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44 AND P38 MAPK.
S64
Poster Abstracts / Cardiovascular Pathology 13 (2004) S17–S79
P148 A NEW THEORY ABOUT CARDIAC LOOPING : OPPOSITIONAL AXIAL ROTATION OF INFLOW AND OUTFLOW OF THE ENDOCARDIAL HEART TUBE. Shahin Ghazali. Islamic Azad University of Sanandaj. Numerous investigations have been carried out to understand the real mechanism of cardiac looping and many ideas such as biomechanical force of dorsal mesocardium, act of the heart tube smooth muscle cells and gene expression are proposed in this context, but there are still some open questions about justification of this phenomenon, The manner in which motions and rotations of the developing endocardial heart tube are happening in x, y, z coordinate have not received enough attention. This theory is based on the oppositional axial rotation of inflow and outflow of the endocardial heart tube. The rotations start in the above mentioned regions and its influence in the middle of the endocardial heart tube are observed as a ventral curvature, and then both, an elongation in the conus cordis and a bulging in the primitive ventricle are emerged. This oppositional axial rotation causes the heart tube to emerge as C shaped loop, S shaped loop, phi shaped loop and finally P shaped loop. This theory may also solve some problems such as remodeling of inflow, septation of outflow, reposition of the atrioventricular canal to the right and even the manner of locating septation of primitive ventricle, atrium and cushions. Keywords: cardiac looping; axial rotation; heart tube; morphogenesis
P149 NOVEL HEPARIN NONBINDING ENDOTHELIAL CELL-DERIVED SOLUBLE FACTOR(S) PROMOTES GROWTH AND INHIBITS APOPTOSIS OF PORCINE AORTIC SMOOTH MUSCLE CELLS. Elena V. Lyubimov, Avrum I. Gotlieb. Toronto General Hospital, Dept. of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. Endothelial cells appear to regulate, at least in part, neointimal formation in aortic tissue. We reported that partially purified endothelial cell-derived soluble factor(s) (ECDSF) induced smooth muscle cell (SMC) proliferation in monolayer culture and intimal SMC (iSMC) accumulation in organ cultures. We now characterize ECDSF. It is a glycoprotein, as shown by trypsin digest and glycoprotein immunoblot staining, and shows a prominent 70 kDa band in SDS-NuPAGE. Using BrdU labeling and TUNEL labeling in organ cultures we showed that ECDSF promotes iSMC proliferation, especially within the first 24 hours after initiation of organ culture and then over 7 days, and also inhibits intimal SMC apoptosis. Endothelial derived soluble factor(s) has a molecular weight higher then other known growth factors and does not have affinity to heparin, as shown by heparin column affinity chromatography. Thus ECDSF increases iSMC number due to increased proliferation and suppression of apoptosis, leading to neointimal formation.
P150 EFFECT OF PPAR GAMMA ACTIVATION ON ANGIOGENESIS PROCESS: INVOLVEMENT OF AKT, P42/44 AND P38 MAPK. Joanna Grzybowska, Beata Kiec-Wilk, Ewa Piatkowska, Anna Polus, Aldona Dembinska-Kiec, Karsten Kristiansen. Department of Clinical Biochemistry, Jagiellonian University College of Medicine, Krakow, Poland, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark. Background and objective: The Jagged-Notch system plays a crucial role in endothelial cell differentiation and angiogenesis. High Notch4 expression characterizes proliferating endothelial cells, and Jagged/Notch interaction may inhibit angiogenesis by preventing cell differentiation. VEGF, a potent stimulator of proliferation, induces the expression of Jagged 1 and Notch 4 in
endothelial cells whereas bFGF, a known stimulator of differentiation, downregulates expression of Notch 4 and Jagged 1. The fatty acid activated peroxisome proliferation-activated receptors (PPARs) are important regulators of cellular growth and differentiation, including endothelial cell differentiation and angiogenesis. Administration of the PPAR-gamma agonist ciglitazone was found to down-regulate expression of Jagged1 and Notch4 in endothelial cells and to inhibit angiogenesis and branching of tubules in the 3D matrigel model of angiogenesis. The present work aims at elucidating the signaling pathways involved in the regulation of Jagged1/Notch4 expression in endothelial cells in response to treatment with VEGF, bFGF and PPAR-gamma agonists. Methods: HUVEC (70% of confluence) were stimulated with VEGF, bFGF or PPAR-gamma activators for 24 hours. Total RNA was isolated and used for real-time RT-PCR analyse (DNA Engine Opticon) to determine Jagged1 and Notch-4 gene expression. Studies of cell differentiation were performed using the 3D matrigel tubulogenesis model system. The influence of proangiogenic factors (VEGF, bFGF) and PPAR agonists on Jagged1/ Notch4 protein expression and phosphorylation-dependent activation of kinases of proteins involved in intracellular signaling pathways were detected by Western Blotting. Results and conclusions: We have investigated the effect of VEGF, bFGF and PPAR agonists on differentiation of endothelial cells and angiogenesis using human umbilical endothelial cells (HUVEC). Incubation of HUVEC for 24 hours with bFGF and the PPAR-gamma agonist ciglitazone, but not VEGF, down-regulated the expression of Jagged 1 and Notch-4. We have observed in 3D matrigel tubulogenesis model system that VEGF and bFGF after 24 hours induced tubulogenesis, whereas the PPAR-gamma agonist ciglitazone inhibited HUVEC differentiation. Treatment with VEGF and bFGF activated p42/44 MAPK cascades whereas PPAR agonist (ciglitazone) activated p38 MAPK pathway. In this study we will also present the effect of specific inhibitors of these kinases as well as the effects of different PPAR agonists on Jagged1/Notch4 expression. Supported by European Commission 5th Framework Program-project DLARFID Nr QLTR-2001-00183 and The Polish State Research Grant Nr 3 P05A 011 24
