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P373 PROTEOMIC CHARACTERIZATION OF EXTRACELLULAR ENVIRONMENT COMPONENTS REVEALS NOVEL PROTEINS IN THE HUMAN AORTA
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Atherosclerosis Supplements 11, no. 2 (2010) 17–108
P373 PROTEOMIC CHARACTERIZATION OF EXTRACELLULAR ENVIRONMENT COMPONENTS REVEALS NOVEL PROTEINS IN THE HUMAN AORTA A. Didangelos1 , K. Mandall2 , M. Jahangiri3 , M. Mayr1 . 1 British Heart Foundation, James Black Center, King’s College London, London, UK , 2 John Hopkins University, Baltimore, MD, USA, 3 St. George’s Healthcare NHS Trust, London, UK Although proteomics has been previously applied to vascular tissues, few studies have specifically targeted the proteins of the extracellular environment. Thus, its detailed composition remains to be characterized. In this study, our objective was to develop a methodology for the extraction and enrichment of extracellular proteins from human aortas. We designed a biochemical subfractionation which achieved minimum contamination with cellular proteins and very effective solubilisation of the proteins of the extracellular environment. Our methodology resulted in the identification by state-of-the-art mass spectrometry of 110 extracellular proteins, of which one third have never been reported in the proteomic literature of vascular tissues so far. In particular, our study revealed the presence of 4 novel glycoproteins in human aortas (podocan, sclerostin, agrin and asporin) and we were able to localize them on the aortic ECM. Their function in the vasculature is currently unknown. Interestingly, we found that cholesterol loading regulates podocan and agrin expression in human aortic smooth muscle cells. More detailed studies are currently in progress. Finally, our methodology allowed us to screen for pathological proteolysis in the aortic extracts. We were able to accurately detect and confirm extensive degradation of fibronectin and relate it to the presence of MMP-9. In conclusion, we expect this proteomic methodology to further our understanding in the composition of the extracellular environment, shed light on ECM remodelling and degradation and provide insights into important pathological processes, such as plaque rupture, aneurysm formation and restenosis. P374 EPIGENETIC MODIFICATIONS IN HUMAN CAROTID ATHEROMATOUS PLAQUES C. Kroupis1 , S. Georgopoulos2 , N. Poumpouridou1 , S.C. Oliveira1 , I. Chatziioannidou1 , A. Dionyssiou-Asteriou1 . 1 Attikon University Hospital, University of Athens Medical School, Haidari, 2 Vascular Unit, First Surgical Dept, University of Athens Medical School, Athens, Greece Objective: DNA promoter hypermethylation is the most common epigenetic mechanism and responsible for loss of critical gene expression. There is increasing evidence that DNA methylation plays a significant role in many pathways affecting pathogenesis and progression of atherosclerosis but it is mainly derived from experimental models and cell lines. Our goal was to investigate DNA methylation in human atherosclerotic plaques starting with the methylation status of CpG islands in the promoter of DAPK gene which is involved in apoptosis and therefore plays a critical role in the remodeling of the plaque. Patients and Methods: Fifteen patients with vascular occlusive disease had their carotid atheromatous plaque surgically removed (>70% stenosis, clear evidence of atherosclerosis in the patients, all with abnormal blood lipid profile). After obtaining informed consent, we thawed part of the frozen carotid tissue and DNA was extracted (Macherey-Nagel, Germany). It was then subjected to sodium-bisulfite conversion (Zymo, USA). Methylation status was examined with methylation-specific PCR (MSP) by using gene specific primers for the promoter of DAPK gene (IDT, USA). Results: According to our results, we did not detect methylation in the promoter of the DAPK gene. All converted samples tested positive for unmethylated sequence. Negative and positive controls for both fully methylated and unmethylated MSP reactions were as expected. Conclusions: In our pilot study, no DNA methylation of DAPK gene promoter was detected however we plan to expand our sample size and examine also other genes that are critical for smooth muscle cell migration and mitotic switch. P375 MONOCYTIC NOX4, A NOVEL SOURCE OF INTRACELLULAR ROS, LOCALIZES TO REDOX SIGNALING COMPLEXES AND IS REQUIRED FOR OxLDL-INDUCED MACROPHAGE DEATH C.F. Lee1 , R. Asmis1,2 . 