- Email: [email protected]
Tu-P7:32 Application of vasohibin for prevension of adventitial angiogenesis and arterial neoinyimal formation
Tues&ty, June 20, 2006: Poster Session P7 Basic science ( lst part) ~
THERAPEUTIC ANGIOGENESIS USING NOVEL VASCULAR ENDOTHELIAL GROWTH F A C T O R - E / H U M A N P L A C E N T A L GROWTH FACTOR CHIMERA GENES
N. Inoue 1, T. Kondo 1, Y. Numaguchi 1 , T. Murohara 1 , M. Shibuya 2 . 1The
Universi~ of Nagoya, Graduate School of Medicine, Nagoya, Japan: 2The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Vascular endothelial growth factor-A (VEGF-A) can promote angiogenesis but causes adverse side effects such as edema, atherosclerotic plaque development, or tissue inflammation. VEGF-E, which was found in the genome of the Off virus, specifically binds to VEGF receptor-2 and shows mitotic activity on endothelial cells. Recently, we created two forms of VEGF-E and human Placental Growth Factor (P1GF) chimera genes (VEGF-Echimera#9 and VEGF-Echimera#33) which are humanized genes holding VEGF-E function. We examined proangiogenic activities of these genes. Four types of plasmid (pCDNA3.1-LacZ, phVEGF-A, pVEGF-Echimera#9 and pVEGFEchimera#33, n=8 each) were administered in a rat hindlimb ischemia model, pVEGF-Echimera#9, pVEGF-Echimera#33 and pVEGF-A significantly increased ischemic/normal hindlimbs laser doppler blood flow (LDBF) compared with the control pCDNA3.1- LacZ treated group (by 1.4 fold, 1.5fold, and 1.5fold respectively, p<0.05). Histochemical staining by alkaline phosphatase also revealed that pVEGF-Echimera#9, pVEGF-Echimera#33 and pVEGF-A treated groups had increased capillary density compared with the pCDNA3.1- LacZ treated group(1.4 fold, 1.5fold and 1.5fold respectively, p<0.05). Furthermore, immunostaJning for anti-ED1 revealed that lower number of macrophages were infiltrated in both pVEGF-Echimera#9 and pVEGF-Echimera#33 groups compared with the pVEGF-A group. In conclusion, novel chimera genes, pVEGF-Echimera#9 and pVEGF-Echimera#33, significantly induced angiogenesis in response to tissue ischemia, in addition, may avoid tissue inflammation often observed in VEGF-A-induced angiogenesis.
•
NITROPRAVASTATIN, N C X 6550, S T I M U L A T E S THERAPEUTIC N E O V A S C U L A R I Z A T I O N AND IMPROVES CLINICAL RECOVERY IN LIMB ISCHEMIA IN STZ-INDUCED DIABETIC MICE
C. Emanueli 1 , A. Monopoli 2, M. Melloni 1 , S. Gadau 3, I. Campesi 3, E. Ongini 2, P. Madeddu 1 . 1Bristol Heart Institute, Universi~ of Bristol,
Bristol, United Kingdom." 2Nicox Research Institute, Milan, Italy: 3National Institute of Biostructures arm Biosystems (btbb), Osilo and Alghero, Italy Diabetes increases the risk of peripheral ischemia. Moreover, post-ischemic neovasculaxisation is impaired by diabetes. We evaluated whether NCX 6550, a nitric oxide (NO)-releasing pravastatin derivative, supports angiogenesis in the ischemic limb muscles of normoglycemic and streptozotocin (STZ)-induced type-1 diabetic mice. Normal or STZ-diabetic mice received either regular (vehicle) or medicated diet containing pravastatin (10 mg/kg B W daily) or NCX 6550 (12 mg/kg B W daily) starting 4 days before unilateral limb ischemia and for the following 2 weeks. Major end-points were: the clinical outcome, hindlimb blood flow recovery, muscular neovasculaxization and blood circulating endothelial progenitor cells 0EPCs) levels. NCX 6550 stimulated reparative angiogenesis of the ischemic adductor muscle (p<0.01 vs vehicle and pravastatin) and mobilized circulating EPCs in normoglycemic and STZ-diabetic mice (p=NS vs normoglycemic mice, p<0.01 as vehicle-treated diabetic mice, p<0.05 vs pravastatin). In the same models, NCX 6550 improved limb reperfusion (p<0.01 vs vehicle; p<0.05 vs pravastatin) and foot salvage better than pravastatin. In conclusion, our data show that NCX 6550 is superior to pravastatin in counteracting the consequences of limb ischemia in both normoglycemic and STZ-diabetic mice. Thus, the incorporation of a bioactive NO moiety greatly improve statins therapeutic potential for the treatment of peripheral vascular disease.
