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-mice
Monday September 29, 2003: Poster Session Growth factors, cytokines and their receptors in mice SMC. Therefore it is suggested that targeted disruption of Smad3 caused enhanced migration from media to intima, less ECM deposition, and accelerated growth of SMC, which resulted in marked neointimal hyperplasia. 1P-0206
Discovery and profile of β-keto-sulfone analogs: Potent, selective, and novel CCR2 receptor antagonists
Y. Song, K.-L. Sun, L. Bratton, P. Unangst, S. Miller, M. Millar, J. Low, H. Gong, D. Heilig, B.K. Trivedi. Pfizer Inc, USA
1P-0207
Interleukin-12 deficiency enhances atherosclerosis in LDLR-/-, Rag1-/- mice
C. Nakajima, Y. Nakajima, J. Counselman, G. Bradshaw, W. Boisvert. Scripps Research Institute, La Jolla, CA, USA Objective:-Atherosclerotic lesions contain immune cells that can orchestrate and affect inflammatory responses. Several studies have shown that cytokines associated with pro-inflammatory T helper (Th)1 response such as interferon-gamma (IFN-γ) and tumor necrosis factor alpha are important in atherogenesis, while cytokines associated with anti-inflammatory Th2 response such as interleukin 10 (IL-10) are important atheroprotective agents. Amongst other pro-inflammatory cytokines interleukin 12 (IL-12) plays a vital role for the induction of IFN-γ by the leukocytes. Accordingly, the goal of this study was to investigate the role of IL-12 in the development of atherosclerosis in low-density lipoprotein (LDL) receptor-/- mice. Methods: Bone marrow cells from either IL-12-/- or IL-12+/+ mice were transplanted into irradiated male LDL receptor-/-, Rag1-/- mice. Rag1-/- mice, which have no endogenous lymphocytes, were used to ensure a complete engraftment of the donor lymphocyte phenotype. After an interval sufficient to allow engraftment, mice were placed on a high-fat diet for 16 weeks to induce atherosclerosis. Results: The deficiency of IL-12 in bone marrow-derived cells had no effect on plasma cholesterol concentrations. Mice transplanted with the IL-12-/marrow had significantly increased lesion size in the aortic valve. Intracellular cytokine staining of splenocytes showed a decrease in IL-10- and IL-4producing cells. A reduction in IL-10 mRNA as well as IFN-γ mRNA by real-time polymerase chain reaction was also observed. Conclusions: Despite reduction in both Th1- and Th2-type cytokines, enhanced atherosclerosis in IL-12-deficient mice indicates that atheroprotection offered by Th2-type cytokine is more important than the known proatherogenic effects of Th1-type cytokine in lesion development. 1P-0208
Macrophage inflammatory protein-related protein-2 (MRP-2), a novel CC chemokine, modulates adipocyte development and function as an adipocytokine
T. Kawada 1 , C.-S. Kim 2 , H. Yoon 3 , B.-S. Kwon 2 , R. Yu 2 . 1 Kyoto University, Japan; 2 University of Ulsan; 3 Chosun University, Republic of Korea Adipocyte not only stores energy, but also secretes biologically active molecules (adipocytokines), which act as contributing factors to the obesityrelated inflammatory diseases such as atherosclerosis and diabetes. Our previous studies have shown that the levels of macrophage inflammatory protein-related protein-2 (MRP-2), a novel CC chemokine, are enhanced in mice fed high fat diet as well as human atherosclerosis patients. In the
present study, we investigated the potential of MRP-2 to induce preadipocyte migration and to modulate adipocyte function. MRP-2 and its receptors (CCR1/-3) were highly expressed in preadiocytes and differentiated adipocytes as well as in the mouse fat pad. The level of MRP-2 expression in adipose tissue was enhanced in obese mice compared to lean mice. Chemotaxis assay revealed that MRP-2 is a specific chemotactic regulator in preadipocyte migration. The treatment with the anti-MRP-2 or anti-CCR-1/-3 antibody blocked MRP-2 induced migration. The secretion of MRP-2 by adipocytes was markedly increased by the stimulation with proinflammatory cytokine (TNF-alpha). MRP-2 suppressed the adipocyte differentiation in 3T3-L1 cells. Taken together, our data suggest that MRP-2 plays an important role in the adipocyte-related inflammatory diseases and adipose tissue development. 1P-0209
Critical functional role of IL-1β for expression and biological function of VEGF revealed by analysis of IL-1β knock-out mice