1 Department of Biochemistry, 2 Department of Clinical Laboratory Sciences, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Cell death of human monocyte-derived macrophages (HMDM) induced by OxLDL is caspase-independent and requires the activation of two independent pathways: depletion of intracellular glutathione and collapse of the glutathione redox state and the increased formation of intracellular ROS. The source of these ROS, however, was not known. We now identified a new member of the Nox family, Nox4, in human monocytes and HMDM. OxLDL upregulated Nox4 mRNA expression but not Nox1, 2, 3 or 5 mRNA. Using a novel monoclonal antibody directed against Nox4, we showed that OxLDL concomitantly up-regulates Nox4 and p22phox protein levels. Confocal
Poster Presentations
microscopy and immunoprecipitation studies showed that Nox4 co-localize with p22phox , suggesting that an active Nox4/p22phox complex is present in HMDM. Nox4 also colocalizes with components of the active focal adhesion complex, indicating that under physiological conditions Nox4 may play a role in macrophage adhesion and migration. Inhibition of MEK but not p38MAPK, JNK or Jak2 prevented the up-regulation of Nox4 induced by OxLDL. Inhibition of MEK also protected against OxLDL-induced increases in ROS formation and OxLDL-mediated macrophage death. In contrast, inhibitors of p38-MAPK or JNK did not block OxLDL-induced ROS formation, and showed no protection against OxLDL. Adenovirus-delivered siRNA directed against Nox4 suppressed OxLDL-induced ROS formation and macrophage death while Nox4 overexpression enhanced ROS formation and accelerated macrophage death induced by OxLDL. Our data demonstrate that the Nox4/p22phox complex is induced in HMDM via the MEK/ERK pathway and mediates OxLDL-induced macrophage death, implicating monocytic Nox4 in the development and progression of atherosclerotic lesions. P376 VASCULAR ENDOTHELIAL GROWTH FACTOR INDUCES EXPRESSION OF POLY(ADP-RIBOSE)-POLYMERASE AND INHIBITOR OF CASPASE-ACTIVATED DEOXYRIBONUCLEASE IN ENDOTHELIAL CELLS: A NOVEL ANTI-APOPTOTIC MECHANISM 1 N. Al-Fakhri1 , M. Hormann ¨ , L. Mey1 , Z. Kharip1 , K. Nemeth1 , H. Renz1 , M. Heidt2 . 1 Department of Clinical Chemistry and Molecular Diagnostics, 2 Department of Cardiology, University Clinic Marburg, Marburg, Germany Vascular endothelial growth factor (VEGF) exerts a potent anti-apoptotic effect on endothelial cells, however, few data exist on the underlying mechanisms. Here, we present a novel mechanism by which VEGF mediates cell survival through the regulation of poly(ADP-ribose)-polymerase (PARP) and inhibitor of caspase-activated deoxyribonuclease (ICAD). Endothelial cells (HUVEC, huPAEC and cell lines) were incubated with VEGF 100 pg/ml–1 mg/ml and apoptosis was induced by integrin inhibition. PARP and ICAD production and function as well as signal transduction pathways were analyzed by Western blot, real-time RT-PCR and enzyme activity assays. Apoptosis was quantified by annexin V-flow cytometry. These experiments were corroborated by PARP and ICAD gene silencing experiments. VEGFR-2 and neuropilin-1 (NP-1) were analyzed by fluorescence microscopy and inhibitor experiments. VEGF dose-dependently induced PARP and ICAD on the protein and mRNA level. Endothelial apoptosis was induced by caspase activation resulting in PARP and ICAD degradation. Preincubation with VEGF dose-dependently reduced the sensitivity to apoptosis induction by 60−90% compared to control. VEGFR-2, that could be detected together with NP-1 on the cell surface, was involved in signal transduction together with JNK and Akt. mRNA knockdown revealed that the anti-apoptotic effect of VEGF was exerted through PARP and ICAD regulation. PARP and ICAD are targets of the caspase cascade in apoptosis induction, their increased expression may also counteract apoptosis. VEGF inhibits endothelial apoptosis by increasing PARP and ICAD gene expression thereby reducing the effects of caspase activation. In arteriosclerosis, VEGF may promote endothelial cell survival and inhibit neointima development and progression through these mechanisms. P377 ROLE OF GROWTH-DIFFERENTIATION FACTOR 15 (GDF-15) IN A MOUSE-MODEL OF ATHEROSCLEROSIS G.A. Bonaterra1 , J. Thogersen2 , S. Zugel ¨ 1 , G. Bendner2 , S. Vorwald2 , 2 J. Fey , U. Traut2 , J. Strelau2 , R. Kinscherf1 . 