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DIVERGENT EFFECTS OF LIPOSOMAL - AND ADENOVIRAL-MEDIATED VASCULAR ENDOTHELIAL GROWTH FACTOR GENE TRANSFER ON NEOINTIMAL MACROPHAGE ACCUMULATION IN HYPERCHOLESTEROLEMIC RABBITS
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Poster Abstracts / Cardiovascular Pathology 13 (2004) S80–S138
IFN-gamma, TNF-alpha, TWEAK and TGF-beta induced an intracellular redistribution of HMGB1 and stimulated secretion by the monocyte/ macrophages. Inhibitors of MEK1/MEK2, protein kinase C and PI-3/ Akt, which inhibit lysosomal degranulation and mRNA translation, attenuated cytokine induced MHGB1 secretion. Macrophage is the major cell type responsible for HMGB1 production in human atherosclerotic lesions. Inflammatory cytokines and transforming growth factor-beta increase HMGB1 expression and secretion by monocyte/ macrophages. HMGB1 appears to be a common mediator of inflammation induced by inflammatory cytokines and is likely to contribute to lesion progression and chronic inflammation.
P255 THE COMPLEMENT COMPONENT C5A INDUCES THE EXPRESSION OF MATRIX METALLOPROTEINASE-1 AND PLASMINOGEN ACTIVATOR INHIBITOR-1 IN HUMAN MACROPHAGES. S. Kastl, W. S. Speidl, C. Kaun, G. Maurer, K. Huber, J. Wojta. Department of Internal Medicine II, University of Vienna, 3rd Department of Medicine, Wilhelminenspital, Vienna, Austria. Background: Activation of the complement cascade is a major aspect of chronic inflammatory diseases. Accumulating evidence suggests that chronic inflammation is a key event in the development of atherosclerosis. Furthermore components of the complement system have been identified in atherosclerotic plaques. Rupture of such plaques usually occurs at their shoulder where macrophages accumulate and may be caused by activation of these cells, which release matrix metalloproteinases (MMP), that cause degradation of the matrix, and plasminogen activator inhibitor (PAI), that modulates tissue injury, repair processes and contributes to thrombus formation. Here we examined the effect of components of the complement system on the production of MMP-1 and PAI- 1 in human monocyte derived macrophages (MDM). Methods: Peripheral blood monocytes were isolated using Ficoll-Paque and CD14 MicroBeadsaˆ. For macrophage transformation cells were cultivated for 8 – 10 days in the presence of human serum. After incubation with the complement components C1q, C3a, C4, C5a, C6, C7, C8 and C9, MMP-1, TIMP-1 and PAI-1 antigen was determined by specific ELISAs. MMP-1 activity was determined by a specific activity assay in the conditioned media of MDM. MMP-1, TIMP-1, and PAI-1 specific mRNA was quantitated by RealTime-PCR. Results: C5a increased MMP-1 antigen concentration and time-dependently up to 6-fold. MMP-1 activity increased approximately 3-fold. TIMP-1 antigen was up-regulated 2-fold. These results could be confirmed on specific mRNA level. C5a also increased PAI-1 antigen concentration and time dependently up to 10 fold. The other complement components had no effect on the expression of MMP-1 and PAI-1. Conclusion: The complement component C5a induces the expression of MMP-1 and PAI-1 in human macrophages in vitro. If this effect is also present in vivo it suggests a new role for this complement component in destabilization of atherosclerotic plaques, and in the modulation of tissue injury and repair processes and thrombus formation associated with the development of atherosclerotic lesions.
P256 DIRECT RECIPROCAL EFFECT OF RESISTIN AND ADIPONECTIN ON VASCULAR ENDOTHELIAL CELLS. Daiji Kawanami, Koji Maemura, Norihiko Takeda, Tomohiro Harada, Takefumi Nojiri, Tetsuya Saito, Yasushi Imai, Ryozo Nagai. Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo. Background: Increasing evidence has recently demonstrated that the adipocyte is not merely a tissue that stores fat, but also secretes various
molecules that modulate insulin resistance, named adipocytokines. Resistin is a recently identified adipocytokine, implicated in development of obesity and insulin resistance. In this study, we investigated the direct effects of resistin and adiponectin, an adipocytokine that improves insulin resistance, on expression of markers of inflammation in vascular endothelial cells. Methods and Results: Northern blot and Western blot analysis revealed that resistin induced vascular cellular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) gene expression in human aortic endothelial cells (HAECs), both at mRNA and protein levels. Furthermore, resistin directly induced expression of pentraxin 3 (PTX3), a marker of inflammation that shares high homology to C-reactive protein (CRP), in HAECs. The induction of VCAM-1 by resistin was inhibited partially by pitavastatin. Moreover, the induction of VCAM-1 and ICAM-1 by resistin was inhibited completely by adiponectin. Conclusions: In this study, we have demonstrated the direct proinflammatory effect of resistin on vascular endothelial cells, which was inhibited by adiponectin. Our findings raise the possibility that the balance in the concentrations of adipocytokines such as resistin and adiponectin determines the inflammation status of vasculature, and the progress of atherosclerosis as part of the metabolic syndrome.
