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OXYGEN REGULATION OF HUMAN AORTIC SMOOTH MUSCLE CELL CYCLE ASSOCIATED GENE EXPRESSION
Cardiovascular Pathology 13 (2004) S139 – S200
Poster Abstracts–Friday June 4, 2004 Hypoxia and Vessel Dysfunction P382 HYPERBARIC OXYGEN INCREASES PRODUCTION OF ANGIOGENIC GROWTH FACTORS IN ISCHEMIC HINDLIMBS OF MICE. Tetsuich Asano, Yutaka Imai, Eiji Kaneko, Yuko Mikami, Tsuyoshi Chiba, Toru Nakayama, Yoshihiro Mano, Kentaro Shimokado. Tokyo Medical and Dental University Graduate School. Background and purpose: Hyperbaric Oxygen (HBO) has been used to treat peripheral arterial disease, but mechanisms how HBO treatment improves ischemia are not clear. We studied whether HBO affects production of angiogenic factors and their receptors in ischemic hindlimbs of mice. Methods and Results: Male BALB/c mice (7 weeks) were divided into 4 groups: ischemia with or without HBO and sham operation with or without HBO. To make hindlimb ishchemia, the left femoral artery and its branches were ligated and excised. For HBO treatment, 100% oxygen was administered under 3 atm for 60 miniutes in a hyperbaric chamber designed for animals for 14 days after ligation of the femoral arteries. At various time points, mice were sacrificed, bilateral gastrocnemic muscle was excised, and the amount of mRNA for angiogenic factors and their receptors were determined by a real-time PCR. HBO improved voluntary movement of ischemic hindlimbs. Histological examination revealed that HBO increased the number of capillaries and prevented the atrophy of gastrocnemic muscle in ischemic hindlimbs. HBO increased the amount of mRNA for bFGF, FGF receptor and HGF in ischemic hindlimbs as compared to that in shamoperated hindlimbs. HBO did not affect the amount of the mRNA in animals with sham-operation. Ischemia itself minimally affected the amount of mRNA for angiogenic factors. Conclusions: These findings suggest that HBO improves the prognosis of ischemic limbs by increasing the local production of angiogenic factors.
P383 OXYGEN REGULATION OF HUMAN AORTIC SMOOTH MUSCLE CELL CYCLE ASSOCIATED GENE EXPRESSION. Julie Basu Ray, Sara Arab, Peter Liu, Michael Ward. St. Michael’s Hospital, University of Toronto, Toronto, Ontario, University Health Network, University of Toronto, Toronto, Ontario. Vascular wall hypoxia occurs following arterial injury, in atherosclerosisaffected arteries and in the hypoxic centers of malignant tumors. In these settings, oxygen regulates the expression of genes that govern vascular cell replication and death. These hypoxic responses are therefore, relevant to the physiological adaptation to changing environmental conditions and the pathophysiology of a wide spectrum of human disease. This study aims to determine the effects of hypoxia on the proliferative responses of human aortic smooth muscle cells (HASMC) in order to understand how oxygen regulates the structure and function of the systemic circulation. We found (by cell counting) that, whereas cells exposed to 3% O2 (moderate hypoxia) proliferate more rapidly than those incubated under normoxic (21% O2) conditions, cells incubated at 1% O2 (severe hypoxia) demonstrate a reduced growth rate. Microarray studies, confirmed by western 1054-8807/04/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi: 10.1016/j.carpath.2004.03.416
blotting, indicate increased expression of markers of proliferation including CDC6, MCM proteins, PCNA and MKi67, in cells exposed to 3% O2 and down regulation of these genes following incubation at 1% O2. Conversely, expression of CHK1 which promotes cell cycle arrest was markedly increased after 48 hours of incubation at 1% O2. Expression of TERF1, part of the telomerase complex and a determinant of cell survival, was at enhanced after 48 hours of hypoxic incubation at 3% O2, in contrast, to its consistent down regulation in 1% O2 further supporting the aggregation of mitotic cells during incubation at 3% O2 and growth arrested cells at 1% O2. We conclude that the differential response to these two conditions is mediated at multiple levels including alterations in the expression of genes regulating both the rate of cell cycle progression and cell survival. Insight into the molecular mechanisms that underlie these contrasting responses would aid development of pharmacological tools to manipulate smooth muscle cell proliferation, and hence vascular remodeling in disease states. Canadian Institutes of Health Research
