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NOVEL REGULATORY PATHWAY OF PDGF-A CHAIN AS MEDIATED BY TRANSCRIPTION FACTOR KLF5 THROUGH COOPERATIVE ACTIVATION WITH NF-KB AND PHORBOL ESTER
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200 encompassing the discoidin domain physically associate with MMP-2, but not with MMP-9 in vitro and in cultured vascular smooth muscle cells. Supported by CIHR, HSFC. Pamela J. Boyd was supported by an OGSST Scholarship
P441 MT1-MMP IS INCLUDED INTO TETRASPANIN MICRODOMAINS AT ENDOTELIAL LATERAL JUNCTIONS. Marı´a Ya´n˜ez-Mo´, Olga Barreiro, Beatriz G. Ga´lvez, Alicia G. Arroyo, Francisco Sa´nchez-Madrid. Servicio de Inmunologı´a Hospital Universitario de la Princesa, U.A.M. Madrid, C.I.B., C.S.I.C., Madrid. MT1-MMP transmembrane metalloproteinase is expressed in endothelial cells and knock out mice for its gene show severe angiogenic defects. In HUVEC cells MT1-MMP is localized at two different subcellular compartments depending on RhoA activity: it is present at caveolae, where it associates to alphavbeta3 integrin and actively participates in cell motility, and, when cells are quiescent onto beta1 ECM substrates, it relocalizes to lateral junctions. Here we show that MT1-MMP on the plasma membrane is constitutively associated to tetraspanin CD9 and, when localized to cell-cell junctions, it becomes associated to tetraspanin CD151 and to alpha3beta1 integrin. Tetraspanins are low molecular weight proteins that organize membrane microdomains by association through their extracellular second large loop (LEL) to different transmembrane proteins, including beta1 integrins. Inclusion into tetraspanin microdomains can be essential for the proper function of some receptors that depend on clustering regulation. MT1-MMP dimerization has proven to be essential for MMP2 activation so clustering mechanisms might be functionally relevant. Tetraspanin microdomains were altered in vitro by incubation of HUVEC cells with soluble LEL-GST peptides. Alternatively siRNA specific for CD9 or CD151 were used to downregulate their expression on the membrane. The subcellular localization of MT1-MMP, its internalization rate and activity on MMP2 was measured. The effect on other MT1-MMP mediated functions such as cell migration or angiotube formation was also assessed.
P442 THE EFFECTS OF DOXYCYCLINE ON SMOOTH MUSCLE CELL ADHESION AND AGGREGATION. Bernard KS Ho, Katey Donaldson, Diane Mulholland, Michelle P Bendeck. Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, ON. Matrix metalloproteinases (MMPs) are matrix-degrading enzymes that are involved in the pathogenesis of many vascular diseases. In atherosclerosis and restenosis, they mediate smooth muscle cell (SMC) proliferation and migration, ultimately contributing to intimal thickening and inward constrictive vessel remodeling. Doxycycline, a synthetic tetracycline, has been found to significantly inhibit MMP activity independent of its antibiotic activity. We have recently reported that treatment with doxycycline significantly attenuated intimal thickening in balloon catheter-injured rat carotid arteries, and inward remodeling in the injured rabbit aorta. This was accompanied by decreases in SMC proliferation, migration, MMP activity and matrix production in vivo. To determine the molecular mechanisms behind these inhibitory effects, we are now studying the effect of doxycycline on SMCs in vitro. Migration and proliferation are influenced by the degree to which cells are attached via cell-cell and cell-matrix adhesions. SMCs were harvested from the intimal or medial layers of rat carotid arteries, and propagated in culture. SMCs were treated with 15 ug/ ml doxycycline; this dose was chosen because it inhibits the growth of several cell types in vitro and is within the range of concentration measured in the plasma after oral administration in rats. We found that SMCs treated with doxycycline were very resistant to trypsinization. We estimated that only half as many cells were released from doxycycline-treated cultures compared to control cultures. We next measured the number of cells left on
S157