P257 DIVERGENT EFFECTS OF LIPOSOMAL - AND ADENOVIRAL-MEDIATED VASCULAR ENDOTHELIAL GROWTH FACTOR GENE TRANSFER ON NEOINTIMAL MACROPHAGE ACCUMULATION IN HYPERCHOLESTEROLEMIC RABBITS. Rohit Khurana, Shahida Shafi, John F Martin, Ian Zachary. Centre for Cardiovascular Biology & Medicine, Dept. of Medicine, University College London, UK. Introduction: We previously showed that periadventitial liposome-mediated VEGF gene transfer inhibits intimal thickening in normal rabbits. However, the effect of VEGF on the formation of macrophage-rich lesions in hypercholesterolaemic animals is not known. Method: Macrophage-rich lesion formation was induced by collar placement around the left carotid artery of male NZW rabbits, fed on a high cholesterol diet. The endothelium remained intact throughout the experiment. At day 5, either liposome-mediated VEGF-A (n = 10) and LacZ (n = 10) plasmids (25 mg), or adenoviruses (Ad-) encoding either VEGF-A (n = 8) or LacZ (n = 8) genes (5 109 pfu), were transferred to the periadventitial space within the collar. Carotid arteries were analysed 9 days after gene transfer. Results: Transgene expression was confirmed by RT-PCR. Ad-LacZ gene transfer resulted in markedly enhanced transduction efficiency compared to liposome-LacZ, as determined by b-gal staining. Liposome-VEGF caused a 67% reduction in intimal thickening ( p < .001) and markedly reduced neointimal macrophage recruitment (224 ± 66 vs 453 ± 88 / mm2 neointima, p < .02) without significant adventitial neovascularization as evaluated by CD31 immunostaining, compared to liposome-LacZ. Inhibition of neointimal macrophage accumulation was associated with decreased endothelial VCAM-1 expression ( p < .001), but not ICAM-1 expression. In contrast, Ad-VEGF exerted no significant effect on either intimal thickening or macrophage recruitment, compared to Ad-LacZ transfer, but induced a significant increase in adventitial neovascularization ( p < .01). Conclusion: These results show for the first time that low level VEGF expression is arterioprotective and may be therapeutically useful against pathophysiological intimal thickening in human disease. However, the effects of VEGF on intimal thickening and macrophage infiltration are markedly concentration-dependent; VEGF does not have arterioprotective properties at higher, Ad- mediated, levels of expression at which angiogenic effects predominate. British Heart Foundation
Poster Abstracts / Cardiovascular Pathology 13 (2004) S80–S138
IFN-gamma, TNF-alpha, TWEAK and TGF-beta induced an intracellular redistribution of HMGB1 and stimulated secretion by the monocyte/ macrophages. Inhibitors of MEK1/MEK2, protein kinase C and PI-3/ Akt, which inhibit lysosomal degranulation and mRNA translation, attenuated cytokine induced MHGB1 secretion. Macrophage is the major cell type responsible for HMGB1 production in human atherosclerotic lesions. Inflammatory cytokines and transforming growth factor-beta increase HMGB1 expression and secretion by monocyte/ macrophages. HMGB1 appears to be a common mediator of inflammation induced by inflammatory cytokines and is likely to contribute to lesion progression and chronic inflammation.
P255 THE COMPLEMENT COMPONENT C5A INDUCES THE EXPRESSION OF MATRIX METALLOPROTEINASE-1 AND PLASMINOGEN ACTIVATOR INHIBITOR-1 IN HUMAN MACROPHAGES. S. Kastl, W. S. Speidl, C. Kaun, G. Maurer, K. Huber, J. Wojta. Department of Internal Medicine II, University of Vienna, 3rd Department of Medicine, Wilhelminenspital, Vienna, Austria. Background: Activation of the complement cascade is a major aspect of chronic inflammatory diseases. Accumulating evidence suggests that chronic inflammation is a key event in the development of atherosclerosis. Furthermore components of the complement system have been identified in atherosclerotic plaques. Rupture of such plaques usually occurs at their shoulder where macrophages accumulate and may be caused by activation of these cells, which release matrix metalloproteinases (MMP), that cause degradation of the matrix, and plasminogen activator inhibitor (PAI), that modulates tissue injury, repair processes and contributes to thrombus formation. Here we examined the effect of components of the complement system on the production of MMP-1 and PAI- 1 in human monocyte derived macrophages (MDM). Methods: Peripheral blood monocytes were isolated using Ficoll-Paque and CD14 MicroBeadsaˆ. For macrophage transformation cells were cultivated for 8 – 10 days in the presence of human serum. After incubation with the complement components C1q, C3a, C4, C5a, C6, C7, C8 and C9, MMP-1, TIMP-1 and PAI-1 antigen was determined by specific ELISAs. MMP-1 activity was determined by a specific activity assay in the conditioned media of MDM. MMP-1, TIMP-1, and PAI-1 specific mRNA was quantitated by RealTime-PCR. Results: C5a increased MMP-1 antigen concentration and time-dependently up to 6-fold. MMP-1 activity increased approximately 3-fold. TIMP-1 antigen was up-regulated 2-fold. These results could be confirmed on specific mRNA level. C5a also increased PAI-1 antigen concentration and time dependently up to 10 fold. The other complement components had no effect on the expression of MMP-1 and PAI-1. Conclusion: The complement component C5a induces the expression of MMP-1 and PAI-1 in human macrophages in vitro. If this effect is also present in vivo it suggests a new role for this complement component in destabilization of atherosclerotic plaques, and in the modulation of tissue injury and repair processes and thrombus formation associated with the development of atherosclerotic lesions.
