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BONE MORPHOGENIC PROTEIN 4 (BMP4) PRODUCED IN ENDOTHELIAL CELLS (EC) BY OSCILLATORY SHEAR (OS) INDUCES MONOCYTE ADHESION BY A SUPEROXIDE-DEPENDENT MECHANISM.
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200 and the protein kinase G (PKG) inhibitor, KT-5823 (1 mM), thus excluding a role for GC/cGMP/PKG-dependent pathways. In conclusion, we have demonstrated that NO donors suppress NADPH oxidase activity in endothelial cells. This anti-oxidative effect of NO may reflect an endogenous protective mechanism in arteries that serves to preserve endothelial function. National Health and Medical Research Council of Australia.
P422 OXIDIZED LOW DENSITY LIPOPROTEINS REDUCES STORAGE AND DEPOSITION OF PLASMINOGEN ACTIVATOR INHIBITOR-1 IN VASCULAR ENDOTHELIAL CELL CULTURES. Garry Shen, Guoping M. Ma, Andrew J. Halayko, Gerald L. Stelmack, Craig T. Hillier, Ruozhi Zhao. University of Manitoba. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of tissue and urokinase plasminogen activator. Increased levels of PAI-1 have been considered as a non-traditional risk factor for atherosclerotic coronary heart disease (CHD) and diabetic vascular complications. Low density lipoprotein (LDL) is a classical risk factor for CHD. Oxidation or glycation increases the atherogencity of LDL. Elevated levels of LDL and its modified forms have been found in blood circulation of patients with CAD or diabetes. Previous studies in our laboratory demonstrated that oxidized or glycated LDL increased the generation of PAI-1 from cultured vascular endothelial cells (EC). The present study examined the effects of oxidized and glycated LDL on the expression, release, storage and deposition of PAI-1 in cultured human umbilical vein EC (HUVEC) using biochemical, immunocytochemical and confocal fluorescence microscopy approaches. Native, oxidized and glycated LDL increased the release of PAI-1 following over 24 h of treatment, and the steady state levels of PAI-1 mRNA after 48 h of treatment compared to controls. Oxidized LDL induced greater increases in PAI-1 release from EC than native or glycated LDL. Intracellular regions with high intensity of PAI-1 were co-localized with giantin, a membrane protein of the Golgi apparatus. Diffused distribution of PAI-1 was detected in cytoplasmic and extracellular regions in EC cultures. Moderately increased levels of cell-associated PAI-1 were detected in EC treated with oxidized or glycated LDL for 6 h compared to controls or native LDL-treated cultures. After 48 h of incubation, cell-associated PAI-1 in Golgi apparatus, cytoplasmic and extracellular space in oxidized LDL-treated EC was significantly reduced compared to control, native or glycated LDL-treated EC. The results indicate that prolonged exposure to oxidized LDL reduced PAI-1 accumulation in intracellular and extracellular pools with a pattern distinct from native or glycated LDL. This may contribute to increased PAI-1 in blood circulation, reduced fibrinolytic activity and thrombotic tendency in patients with dyslipidemia. CDA, CIHR
1528/+55 bp of the PAI-1 promoter/luciferase reporter gene transiently transfected in HUVEC. OxLDL induced significantly greater activation of the PAI-1 promoter compared to native LDL. Native and oxidized LDL, but not very low density lipoprotein (VLDL), activated a VLDL-responsive element ( 672/ 657 bp)-free mutant PAI-1 promoter. Native and oxidized LDL activated 1528/+55 bp and 1197/+55 bp, but not 1105/+55 bp or 804/+55 bp PAI-1 promoter. Gel shift assay demonstrated that the binding of a nuclear protein to oligonucleotides corresponding to the targeted region of the PAI-1 promoter was increased by oxLDL treatment. Mutagenesis suggested a 10 – 12 bp fragment within the targeted region of the PAI-1 promoter suggested by transfection and gel shift assays was responsible to oxLDL-induced PAI-1 promoter activation in EC. The results suggest that LDL, particularly its oxidized form, may activate PAI-1 promoter transfected in EC through the interaction between a nuclear protein and a responsive element distinct from previously reported VLDL-responsive element. CIHR
P424 BONE MORPHOGENIC PROTEIN 4 (BMP4) PRODUCED IN ENDOTHELIAL CELLS (EC) BY OSCILLATORY SHEAR (OS) INDUCES MONOCYTE ADHESION BY A SUPEROXIDE-DEPENDENT MECHANISM. George P Sorescu, Jinah Hwang, Sergey Dikalov, Debra A Smith, Sarah L Tressel, Hanjoong Jo. Biomedical Engineering-GA Tech, Emory University-Division of Cardiology. Atherosclerosis is an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions including OS. Recently, we have shown that OS triggers BMP4 expression in EC (Sorescu et al. J Biol Chem 278-31128, 2003). OS-induced BMP4 was then responsible for monocyte adhesion (a critical early step in atherogenesis) by inducing intercellular adhesion molecule-1 (ICAM-1) expression. Here, we tested the hypothesis that BMP4 stimulates the inflammatory response by reactive oxygen species (ROS)-dependent mechanisms. Treatment of EC with ROS scavengers (PEG-Catalase, Tiron, and N-Acetyl Cysteine) blocked ICAM-1 expression and monocyte adhesion induced by either BMP4 or OS. Both OS and BMP4 stimulated H2O2 (DCF-DA assay) and O2- (ESR assay using CMH) production in EC. Moreover, OS-induced O2- production was blocked by pre-treating EC with noggin (a BMP4 antagonist), suggesting a role for BMP4. To further confirm the identity and source of ROS, EC were obtained from p47phox NADH oxidase knockout mice (MAE-p47 / ). BMP4 failed to induce ICAM-1 expression and monocyte adhesion in MAE-p47 / . Both inflammatory responses were restored by transfecting them with p47phox plasmid. These results demonstrate that BMP4 is a mechanosensitive, pro-inflammatory cytokine inducing monocyte adhesion by stimulating ROS production from NADPH oxidase in EC.