P384 VASCULAR GENE EXPRESSION IN PATIENTS WITH CRITICAL LEG ISCHEMIA: OVEREXPRESSION OF THROMBOSPONDIN 1. Judith Favier, Ste´phane Germain, Joseph Emmerich, Pierre Corvol, Jean-Marie Gasc. INSERM U36, Colle`ge de France, Paris, France , INSERM U36, Colle`ge de France, Paris, France, Service de Me´decine Vasculaire -HTA, Hoˆpital Europe´en Georges Pompidou INSERM U428 and Centre Claude Bernard, Paris, France. Critical limb ischemia (CLI) is the late stage of peripheral arterial disease. It is characterized by severe rest pain ischemic ulcers, gangrene, and is estimated to affect 500 to 1000 individuals per million per year. Despite revascularization strategies by surgery or angioplasty, the disease often leads to lower limb amputation. Clinical trials are currently underway to induce therapeutic angiogenesis in patients suffering from CLI. However, very little is known on vascular gene expression in human affected muscles. The objective of this study was thus to better characterize gene expression governing the balance of angiogenic and angiostatic forces in human ischemic tissues. Using in situ hybridization we have studied the expression of angiogenesis-related genes in tissues from 13 amputated patients suffering from CLI. We analyzed mRNA expression of two hypoxia-inducible transcription factors HIF1alpha and HIF2alpha/EPAS1; vascular endothelial growth factor (VEGF) and both its receptors VEGFR-1 and VEGFR-2; the angiopoietin receptor Tie2; angiotensinogen and thrombospondin 1. We compared their expression levels in proximal, healthy muscles versus their distal, ischemic counterparts. Surprisingly, none of the angiogenic factors studied was noticeably differentially expressed in one or the other tissue type. In contrast,the antiangiogenic gene thrombospondin 1 appeared as a critical marker of all ischemic tissues. Quantification of thrombospondin 1 levels using real-time RT-PCR revealed a 22-fold overexpression of its mRNA and the presence of its encoded protein was confirmed by western blot. We studied the vascular density in these tissues after immunostaining endothelial cells (anti-CD34 antibody) and
Poster Abstracts–Friday June 4, 2004 Hypoxia and Vessel Dysfunction P382 HYPERBARIC OXYGEN INCREASES PRODUCTION OF ANGIOGENIC GROWTH FACTORS IN ISCHEMIC HINDLIMBS OF MICE. Tetsuich Asano, Yutaka Imai, Eiji Kaneko, Yuko Mikami, Tsuyoshi Chiba, Toru Nakayama, Yoshihiro Mano, Kentaro Shimokado. Tokyo Medical and Dental University Graduate School. Background and purpose: Hyperbaric Oxygen (HBO) has been used to treat peripheral arterial disease, but mechanisms how HBO treatment improves ischemia are not clear. We studied whether HBO affects production of angiogenic factors and their receptors in ischemic hindlimbs of mice. Methods and Results: Male BALB/c mice (7 weeks) were divided into 4 groups: ischemia with or without HBO and sham operation with or without HBO. To make hindlimb ishchemia, the left femoral artery and its branches were ligated and excised. For HBO treatment, 100% oxygen was administered under 3 atm for 60 miniutes in a hyperbaric chamber designed for animals for 14 days after ligation of the femoral arteries. At various time points, mice were sacrificed, bilateral gastrocnemic muscle was excised, and the amount of mRNA for angiogenic factors and their receptors were determined by a real-time PCR. HBO improved voluntary movement of ischemic hindlimbs. Histological examination revealed that HBO increased the number of capillaries and prevented the atrophy of gastrocnemic muscle in ischemic hindlimbs. HBO increased the amount of mRNA for bFGF, FGF receptor and HGF in ischemic hindlimbs as compared to that in shamoperated hindlimbs. HBO did not affect the amount of the mRNA in animals with sham-operation. Ischemia itself minimally affected the amount of mRNA for angiogenic factors. Conclusions: These findings suggest that HBO improves the prognosis of ischemic limbs by increasing the local production of angiogenic factors.