1 Anatomy and Cell Biology, Philipps-University, Marburg, 2 Anatomy and Cell Biology III, Ruprecht-Karls University, Heidelberg, Germany GDF-15 is expressed in macrophages after stimulation by several biological mediators. Interactions between peripheral blood mononuclear cells (PBMCs) and those within plaque are suggested to be of pathophysiological relevance for lipid-induced atherosclerosis. We therefore postulated that the expression of GDF-15 is associated with the development and progression of plaque, possibly through the regulation of apoptotic processes. To analyze the physiological functions of GDF-15, we generated GDF-15 deficient mice and crossbred them with apoE-deficient mice. After 10 weeks the offspring were feed for 12 weeks with a cholesterol-enriched western diet. Immunohistomorphometry of CD68 (macrophages) and a-actin (smooth muscle cells) were performed on cryostat cross-sections of the innominate artery (=brachiocephalic trunk). Lumen stenosis (LS), cell density (CD) and apoptosis (TUNEL) were also measured. Additionally, peritoneal macrophages were obtained from the apoE−/− /GDF-15+/+ and apoE−/− /GDF-15−/− mice and incubated with 100mg/ml native or oxidized LDL for 24 h. The mRNA was analyzed by real-time PCR. ApoE−/− /GDF-15−/− compared to apoE−/− /GDF-15+/+ mice showed a reduction of LS (15%), as well as in plaque a decrease of TUNEL positive-cells (10%) and an increase of the CD (35%). However, the percentage of macrophages and smooth muscle cells was similar in both groups of mice. Treatment of peritoneal macrophages of apoE−/− /GDF-15−/− mice with ox-LDL, compared with treatment of native LDL, showed changes in the expression of several genes associated
Atherosclerosis Supplements 11, no. 2 (2010) 17–108
P373 PROTEOMIC CHARACTERIZATION OF EXTRACELLULAR ENVIRONMENT COMPONENTS REVEALS NOVEL PROTEINS IN THE HUMAN AORTA A. Didangelos1 , K. Mandall2 , M. Jahangiri3 , M. Mayr1 . 1 British Heart Foundation, James Black Center, King’s College London, London, UK , 2 John Hopkins University, Baltimore, MD, USA, 3 St. George’s Healthcare NHS Trust, London, UK Although proteomics has been previously applied to vascular tissues, few studies have specifically targeted the proteins of the extracellular environment. Thus, its detailed composition remains to be characterized. In this study, our objective was to develop a methodology for the extraction and enrichment of extracellular proteins from human aortas. We designed a biochemical subfractionation which achieved minimum contamination with cellular proteins and very effective solubilisation of the proteins of the extracellular environment. Our methodology resulted in the identification by state-of-the-art mass spectrometry of 110 extracellular proteins, of which one third have never been reported in the proteomic literature of vascular tissues so far. In particular, our study revealed the presence of 4 novel glycoproteins in human aortas (podocan, sclerostin, agrin and asporin) and we were able to localize them on the aortic ECM. Their function in the vasculature is currently unknown. Interestingly, we found that cholesterol loading regulates podocan and agrin expression in human aortic smooth muscle cells. More detailed studies are currently in progress. Finally, our methodology allowed us to screen for pathological proteolysis in the aortic extracts. We were able to accurately detect and confirm extensive degradation of fibronectin and relate it to the presence of MMP-9. In conclusion, we expect this proteomic methodology to further our understanding in the composition of the extracellular environment, shed light on ECM remodelling and degradation and provide insights into important pathological processes, such as plaque rupture, aneurysm formation and restenosis. P374 EPIGENETIC MODIFICATIONS IN HUMAN CAROTID ATHEROMATOUS PLAQUES C. Kroupis1 , S. Georgopoulos2 , N. Poumpouridou1 , S.C. Oliveira1 , I. Chatziioannidou1 , A. Dionyssiou-Asteriou1 . 