~
IDENTIFICATION OF MEMBRANE A D A P T O R MOLECULES FOR ATYPICAL GPI-ANCHORED T-CADHERIN
D. Ivanov I , M. Philippova I , P. Erne 2, T. Resink I . 1Department of Research, Basel Universi~ Hospital, Basel, Switzerland." 2Division of Cardiology, Kantonsspital LucetTt, LucetTt, Switzerland T-cadherin (T-cad) is an atypical GPI-anchored member of the cadherin super-
191
family. In the cardiovascular system T-cad is upregulated on endothelial and smooth muscle cells during pathological vascular tissue remodelling. In vitro T-cad overexpression and ligation stimulate migration, proliferation, survival and angiogenesis via activation of PI3 kinase/Akt/mTOR pathway. Membrane adaptors transmitting signals from the cell surface T-cad to its intracellular taxgets are not known. The aim of this study was to identify membrane signalling partners of T-cad by coprecipitation. HUVEC were infected with adenoviral vector encoding chimeric T-cad with c-myc tag on the N-terminus, lysed with Triton X-114, and T-cad/c-myc protein was precipitated from lysates using anti-c-myc antibodies. The samples were applied on gradient SDS-PAGE and proteins coprecipitated with T-cad/c-myc were visualized by silver or SYPRO Orange staining. 7 specific bands (200, 150, 85, 75, 70, 65 and 60 kDa) present in the precipitate were excised and subjected to Nanoflow liquid chromatography-tandem mass spectrometry (NanoLC-MS). The following proteins were identified: glucose-related protein GRP-78, GABA receptor alfa-1 subunit, integrin beta-3 and two hypothetical proteins LOC124245 and FLJ32070. We also show that blocking anti-GRP78 antibodies reduce phosphorylation levels of Akt and GSK3 proteins suggesting that GRP78 may mediate T-cad effects on Akt pathway. Since integrin beta-3 and GRP-78 are involved in control of angiogenesis and survival, these proteins are good candidates for the role of membrane adaptors of T-cad-dependent signalling.
~
DIRECT EFFECT OF HMG-COA REDUCTASE INHIBITORS TO HUMAN ENDOTHELIAL CELLS A N D ON ANGIOGENESIS DEPENDS ON KINDS OF STATIN
T. Shingu, M. Katsumoto, S. Nomura, A. Nakata, R. Kuwashima, K. Chayama. Departnwttt of Medicine attd Molecular Sciences, Hiroshima
Universi~, Hiroshima, Japan B a c k g r o u n d a n d P u r p o s e : HMG-CoA Reductase Inhibitors (statins) have direct effects to vascular endothelial cells 0ECs) and on angiogenesis. However, it is unclear whether the effect is statin-specific or not. We therefore designed the present study to compare effect of statins on vascular ECs and on angiogenesis among pitavastatin, atorvastatin and pravastatin. Methods a n d Results: We used human epidermal microvessel ECs (HMVECs) and added statins ranging from 0.005 to 1 umol/1. More than 0.1 umol/1 of pitavastatin inhibited FGF-2-induced migration and proliferation of HMVECs as examined by scratch wound assay, chemotaxis assay and measurement of mitochondrial metabolic activity, and increased cell death assessed by trypan blue dye exclusion test and by DNA staining with bisbenzimide. In contrast, less than 0.01 umol/1 of pitavastatin increased proliferation and migration of HMVECs, and reduced cell death significantly. Atorvastatin tended to increase migration of HMVECs, and significantly reduced cell death at relatively higher concentration (0.1 umol/1), whereas pravastatin had no effect to HMVECs. Effect of statins on angiogenesis was examined by quail chorioallantoic membrane (CAM) assay. High dose of statins except for pravastatin significantly reduced angiogenesis in C A M (about -25% versus FGF-2 only). In contrast, low dose of statins tended to increase FGF-2-induced angiogenesis(about + 12% versus FGF-2 only). Pravastatin did not affected the angiogenesis. C o n d u s i o n s : These results suggest that both responses of ECs and angiogenesis to statins depend on not only concentration but also kinds of statins.
