458A ABSTRACTS - Vascular Disease, Hypertension, and Prevention JACC March 3, 2004 Vascular Disease, Hypertension, and Prevention prevents the expected reduction in collateral flow after PCI of a stenotic vessel. There is a direct dose-response relation between exercise capacity gained and coronary collateral flow augmentation in the angiographically normal vessel. 1045-191 High-Density Lipoprotein-Induced Tube Formation Requires the Activation of Ras/Raf/Mitogen-Activated Protein Kinase in Human Coronary Artery Endothelial Cells Shin-ichiro Miura , Masahiro Fujino, Hiroyuki Tanigawa, Yoshino Matsuo, Akira Kawamura, Hiroaki Nishikawa, Keijiro Saku, Fukuoka University School of Medicine, Fukuoka, Japan Objectives: High density lipoprotein (HDL) levels have been shown to be inversely corre- lated with coronary artery disease, but the mechanisms of the direct protective effect of HDL on endothelial cells (ECs) are not fully understood. In this study, we investigated the role of the HDL–mediated promotion of angiogenesis in human coronary artery ECs (HCECs). Methods and Results: We developed an in vitro model of HCEC tube formation on a matrix gel. HDL induced tube formation, in which the dose-response showed that the maximum effective dose of HDL was 100 μg/ml. We also examined the effect of sphin- gosine-1-phosphate (S1P), which is a carrier of bioactive lipids of HDL, to analyze tube formation. Since HDL contains S1P (about 180 pmol/mg protein), 100 μg/ml of HDL con- tains about 0.018 μM of S1P. Although we observed 0.02 μM of S1P significantly induced tube formation, it was only 20 % of 100 μg/ml HDL-induced tube formation. HDL-induced tube formation may be partly mediated by S1P. PD98059, an inhibitor of p42/44 mitogen- activated protein kinase (MAPK) activity, but not SB203580, an inhibitor of p38 MAPK activity, suppressed HDL-induced tube formation. Dominant-negative Ras N17 inhibited HDL-induced tube formation. HDL activated Ras by ras pull-down assay and its effect was inhibited by pertusis toxin (PTX). Moreover, HDL activated phospho(p)-p42/44 MAPK, while Ras N17 blocked HDL-induced pp42/44 MAPK. Conclusions: These results indicate that HDL induced a potent signal through a Ras/ MAPK pathway mediated by PTX-sensitive G-protein coupled receptor to the angiogenic phenotype in HCECs. 1045-192 Monocytes, but Not Neutrophils or Lymphocytes Are Essential Mediators of Arteriogenesis Sebastian Grundmann, Imo Hoefer , Niels van Royen, Stephan Schirmer, Susann Ulusans, Ivo Buschmann, University Hospital, Freiburg, Germany, Perfusion Technologies, Freiburg, Germany Background: Blood vessel growth following arterial occlusion is mediated by infiltrating leukocytes. While all three leukocyte subpopulations (neutrophils, lymphocytes, mono- cytes) are known to play an important role in angiogenesis, only monocytes have been shown to influence the growth of collateral arteries (arteriogenesis). In this study we examined the importance of neutrophils and lymphocytes in a rabbit hindlimb model of arteriogenesis. Methods: 36 Rabbits received either Interleukin-8 (IL-8), Neutrophil-acti- vating-protein-2 (NAP-2) or Lymphotactin (Ltn) via osmotic minipumps directly into the collateral circulation after femoral artery ligation. NAP-2 is a relatively selective activator of neutrophils, Ltn chemoattracts lymphocytes and Il-8 has an effect on both celltypes. PBS and MCP-1 treated groups served as controls. After one week, leukocytes around growing collateral arteries were quantified via immunohistology and effects on integrin markers of leukocytes activation (Mac-1, LFA-1) were assessed by flow-cytometry. Col- lateral conductance was assessed using fluorescent microspheres. Results: Although a significant increase in neutrophil accumulation after IL-8 and NAP-2 treatment was detected in-vivo (cells/mm 2 : PBS:8,33±2,87,MCP-1:25,23±10,81,IL-8:36,06±12,65,NAP- 2:20,67±7,41,Ltn:8,27±5,44) and Il-8 and NAP-2 resulted in neutrophil activation in flow- cytometry (Mac-1 Expression: PBS:21,12±2,11,MCP-1:24,11±2,64,IL- 8:32,65±2,10,NAP-2:27,30±2,25, Ltn:17,30±2,16), no significant increase in collateral conductance was observed. Ltn treatment resulted in lymphocyte accumulation (cells/ mm 2 : PBS:2,48±1,24,MCP-1:4,26±1,16,IL-8:6,88±5,28,NAP- 2:2,70±0,57,Ltn:12,80±3,50), but not in collateral artery growth. Collateral conductance: [ml/min/100mmHg]: PBS:50,70±5,15, MCP-1:339,60±19,6, IL-8:58,91±5,56, NAP- 2:66,83±8,72, Ltn:52,80±5,37. Conclusion: While lymphocytes and neutrophils are known to participate in angiogenesis, their importance for arteriogenesis seems to be neglectible. These findings support the hypothesis of monocytes/macrophages as key mediators of collateral artery growth. 