the plate after trypsinizing by toluidine blue staining. Staining of the cells left behind in the doxycycline-treated wells (A595nm 0.366 ± 0.19) was much greater than in controls (A595nm 0.011 ± 0.01). To determine whether the matrix produced by cells treated with doxycycline was more adhesive, SMCs were incubated ± 15 ug/ml doxycycline for one week, then cells were removed from the matrix by treating with Triton, and fresh naı¨ve SMCs were plated on the conditioned matrix. The doxycycline-conditioned matrix supported greater cell attachment than the control matrix. After doxycycline treatment there were 39.2 ± 2 cells/200X field, compared to 31.6 ± 1 cells/ field for control. To assay for cell aggregation, SMCs were treated ± 15 ug/ ml doxycycline for 24 hours, then suspended in agarose for one hour and allowed to aggregate. Doxycycline treatment resulted in the formation of large multi-cellular aggregates as visualized by phase-contrast microscopy. We conclude that doxycycline treatment results in greater SMC aggregation and attachment to substrate, mechanisms by which doxycycline might inhibit the ability of cells to detach and proliferate or migrate. Heart and Stroke Foundation of Ontario
Regulation of Smooth Muscle Cell Phenotype and Heterogeneity P443 HUMAN ARTERIAL SMOOTH MUSCLE CELL SUB-POPULATIONS ARE DIFFERENTIALLY CONVERTED INTO FOAM CELLS BY NATIVE AND OXIDIZED LIPOPROTEINS. Carmen A. Argmann, Cynthia G. Sawyez, Shaohua LI, Robert A Hegele, J. Geoffrey Pickering, Murray W. Huff. Robarts Research Institute, London, Ontario. Vascular SMCs manifest diverse phenotypes and emerging evidence suggests this is due to inherently distinct SMC subtypes. Recently, Li et al (Circ Res 2001;89:517-525) successfully cloned two uniquely responsive SMC sub-populations from a single human artery and we utilized these unique clones to test the hypothesis that distinct SMC subtypes display different susceptibilities to foam cell formation. When challenged with human atherogenic native or oxidized HTG-VLDL, the larger, slowergrowing, spindle-shaped HITB5 SMC clone accumulated significantly more lipid as evidenced by substantial oil-red-O staining. Furthermore, these cells deposited much more cholesteryl ester (CE) and triglyceride (TG) than the smaller, faster-growing epithelioid-shaped HITA2 SMC clone (10 versus 1 mg CE/mg cell protein (PN) and 60 versus 7 mg TG/mg PN, P < .05). Lipoprotein lipase (LPL), a key enzyme involved in cellular lipoprotein uptake, was identified as one differentially expressed protein that altered the predisposition of HITA2 SMCs for foam cell formation. Although HITB5 SMCs secreted significantly more LPL than did HITA2 SMCs (0.7 versus 0.2 U/mL media, P < .05), the addition of bovine milk LPL to HITA2 SMCs, significantly increased native and oxidized HTGVLDL-induced lipid accumulation. This was blocked by the addition of the lipase inhibitor, tetrahydrolipstatin, demonstrating the requirement for LPL activity. Therefore, inherently distinct SMC subsets are differentially predisposed to convert to foam cells. Moreover, the response of SMC subsets to atherogenic lipoproteins can be influenced by the environment. Canadian Institutes for Health Research
P444 NOVEL REGULATORY PATHWAY OF PDGF-A CHAIN AS MEDIATED BY TRANSCRIPTION FACTOR KLF5 THROUGH COOPERATIVE ACTIVATION WITH NF-KB AND PHORBOL ESTER. Kenichi Aizawa, Toru Suzuki, Nanae Kada, Atsushi Ishihara, Masahiko Kurabayashi, Ryozo Nagai. The University of Tokyo, Tokyo, Japan, Gunma University, Gunma, Japan. Background: The transcription factor KLF5 and its genetically downstream target gene platelet-derived growth factor (PDGF) A-chain are key
S158
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200
factors in regulating cardiovascular remodeling. Here we identify a novel regulatory pathway in which PDGF-A chain gene expression, under the control of KLF5, is cooperatively activated by the Nuclear Factor-kB (NFkB) p50 subunit in response to a pathologic stimulus. Results and Discussion: KLF5 transcriptionally activates PDGF A-chain through a cis-element previously shown to mediate phorbol ester induction through the early response factor, Egr-1. KLF5 mediated delayed and persistent expression of PDGF-A chain. Interestingly, the NF-kB p50 subunit further cooperatively activated PDGF-A chain by KLF5. Gel-shift assays showed that p50 acts through protein-protein interaction requiring DNA-binding of KLF5 on the promoter. This synergy was seen for KLF5 but not Egr-1. Co-immunoprecipitation studies confirmed interaction. RNA interference analysis further showed that KLF5 and p50 are important for induction of PDGF-A chain. Conclusions: We show a new transcriptional regulatory pathway of pathologic induction of PDGF-A chain as regulated by KLF5. To our knowledge, this is the first report implicating transcriptional regulation of delayed induction of PDGF-A chain, and the first to implicate a pathway of NF-kB signaling on PDGF-A chain as well. This new pathway is a tempting target for therapeutic intervention aimed at modulating PDGF-A chain and its associated pathologies in the cardiovascular system.