1 Attikon University Hospital, University of Athens Medical School, Haidari, 2 Vascular Unit, First Surgical Dept, University of Athens Medical School, Athens, Greece Objective: DNA promoter hypermethylation is the most common epigenetic mechanism and responsible for loss of critical gene expression. There is increasing evidence that DNA methylation plays a significant role in many pathways affecting pathogenesis and progression of atherosclerosis but it is mainly derived from experimental models and cell lines. Our goal was to investigate DNA methylation in human atherosclerotic plaques starting with the methylation status of CpG islands in the promoter of DAPK gene which is involved in apoptosis and therefore plays a critical role in the remodeling of the plaque. Patients and Methods: Fifteen patients with vascular occlusive disease had their carotid atheromatous plaque surgically removed (>70% stenosis, clear evidence of atherosclerosis in the patients, all with abnormal blood lipid profile). After obtaining informed consent, we thawed part of the frozen carotid tissue and DNA was extracted (Macherey-Nagel, Germany). It was then subjected to sodium-bisulfite conversion (Zymo, USA). Methylation status was examined with methylation-specific PCR (MSP) by using gene specific primers for the promoter of DAPK gene (IDT, USA). Results: According to our results, we did not detect methylation in the promoter of the DAPK gene. All converted samples tested positive for unmethylated sequence. Negative and positive controls for both fully methylated and unmethylated MSP reactions were as expected. Conclusions: In our pilot study, no DNA methylation of DAPK gene promoter was detected however we plan to expand our sample size and examine also other genes that are critical for smooth muscle cell migration and mitotic switch. P375 MONOCYTIC NOX4, A NOVEL SOURCE OF INTRACELLULAR ROS, LOCALIZES TO REDOX SIGNALING COMPLEXES AND IS REQUIRED FOR OxLDL-INDUCED MACROPHAGE DEATH C.F. Lee1 , R. Asmis1,2 . 1 Department of Biochemistry, 2 Department of Clinical Laboratory Sciences, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Cell death of human monocyte-derived macrophages (HMDM) induced by OxLDL is caspase-independent and requires the activation of two independent pathways: depletion of intracellular glutathione and collapse of the glutathione redox state and the increased formation of intracellular ROS. The source of these ROS, however, was not known. We now identified a new member of the Nox family, Nox4, in human monocytes and HMDM. OxLDL upregulated Nox4 mRNA expression but not Nox1, 2, 3 or 5 mRNA. Using a novel monoclonal antibody directed against Nox4, we showed that OxLDL concomitantly up-regulates Nox4 and p22phox protein levels. Confocal
Poster Presentations
microscopy and immunoprecipitation studies showed that Nox4 co-localize with p22phox , suggesting that an active Nox4/p22phox complex is present in HMDM. Nox4 also colocalizes with components of the active focal adhesion complex, indicating that under physiological conditions Nox4 may play a role in macrophage adhesion and migration. Inhibition of MEK but not p38MAPK, JNK or Jak2 prevented the up-regulation of Nox4 induced by OxLDL. Inhibition of MEK also protected against OxLDL-induced increases in ROS formation and OxLDL-mediated macrophage death. In contrast, inhibitors of p38-MAPK or JNK did not block OxLDL-induced ROS formation, and showed no protection against OxLDL. Adenovirus-delivered siRNA directed against Nox4 suppressed OxLDL-induced ROS formation and macrophage death while Nox4 overexpression enhanced ROS formation and accelerated macrophage death induced by OxLDL. Our data demonstrate that the Nox4/p22phox complex is induced in HMDM via the MEK/ERK pathway and mediates OxLDL-induced macrophage death, implicating monocytic Nox4 in the development and progression of atherosclerotic lesions. P376 VASCULAR ENDOTHELIAL GROWTH FACTOR INDUCES EXPRESSION OF POLY(ADP-RIBOSE)-POLYMERASE AND INHIBITOR OF CASPASE-ACTIVATED DEOXYRIBONUCLEASE IN ENDOTHELIAL CELLS: A NOVEL ANTI-APOPTOTIC MECHANISM 1 N. Al-Fakhri1 , M. Hormann ¨ , L. Mey1 , Z. Kharip1 , K. Nemeth1 , H. Renz1 , M. Heidt2 . 