- Email: [email protected]
MOLECULAR IDENTIFICATION OF HETEROTETRAMERIC K+ CHANNELS IN HUMAN AORTIC ENDOTHELIAL CELLS: A DOMINANT CONTRIBUTION OF KIR2.1 AND KIR2.2
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200 administered under normoxemic conditions without and after precontraction of the vascular bed. Results: The integrity of vascular endothelium in the created model of an isolated perfused mouse hind limb was proven, as the acetylcholine-relaxation of the epinephrine-precontracted vascular bed [74.6 + 6.4%(x + SEM, n = 6)]. When adenosine was administered even at the lowest dosage of 106 mM a statistically significant reduction in vascular resistance could be achieved. These results were even more pronounced after precontraction with 1 mM epinephrine. The reduction of vascular resistance at the highest dosage of 10-3 mM adensoine was 87.8 + 15.1% (x + SEM, n = 6). Conclusion: The model of the isolated perfused mouse hind limb seems to be an effective model for the evaluation of different questions concerning vascular resistance. With the addition of adenosine we could achieve a significant vasodilatative effect even at the lowest dosage of 10-6 mM under normoxemic conditions.
P557 THE EVALUATION OF THE ADENOSINE RECEPTORS IN THE ISOLATED PERFUSED MOUSE HIND LIMB. Marianne Brodmann, Ulrike Lischnig, Andreas Lueger, Gerhard Stark, Ernst Pilger. Div of Angiology, Dep of Internal Medicine, University Hospital Graz, Austria, Div of Pharmacotherapy, Dep of Internal Medicine, University Hospital Graz, Austria. Purpose: Adenosine exerts its action through different receptors. The distribution of adenosine receptors is very inhomogeneous throughout the organism. The adenosine mediated peripheral vasodilatation is said to be adenosine A2 receptor mediated. The purpose of this study was to evaluate if the vasodilatative effect in the peripheral vessels is adenosine A2 receptor mediated and which receptor subgroup (A2Aor A2B) plays the important role. Methods: After equilibration with Tyrode´s solution and precontraction with epinephrine 1mM, in the first series of experiments adenosine in dosages from 10-6 to 10-3 mM was administered under normoxemic conditions. In the second series of experiments the vascular bed was precontracted once again with epinephrine 1mM and afterwards CGS 21860, an adenosine A2Areceptor agonist, in dosages from 10-8 to 10-5 mM was administered under normoxemic conditions. In the last series of experiments after precontraction with epinephrine 1mM an adenosine A2B receptor agonist, NECA, was administered in dosages of 10-8 to 10-5, too. Results: With adenosine at the highest dosage of 10-3mM the vasoconstrictive effect of epinephrine could nearly be abolished. The reduction of peripheral vascular resistance was 87.8 + 15.1% (x + SEM, n = 6). Under the addition of CGS 21680 vasodilatation was also achieved, but only in a significant way at dosages of 10-6 and 10-5, but the effect was less expressed compared to adenosine 10-3mM [47.7 + 31.0%versus 87.8 + 15.1% (x + SEM, n = 6)]. When NECA, the adenosine A2B receptor agonist was added the vasodilatative effect was nearly the same as with adenosine alone at all dosages. At the highest dosage of 10-5 mM a reduction of peripheral vascular resistance of 90.1 + 19.9% could be achieved. Conclusion: The vasodilatative effect of adenosine in peripheral vessels seems to be adenosine A2 receptor mediated, whereby the A2B receptor seems to play the key role.
