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ARTERIAL REMODELING INDUCED BY EITHER REDUCED FLOW OR INCREASED FLOW BOTH REQUIRE TROPHIC ACTION OF CATECHOLAMINES
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Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200
pathological processes in the vascular wall, however, a larger study is necessary for clarification of these possibilities.
P463 ARTERIAL REMODELING INDUCED BY EITHER REDUCED FLOW OR INCREASED FLOW BOTH REQUIRE TROPHIC ACTION OF CATECHOLAMINES. Cauveh Erami, Hua Zhang, Tina Bleeke, Akito Tanoue, Gozoh Tsujimoto, Steven Thomas, James Faber. Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, NC, USA, Department of Molecular and Cellular Pharmacology, National Children’s Medical Research Center, Tokyo, Japan, Department of Molecular and Cellular Pharmacology, National Children’s Medical Research Center, Tokyo, Japan, Department of Pharmacology, University of Pennsylvania, Philadelphia, PA, USA. Alpha1-adrenoceptor (AR) stimulation induces proliferation, hypertrophy and migration of vascular smooth muscle cells and adventitial fibroblasts in cell culture and in organ culture of aorta and pulmonary artery. We have also shown in in vivo studies of carotid artery that this trophic effect of endogenous catecholamines is augmented by over-distension injury and contributes significantly to wall thickening and lumen loss. The trophic activity is mediated by alpha1A-ARs in rat and alpha1B-ARs in mice, but not by alpha1D-ARs that instead mediate contraction of the above arteries. The aim of this study was to determine whether catecholamines also contribute to flow-mediated arterial wall growth and remodeling. In wildtype mice or mice devoid of norepinephrine and epinephrine synthesis [dopamine beta-hydroxylase knockout (DBH-KO)], flow in the left common carotid (LC) was reduced -80% by ligation of its distal branches (except the thyroid artery); this increased flow in the right common carotid (RC) by + 80 %. Vessels were removed 3 weeks later for digital morphometry by blinded observer. Outward eutrophic remodeling of RC and hypertrophic remodeling of LC were both strongly reduced in DBH-knockouts (see table below). This was associated with inhibition ( p < 0.05) of: proliferation in RC (Ki67), apoptosis in RC and LC (cleaved caspase 3), and leukocyte accumulation in RC and LC (CD45), when intima, media and adventitia were examined 5 days after ligation. Although mean arterial pressure is reportedly unaltered in DBHKOs, as a control for this possibility we also examined alpha1D-AR-KO mice where arterial pressure is reduced by 8%. Left and right carotid remodeling were unaffected in alpha1D-AR-KOs. Since expression of alpha1D- and alpha1B-ARs, but not alpha1A-ARs, are detected in murine carotid, these data are consistent with the hypothesis that catecholamines, acting through trophic alpha1B-ARs, contribute significantly to both reduced-flow ‘‘pathological’’ hypertrophic remodeling and to ‘‘physiological’’ expansive remodeling.
P464 THE TIE2-LIGAND ANGIOPOIETIN-2 IS STORED IN AND RAPIDLY RELEASED UPON STIMULATION FROM ENDOTHELIAL CELL WEIBEL-PALADE BODIES. Ulrike Fiedler, Marion Scharpfenecker, Stefanie Koidl, Anja Hegen, Hellmut G. Augustin. Tumor Biology Center Freiburg, Dept. of Vascular Biology and Angiogenesis Research, Breisacher Str. 117, D-79106 Freiburg, Germany. The angiopoietin-Tie ligand-receptor system plays a crucial role in physiological and pathological angiogenesis by mediating vascular remodelling
and maturation processes. Ang-1 acts antiapoptotic and controls the quiescent endothelial cell phenotype. It also has anti-inflammatory and vesselsealing functions. Ang-2 is the functional antagonist of Ang-1 by binding Tie-2 without inducing receptor phosphorylation and subsequent signal transduction. As such, it is believed to act as a vessel destabilizing cytokine facilitating the functions of other cytokines such as VEGF. Ang-1 is expressed by many cell types and constitutive Ang 1/Tie 2 signaling has been described in the quiescent vasculature in vivo. In contrast, Ang-2 is almost exclusively expressed by endothelial cells and appears to act as dynamically-regulated autocrine antagonist of the Ang 1/Tie 2 interaction. In order to shed light into the autocrine antagonistic functions of Ang-2, we have studied Ang-2 synthesis and secretion in different populations of endothelial cells. We found a characteristic granular staining of endogenous and endothelial cell overexpressed Ang-2 indicating storage in cytoplasmic granules. The primary endothelial cell storage granules are Weibel-Palade bodies, which are composed of von Willebrand factor and store in addition to von Willebrand factor, P-selectin, CD63, t-PA and IL-8. Light and electron microscopic double stainings revealed Ang-2 colocalisation with von Willebrand factor, identifying Ang-2 as a novel Weibel-Palade body molecule. Interestingly, Ang-2 and P-selectin do not co-localize indicating that Ang-2 and P-selectin storage are mutually exclusive. Ang-2 could be released from Weibel-Palade bodies within 10 minutes by challenging endothelial cells with PMA, thrombin, histamine, or the calcium ionophore ionomycin. New-synthesised Ang-2 is than distributed into Weibel-Palade bodies and has a half-life of at least 18 hours. Collectively we have shown that the autocrine antagonist of Ang-1, Ang-2 is a stored and rapidly available molecule in endothelial cells, suggesting functions of the angiopoietin/Tie2 system beyond the established roles during angiogenesis likely to be involved in rapid vascular homeostatic reactions such as inflammation and coagulation.