1 Department of Clinical Chemistry and Molecular Diagnostics, 2 Department of Cardiology, University Clinic Marburg, Marburg, Germany Vascular endothelial growth factor (VEGF) exerts a potent anti-apoptotic effect on endothelial cells, however, few data exist on the underlying mechanisms. Here, we present a novel mechanism by which VEGF mediates cell survival through the regulation of poly(ADP-ribose)-polymerase (PARP) and inhibitor of caspase-activated deoxyribonuclease (ICAD). Endothelial cells (HUVEC, huPAEC and cell lines) were incubated with VEGF 100 pg/ml–1 mg/ml and apoptosis was induced by integrin inhibition. PARP and ICAD production and function as well as signal transduction pathways were analyzed by Western blot, real-time RT-PCR and enzyme activity assays. Apoptosis was quantified by annexin V-flow cytometry. These experiments were corroborated by PARP and ICAD gene silencing experiments. VEGFR-2 and neuropilin-1 (NP-1) were analyzed by fluorescence microscopy and inhibitor experiments. VEGF dose-dependently induced PARP and ICAD on the protein and mRNA level. Endothelial apoptosis was induced by caspase activation resulting in PARP and ICAD degradation. Preincubation with VEGF dose-dependently reduced the sensitivity to apoptosis induction by 60−90% compared to control. VEGFR-2, that could be detected together with NP-1 on the cell surface, was involved in signal transduction together with JNK and Akt. mRNA knockdown revealed that the anti-apoptotic effect of VEGF was exerted through PARP and ICAD regulation. PARP and ICAD are targets of the caspase cascade in apoptosis induction, their increased expression may also counteract apoptosis. VEGF inhibits endothelial apoptosis by increasing PARP and ICAD gene expression thereby reducing the effects of caspase activation. In arteriosclerosis, VEGF may promote endothelial cell survival and inhibit neointima development and progression through these mechanisms. P377 ROLE OF GROWTH-DIFFERENTIATION FACTOR 15 (GDF-15) IN A MOUSE-MODEL OF ATHEROSCLEROSIS G.A. Bonaterra1 , J. Thogersen2 , S. Zugel ¨ 1 , G. Bendner2 , S. Vorwald2 , 2 J. Fey , U. Traut2 , J. Strelau2 , R. Kinscherf1 . 1 Anatomy and Cell Biology, Philipps-University, Marburg, 2 Anatomy and Cell Biology III, Ruprecht-Karls University, Heidelberg, Germany GDF-15 is expressed in macrophages after stimulation by several biological mediators. Interactions between peripheral blood mononuclear cells (PBMCs) and those within plaque are suggested to be of pathophysiological relevance for lipid-induced atherosclerosis. We therefore postulated that the expression of GDF-15 is associated with the development and progression of plaque, possibly through the regulation of apoptotic processes. To analyze the physiological functions of GDF-15, we generated GDF-15 deficient mice and crossbred them with apoE-deficient mice. After 10 weeks the offspring were feed for 12 weeks with a cholesterol-enriched western diet. Immunohistomorphometry of CD68 (macrophages) and a-actin (smooth muscle cells) were performed on cryostat cross-sections of the innominate artery (=brachiocephalic trunk). Lumen stenosis (LS), cell density (CD) and apoptosis (TUNEL) were also measured. Additionally, peritoneal macrophages were obtained from the apoE−/− /GDF-15+/+ and apoE−/− /GDF-15−/− mice and incubated with 100mg/ml native or oxidized LDL for 24 h. The mRNA was analyzed by real-time PCR. ApoE−/− /GDF-15−/− compared to apoE−/− /GDF-15+/+ mice showed a reduction of LS (15%), as well as in plaque a decrease of TUNEL positive-cells (10%) and an increase of the CD (35%). However, the percentage of macrophages and smooth muscle cells was similar in both groups of mice. Treatment of peritoneal macrophages of apoE−/− /GDF-15−/− mice with ox-LDL, compared with treatment of native LDL, showed changes in the expression of several genes associated