1045-193 Granulocyte-Colony Stimulating Factor Mobilizes and Activates Endothelial Progenitor Cells in Patients With Coronary Artery Disease Tiffany M. Powell , Jonathan D. Paul, Jonathan M. Hill, Elizabeth J. Read, Philip J. McCoy, Richard O. Cannon, III, National Institutes of Health, Bethesda, MD Background: Circulating endothelial progenitor cells may function to repair cardiovascu- lar injury, but are reduced in patients with coronary artery disease. Granulocyte-colony stimulating factor (G-CSF) mobilizes hematopoietic stem cells (CD34+) in healthy sub- jects, but whether this cytokine mobilizes endothelial progenitor cells capable of endothe- lial maturation in coronary artery disease patients is unknown. Methods: G-CSF (10 mcg/kg) was administered daily for 5 days to 12 coronary artery disease patients, and circulating CD34+ cells, endothelial progenitor cells (CD133/ VEGFR-2+), and mature endothelial cells [CD144 (VE-cadherin), CD31 (PECAM), CD51/61 (alpha v beta III inte- grin)] were measured by flow cytometry. Results: G-CSF increased CD34+ cells from <1 cell/microL of blood at baseline to 60±18 cells/microL within 24 hours of the last dose (p<0.001), similar to CD34+ mobilization achieved in 28 healthy donors >40 years of age at our hospital (78±8 cells/microL). Endothelial progenitor cells were also mobilized, although to low levels (from <1 cell/microL at baseline to 6±3 cells/microL within 24 hours of last G-CSF dose, p<0.001). Mature circulating endothelial cells also increased post-G- CSF: CD34/CD144+ from 16±16 to 107±90 cells/microL, CD34/CD31+ from 54±28 to 224±60 cells/microL, CD34/CD51/61 from 14±14 to 81±64 cells/microL (all p<0.05 vs baseline). One week following completion of treatment, CD34+ cells and endothelial pro- genitor cells had returned to baseline, but levels of mature endothelial cells remained increased over baseline (p<0.05). Conclusion: These findings establish that G-CSF administration to coronary artery disease patients mobilizes CD34+ cells, which includes the endothelial progenitor cell subset, into the circulation. Mobilization is associated with increased cells expressing mature endothelial markers, which persist even at one week following the last dose of G-CSF. This suggests sustained G-CSF-stimulated differentia- tion along an endothelial cell lineage, with potential therapeutic implications for neovas- cularization of ischemic myocardium. 1045-194 Impaired Arteriogenic Response to Acute Hindlimb Ischemia in CD8 Knockout Mice Eugenio Stabile , Mary Susan Burnett, Timothy D. Kinnaird, Craig Watkins, Cheol W. Lee, Matie Shou, Andrea la Sala, Schmuel Fuchs, Stephen E. Epstein, Washington Hospital Center, Washington, DC Background: CD8 + cytotoxic T lymphocytes regulate cellular responses of the immune system, which play a pivotal role in modulating collateral vessel development. The aim of our study was to investigate if the absence of circulating CD8 + T-cells impairs collateral development after femoral artery ligation in CD8 -/- mice. Methods and Results: After surgical excision of the femoral artery, Laser Doppler Perfu- sion Imaging demonstrated reduced collateral flow induction in CD8 -/- mice compared to control mice (C57BL/6J) at day 3 (0.21±0.01 vs 0.29±0.03, p< 0.01) which persisted to day 28 (0.69±0.04 vs 0.90±0.04, p< 0.01). In CD8 -/- mice , when compared to controls, the biological importance of the reduced collateral flow was evident by diminished recov- ery of hindlimb function (ambulatory impairment score: 1.73 ± 0.18 vs 0.86 ± 