P465 RAT MESENTERIC COLLATERAL ARTERY REMODELING IN RESPONSE TO FLOW INCLUDES PRODUCTION AND ACTIVATION OF MATRIX METALLOPROTEINASE-2. Tara L. Haas, Kevin Sheridan, Gagan Dhanota, Lia Tsotsos, Joseph L. Unthank. Dept. of Kinesiology and Health Science, York University, Toronto, ON, Dept. of Surgery, Indiana University, Indianapolis, IN. Chronic increases in flow through collateral arteries adjacent to a site of arterial occlusion result in luminal expansion with significant remodeling of both intimal and medial layers of the collateral arteries (Circ Res. 79:101523, 1996; Am J Physiol 283: H146-H155, 2002). We hypothesized that collateral remodeling would involve upregulation of matrix metalloproteinases. Sequential ileal arteries were ligated to create a collateral pathway as previously described. Two to seven days after model creation, the mesenteric vasculature was perfused with cold phosphate buffered saline, the mesentery excised and collateral and control arteries were dissected then quick frozen on dry ice. Arteries were pulverized and protein extracts were analyzed by gelatin zymography. Control arteries had basal levels of MMP2 protein (1.1 +/ 0.06), but this was increased significantly by 4 days post-occlusion (2.34 +/ 0.4, p < .05 vs. control), and remained elevated at 7 days post-occlusion (2.56 +/ 0.71). Percent active MMP-2 protein was minimal in control and 2 day arteries(2.4 +/ 1%), significantly elevated in collaterals at 4 days (21 +/ 5%, p < .05 compared to control), and diminished again at 7 days post-occlusion (10.4 +/ 2.0%, ns). These results show that MMP-2 production and activation is elevated during the most rapid phase of collateral artery expansion and suggest that this protease may contribute to the remodeling process. Funded by NSERC and NIH HL42898.
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200
pathological processes in the vascular wall, however, a larger study is necessary for clarification of these possibilities.
P463 ARTERIAL REMODELING INDUCED BY EITHER REDUCED FLOW OR INCREASED FLOW BOTH REQUIRE TROPHIC ACTION OF CATECHOLAMINES. Cauveh Erami, Hua Zhang, Tina Bleeke, Akito Tanoue, Gozoh Tsujimoto, Steven Thomas, James Faber. Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, NC, USA, Department of Molecular and Cellular Pharmacology, National Children’s Medical Research Center, Tokyo, Japan, Department of Molecular and Cellular Pharmacology, National Children’s Medical Research Center, Tokyo, Japan, Department of Pharmacology, University of Pennsylvania, Philadelphia, PA, USA. Alpha1-adrenoceptor (AR) stimulation induces proliferation, hypertrophy and migration of vascular smooth muscle cells and adventitial fibroblasts in cell culture and in organ culture of aorta and pulmonary artery. We have also shown in in vivo studies of carotid artery that this trophic effect of endogenous catecholamines is augmented by over-distension injury and contributes significantly to wall thickening and lumen loss. The trophic activity is mediated by alpha1A-ARs in rat and alpha1B-ARs in mice, but not by alpha1D-ARs that instead mediate contraction of the above arteries. The aim of this study was to determine whether catecholamines also contribute to flow-mediated arterial wall growth and remodeling. In wildtype mice or mice devoid of norepinephrine and epinephrine synthesis [dopamine beta-hydroxylase knockout (DBH-KO)], flow in the left common carotid (LC) was reduced -80% by ligation of its distal branches (except the thyroid artery); this increased flow in the right common carotid (RC) by + 80 %. Vessels were removed 3 weeks later for digital morphometry by blinded observer. Outward eutrophic remodeling of RC and hypertrophic remodeling of LC were both strongly reduced in DBH-knockouts (see table below). This was associated with inhibition ( p < 0.05) of: proliferation in RC (Ki67), apoptosis in RC and LC (cleaved caspase 3), and leukocyte accumulation in RC and LC (CD45), when intima, media and adventitia were examined 5 days after ligation. Although mean arterial pressure is reportedly unaltered in DBHKOs, as a control for this possibility we also examined alpha1D-AR-KO mice where arterial pressure is reduced by 8%. Left and right carotid remodeling were unaffected in alpha1D-AR-KOs. Since expression of alpha1D- and alpha1B-ARs, but not alpha1A-ARs, are detected in murine carotid, these data are consistent with the hypothesis that catecholamines, acting through trophic alpha1B-ARs, contribute significantly to both reduced-flow ‘‘pathological’’ hypertrophic remodeling and to ‘‘physiological’’ expansive remodeling.