P383 OXYGEN REGULATION OF HUMAN AORTIC SMOOTH MUSCLE CELL CYCLE ASSOCIATED GENE EXPRESSION. Julie Basu Ray, Sara Arab, Peter Liu, Michael Ward. St. Michael’s Hospital, University of Toronto, Toronto, Ontario, University Health Network, University of Toronto, Toronto, Ontario. Vascular wall hypoxia occurs following arterial injury, in atherosclerosisaffected arteries and in the hypoxic centers of malignant tumors. In these settings, oxygen regulates the expression of genes that govern vascular cell replication and death. These hypoxic responses are therefore, relevant to the physiological adaptation to changing environmental conditions and the pathophysiology of a wide spectrum of human disease. This study aims to determine the effects of hypoxia on the proliferative responses of human aortic smooth muscle cells (HASMC) in order to understand how oxygen regulates the structure and function of the systemic circulation. We found (by cell counting) that, whereas cells exposed to 3% O2 (moderate hypoxia) proliferate more rapidly than those incubated under normoxic (21% O2) conditions, cells incubated at 1% O2 (severe hypoxia) demonstrate a reduced growth rate. Microarray studies, confirmed by western 1054-8807/04/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi: 10.1016/j.carpath.2004.03.416
blotting, indicate increased expression of markers of proliferation including CDC6, MCM proteins, PCNA and MKi67, in cells exposed to 3% O2 and down regulation of these genes following incubation at 1% O2. Conversely, expression of CHK1 which promotes cell cycle arrest was markedly increased after 48 hours of incubation at 1% O2. Expression of TERF1, part of the telomerase complex and a determinant of cell survival, was at enhanced after 48 hours of hypoxic incubation at 3% O2, in contrast, to its consistent down regulation in 1% O2 further supporting the aggregation of mitotic cells during incubation at 3% O2 and growth arrested cells at 1% O2. We conclude that the differential response to these two conditions is mediated at multiple levels including alterations in the expression of genes regulating both the rate of cell cycle progression and cell survival. Insight into the molecular mechanisms that underlie these contrasting responses would aid development of pharmacological tools to manipulate smooth muscle cell proliferation, and hence vascular remodeling in disease states. Canadian Institutes of Health Research
P384 VASCULAR GENE EXPRESSION IN PATIENTS WITH CRITICAL LEG ISCHEMIA: OVEREXPRESSION OF THROMBOSPONDIN 1. Judith Favier, Ste´phane Germain, Joseph Emmerich, Pierre Corvol, Jean-Marie Gasc. INSERM U36, Colle`ge de France, Paris, France , INSERM U36, Colle`ge de France, Paris, France, Service de Me´decine Vasculaire -HTA, Hoˆpital Europe´en Georges Pompidou INSERM U428 and Centre Claude Bernard, Paris, France. Critical limb ischemia (CLI) is the late stage of peripheral arterial disease. It is characterized by severe rest pain ischemic ulcers, gangrene, and is estimated to affect 500 to 1000 individuals per million per year. Despite revascularization strategies by surgery or angioplasty, the disease often leads to lower limb amputation. Clinical trials are currently underway to induce therapeutic angiogenesis in patients suffering from CLI. However, very little is known on vascular gene expression in human affected muscles. The objective of this study was thus to better characterize gene expression governing the balance of angiogenic and angiostatic forces in human ischemic tissues. Using in situ hybridization we have studied the expression of angiogenesis-related genes in tissues from 13 amputated patients suffering from CLI. We analyzed mRNA expression of two hypoxia-inducible transcription factors HIF1alpha and HIF2alpha/EPAS1; vascular endothelial growth factor (VEGF) and both its receptors VEGFR-1 and VEGFR-2; the angiopoietin receptor Tie2; angiotensinogen and thrombospondin 1. We compared their expression levels in proximal, healthy muscles versus their distal, ischemic counterparts. Surprisingly, none of the angiogenic factors studied was noticeably differentially expressed in one or the other tissue type. In contrast,the antiangiogenic gene thrombospondin 1 appeared as a critical marker of all ischemic tissues. Quantification of thrombospondin 1 levels using real-time RT-PCR revealed a 22-fold overexpression of its mRNA and the presence of its encoded protein was confirmed by western blot. We studied the vascular density in these tissues after immunostaining endothelial cells (anti-CD34 antibody) and