P256 DIRECT RECIPROCAL EFFECT OF RESISTIN AND ADIPONECTIN ON VASCULAR ENDOTHELIAL CELLS. Daiji Kawanami, Koji Maemura, Norihiko Takeda, Tomohiro Harada, Takefumi Nojiri, Tetsuya Saito, Yasushi Imai, Ryozo Nagai. Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo. Background: Increasing evidence has recently demonstrated that the adipocyte is not merely a tissue that stores fat, but also secretes various
molecules that modulate insulin resistance, named adipocytokines. Resistin is a recently identified adipocytokine, implicated in development of obesity and insulin resistance. In this study, we investigated the direct effects of resistin and adiponectin, an adipocytokine that improves insulin resistance, on expression of markers of inflammation in vascular endothelial cells. Methods and Results: Northern blot and Western blot analysis revealed that resistin induced vascular cellular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) gene expression in human aortic endothelial cells (HAECs), both at mRNA and protein levels. Furthermore, resistin directly induced expression of pentraxin 3 (PTX3), a marker of inflammation that shares high homology to C-reactive protein (CRP), in HAECs. The induction of VCAM-1 by resistin was inhibited partially by pitavastatin. Moreover, the induction of VCAM-1 and ICAM-1 by resistin was inhibited completely by adiponectin. Conclusions: In this study, we have demonstrated the direct proinflammatory effect of resistin on vascular endothelial cells, which was inhibited by adiponectin. Our findings raise the possibility that the balance in the concentrations of adipocytokines such as resistin and adiponectin determines the inflammation status of vasculature, and the progress of atherosclerosis as part of the metabolic syndrome.
P257 DIVERGENT EFFECTS OF LIPOSOMAL - AND ADENOVIRAL-MEDIATED VASCULAR ENDOTHELIAL GROWTH FACTOR GENE TRANSFER ON NEOINTIMAL MACROPHAGE ACCUMULATION IN HYPERCHOLESTEROLEMIC RABBITS. Rohit Khurana, Shahida Shafi, John F Martin, Ian Zachary. Centre for Cardiovascular Biology & Medicine, Dept. of Medicine, University College London, UK. Introduction: We previously showed that periadventitial liposome-mediated VEGF gene transfer inhibits intimal thickening in normal rabbits. However, the effect of VEGF on the formation of macrophage-rich lesions in hypercholesterolaemic animals is not known. Method: Macrophage-rich lesion formation was induced by collar placement around the left carotid artery of male NZW rabbits, fed on a high cholesterol diet. The endothelium remained intact throughout the experiment. At day 5, either liposome-mediated VEGF-A (n = 10) and LacZ (n = 10) plasmids (25 mg), or adenoviruses (Ad-) encoding either VEGF-A (n = 8) or LacZ (n = 8) genes (5 109 pfu), were transferred to the periadventitial space within the collar. Carotid arteries were analysed 9 days after gene transfer. Results: Transgene expression was confirmed by RT-PCR. Ad-LacZ gene transfer resulted in markedly enhanced transduction efficiency compared to liposome-LacZ, as determined by b-gal staining. Liposome-VEGF caused a 67% reduction in intimal thickening ( p < .001) and markedly reduced neointimal macrophage recruitment (224 ± 66 vs 453 ± 88 / mm2 neointima, p < .02) without significant adventitial neovascularization as evaluated by CD31 immunostaining, compared to liposome-LacZ. Inhibition of neointimal macrophage accumulation was associated with decreased endothelial VCAM-1 expression ( p < .001), but not ICAM-1 expression. In contrast, Ad-VEGF exerted no significant effect on either intimal thickening or macrophage recruitment, compared to Ad-LacZ transfer, but induced a significant increase in adventitial neovascularization ( p < .01). Conclusion: These results show for the first time that low level VEGF expression is arterioprotective and may be therapeutically useful against pathophysiological intimal thickening in human disease. However, the effects of VEGF on intimal thickening and macrophage infiltration are markedly concentration-dependent; VEGF does not have arterioprotective properties at higher, Ad- mediated, levels of expression at which angiogenic effects predominate. British Heart Foundation