P445 IKK2 MEDIATED NF-KB ACTIVATION INCREASES NEOINTIMAL HYPERPLASIA AND INFLAMMATION. Bu De-xiu, Erl Wolfgang, Martin Rainer de, Hansson Go¨ran K, Yan Zhong-qun. Cardiovascular Research Unit L8:03, Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden, Institut fu¨r Prophylaxe der Kreislaufkrankheiten, Ludwig-Maximilians-University, Munich, Germany , Department of Vascular Biology, University of Vienna, Austria. Vascular inflammatory reaction with activation of NF-kB is a prominent feature of neointima formation. However, the impact of NF-kB activation on vascular inflammation and repair remain unclear. In the present study we characterized the role NF-kB activation in neointima formation after vascular injury. Methods and Results: Rat carotid arteries were denuded with a balloon catheter. To modulate NF-kB, a recombinant adenoviral vector carrying a dominant negative mutant of IKK2(dnIKK2) or E coli b-galactosidase (bGal) as a reporter gene, was applied to the ballooned artery. In other rats, the NF-kB inhibitor, antioxidant PDTC was administered perivascularly. Neointima and media were dissected from rat carotid arteries two weeks after ballooning and analysed for lesion size, inflammation, and NF-kB activation. Neointimal lesions displayed augmented NF-kB activation in conjunction with elevated IKK activity. Persistent NF-kB inhibition by overexpression of dnIKK2 significantly suppressed activation of NF-kB, diminished expression of iNOS, TNFa and MCP-1 and reduced neointimal hyperplasia. In contrast, transient NF-kB activation by PDTC application reduced the initial inflammatory response in medial SMC but did not affect neointimal formation. Conclusions: Enhanced NF-kB activation is due to increased IKK activity in the neointimal smooth muscle cells. IKK2 mediated NF-kB activation promotes the development of neointimal hyperplasia and inflammation. IKK2 might therefore be a potential target for therapeutic intervention in restenosis after angioplasty.
P446 VEGF IN FUNCTIONAL MATURATION AND SYMPATHETIC INNERVATION OF ARTERIES. Jo De Mey, Gregorio Fazzi, Erik Storkebaum, Peter Carmeliet. Dept. Pharmacology & Toxicology, Cardiovascular Research Institute Maastricht (CARIM), Universiteit Maastricht, Maastricht, The Netherlands., Center for Transgene Technology and Gene Therapy, Katholieke Universiteit Leuven, Louvain, Belgium. Background: Previous findings indicated that vascular endothelial growth factor is intimately involved in vasculogenesis and angiogenesis and that this VEGF might be involved in arteriogenesis as well1. Aim: Evaluate whether VEGF is involved in the establishment and/or maintenance of arterial contractile reactivity and its modulation by sympathetic nerves. Material and Methods: Carotid (CA), 1st order mesenteric (MrA) and saphenous arteries (SA) were isolated from wild type mice and littermates lacking the hypoxia-responsive element in the promoter sequence of their VEGF gene (VEGFdd)2. Standard arterial morphometry and myograph studies involving direct and indirect sympatho-mimetic stimuli, were used to quantify arterial structure and contractile reactivity3. Results: Structure and reactivity were not modified in CA of VEGFdd. In MrA and SA of VEGFdd lumen diameter and medial surface area were significantly reduced. Contractile responses to potassium, norepinephrine (NE), angiotensin II, and the thromboxane analogue U46619 as well as those to electrical field stimulation of the sympathetic nerve endings (EFS) and tyramine (TYR), were all markedly reduced in MrA and SA of VEGFdd compared to wild type. Responses to EFS and TYR were decreased more than those to exogenous NE. Also, the density of periarterial sympathetic nerve fibers, highlighted by glyoxylic acid staining, was lower in MrA