K. Amano 1 , M. Okigaki 1 , T. Imada 1 , O. Iba 1 , S. Fujiyama 1 , H. Masaki 2 , Y. Adachi 3 , H. Matsubara 4 , T. Iwasaka 1 . 1 Department of Medicine II and Cardiovascular Center, Kansai Medical University; 2 Department of Clinical Sciences and Laboratory Medicine; 3 First Department of Pathology, Kansai Medical University; 4 Department of Cardiology and Vascular Regenerative Medicine, Kyoto Prefectural University School of Medicine, Japan Cytokines have been shown to play critical role in neovascularization. We previously reported that interleukin-1β (IL-1β) up-regulates cardiac expression of vascular endothelial growth factor (VEGF) and VEGF receptor2. This evidence indicates the possibility that IL-1β may play important roles in neovascularization via VEGF induction. In order to address this question, we analyzed the IL-1β Knock-out mice (IL-1β -/- mice) using a limb muscle ischemia model. Result: IL-1β-/- mice showed following phenotypes. 1) Recovery of blood perfusion as well as proliferation of endothelial cells in surgically induced ischemic limb was impaired. 2) Expression of hypoxia inducible factor-1α (HIF-1α) as well as VEGF, hepatocyte growth factor (HGF) and also monocyte chemoattractant protein-1(MCP-1) were reduced. 3) Mobilization of Flk-1+ cells, which were confirmed to differentiate into endothelial cells, into peripheral blood and also increase in the number of endothelial cells in ischemic tissues were almost disappeared. 4) Activation of vascular cell adhesion molecule 1 (VCAM1) and expression of matrix metalloproteinase9 (MMP9) in ischemic tissues was impaired. 5) Expression of laminin was increased. 6) In addition, IL-1β injection into muscle induces VEGF expression there, followed by mobilization of Flk-1+ cells into peripheral blood and proliferation of endothelial cells in injected tissue. Also, this change is blocked with injection of anti-VEGF antibody. Conclusion: IL-1β production in response to tissue ischemia enhances angiogenesis by HIF-1α-mediated VEGF induction and mobilization of endothelial progenitor cells. 1P-0210
Effects of endogenous placental growth factor on vascular permeability in pathological conditions
S. Ueshima 1 , A. Luttun 2 , K. Brusselmans 2 , M. Tjwa 2 , J.-M. Herbert 3 , O. Matsuo 1 , D. Collen 2 , P. Carmeliet 2 , L. Moons 2 . 1 Department of Physiology, Kinki University School of Medicine, Japan; 2 The Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, KU Leuven, Belgium; 3 Cardiovascular/Thrombosis Research Department, Synthelabo, France The increased vascular leakage causes the pathogenesis of numerous disorders, such as intracranial hypertension in patients with brain tumors, obstruction of respiratory airways during asthma, intravitreous scar formation in diabetic retinopathy and circulatory collapse in sepsis. The placental growth factor (PlGF) is a homologue of vascular endothelial growth factor (VEGF), that is a potent inducer of plasma extravasation. To investigate the role of PlGF in vivo, mice deficient for PlGF (PlGF-/-) were generated by homologous recombination in embryonic stem cells. Extravasation of wound fluid was examined by subcutaneous implantation of a perforated polyethylene capsule. The wound fluid aspirated from the capsule which had been implanted in wild-type mice (PlGF+/+) was larger than that in PlGF-/-. However, a density of new vessels observed in the granulation tissue infiltrated into the capsule revealed no significant difference between PlGF-/- and PlGF+/+. Therefore, it was suggested that the reduced vascular leakage was not due to the reduced neovascular formation in PlGF-/-. Furthermore, the absence of PlGF reduced vascular leakage that was induced by allergens or inflammation. These find-
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
MONDAY
Emerging evidence has shown that monocyte chemoattractant protein-1 (MCP1) plays a central role in inflammatory responses to arterial injury by recruiting monocytes/macrophages to the vessel wall, and this process leads to restenosis and atherogenesis. Preclinical studies in this field have suggested that blocking monocytes/macrophages recruitment to target tissue would be a highly effective therapeutic intervention of arteriosclerosis. Due to our continued interest in development of anti-atherosclerotic agents, we screened our library of compounds in a ligand-binding assay for CCR2 receptor antagonists and identified a number of chemical leads. We have developed SAR around a chemical lead that led to the identification of a series of β-keto-sulfone as the potent, selective and novel small molecule CCR2 receptor antagonists. One compound from this chemical class has an IC50 value of 15 nM in the binding assay. It is also active in chemotaxis assay with an IC50 value of 0.43 µM. Our studies showed that the interaction between MCP-1 and CCR2 receptors can be effectively blocked by small molecules, and monocyte migration can be inhibited by potent CCR2 antagonists in vitro. This paper will present the SAR studies on this class of compounds, focusing on in vitro profile, along with PK properties of selected compounds.