Tu-P7:32
APPLICATION OF VASOHIBIN FOR PREVENSION OF ADVENTITIAL ANGIOGENESIS AND ARTERIAL NEOINYIMAL FORMATION
H. Yamashita, M. Abe, T. Shibuya, K. Watanabe, K. Shimizu, Y. Sato.
Institute of Development, Agbtg, and Cancer; Tohoku Universi~, Sendai, Miyagi, Japan B a c k g r o u n d : We previously reported a novel angiogenesis inhibitor, vasohibin. Vasohibin is regarded as an endothelium-derived negative feedback regulator of angiogenesis. In this study, we introduced human vasohibin gene into a replication-defective adenovirus vector (AdVh) and examined whether vasohibin exhibit any effects on arterial neointimal formation. Methods a n d Results: We injected 109 PFU of AdVh in the mouse tail vein. This procedure caused efficient transfection of the human vasohibin gene in the mouse liver. The expression of human vasohibin was observed for at least 2 weeks in the liver, and human vasohibin protein was present in the plasma during this period. More importantly, we could observe the significant anti-angiogenic effect at remote sites such as the cornea during this period. By using this procedure, we examined the effect of vasohibin on arterial
XIV bztetTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
192
Tues&ty, June 20, 2006: Poster Session P7 Basic science ( lst part)
neointimal formation. We applied arterial cuff placement model to analyze arterial neointimal formation. We observed that vasohibin gene transfection in the liver significantly inhibited adventitial angiogenesis and neointimal formation of the femoral artery after the placement of an external cuff. Vasohibin exhibited no direct effect on the SMC migration and proliferation. Conclusions: Vasohibin is effective for the prevention of arterial neointimal formation by inhibiting adventitial angiogenesis. Funding: This work was supported by the 21st Century COE Program Special Research Grant, the Center for Innovative Therapeutic Development for Common Diseases, from the Ministry of Education, Sdence, Sports and Culture of Japan
~
REGULATION OF ANGIOGENESIS A N D ANGIOGENIC FACTORS BY CARDIOVASCULAR MEDICATIONS
S. Miura, H. Nishikawa, A. Iwata, S. ImaJzumi, Y. Matsuo, K. Saku.
Department of Cardiology, Fukuoka Universi~ Hospital, Fukuoka, Japan Coronary artery disease (CAD) is the most important cause of death in the industrialized world. After experimental myocardial infaxctiom numerous dilated vessels appear in the border zone between the infarct and noninfarct areas. Angiogenic therapy has been widely regarded as an attractive approach both for treating CAD and for enhancing arterioprotective functions of the endothelium. In this report, we critically review the evidence supporting the regulation of angiogenesis and angiogenic factors by newly developed cholesterol ester transfer protein inhibitor and other medical treatments such as statins, angiotensin II receptor blocker, angiotensin converting enzyme inhibitor (ACE-I) and calcium channel blocker. Furthermore, in patients with CAD, the development of collateral artery growth (arteriogenesis) or capillary network growth (angiogenesis), may be important. Current evidence from clinical trials on these therapies suggests that the development of coronary collateral circulation is likely to be a viable therapeutic strategy for cardiovasculax disease, while adaptation to chronic coronary stenosis can proceed. Our assessment of the evidence leads us to conclude that, although many studies have suggested that the administration of angiogenic growth factors and the transplantation of bone marrow-derived angioblasts may be beneficial for the infaxcted heart, the development of collateral circulation using statins and ACE-I may play a critical role in patients with CAD.
~
MICROPARTICLES DERIVED FROM BONE MARROW ISOLATED CELLS REGULATE ANGIOGENESIS IN VITRO
J. Grzybowska 1'4 , L. Paxtyka I , T. Petriczek 2 , Z. Walter 3, A. Polus I , R. N l• z a n k o w s k"i~- , A . Dembinska-Kiec 1 . / Department of Clinical
Biochemistry, The Jagiellonian Unh,ersi~ Medical College, Cracow, PolatM: 2Departnwnt of Angiology, _rid Chair of bttetytal Medicine, The Jagiellonian Universi~ Medical College, Cracow, Poland: ~Department of Hematology, The Jagiellonian University, Medical College, Cracow, PolatM,"4PostgrcMuate School of Molecular Medicine, Medical Universi~ of Warsaw, Warsaw, PolatM Background: Micropaxticles are small-membrane vesicles (below lure) that axe release by almost all activated cells. Microparticles derived from THcells (CD4+), monocytes (CD14+), platelets (CD61+), endothelium (CD62E+), granulocytes (CD66b+) and erythrocytes (glycophorin A+) are identified in blood and axe believed to play an important role in many physiological and pathological processes. Recently the proangiogenic activity of PMP was demonstrated. Study aimed to demonstrate that micropaxticles present in bone marrow mononudeax cells action (BMP) used for critical