P151 A NOVEL RHOGAP PROTEIN, P74, THAT IS INVOLVED IN ANGIOGENESIS AND CAPILLARY TUBE FORMATION. Christopher Hahn, Zhi-Jian Su, Mathew Vadas, Jennifer Gamble. Human Immunology, Hanson Institute, Frome Rd, Adelaide, SA, Australia. The process of angiogenesis involves a complex orchestration of cellular events driven by soluble factors, cell-cell and cell-ECM interactions. Remarkably, pure cultures of endothelial cells, when given the appropriate soluble and ECM signals, are able to form capillary tubes with a lumen, resembling the in vivo angiogenic situation. The extracellular cues are processed by the endothelial cells via signal transduction pathways resulting in both progressive structural and functional changes as well as changes in the transcriptome. To comprehensively study expression profiles of known and novel genes, a 3-D collagen gel/HUVEC model of angiogenesis was used, in which we have defined the timing and series of distinct morphological events leading to capillary tube formation. RNA was harvested at various time points and used to make subtracted libraries. One differentially regulated clone corresponds to a novel gene, belonging to the RhoGAP family, which we have called p74. Its mRNA was upregulated at 3-6 hours in the capillary tube assay and returned to basal levels by 24 hours. Expression of antisense mRNA using an adenoviral vector, markedly inhibited capillary tube formation on matrigel, possibly via its detrimental effects on proliferation and migration or due to an increase in the percentage of cells undergoing apoptosis. RhoGAP activity studies demonstrated a specificity to Rho, but not Rac or Cdc42. Consistent with this, stress fibre
Poster Abstracts / Cardiovascular Pathology 13 (2004) S17–S79
P148 A NEW THEORY ABOUT CARDIAC LOOPING : OPPOSITIONAL AXIAL ROTATION OF INFLOW AND OUTFLOW OF THE ENDOCARDIAL HEART TUBE. Shahin Ghazali. Islamic Azad University of Sanandaj. Numerous investigations have been carried out to understand the real mechanism of cardiac looping and many ideas such as biomechanical force of dorsal mesocardium, act of the heart tube smooth muscle cells and gene expression are proposed in this context, but there are still some open questions about justification of this phenomenon, The manner in which motions and rotations of the developing endocardial heart tube are happening in x, y, z coordinate have not received enough attention. This theory is based on the oppositional axial rotation of inflow and outflow of the endocardial heart tube. The rotations start in the above mentioned regions and its influence in the middle of the endocardial heart tube are observed as a ventral curvature, and then both, an elongation in the conus cordis and a bulging in the primitive ventricle are emerged. This oppositional axial rotation causes the heart tube to emerge as C shaped loop, S shaped loop, phi shaped loop and finally P shaped loop. This theory may also solve some problems such as remodeling of inflow, septation of outflow, reposition of the atrioventricular canal to the right and even the manner of locating septation of primitive ventricle, atrium and cushions. Keywords: cardiac looping; axial rotation; heart tube; morphogenesis
P149 NOVEL HEPARIN NONBINDING ENDOTHELIAL CELL-DERIVED SOLUBLE FACTOR(S) PROMOTES GROWTH AND INHIBITS APOPTOSIS OF PORCINE AORTIC SMOOTH MUSCLE CELLS. Elena V. Lyubimov, Avrum I. Gotlieb. Toronto General Hospital, Dept. of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. Endothelial cells appear to regulate, at least in part, neointimal formation in aortic tissue. We reported that partially purified endothelial cell-derived soluble factor(s) (ECDSF) induced smooth muscle cell (SMC) proliferation in monolayer culture and intimal SMC (iSMC) accumulation in organ cultures. We now characterize ECDSF. It is a glycoprotein, as shown by trypsin digest and glycoprotein immunoblot staining, and shows a prominent 70 kDa band in SDS-NuPAGE. Using BrdU labeling and TUNEL labeling in organ cultures we showed that ECDSF promotes iSMC proliferation, especially within the first 24 hours after initiation of organ culture and then over 7 days, and also inhibits intimal SMC apoptosis. Endothelial derived soluble factor(s) has a molecular weight higher then other known growth factors and does not have affinity to heparin, as shown by heparin column affinity chromatography. Thus ECDSF increases iSMC number due to increased proliferation and suppression of apoptosis, leading to neointimal formation.