65
Discovery and profile of β-keto-sulfone analogs: Potent, selective, and novel CCR2 receptor antagonists
Y. Song, K.-L. Sun, L. Bratton, P. Unangst, S. Miller, M. Millar, J. Low, H. Gong, D. Heilig, B.K. Trivedi. Pfizer Inc, USA
1P-0207
Interleukin-12 deficiency enhances atherosclerosis in LDLR-/-, Rag1-/- mice
C. Nakajima, Y. Nakajima, J. Counselman, G. Bradshaw, W. Boisvert. Scripps Research Institute, La Jolla, CA, USA Objective:-Atherosclerotic lesions contain immune cells that can orchestrate and affect inflammatory responses. Several studies have shown that cytokines associated with pro-inflammatory T helper (Th)1 response such as interferon-gamma (IFN-γ) and tumor necrosis factor alpha are important in atherogenesis, while cytokines associated with anti-inflammatory Th2 response such as interleukin 10 (IL-10) are important atheroprotective agents. Amongst other pro-inflammatory cytokines interleukin 12 (IL-12) plays a vital role for the induction of IFN-γ by the leukocytes. Accordingly, the goal of this study was to investigate the role of IL-12 in the development of atherosclerosis in low-density lipoprotein (LDL) receptor-/- mice. Methods: Bone marrow cells from either IL-12-/- or IL-12+/+ mice were transplanted into irradiated male LDL receptor-/-, Rag1-/- mice. Rag1-/- mice, which have no endogenous lymphocytes, were used to ensure a complete engraftment of the donor lymphocyte phenotype. After an interval sufficient to allow engraftment, mice were placed on a high-fat diet for 16 weeks to induce atherosclerosis. Results: The deficiency of IL-12 in bone marrow-derived cells had no effect on plasma cholesterol concentrations. Mice transplanted with the IL-12-/marrow had significantly increased lesion size in the aortic valve. Intracellular cytokine staining of splenocytes showed a decrease in IL-10- and IL-4producing cells. A reduction in IL-10 mRNA as well as IFN-γ mRNA by real-time polymerase chain reaction was also observed. Conclusions: Despite reduction in both Th1- and Th2-type cytokines, enhanced atherosclerosis in IL-12-deficient mice indicates that atheroprotection offered by Th2-type cytokine is more important than the known proatherogenic effects of Th1-type cytokine in lesion development. 1P-0208
Macrophage inflammatory protein-related protein-2 (MRP-2), a novel CC chemokine, modulates adipocyte development and function as an adipocytokine
T. Kawada 1 , C.-S. Kim 2 , H. Yoon 3 , B.-S. Kwon 2 , R. Yu 2 . 1 Kyoto University, Japan; 2 University of Ulsan; 3 Chosun University, Republic of Korea Adipocyte not only stores energy, but also secretes biologically active molecules (adipocytokines), which act as contributing factors to the obesityrelated inflammatory diseases such as atherosclerosis and diabetes. Our previous studies have shown that the levels of macrophage inflammatory protein-related protein-2 (MRP-2), a novel CC chemokine, are enhanced in mice fed high fat diet as well as human atherosclerosis patients. In the
present study, we investigated the potential of MRP-2 to induce preadipocyte migration and to modulate adipocyte function. MRP-2 and its receptors (CCR1/-3) were highly expressed in preadiocytes and differentiated adipocytes as well as in the mouse fat pad. The level of MRP-2 expression in adipose tissue was enhanced in obese mice compared to lean mice. Chemotaxis assay revealed that MRP-2 is a specific chemotactic regulator in preadipocyte migration. The treatment with the anti-MRP-2 or anti-CCR-1/-3 antibody blocked MRP-2 induced migration. The secretion of MRP-2 by adipocytes was markedly increased by the stimulation with proinflammatory cytokine (TNF-alpha). MRP-2 suppressed the adipocyte differentiation in 3T3-L1 cells. Taken together, our data suggest that MRP-2 plays an important role in the adipocyte-related inflammatory diseases and adipose tissue development. 1P-0209
Critical functional role of IL-1β for expression and biological function of VEGF revealed by analysis of IL-1β knock-out mice