limb ischemia therapy are able do stimulate angiogenesis. Methods: BMP were isolated from bone marrow of patients with critical limb ischemia. BMP concentration and cellular source was determined using flow cytometry. The influence of BMP on tubular structure formation by HUVEC was investigated in matrigel. To determine the effect on cell proliferation the BrdU method was used. Chemotactic properties of BMP was investigated using Fluoro-Block chambers (NUNC). Results and Conclusions: BMP used for critical limb ischemia therapy affect differentiation of endothelial cells and angiogenesis in vitro. Incubation of HUVEC with BMP for 24 hours stimulated proliferation and tubulogenesis. Funding: Supported by the E U FP-6-2002-LIFESCIHEALTH-502988:SCCR, and Polish State Research Grant No CR 90/2003, 501/KL/438/L
ITu-P7:35
I
I THE EFFECT OF ASPIRIN ON SERUM VASCULAR i
ENDOTHELIAL GROWTH FACTOR AND NITRIC OXIDE CONCENTRATIONS IN HIGH-CHOLESTEROL FED RABBITS
M. Khazaei 1, J.I. Mobaxake 2, M. Nematbakhsh 3 , H. Ahmadi 3. 1Department
of Physiology and Pharmacology, Medical Universi~ of BitjatM, Bitjand, h'an; 2Department of Physiology, Pasteur Institute of h'an, Teheran, h'an," 3Department of Physiology, Medical Universi~ of lsfahan, Isfahan, h'an Objectives: Hypercholesterolemia is one of the major risk factors for atherosclerosis which is chaxacterized by endothelial dysfunction. This study was designed to investigate the effect of aspirin on serum Vascular Endothelial Growth Factor (VEGF) and Nitric Oxide (NO) concentrations in hypercholesterolemic animals. Methods: Sixteen male rabbits were randomly divided into two groups: aspirin treated and control. Aspirin (10 mg/kg/day) was administered orally. All animals were fed with high-cholesterol diet (1%) during the experiment. After five weeks, blood pressure, serum lipid and lipoprotein profiles, VEGF and NO concentrations were measured. Results: Results showed that aspirin did not change blood pressure. Aspirin significantly decreased serum LDL (1276-t-72.1 vs. 1505+68.03mg/dl) and triglyceride (477.5-t-8.3 vs. 649.1+15.2mg/dl) (p<0.05). High-cholesterol diet significantly decreased serum V E G F level in both groups (Control: 24.59-t-0.42 vs. 38.09-t-2.49 pg/ml; Aspirin: 24.72-t-0.84 vs. 42.29-t-2.03 pg/ml) (P<0.05) and aspirin could not change serum VEGF level in hypercholesterolemic animals (24.72-t-0.84 vs. 24.59-t-0.42 pg/ml) (p>0.05). Serum NO concentration was also significantly decreased after five weeks high-cholesterol diet (Control: 5.87-t-0.33 vs. 8.67-t-0.68 itmol/lit; Aspirin: 5.66-4-0.33 vs. 8.58-4-0.60 I~mol/lit) (P<0.05). Aspirin did not change serum NO level (5.66-4-0.33 vs. 5.87-4-0.33 i~mol/lit) (p>0.05). Conclusion: We conclude that, under the condition of this study, aspirin cannot change serum V E G F and NO concentrations in high-cholesterol fed animals. Funding: Funding from Medical University of IsfaJaan, Iran.
JTu-P7:36 J FATTY A C I D S A N D B E T A - C A R O T E N E
IN PROGENITOR CELL DIFFERENTIATION
A.M. Dembinska-Kiec 1 A. Polus 1 B. Kiec-Wilk 1, T. Langmann 2, G. Schmitz 2 . 1Department of Clinical Biochemistt T, The Jagiellonian
Universi~ Medical College, Cracow, Poland: : bzstitut Fuer Klinische Chemie Und [xtboratoriumsmedizin, Klinikum Der Universitaet Regensburg, Regensburg, Germany Introduction: The lipid- soluble Beta-carotene (BC), retinoic acid (RA) in concert with fatty acids (FFA) axe essential for cell differentiation. A i m was to analyze the effect of BC and the selected FFA on the human umbilical cord blood progenitor cell (EPC), adipose tissue - derived progenitors (APC), and HUVEC angiogenic activity Methods: CD34/AC133 were isolated by magnetic microbits. APC were obtained as the adherent fraction of the collagenase separated adipose tissue. Cells were cultured with SDF and VEGF. After 6 days cells expressing VE-cadherin (EPC) were used. Influence of BC as well as palmitic, linoleic, arachidonic acids, on gene expression was estimated by Real-Time PCR or by oligonucleotide chips (Affymetrix HG-U133A), when protein synthesis by flow-cytometry, cell proliferation by BrdU incorporation. Influence on angiogenesis - the tube formation was investigated in the in vitro Matrigel model. SDF or VEGF- induced chemotaxis was measured by BD Falcon Fluoro Bloc System. Results: On the contrary to VEGF, bFGF BC or FFA did not significantly influence the UPC proliferation and the tube formation, however BC > V E G F > SDF-induced chemotaxis of EPC, APC and HUVEC. Microarray and real-time PCR revealed the up-regulation of the extracellular matrix, VEGF signalling pathway, G protein coupled receptors (and subsequently Ras/Rho signaling pathway), genes confirming the BC biological effect. The regulation of SDF/CXCR4 axis was evidenced. Funding: Supported by the EU F6 SCCR FP-6-2002-LIFESCIHEALTH502988; and Polish State Research Grant No CR 90/2003, 501/KL/438/L projects.