0.19, p< 0.01), greater calf muscle atrophy (mean fiber area 767 ± 68 vs 1067 ± 69, μm 2 , p< 0.01), and increased fibrotic tissue content (14 ± 1% vs 7 ± 1%, p< 0.01). Exogenous CD8 + T-cells, when infused into CD8-/- mice immediately after ischemia induction, selec- tively home at the site of collateral formation and, over time, significantly increased collat- eral flow, improved hindlimb functional recovery, and reduced muscle atrophy/fibrosis. Conclusions: These results demonstrate that CD8+ T-cells are a critical component of the immune system in regulating the early phase of normal collateral development. CD8 - /- mice demonstrated both delayed and impaired blood flow recovery after femoral artery ligation, and infusion of CD8 + T cells immediately after surgery rescued the phenotype. Our study provides further evidence that the immune system is critical in modulating col- lateral development in response to peripheral ischemia. 1045-195 Monocyte Chemoattractant Protein-1 Activates Vascular Endothelial Growth Factor- and Tumor Necrosis Factor- Alpha-Mediated Angiogenesis in Ischemic Hindlimbs of Mice Hiroshi Niiyama , Hisashi Kai, Hironobu Tahara, Toyoaki Murohara, Tsutomu Imaizumi, Kurume Univeristy, Kurume, Japan Recently, we and others have suggested that macrophage accumulation plays a role in angiogenesis in hindlimb ischemia model. Macrophage chemoattractant protein (MCP)-1 is a key molecule to trigger inflammatory changes in various diseases. Thus, we sought to determine the role of the endogenous MCP-1 in ischemia-induced angiogenesis. At day 0, unilateral hindlimb ischemia was induced by excising surgically entire left femoral artery and vein in mice. Immediately after operation, plasmid DNA encoding 7ND, a dom- inant negative mutant of MCP-1, or the empty plasmid (as control) was injected into the ipsilateral thigh adductor muscle. Serial laser Doppler blood flow analysis showed an abrupt decrease in blood flow, followed by a remarkable recovery, in ischemic hindlimbs of controls. Control mice showed well-developed collateral vessels and capillary forma- tion as assessed by postmortem angiography and immunohistostaining for CD31, respectively, at day 21 after induction of ischemia. In 7ND-treated mice, although the extent of the early decrease in laser Doppler blood flow was similar to that in controls, the recovery was impaired. At day 3, macrophage infiltration and inductions of vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF)-alpha, known angio- genic factors, were prominent in the adductor muscle of ischemic hindlimbs in controls. 7ND treatment significantly reduced the number of infiltrated macrophages and repressed VEGF and TNF-alpha inductions in response to ischemia at day 3. Moreover, the number of angiographically visible collateral vessels and the capillary density were significantly decreased in ischemic hindlimbs of 7ND-treated mice at day 21. In conclu- sion, MCP-1-mediated macrophage accumulation may play an important role in ischemia-induced angiogenesis at least in part by activating induction of angiogenic fac- tors such as VEGF and TNF-alpha in ischemic hindlimbs. 1045-196 Fiber Type-Specific Angiogenic Dysregulation in a Genetic Mouse Model of Heart Failure Richard E. Waters, II , Brian Annex, Zhen Yan, Duke University Medical Center, Durham, NC Introduction. Chronic heart failure (CHF) leads to intrinsic skeletal muscle abnormalities including slow-oxidative to fast-glycolytic fiber type switching, decreased capillary den- sity, and reduced mitochondrial function. These skeletal muscle abnormalities contribute to clinical exercise intolerance. Methods. A genetic mouse model of heart failure induced through cardiac-targeted overexpression of the sarcoplasmic reticulum Ca 2+ storage pro- tein calsequestrin (CSQ) has been recently characterized. Skeletal muscle (plantaris) from CSQ/CHF mice and wild type (WT) mice was analyzed with triple color immunof- lourescence using antibodies specific for myosin heavy chain I, IIa, IIb, and endothelial cells. Results. A decrease in oxidative myofibers (I + IIa), a concurrent increase in glyco-