K. Amano 1 , M. Okigaki 1 , T. Imada 1 , O. Iba 1 , S. Fujiyama 1 , H. Masaki 2 , Y. Adachi 3 , H. Matsubara 4 , T. Iwasaka 1 . 1 Department of Medicine II and Cardiovascular Center, Kansai Medical University; 2 Department of Clinical Sciences and Laboratory Medicine; 3 First Department of Pathology, Kansai Medical University; 4 Department of Cardiology and Vascular Regenerative Medicine, Kyoto Prefectural University School of Medicine, Japan Cytokines have been shown to play critical role in neovascularization. We previously reported that interleukin-1β (IL-1β) up-regulates cardiac expression of vascular endothelial growth factor (VEGF) and VEGF receptor2. This evidence indicates the possibility that IL-1β may play important roles in neovascularization via VEGF induction. In order to address this question, we analyzed the IL-1β Knock-out mice (IL-1β -/- mice) using a limb muscle ischemia model. Result: IL-1β-/- mice showed following phenotypes. 1) Recovery of blood perfusion as well as proliferation of endothelial cells in surgically induced ischemic limb was impaired. 2) Expression of hypoxia inducible factor-1α (HIF-1α) as well as VEGF, hepatocyte growth factor (HGF) and also monocyte chemoattractant protein-1(MCP-1) were reduced. 3) Mobilization of Flk-1+ cells, which were confirmed to differentiate into endothelial cells, into peripheral blood and also increase in the number of endothelial cells in ischemic tissues were almost disappeared. 4) Activation of vascular cell adhesion molecule 1 (VCAM1) and expression of matrix metalloproteinase9 (MMP9) in ischemic tissues was impaired. 5) Expression of laminin was increased. 6) In addition, IL-1β injection into muscle induces VEGF expression there, followed by mobilization of Flk-1+ cells into peripheral blood and proliferation of endothelial cells in injected tissue. Also, this change is blocked with injection of anti-VEGF antibody. Conclusion: IL-1β production in response to tissue ischemia enhances angiogenesis by HIF-1α-mediated VEGF induction and mobilization of endothelial progenitor cells. 1P-0210
Effects of endogenous placental growth factor on vascular permeability in pathological conditions
S. Ueshima 1 , A. Luttun 2 , K. Brusselmans 2 , M. Tjwa 2 , J.-M. Herbert 3 , O. Matsuo 1 , D. Collen 2 , P. Carmeliet 2 , L. Moons 2 . 1 Department of Physiology, Kinki University School of Medicine, Japan; 2 The Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, KU Leuven, Belgium; 3 Cardiovascular/Thrombosis Research Department, Synthelabo, France The increased vascular leakage causes the pathogenesis of numerous disorders, such as intracranial hypertension in patients with brain tumors, obstruction of respiratory airways during asthma, intravitreous scar formation in diabetic retinopathy and circulatory collapse in sepsis. The placental growth factor (PlGF) is a homologue of vascular endothelial growth factor (VEGF), that is a potent inducer of plasma extravasation. To investigate the role of PlGF in vivo, mice deficient for PlGF (PlGF-/-) were generated by homologous recombination in embryonic stem cells. Extravasation of wound fluid was examined by subcutaneous implantation of a perforated polyethylene capsule. The wound fluid aspirated from the capsule which had been implanted in wild-type mice (PlGF+/+) was larger than that in PlGF-/-. However, a density of new vessels observed in the granulation tissue infiltrated into the capsule revealed no significant difference between PlGF-/- and PlGF+/+. Therefore, it was suggested that the reduced vascular leakage was not due to the reduced neovascular formation in PlGF-/-. Furthermore, the absence of PlGF reduced vascular leakage that was induced by allergens or inflammation. These find-
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
MONDAY
Emerging evidence has shown that monocyte chemoattractant protein-1 (MCP1) plays a central role in inflammatory responses to arterial injury by recruiting monocytes/macrophages to the vessel wall, and this process leads to restenosis and atherogenesis. Preclinical studies in this field have suggested that blocking monocytes/macrophages recruitment to target tissue would be a highly effective therapeutic intervention of arteriosclerosis. Due to our continued interest in development of anti-atherosclerotic agents, we screened our library of compounds in a ligand-binding assay for CCR2 receptor antagonists and identified a number of chemical leads. We have developed SAR around a chemical lead that led to the identification of a series of β-keto-sulfone as the potent, selective and novel small molecule CCR2 receptor antagonists. One compound from this chemical class has an IC50 value of 15 nM in the binding assay. It is also active in chemotaxis assay with an IC50 value of 0.43 µM. Our studies showed that the interaction between MCP-1 and CCR2 receptors can be effectively blocked by small molecules, and monocyte migration can be inhibited by potent CCR2 antagonists in vitro. This paper will present the SAR studies on this class of compounds, focusing on in vitro profile, along with PK properties of selected compounds.
65