XIV bztetTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
THERAPEUTIC ANGIOGENESIS USING NOVEL VASCULAR ENDOTHELIAL GROWTH F A C T O R - E / H U M A N P L A C E N T A L GROWTH FACTOR CHIMERA GENES
N. Inoue 1, T. Kondo 1, Y. Numaguchi 1 , T. Murohara 1 , M. Shibuya 2 . 1The
Universi~ of Nagoya, Graduate School of Medicine, Nagoya, Japan: 2The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Vascular endothelial growth factor-A (VEGF-A) can promote angiogenesis but causes adverse side effects such as edema, atherosclerotic plaque development, or tissue inflammation. VEGF-E, which was found in the genome of the Off virus, specifically binds to VEGF receptor-2 and shows mitotic activity on endothelial cells. Recently, we created two forms of VEGF-E and human Placental Growth Factor (P1GF) chimera genes (VEGF-Echimera#9 and VEGF-Echimera#33) which are humanized genes holding VEGF-E function. We examined proangiogenic activities of these genes. Four types of plasmid (pCDNA3.1-LacZ, phVEGF-A, pVEGF-Echimera#9 and pVEGFEchimera#33, n=8 each) were administered in a rat hindlimb ischemia model, pVEGF-Echimera#9, pVEGF-Echimera#33 and pVEGF-A significantly increased ischemic/normal hindlimbs laser doppler blood flow (LDBF) compared with the control pCDNA3.1- LacZ treated group (by 1.4 fold, 1.5fold, and 1.5fold respectively, p<0.05). Histochemical staining by alkaline phosphatase also revealed that pVEGF-Echimera#9, pVEGF-Echimera#33 and pVEGF-A treated groups had increased capillary density compared with the pCDNA3.1- LacZ treated group(1.4 fold, 1.5fold and 1.5fold respectively, p<0.05). Furthermore, immunostaJning for anti-ED1 revealed that lower number of macrophages were infiltrated in both pVEGF-Echimera#9 and pVEGF-Echimera#33 groups compared with the pVEGF-A group. In conclusion, novel chimera genes, pVEGF-Echimera#9 and pVEGF-Echimera#33, significantly induced angiogenesis in response to tissue ischemia, in addition, may avoid tissue inflammation often observed in VEGF-A-induced angiogenesis.
•
NITROPRAVASTATIN, N C X 6550, S T I M U L A T E S THERAPEUTIC N E O V A S C U L A R I Z A T I O N AND IMPROVES CLINICAL RECOVERY IN LIMB ISCHEMIA IN STZ-INDUCED DIABETIC MICE
C. Emanueli 1 , A. Monopoli 2, M. Melloni 1 , S. Gadau 3, I. Campesi 3, E. Ongini 2, P. Madeddu 1 . 1Bristol Heart Institute, Universi~ of Bristol,
Bristol, United Kingdom." 2Nicox Research Institute, Milan, Italy: 3National Institute of Biostructures arm Biosystems (btbb), Osilo and Alghero, Italy Diabetes increases the risk of peripheral ischemia. Moreover, post-ischemic neovasculaxisation is impaired by diabetes. We evaluated whether NCX 6550, a nitric oxide (NO)-releasing pravastatin derivative, supports angiogenesis in the ischemic limb muscles of normoglycemic and streptozotocin (STZ)-induced type-1 diabetic mice. Normal or STZ-diabetic mice received either regular (vehicle) or medicated diet containing pravastatin (10 mg/kg B W daily) or NCX 6550 (12 mg/kg B W daily) starting 4 days before unilateral limb ischemia and for the following 2 weeks. Major end-points were: the clinical outcome, hindlimb blood flow recovery, muscular neovasculaxization and blood circulating endothelial progenitor cells 0EPCs) levels. NCX 6550 stimulated reparative angiogenesis of the ischemic adductor muscle (p<0.01 vs vehicle and pravastatin) and mobilized circulating EPCs in normoglycemic and STZ-diabetic mice (p=NS vs normoglycemic mice, p<0.01 as vehicle-treated diabetic mice, p<0.05 vs pravastatin). In the same models, NCX 6550 improved limb reperfusion (p<0.01 vs vehicle; p<0.05 vs pravastatin) and foot salvage better than pravastatin. In conclusion, our data show that NCX 6550 is superior to pravastatin in counteracting the consequences of limb ischemia in both normoglycemic and STZ-diabetic mice. Thus, the incorporation of a bioactive NO moiety greatly improve statins therapeutic potential for the treatment of peripheral vascular disease.