P464 THE TIE2-LIGAND ANGIOPOIETIN-2 IS STORED IN AND RAPIDLY RELEASED UPON STIMULATION FROM ENDOTHELIAL CELL WEIBEL-PALADE BODIES. Ulrike Fiedler, Marion Scharpfenecker, Stefanie Koidl, Anja Hegen, Hellmut G. Augustin. Tumor Biology Center Freiburg, Dept. of Vascular Biology and Angiogenesis Research, Breisacher Str. 117, D-79106 Freiburg, Germany. The angiopoietin-Tie ligand-receptor system plays a crucial role in physiological and pathological angiogenesis by mediating vascular remodelling
and maturation processes. Ang-1 acts antiapoptotic and controls the quiescent endothelial cell phenotype. It also has anti-inflammatory and vesselsealing functions. Ang-2 is the functional antagonist of Ang-1 by binding Tie-2 without inducing receptor phosphorylation and subsequent signal transduction. As such, it is believed to act as a vessel destabilizing cytokine facilitating the functions of other cytokines such as VEGF. Ang-1 is expressed by many cell types and constitutive Ang 1/Tie 2 signaling has been described in the quiescent vasculature in vivo. In contrast, Ang-2 is almost exclusively expressed by endothelial cells and appears to act as dynamically-regulated autocrine antagonist of the Ang 1/Tie 2 interaction. In order to shed light into the autocrine antagonistic functions of Ang-2, we have studied Ang-2 synthesis and secretion in different populations of endothelial cells. We found a characteristic granular staining of endogenous and endothelial cell overexpressed Ang-2 indicating storage in cytoplasmic granules. The primary endothelial cell storage granules are Weibel-Palade bodies, which are composed of von Willebrand factor and store in addition to von Willebrand factor, P-selectin, CD63, t-PA and IL-8. Light and electron microscopic double stainings revealed Ang-2 colocalisation with von Willebrand factor, identifying Ang-2 as a novel Weibel-Palade body molecule. Interestingly, Ang-2 and P-selectin do not co-localize indicating that Ang-2 and P-selectin storage are mutually exclusive. Ang-2 could be released from Weibel-Palade bodies within 10 minutes by challenging endothelial cells with PMA, thrombin, histamine, or the calcium ionophore ionomycin. New-synthesised Ang-2 is than distributed into Weibel-Palade bodies and has a half-life of at least 18 hours. Collectively we have shown that the autocrine antagonist of Ang-1, Ang-2 is a stored and rapidly available molecule in endothelial cells, suggesting functions of the angiopoietin/Tie2 system beyond the established roles during angiogenesis likely to be involved in rapid vascular homeostatic reactions such as inflammation and coagulation.
P465 RAT MESENTERIC COLLATERAL ARTERY REMODELING IN RESPONSE TO FLOW INCLUDES PRODUCTION AND ACTIVATION OF MATRIX METALLOPROTEINASE-2. Tara L. Haas, Kevin Sheridan, Gagan Dhanota, Lia Tsotsos, Joseph L. Unthank. Dept. of Kinesiology and Health Science, York University, Toronto, ON, Dept. of Surgery, Indiana University, Indianapolis, IN. Chronic increases in flow through collateral arteries adjacent to a site of arterial occlusion result in luminal expansion with significant remodeling of both intimal and medial layers of the collateral arteries (Circ Res. 79:101523, 1996; Am J Physiol 283: H146-H155, 2002). We hypothesized that collateral remodeling would involve upregulation of matrix metalloproteinases. Sequential ileal arteries were ligated to create a collateral pathway as previously described. Two to seven days after model creation, the mesenteric vasculature was perfused with cold phosphate buffered saline, the mesentery excised and collateral and control arteries were dissected then quick frozen on dry ice. Arteries were pulverized and protein extracts were analyzed by gelatin zymography. Control arteries had basal levels of MMP2 protein (1.1 +/ 0.06), but this was increased significantly by 4 days post-occlusion (2.34 +/ 0.4, p < .05 vs. control), and remained elevated at 7 days post-occlusion (2.56 +/ 0.71). Percent active MMP-2 protein was minimal in control and 2 day arteries(2.4 +/ 1%), significantly elevated in collaterals at 4 days (21 +/ 5%, p < .05 compared to control), and diminished again at 7 days post-occlusion (10.4 +/ 2.0%, ns). These results show that MMP-2 production and activation is elevated during the most rapid phase of collateral artery expansion and suggest that this protease may contribute to the remodeling process. Funded by NSERC and NIH HL42898.