and SA of VEGFdd; than wild type. Maximal contractile responses normalized to media surface area (active wall stress) were larger in MrA and SA than CA, and were reduced in MrA and SA of VEGFdd compared to wild type. Conclusion: VEGF promotes functional maturation/differentiation and sympathetic innervation of peripheral small muscular arteries. Relative lack of VEGF, which results in amyotrophic lateral sclerosis (ALS) and accelerates ALS development in the presence of mutant SOD12, might do so by impaired (autonomic nervous control of) peripheral arterial vasomotor tone. Conversely, hypoxia driven VEGF production during development might contribute to the peripheral arterial hyper-innervation which we have proposed to link intrauterine growth retardation, resulting from chronic moderated foetal hypoxemia, to increased risk for cardiovascular disease in the adult3. 1 3
Nature Med. 9, 653-660, 2003; 2Nature Genet. 28, 131-138, 2001; Circulation 102, 2892-2897, 2000.
P447 PRO-APOPTOTIC EFFECT OF HYDROXYMETHYLGLUTARYL-COENZYME A REDUCTASE INHIBITORS ON HUMAN MUSCLE CELLS IN VITRO. Svitlana V. Demyanets, Christoph Kaun, J. Pammer, Stefan Pfaffenberger, Tomas W. Weiss, Gerald Maurer, Richard Pacher, Kurt Huber, Johann Wojta. Department of Internal Medicine II, University of Vienna, Austria, Department of Clinical Pathology, University of Vienna, Austria, Wilhelminenspital, 3rd Medical Department, Austria. Background: A major side effect associated with the therapeutic use of hydroxymethylglutaryl-coenzyme A reductase inhibitors or statins is myopathy, which can progress to life-threatening rhabdomyolysis. Relatively scant recent in vitro reports have shown that statins induce apoptosis. Such induction of apoptosis could contribute to muscle injury and damage. Therefore we investigated possible effects of different statins in vitro in human cells of different muscle origin on the expression of
P441 MT1-MMP IS INCLUDED INTO TETRASPANIN MICRODOMAINS AT ENDOTELIAL LATERAL JUNCTIONS. Marı´a Ya´n˜ez-Mo´, Olga Barreiro, Beatriz G. Ga´lvez, Alicia G. Arroyo, Francisco Sa´nchez-Madrid. Servicio de Inmunologı´a Hospital Universitario de la Princesa, U.A.M. Madrid, C.I.B., C.S.I.C., Madrid. MT1-MMP transmembrane metalloproteinase is expressed in endothelial cells and knock out mice for its gene show severe angiogenic defects. In HUVEC cells MT1-MMP is localized at two different subcellular compartments depending on RhoA activity: it is present at caveolae, where it associates to alphavbeta3 integrin and actively participates in cell motility, and, when cells are quiescent onto beta1 ECM substrates, it relocalizes to lateral junctions. Here we show that MT1-MMP on the plasma membrane is constitutively associated to tetraspanin CD9 and, when localized to cell-cell junctions, it becomes associated to tetraspanin CD151 and to alpha3beta1 integrin. Tetraspanins are low molecular weight proteins that organize membrane microdomains by association through their extracellular second large loop (LEL) to different transmembrane proteins, including beta1 integrins. Inclusion into tetraspanin microdomains can be essential for the proper function of some receptors that depend on clustering regulation. MT1-MMP dimerization has proven to be essential for MMP2 activation so clustering mechanisms might be functionally relevant. Tetraspanin microdomains were altered in vitro by incubation of HUVEC cells with soluble LEL-GST peptides. Alternatively siRNA specific for CD9 or CD151 were used to downregulate their expression on the membrane. The subcellular localization of MT1-MMP, its internalization rate and activity on MMP2 was measured. The effect on other MT1-MMP mediated functions such as cell migration or angiotube formation was also assessed.