~
IDENTIFICATION OF MEMBRANE A D A P T O R MOLECULES FOR ATYPICAL GPI-ANCHORED T-CADHERIN
D. Ivanov I , M. Philippova I , P. Erne 2, T. Resink I . 1Department of Research, Basel Universi~ Hospital, Basel, Switzerland." 2Division of Cardiology, Kantonsspital LucetTt, LucetTt, Switzerland T-cadherin (T-cad) is an atypical GPI-anchored member of the cadherin super-
191
family. In the cardiovascular system T-cad is upregulated on endothelial and smooth muscle cells during pathological vascular tissue remodelling. In vitro T-cad overexpression and ligation stimulate migration, proliferation, survival and angiogenesis via activation of PI3 kinase/Akt/mTOR pathway. Membrane adaptors transmitting signals from the cell surface T-cad to its intracellular taxgets are not known. The aim of this study was to identify membrane signalling partners of T-cad by coprecipitation. HUVEC were infected with adenoviral vector encoding chimeric T-cad with c-myc tag on the N-terminus, lysed with Triton X-114, and T-cad/c-myc protein was precipitated from lysates using anti-c-myc antibodies. The samples were applied on gradient SDS-PAGE and proteins coprecipitated with T-cad/c-myc were visualized by silver or SYPRO Orange staining. 7 specific bands (200, 150, 85, 75, 70, 65 and 60 kDa) present in the precipitate were excised and subjected to Nanoflow liquid chromatography-tandem mass spectrometry (NanoLC-MS). The following proteins were identified: glucose-related protein GRP-78, GABA receptor alfa-1 subunit, integrin beta-3 and two hypothetical proteins LOC124245 and FLJ32070. We also show that blocking anti-GRP78 antibodies reduce phosphorylation levels of Akt and GSK3 proteins suggesting that GRP78 may mediate T-cad effects on Akt pathway. Since integrin beta-3 and GRP-78 are involved in control of angiogenesis and survival, these proteins are good candidates for the role of membrane adaptors of T-cad-dependent signalling.
~
DIRECT EFFECT OF HMG-COA REDUCTASE INHIBITORS TO HUMAN ENDOTHELIAL CELLS A N D ON ANGIOGENESIS DEPENDS ON KINDS OF STATIN
T. Shingu, M. Katsumoto, S. Nomura, A. Nakata, R. Kuwashima, K. Chayama. Departnwttt of Medicine attd Molecular Sciences, Hiroshima
Universi~, Hiroshima, Japan B a c k g r o u n d a n d P u r p o s e : HMG-CoA Reductase Inhibitors (statins) have direct effects to vascular endothelial cells 0ECs) and on angiogenesis. However, it is unclear whether the effect is statin-specific or not. We therefore designed the present study to compare effect of statins on vascular ECs and on angiogenesis among pitavastatin, atorvastatin and pravastatin. Methods a n d Results: We used human epidermal microvessel ECs (HMVECs) and added statins ranging from 0.005 to 1 umol/1. More than 0.1 umol/1 of pitavastatin inhibited FGF-2-induced migration and proliferation of HMVECs as examined by scratch wound assay, chemotaxis assay and measurement of mitochondrial metabolic activity, and increased cell death assessed by trypan blue dye exclusion test and by DNA staining with bisbenzimide. In contrast, less than 0.01 umol/1 of pitavastatin increased proliferation and migration of HMVECs, and reduced cell death significantly. Atorvastatin tended to increase migration of HMVECs, and significantly reduced cell death at relatively higher concentration (0.1 umol/1), whereas pravastatin had no effect to HMVECs. Effect of statins on angiogenesis was examined by quail chorioallantoic membrane (CAM) assay. High dose of statins except for pravastatin significantly reduced angiogenesis in C A M (about -25% versus FGF-2 only). In contrast, low dose of statins tended to increase FGF-2-induced angiogenesis(about + 12% versus FGF-2 only). Pravastatin did not affected the angiogenesis. C o n d u s i o n s : These results suggest that both responses of ECs and angiogenesis to statins depend on not only concentration but also kinds of statins.