P150 EFFECT OF PPAR GAMMA ACTIVATION ON ANGIOGENESIS PROCESS: INVOLVEMENT OF AKT, P42/44 AND P38 MAPK. Joanna Grzybowska, Beata Kiec-Wilk, Ewa Piatkowska, Anna Polus, Aldona Dembinska-Kiec, Karsten Kristiansen. Department of Clinical Biochemistry, Jagiellonian University College of Medicine, Krakow, Poland, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark. Background and objective: The Jagged-Notch system plays a crucial role in endothelial cell differentiation and angiogenesis. High Notch4 expression characterizes proliferating endothelial cells, and Jagged/Notch interaction may inhibit angiogenesis by preventing cell differentiation. VEGF, a potent stimulator of proliferation, induces the expression of Jagged 1 and Notch 4 in
endothelial cells whereas bFGF, a known stimulator of differentiation, downregulates expression of Notch 4 and Jagged 1. The fatty acid activated peroxisome proliferation-activated receptors (PPARs) are important regulators of cellular growth and differentiation, including endothelial cell differentiation and angiogenesis. Administration of the PPAR-gamma agonist ciglitazone was found to down-regulate expression of Jagged1 and Notch4 in endothelial cells and to inhibit angiogenesis and branching of tubules in the 3D matrigel model of angiogenesis. The present work aims at elucidating the signaling pathways involved in the regulation of Jagged1/Notch4 expression in endothelial cells in response to treatment with VEGF, bFGF and PPAR-gamma agonists. Methods: HUVEC (70% of confluence) were stimulated with VEGF, bFGF or PPAR-gamma activators for 24 hours. Total RNA was isolated and used for real-time RT-PCR analyse (DNA Engine Opticon) to determine Jagged1 and Notch-4 gene expression. Studies of cell differentiation were performed using the 3D matrigel tubulogenesis model system. The influence of proangiogenic factors (VEGF, bFGF) and PPAR agonists on Jagged1/ Notch4 protein expression and phosphorylation-dependent activation of kinases of proteins involved in intracellular signaling pathways were detected by Western Blotting. Results and conclusions: We have investigated the effect of VEGF, bFGF and PPAR agonists on differentiation of endothelial cells and angiogenesis using human umbilical endothelial cells (HUVEC). Incubation of HUVEC for 24 hours with bFGF and the PPAR-gamma agonist ciglitazone, but not VEGF, down-regulated the expression of Jagged 1 and Notch-4. We have observed in 3D matrigel tubulogenesis model system that VEGF and bFGF after 24 hours induced tubulogenesis, whereas the PPAR-gamma agonist ciglitazone inhibited HUVEC differentiation. Treatment with VEGF and bFGF activated p42/44 MAPK cascades whereas PPAR agonist (ciglitazone) activated p38 MAPK pathway. In this study we will also present the effect of specific inhibitors of these kinases as well as the effects of different PPAR agonists on Jagged1/Notch4 expression. Supported by European Commission 5th Framework Program-project DLARFID Nr QLTR-2001-00183 and The Polish State Research Grant Nr 3 P05A 011 24
P151 A NOVEL RHOGAP PROTEIN, P74, THAT IS INVOLVED IN ANGIOGENESIS AND CAPILLARY TUBE FORMATION. Christopher Hahn, Zhi-Jian Su, Mathew Vadas, Jennifer Gamble. Human Immunology, Hanson Institute, Frome Rd, Adelaide, SA, Australia. The process of angiogenesis involves a complex orchestration of cellular events driven by soluble factors, cell-cell and cell-ECM interactions. Remarkably, pure cultures of endothelial cells, when given the appropriate soluble and ECM signals, are able to form capillary tubes with a lumen, resembling the in vivo angiogenic situation. The extracellular cues are processed by the endothelial cells via signal transduction pathways resulting in both progressive structural and functional changes as well as changes in the transcriptome. To comprehensively study expression profiles of known and novel genes, a 3-D collagen gel/HUVEC model of angiogenesis was used, in which we have defined the timing and series of distinct morphological events leading to capillary tube formation. RNA was harvested at various time points and used to make subtracted libraries. One differentially regulated clone corresponds to a novel gene, belonging to the RhoGAP family, which we have called p74. Its mRNA was upregulated at 3-6 hours in the capillary tube assay and returned to basal levels by 24 hours. Expression of antisense mRNA using an adenoviral vector, markedly inhibited capillary tube formation on matrigel, possibly via its detrimental effects on proliferation and migration or due to an increase in the percentage of cells undergoing apoptosis. RhoGAP activity studies demonstrated a specificity to Rho, but not Rac or Cdc42. Consistent with this, stress fibre