P442 THE EFFECTS OF DOXYCYCLINE ON SMOOTH MUSCLE CELL ADHESION AND AGGREGATION. Bernard KS Ho, Katey Donaldson, Diane Mulholland, Michelle P Bendeck. Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, ON. Matrix metalloproteinases (MMPs) are matrix-degrading enzymes that are involved in the pathogenesis of many vascular diseases. In atherosclerosis and restenosis, they mediate smooth muscle cell (SMC) proliferation and migration, ultimately contributing to intimal thickening and inward constrictive vessel remodeling. Doxycycline, a synthetic tetracycline, has been found to significantly inhibit MMP activity independent of its antibiotic activity. We have recently reported that treatment with doxycycline significantly attenuated intimal thickening in balloon catheter-injured rat carotid arteries, and inward remodeling in the injured rabbit aorta. This was accompanied by decreases in SMC proliferation, migration, MMP activity and matrix production in vivo. To determine the molecular mechanisms behind these inhibitory effects, we are now studying the effect of doxycycline on SMCs in vitro. Migration and proliferation are influenced by the degree to which cells are attached via cell-cell and cell-matrix adhesions. SMCs were harvested from the intimal or medial layers of rat carotid arteries, and propagated in culture. SMCs were treated with 15 ug/ ml doxycycline; this dose was chosen because it inhibits the growth of several cell types in vitro and is within the range of concentration measured in the plasma after oral administration in rats. We found that SMCs treated with doxycycline were very resistant to trypsinization. We estimated that only half as many cells were released from doxycycline-treated cultures compared to control cultures. We next measured the number of cells left on
S157
the plate after trypsinizing by toluidine blue staining. Staining of the cells left behind in the doxycycline-treated wells (A595nm 0.366 ± 0.19) was much greater than in controls (A595nm 0.011 ± 0.01). To determine whether the matrix produced by cells treated with doxycycline was more adhesive, SMCs were incubated ± 15 ug/ml doxycycline for one week, then cells were removed from the matrix by treating with Triton, and fresh naı¨ve SMCs were plated on the conditioned matrix. The doxycycline-conditioned matrix supported greater cell attachment than the control matrix. After doxycycline treatment there were 39.2 ± 2 cells/200X field, compared to 31.6 ± 1 cells/ field for control. To assay for cell aggregation, SMCs were treated ± 15 ug/ ml doxycycline for 24 hours, then suspended in agarose for one hour and allowed to aggregate. Doxycycline treatment resulted in the formation of large multi-cellular aggregates as visualized by phase-contrast microscopy. We conclude that doxycycline treatment results in greater SMC aggregation and attachment to substrate, mechanisms by which doxycycline might inhibit the ability of cells to detach and proliferate or migrate. Heart and Stroke Foundation of Ontario
Regulation of Smooth Muscle Cell Phenotype and Heterogeneity P443 HUMAN ARTERIAL SMOOTH MUSCLE CELL SUB-POPULATIONS ARE DIFFERENTIALLY CONVERTED INTO FOAM CELLS BY NATIVE AND OXIDIZED LIPOPROTEINS. Carmen A. Argmann, Cynthia G. Sawyez, Shaohua LI, Robert A Hegele, J. Geoffrey Pickering, Murray W. Huff. Robarts Research Institute, London, Ontario. Vascular SMCs manifest diverse phenotypes and emerging evidence suggests this is due to inherently distinct SMC subtypes. Recently, Li et al (Circ Res 2001;89:517-525) successfully cloned two uniquely responsive SMC sub-populations from a single human artery and we utilized these unique clones to test the hypothesis that distinct SMC subtypes display different susceptibilities to foam cell formation. When challenged with human atherogenic native or oxidized HTG-VLDL, the larger, slowergrowing, spindle-shaped HITB5 SMC clone accumulated significantly more lipid as evidenced by substantial oil-red-O staining. Furthermore, these cells deposited much more cholesteryl ester (CE) and triglyceride (TG) than the smaller, faster-growing epithelioid-shaped HITA2 SMC clone (10 versus 1 mg CE/mg cell protein (PN) and 60 versus 7 mg TG/mg PN, P < .05). Lipoprotein lipase (LPL), a key enzyme involved in cellular lipoprotein uptake, was identified as one differentially expressed protein that altered the predisposition of HITA2 SMCs for foam cell formation. Although HITB5 SMCs secreted significantly more LPL than did HITA2 SMCs (0.7 versus 0.2 U/mL media, P < .05), the addition of bovine milk LPL to HITA2 SMCs, significantly increased native and oxidized HTGVLDL-induced lipid accumulation. This was blocked by the addition of the lipase inhibitor, tetrahydrolipstatin, demonstrating the requirement for LPL activity. Therefore, inherently distinct SMC subsets are differentially predisposed to convert to foam cells. Moreover, the response of SMC subsets to atherogenic lipoproteins can be influenced by the environment. Canadian Institutes for Health Research