P423
Am Heart Assn, SouthEast, NIH HL7107 and HL67413
OXIDIZED LDL ACTIVATED A RESPONSIVE ELEMENT IN PLAMISNOGEN ACTIVATOR INHIBITOR-1 PROMOTER IN VASCULAR ENDOTHELIAL CELLS. Garry Shen , Ruozhi Zhao, Lin Lu, Xiuli Ma, Limei Hu. University of Manitoba.
P425
Increased levels of plasminogen activator inhibitor-1 (PAI-1) are linked to coronary heart disease (CHD) and diabetes. Low density lipoprotein (LDL) is a classical risk factor of CHD. Oxidized LDL (oxLDL) was found in circulation and atherosclerotic lesions in humans. Our previous studies demonstrated that oxidation or glycation enhanced the effects of LDL on the increases in PAI-1 and its mRNA level in human umbilical vein EC (HUVEC). The present study aims to determine responsive element involved in oxidized LDL (oxLDL)-induced activation of PAI-1 promoter in HUVEC. Treatment with native LDL (0.1 mg/ml) for 24 h activated
S151
X-RAYS PRODUCE UNUSUAL PROLIFERATIVE RESPONSES IN HUMAN, PORCINE AND BOVINE AORTIC ENDOTHELIAL CELLS. Pat Thomas, Tilly Ping, Linda Hiebert. University of Saskatchewan, Saskatoon, Saskatchewan. Clonogenic survival assays measure the ability of a known number of irradiated cells to undergo mitosis and form colonies, relative to unirradiated control cells. It is generally thought that irradiation causes cells to undergo DNA damage leading to mitotic death, necrosis or apoptosis, rendering them unable to reproduce and form colonies. In most cell types, survival is minimally affected at low x-ray doses but then declines more
P422 OXIDIZED LOW DENSITY LIPOPROTEINS REDUCES STORAGE AND DEPOSITION OF PLASMINOGEN ACTIVATOR INHIBITOR-1 IN VASCULAR ENDOTHELIAL CELL CULTURES. Garry Shen, Guoping M. Ma, Andrew J. Halayko, Gerald L. Stelmack, Craig T. Hillier, Ruozhi Zhao. University of Manitoba. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of tissue and urokinase plasminogen activator. Increased levels of PAI-1 have been considered as a non-traditional risk factor for atherosclerotic coronary heart disease (CHD) and diabetic vascular complications. Low density lipoprotein (LDL) is a classical risk factor for CHD. Oxidation or glycation increases the atherogencity of LDL. Elevated levels of LDL and its modified forms have been found in blood circulation of patients with CAD or diabetes. Previous studies in our laboratory demonstrated that oxidized or glycated LDL increased the generation of PAI-1 from cultured vascular endothelial cells (EC). The present study examined the effects of oxidized and glycated LDL on the expression, release, storage and deposition of PAI-1 in cultured human umbilical vein EC (HUVEC) using biochemical, immunocytochemical and confocal fluorescence microscopy approaches. Native, oxidized and glycated LDL increased the release of PAI-1 following over 24 h of treatment, and the steady state levels of PAI-1 mRNA after 48 h of treatment compared to controls. Oxidized LDL induced greater increases in PAI-1 release from EC than native or glycated LDL. Intracellular regions with high intensity of PAI-1 were co-localized with giantin, a membrane protein of the Golgi apparatus. Diffused distribution of PAI-1 was detected in cytoplasmic and extracellular regions in EC cultures. Moderately increased levels of cell-associated PAI-1 were detected in EC treated with oxidized or glycated LDL for 6 h compared to controls or native LDL-treated cultures. After 48 h of incubation, cell-associated PAI-1 in Golgi apparatus, cytoplasmic and extracellular space in oxidized LDL-treated EC was significantly reduced compared to control, native or glycated LDL-treated EC. The results indicate that prolonged exposure to oxidized LDL reduced PAI-1 accumulation in intracellular and extracellular pools with a pattern distinct from native or glycated LDL. This may contribute to increased PAI-1 in blood circulation, reduced fibrinolytic activity and thrombotic tendency in patients with dyslipidemia. CDA, CIHR
