knock-in mice harboring point mutations in the 3 integrin cytoplasmic domain thatdisrupt talin- integrin interactions. We introduced a 3(Y747A) substitution that disrupts the



binding of talin, filamin, and other cytoplasmic proteins and a 3(L746A) substitution thatselectively disrupts interactions only with talin. Analysis of platelets from animals homozygousfor each mutation showed decreased agonist-induced fibrinogen binding, providing proof thatthat inside-out signals that activate IIb3 require talin binding to the 3 tail. Injection ofcollagen and epinephrine resulted in the death of 62% of wild type mice from pulmonarythromboembolism; in sharp contrast, both 3(Y747A) and 3(L746A) mice were resistant tothis form of thrombosis. Pathological bleeding, as measured by the presence of fecal blood anddevelopment of anemia, occurred in 53% of 3(Y747A) and virtually all 3 integrin knock-outand PKO mice examined. Remarkably, none of the 3(L746A) animals exhibited this form ofbleeding. These results establish that IIb3 activation in vivo is dependent on the interactionof talin with the 3 integrin cytoplasmic domain. Furthermore, they suggest that modulation of3 integrin-talin interactions may provide an attractive target for anti-thrombotics with reducedrisk of pathological bleeding.

39Mapping Structure-Function Relationships of ABCA1

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