P444 NOVEL REGULATORY PATHWAY OF PDGF-A CHAIN AS MEDIATED BY TRANSCRIPTION FACTOR KLF5 THROUGH COOPERATIVE ACTIVATION WITH NF-KB AND PHORBOL ESTER. Kenichi Aizawa, Toru Suzuki, Nanae Kada, Atsushi Ishihara, Masahiko Kurabayashi, Ryozo Nagai. The University of Tokyo, Tokyo, Japan, Gunma University, Gunma, Japan. Background: The transcription factor KLF5 and its genetically downstream target gene platelet-derived growth factor (PDGF) A-chain are key
S158
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200
factors in regulating cardiovascular remodeling. Here we identify a novel regulatory pathway in which PDGF-A chain gene expression, under the control of KLF5, is cooperatively activated by the Nuclear Factor-kB (NFkB) p50 subunit in response to a pathologic stimulus. Results and Discussion: KLF5 transcriptionally activates PDGF A-chain through a cis-element previously shown to mediate phorbol ester induction through the early response factor, Egr-1. KLF5 mediated delayed and persistent expression of PDGF-A chain. Interestingly, the NF-kB p50 subunit further cooperatively activated PDGF-A chain by KLF5. Gel-shift assays showed that p50 acts through protein-protein interaction requiring DNA-binding of KLF5 on the promoter. This synergy was seen for KLF5 but not Egr-1. Co-immunoprecipitation studies confirmed interaction. RNA interference analysis further showed that KLF5 and p50 are important for induction of PDGF-A chain. Conclusions: We show a new transcriptional regulatory pathway of pathologic induction of PDGF-A chain as regulated by KLF5. To our knowledge, this is the first report implicating transcriptional regulation of delayed induction of PDGF-A chain, and the first to implicate a pathway of NF-kB signaling on PDGF-A chain as well. This new pathway is a tempting target for therapeutic intervention aimed at modulating PDGF-A chain and its associated pathologies in the cardiovascular system.
P445 IKK2 MEDIATED NF-KB ACTIVATION INCREASES NEOINTIMAL HYPERPLASIA AND INFLAMMATION. Bu De-xiu, Erl Wolfgang, Martin Rainer de, Hansson Go¨ran K, Yan Zhong-qun. Cardiovascular Research Unit L8:03, Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden, Institut fu¨r Prophylaxe der Kreislaufkrankheiten, Ludwig-Maximilians-University, Munich, Germany , Department of Vascular Biology, University of Vienna, Austria. Vascular inflammatory reaction with activation of NF-kB is a prominent feature of neointima formation. However, the impact of NF-kB activation on vascular inflammation and repair remain unclear. In the present study we characterized the role NF-kB activation in neointima formation after vascular injury. Methods and Results: Rat carotid arteries were denuded with a balloon catheter. To modulate NF-kB, a recombinant adenoviral vector carrying a dominant negative mutant of IKK2(dnIKK2) or E coli b-galactosidase (bGal) as a reporter gene, was applied to the ballooned artery. In other rats, the NF-kB inhibitor, antioxidant PDTC was administered perivascularly. Neointima and media were dissected from rat carotid arteries two weeks after ballooning and analysed for lesion size, inflammation, and NF-kB activation. Neointimal lesions displayed augmented NF-kB activation in conjunction with elevated IKK activity. Persistent NF-kB inhibition by overexpression of dnIKK2 significantly suppressed activation of NF-kB, diminished expression of iNOS, TNFa and MCP-1 and reduced neointimal hyperplasia. In contrast, transient NF-kB activation by PDTC application reduced the initial inflammatory response in medial SMC but did not affect neointimal formation. Conclusions: Enhanced NF-kB activation is due to increased IKK activity in the neointimal smooth muscle cells. IKK2 mediated NF-kB activation promotes the development of neointimal hyperplasia and inflammation. IKK2 might therefore be a potential target for therapeutic intervention in restenosis after angioplasty.