Tu-P7:32
APPLICATION OF VASOHIBIN FOR PREVENSION OF ADVENTITIAL ANGIOGENESIS AND ARTERIAL NEOINYIMAL FORMATION
H. Yamashita, M. Abe, T. Shibuya, K. Watanabe, K. Shimizu, Y. Sato.
Institute of Development, Agbtg, and Cancer; Tohoku Universi~, Sendai, Miyagi, Japan B a c k g r o u n d : We previously reported a novel angiogenesis inhibitor, vasohibin. Vasohibin is regarded as an endothelium-derived negative feedback regulator of angiogenesis. In this study, we introduced human vasohibin gene into a replication-defective adenovirus vector (AdVh) and examined whether vasohibin exhibit any effects on arterial neointimal formation. Methods a n d Results: We injected 109 PFU of AdVh in the mouse tail vein. This procedure caused efficient transfection of the human vasohibin gene in the mouse liver. The expression of human vasohibin was observed for at least 2 weeks in the liver, and human vasohibin protein was present in the plasma during this period. More importantly, we could observe the significant anti-angiogenic effect at remote sites such as the cornea during this period. By using this procedure, we examined the effect of vasohibin on arterial
XIV bztetTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
192
Tues&ty, June 20, 2006: Poster Session P7 Basic science ( lst part)
neointimal formation. We applied arterial cuff placement model to analyze arterial neointimal formation. We observed that vasohibin gene transfection in the liver significantly inhibited adventitial angiogenesis and neointimal formation of the femoral artery after the placement of an external cuff. Vasohibin exhibited no direct effect on the SMC migration and proliferation. Conclusions: Vasohibin is effective for the prevention of arterial neointimal formation by inhibiting adventitial angiogenesis. Funding: This work was supported by the 21st Century COE Program Special Research Grant, the Center for Innovative Therapeutic Development for Common Diseases, from the Ministry of Education, Sdence, Sports and Culture of Japan
~
REGULATION OF ANGIOGENESIS A N D ANGIOGENIC FACTORS BY CARDIOVASCULAR MEDICATIONS
S. Miura, H. Nishikawa, A. Iwata, S. ImaJzumi, Y. Matsuo, K. Saku.
Department of Cardiology, Fukuoka Universi~ Hospital, Fukuoka, Japan Coronary artery disease (CAD) is the most important cause of death in the industrialized world. After experimental myocardial infaxctiom numerous dilated vessels appear in the border zone between the infarct and noninfarct areas. Angiogenic therapy has been widely regarded as an attractive approach both for treating CAD and for enhancing arterioprotective functions of the endothelium. In this report, we critically review the evidence supporting the regulation of angiogenesis and angiogenic factors by newly developed cholesterol ester transfer protein inhibitor and other medical treatments such as statins, angiotensin II receptor blocker, angiotensin converting enzyme inhibitor (ACE-I) and calcium channel blocker. Furthermore, in patients with CAD, the development of collateral artery growth (arteriogenesis) or capillary network growth (angiogenesis), may be important. Current evidence from clinical trials on these therapies suggests that the development of coronary collateral circulation is likely to be a viable therapeutic strategy for cardiovasculax disease, while adaptation to chronic coronary stenosis can proceed. Our assessment of the evidence leads us to conclude that, although many studies have suggested that the administration of angiogenic growth factors and the transplantation of bone marrow-derived angioblasts may be beneficial for the infaxcted heart, the development of collateral circulation using statins and ACE-I may play a critical role in patients with CAD.
~
MICROPARTICLES DERIVED FROM BONE MARROW ISOLATED CELLS REGULATE ANGIOGENESIS IN VITRO
J. Grzybowska 1'4 , L. Paxtyka I , T. Petriczek 2 , Z. Walter 3, A. Polus I , R. N l• z a n k o w s k"i~- , A . Dembinska-Kiec 1 . / Department of Clinical
Biochemistry, The Jagiellonian Unh,ersi~ Medical College, Cracow, PolatM: 2Departnwnt of Angiology, _rid Chair of bttetytal Medicine, The Jagiellonian Universi~ Medical College, Cracow, Poland: ~Department of Hematology, The Jagiellonian University, Medical College, Cracow, PolatM,"4PostgrcMuate School of Molecular Medicine, Medical Universi~ of Warsaw, Warsaw, PolatM Background: Micropaxticles are small-membrane vesicles (below lure) that axe release by almost all activated cells. Microparticles derived from THcells (CD4+), monocytes (CD14+), platelets (CD61+), endothelium (CD62E+), granulocytes (CD66b+) and erythrocytes (glycophorin A+) are identified in blood and axe believed to play an important role in many physiological and pathological processes. Recently the proangiogenic activity of PMP was demonstrated. Study aimed to demonstrate that micropaxticles present in bone marrow mononudeax cells action (BMP) used for critical limb ischemia therapy are able do