1528/+55 bp of the PAI-1 promoter/luciferase reporter gene transiently transfected in HUVEC. OxLDL induced significantly greater activation of the PAI-1 promoter compared to native LDL. Native and oxidized LDL, but not very low density lipoprotein (VLDL), activated a VLDL-responsive element ( 672/ 657 bp)-free mutant PAI-1 promoter. Native and oxidized LDL activated 1528/+55 bp and 1197/+55 bp, but not 1105/+55 bp or 804/+55 bp PAI-1 promoter. Gel shift assay demonstrated that the binding of a nuclear protein to oligonucleotides corresponding to the targeted region of the PAI-1 promoter was increased by oxLDL treatment. Mutagenesis suggested a 10 – 12 bp fragment within the targeted region of the PAI-1 promoter suggested by transfection and gel shift assays was responsible to oxLDL-induced PAI-1 promoter activation in EC. The results suggest that LDL, particularly its oxidized form, may activate PAI-1 promoter transfected in EC through the interaction between a nuclear protein and a responsive element distinct from previously reported VLDL-responsive element. CIHR
P424 BONE MORPHOGENIC PROTEIN 4 (BMP4) PRODUCED IN ENDOTHELIAL CELLS (EC) BY OSCILLATORY SHEAR (OS) INDUCES MONOCYTE ADHESION BY A SUPEROXIDE-DEPENDENT MECHANISM. George P Sorescu, Jinah Hwang, Sergey Dikalov, Debra A Smith, Sarah L Tressel, Hanjoong Jo. Biomedical Engineering-GA Tech, Emory University-Division of Cardiology. Atherosclerosis is an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions including OS. Recently, we have shown that OS triggers BMP4 expression in EC (Sorescu et al. J Biol Chem 278-31128, 2003). OS-induced BMP4 was then responsible for monocyte adhesion (a critical early step in atherogenesis) by inducing intercellular adhesion molecule-1 (ICAM-1) expression. Here, we tested the hypothesis that BMP4 stimulates the inflammatory response by reactive oxygen species (ROS)-dependent mechanisms. Treatment of EC with ROS scavengers (PEG-Catalase, Tiron, and N-Acetyl Cysteine) blocked ICAM-1 expression and monocyte adhesion induced by either BMP4 or OS. Both OS and BMP4 stimulated H2O2 (DCF-DA assay) and O2- (ESR assay using CMH) production in EC. Moreover, OS-induced O2- production was blocked by pre-treating EC with noggin (a BMP4 antagonist), suggesting a role for BMP4. To further confirm the identity and source of ROS, EC were obtained from p47phox NADH oxidase knockout mice (MAE-p47 / ). BMP4 failed to induce ICAM-1 expression and monocyte adhesion in MAE-p47 / . Both inflammatory responses were restored by transfecting them with p47phox plasmid. These results demonstrate that BMP4 is a mechanosensitive, pro-inflammatory cytokine inducing monocyte adhesion by stimulating ROS production from NADPH oxidase in EC.
P423
Am Heart Assn, SouthEast, NIH HL7107 and HL67413
OXIDIZED LDL ACTIVATED A RESPONSIVE ELEMENT IN PLAMISNOGEN ACTIVATOR INHIBITOR-1 PROMOTER IN VASCULAR ENDOTHELIAL CELLS. Garry Shen , Ruozhi Zhao, Lin Lu, Xiuli Ma, Limei Hu. University of Manitoba.
P425
Increased levels of plasminogen activator inhibitor-1 (PAI-1) are linked to coronary heart disease (CHD) and diabetes. Low density lipoprotein (LDL) is a classical risk factor of CHD. Oxidized LDL (oxLDL) was found in circulation and atherosclerotic lesions in humans. Our previous studies demonstrated that oxidation or glycation enhanced the effects of LDL on the increases in PAI-1 and its mRNA level in human umbilical vein EC (HUVEC). The present study aims to determine responsive element involved in oxidized LDL (oxLDL)-induced activation of PAI-1 promoter in HUVEC. Treatment with native LDL (0.1 mg/ml) for 24 h activated
S151
X-RAYS PRODUCE UNUSUAL PROLIFERATIVE RESPONSES IN HUMAN, PORCINE AND BOVINE AORTIC ENDOTHELIAL CELLS. Pat Thomas, Tilly Ping, Linda Hiebert. University of Saskatchewan, Saskatoon, Saskatchewan. Clonogenic survival assays measure the ability of a known number of irradiated cells to undergo mitosis and form colonies, relative to unirradiated control cells. It is generally thought that irradiation causes cells to undergo DNA damage leading to mitotic death, necrosis or apoptosis, rendering them unable to reproduce and form colonies. In most cell types, survival is minimally affected at low x-ray doses but then declines more