P446 VEGF IN FUNCTIONAL MATURATION AND SYMPATHETIC INNERVATION OF ARTERIES. Jo De Mey, Gregorio Fazzi, Erik Storkebaum, Peter Carmeliet. Dept. Pharmacology & Toxicology, Cardiovascular Research Institute Maastricht (CARIM), Universiteit Maastricht, Maastricht, The Netherlands., Center for Transgene Technology and Gene Therapy, Katholieke Universiteit Leuven, Louvain, Belgium. Background: Previous findings indicated that vascular endothelial growth factor is intimately involved in vasculogenesis and angiogenesis and that this VEGF might be involved in arteriogenesis as well1. Aim: Evaluate whether VEGF is involved in the establishment and/or maintenance of arterial contractile reactivity and its modulation by sympathetic nerves. Material and Methods: Carotid (CA), 1st order mesenteric (MrA) and saphenous arteries (SA) were isolated from wild type mice and littermates lacking the hypoxia-responsive element in the promoter sequence of their VEGF gene (VEGFdd)2. Standard arterial morphometry and myograph studies involving direct and indirect sympatho-mimetic stimuli, were used to quantify arterial structure and contractile reactivity3. Results: Structure and reactivity were not modified in CA of VEGFdd. In MrA and SA of VEGFdd lumen diameter and medial surface area were significantly reduced. Contractile responses to potassium, norepinephrine (NE), angiotensin II, and the thromboxane analogue U46619 as well as those to electrical field stimulation of the sympathetic nerve endings (EFS) and tyramine (TYR), were all markedly reduced in MrA and SA of VEGFdd compared to wild type. Responses to EFS and TYR were decreased more than those to exogenous NE. Also, the density of periarterial sympathetic nerve fibers, highlighted by glyoxylic acid staining, was lower in MrA and SA of VEGFdd; than wild type. Maximal contractile responses normalized to media surface area (active wall stress) were larger in MrA and SA than CA, and were reduced in MrA and SA of VEGFdd compared to wild type. Conclusion: VEGF promotes functional maturation/differentiation and sympathetic innervation of peripheral small muscular arteries. Relative lack of VEGF, which results in amyotrophic lateral sclerosis (ALS) and accelerates ALS development in the presence of mutant SOD12, might do so by impaired (autonomic nervous control of) peripheral arterial vasomotor tone. Conversely, hypoxia driven VEGF production during development might contribute to the peripheral arterial hyper-innervation which we have proposed to link intrauterine growth retardation, resulting from chronic moderated foetal hypoxemia, to increased risk for cardiovascular disease in the adult3. 1 3
Nature Med. 9, 653-660, 2003; 2Nature Genet. 28, 131-138, 2001; Circulation 102, 2892-2897, 2000.
P447 PRO-APOPTOTIC EFFECT OF HYDROXYMETHYLGLUTARYL-COENZYME A REDUCTASE INHIBITORS ON HUMAN MUSCLE CELLS IN VITRO. Svitlana V. Demyanets, Christoph Kaun, J. Pammer, Stefan Pfaffenberger, Tomas W. Weiss, Gerald Maurer, Richard Pacher, Kurt Huber, Johann Wojta. Department of Internal Medicine II, University of Vienna, Austria, Department of Clinical Pathology, University of Vienna, Austria, Wilhelminenspital, 3rd Medical Department, Austria. Background: A major side effect associated with the therapeutic use of hydroxymethylglutaryl-coenzyme A reductase inhibitors or statins is myopathy, which can progress to life-threatening rhabdomyolysis. Relatively scant recent in vitro reports have shown that statins induce apoptosis. Such induction of apoptosis could contribute to muscle injury and damage. Therefore we investigated possible effects of different statins in vitro in human cells of different muscle origin on the expression of