stimulate angiogenesis. Methods: BMP were isolated from bone marrow of patients with critical limb ischemia. BMP concentration and cellular source was determined using flow cytometry. The influence of BMP on tubular structure formation by HUVEC was investigated in matrigel. To determine the effect on cell proliferation the BrdU method was used. Chemotactic properties of BMP was investigated using Fluoro-Block chambers (NUNC). Results and Conclusions: BMP used for critical limb ischemia therapy affect differentiation of endothelial cells and angiogenesis in vitro. Incubation of HUVEC with BMP for 24 hours stimulated proliferation and tubulogenesis. Funding: Supported by the E U FP-6-2002-LIFESCIHEALTH-502988:SCCR, and Polish State Research Grant No CR 90/2003, 501/KL/438/L
ITu-P7:35
I
I THE EFFECT OF ASPIRIN ON SERUM VASCULAR i
ENDOTHELIAL GROWTH FACTOR AND NITRIC OXIDE CONCENTRATIONS IN HIGH-CHOLESTEROL FED RABBITS
M. Khazaei 1, J.I. Mobaxake 2, M. Nematbakhsh 3 , H. Ahmadi 3. 1Department
of Physiology and Pharmacology, Medical Universi~ of BitjatM, Bitjand, h'an; 2Department of Physiology, Pasteur Institute of h'an, Teheran, h'an," 3Department of Physiology, Medical Universi~ of lsfahan, Isfahan, h'an Objectives: Hypercholesterolemia is one of the major risk factors for atherosclerosis which is chaxacterized by endothelial dysfunction. This study was designed to investigate the effect of aspirin on serum Vascular Endothelial Growth Factor (VEGF) and Nitric Oxide (NO) concentrations in hypercholesterolemic animals. Methods: Sixteen male rabbits were randomly divided into two groups: aspirin treated and control. Aspirin (10 mg/kg/day) was administered orally. All animals were fed with high-cholesterol diet (1%) during the experiment. After five weeks, blood pressure, serum lipid and lipoprotein profiles, VEGF and NO concentrations were measured. Results: Results showed that aspirin did not change blood pressure. Aspirin significantly decreased serum LDL (1276-t-72.1 vs. 1505+68.03mg/dl) and triglyceride (477.5-t-8.3 vs. 649.1+15.2mg/dl) (p<0.05). High-cholesterol diet significantly decreased serum V E G F level in both groups (Control: 24.59-t-0.42 vs. 38.09-t-2.49 pg/ml; Aspirin: 24.72-t-0.84 vs. 42.29-t-2.03 pg/ml) (P<0.05) and aspirin could not change serum VEGF level in hypercholesterolemic animals (24.72-t-0.84 vs. 24.59-t-0.42 pg/ml) (p>0.05). Serum NO concentration was also significantly decreased after five weeks high-cholesterol diet (Control: 5.87-t-0.33 vs. 8.67-t-0.68 itmol/lit; Aspirin: 5.66-4-0.33 vs. 8.58-4-0.60 I~mol/lit) (P<0.05). Aspirin did not change serum NO level (5.66-4-0.33 vs. 5.87-4-0.33 i~mol/lit) (p>0.05). Conclusion: We conclude that, under the condition of this study, aspirin cannot change serum V E G F and NO concentrations in high-cholesterol fed animals. Funding: Funding from Medical University of IsfaJaan, Iran.
JTu-P7:36 J FATTY A C I D S A N D B E T A - C A R O T E N E
IN PROGENITOR CELL DIFFERENTIATION
A.M. Dembinska-Kiec 1 A. Polus 1 B. Kiec-Wilk 1, T. Langmann 2, G. Schmitz 2 . 1Department of Clinical Biochemistt T, The Jagiellonian
Universi~ Medical College, Cracow, Poland: : bzstitut Fuer Klinische Chemie Und [xtboratoriumsmedizin, Klinikum Der Universitaet Regensburg, Regensburg, Germany Introduction: The lipid- soluble Beta-carotene (BC), retinoic acid (RA) in concert with fatty acids (FFA) axe essential for cell differentiation. A i m was to analyze the effect of BC and the selected FFA on the human umbilical cord blood progenitor cell (EPC), adipose tissue - derived progenitors (APC), and HUVEC angiogenic activity Methods: CD34/AC133 were isolated by magnetic microbits. APC were obtained as the adherent fraction of the collagenase separated adipose tissue. Cells were cultured with SDF and VEGF. After 6 days cells expressing VE-cadherin (EPC) were used. Influence of BC as well as palmitic, linoleic, arachidonic acids, on gene expression was estimated by Real-Time PCR or by oligonucleotide chips (Affymetrix HG-U133A), when protein synthesis by flow-cytometry, cell proliferation by BrdU incorporation. Influence on angiogenesis - the tube formation was investigated in the in vitro Matrigel model. SDF or VEGF- induced chemotaxis was measured by BD Falcon Fluoro Bloc System. Results: On the contrary to VEGF, bFGF BC or FFA did not significantly influence the UPC proliferation and the tube formation, however BC > V E G F > SDF-induced chemotaxis of EPC, APC and HUVEC. Microarray and real-time PCR revealed the up-regulation of the extracellular matrix, VEGF signalling pathway, G protein coupled receptors (and subsequently Ras/Rho signaling pathway), genes confirming the BC biological effect. The regulation of SDF/CXCR4 axis was evidenced. Funding: Supported by the EU F6 SCCR FP-6-2002-LIFESCIHEALTH502988; and Polish State Research Grant No CR 90/2003, 501/KL/438/L projects.
XIV bztetTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006