S195
P559 INCREASED CONTRACTILITY OF ANG II IN THE AORTA OF CARDIOMYOPATHIC HAMSTERS IS MEDIATED BY AN INCREASED ANG II-BINDING CAPACITY AND RELEASE OF ET-1. Maria J. Crespo, Pablo I. Altieri, Nelson Escobales. Department of Physiology. University of Puerto Rico, School of Medicine, GPO Box 365067, San Juan, Puerto Rico 00936-5067, Department of Physiology. University of Puerto Rico, School of Medicine, GPO Box 365067, San Juan, Puerto Rico 00936. Heart failure (HF) is a multifactorial and progressive disease that has been associated with multiple systemic and vascular alterations. Previous reports from our laboratory showed that in 2-month-old Bio-To2 Syrian cardiomyopathic hamsters (SCH) that have not yet developed the clinical manifestations of HF, the vascular contractility induced by 0.1 microM angiotensin II (Ang. II) was approximately 44% greater than in control animals (1.3 ± 0.1g vs. 0.9 ± 0.1 g respectively, n = 9, P < .05 ). This finding was observed concomitantly with an increased aortic ACE activity. To further evaluate the mechanisms underlying Ang II-enhanced vascular contraction, concentration-response curves for Ang II (0.01 nM- 10 microM) were constructed in the presence and absence of prazosin (alpha-1 blocker), NS-398 (selective COX-2 blocker) and BQ-123 (ETAreceptor antagonist) in aortic rings from 2-month-old SCH. Age-matched golden hamsters were used as controls (CTL). The binding capacity and affinity of the AT1 receptor were also evaluated in aortic homogenates using 125I-Ang II. Our results indicate that incubation with neither 10 microM prazosin or 10 microM NS-398 modify EC50 or Emax values for Ang II indicating that norepinephrine and prostaglandins are not involved in the contractile action of Ang II. However, 10 microM BQ123 reduced by 40% the contraction induced by 1.0 microM Ang II (from 1.05 ± 0.04 to 0.6475 ± 0.06 g/mg tissue, n = 5, P < .05), suggesting that the action of Ang II is mediated in part, by ET-1 release. The ET-1 dependent contraction decreases to 29% at 0.1 microM Ang II concentration. Although dissociation constants for labeled Ang II were found to be similar in the aorta of SCH and CTL animals (Kd : CTL = 2 nM and SCH = 8 nM), 125IAng II binding capacity was about 10-fold greater in SCH than in CTL (Bmax: CTL = 140 and SCH = 1270 fmol/mg protein). Altogether these results suggest that in 2-month-old SCH the action of Ang II is mediated both, by an increased binding capacity for the hormone and ET-1 release. Supported by NIH Grant 2 SO6 GM08224 MBRS-SCORE
P560 MOLECULAR IDENTIFICATION OF HETEROTETRAMERIC K+ CHANNELS IN HUMAN AORTIC ENDOTHELIAL CELLS: A DOMINANT CONTRIBUTION OF KIR2.1 AND KIR2.2. Yun Fang, Victor Romanenko, Congzhu Shi, Gernot Schram, Stanley Nattel, Peter F. Davies, Irena Levitan. Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, USA, Montreal Heart Institute, Montreal, PQ, Canada. Numerous studies have shown that endothelial K+ channels play an important role in endothelium-dependent regulation of vascular tone. Indeed, K+ channels were shown to regulate Ca2+ signaling and NO release, and are implicated in the regulation of endothelial cell proliferation. In addition, endothelial K+ channels were shown to be one of the fastest endothelial responses to flow suggesting an active role in endothelium mechanotransduction. Recently, we have shown that endothelial K+ channels are significantly suppressed by enriching the cells with cholesterol, an effect that may play a major role in the development of hypercholesterolemia-induced endothelial injury. To investigate this hypothesis further, however, it is essential to identify the molecular nature of the endothelial K+ channels. Several complementary strategies were applied, therefore, to determine the molecular identity of K+ channels in human aortic endothelial
S196
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200
cells (HAECs). First, using RT-PCR analysis we have identified four types of inwardly-rectifying K+ channels belonging to a family of Kir2.x inward rectifiers: Kir2.1, 2.2, 2.3, and 2.4. The expression of all four channels was confirmed by sequencing. The relative transcript abundance of the different Kir2.x channels was determined using LightcyclerTM technology. The levels of 2.1, 2.2 and 2.4 mRNAs were found to be roughly similar whereas the level of Kir2.3 mRNA was significantly lower. Second, we performed functional analyses of the endothelial Kir current by testing the sensitivity of the current to the blocking effect of Ba2+ and to pH, and by characterizing the single channel basis of the current. The sensitivity of the whole cell current to Ba2+ was most similar to that of Kir2.1 whereas the contribution of Kir2.4 could be virtually excluded. We also observed that the current was not sensitive to pH, thereby, excluding significant contributions from Kir2.3 and Kir2.4 channels to the overall current. Consistent with these observations, the distribution of the unitary conductances suggested that Kir2.1 and Kir2.2 channels account for most, if not all, of the current. To probe whether the cells express heterotetrameric Kir channels, HAECs were transfected with dominant negative mutants of Kir2.1, 2.2,2.3 and 2.4 channels. The endogenous current was strongly suppressed (80-85%) by all four dominant negative mutants indicating that endogenous Kir subunits are capable of forming heterotetramers. On the basis of these observations, we suggest that endogenous Kir current in endothelial cells is formed by Kir2.1, Kir2.2 and their heterotetramers.
P561 NF-KB INHIBITION POTENTIATES ANTIPROLIFERATIVE EFFECTS OF RADIATION ON VASCULAR SMOOTH MUSCLE CELLS. Kyung-Woo Park, Hyun-Jai Cho, Seil Oh, Jin-Shik Park, Joong-Wha Chung, Young-Seok Cho, Hyo-Soo Kim, Myoung-Mook Lee, Young-Bae Park. Department of Internal Medicine, Seoul National University College of Medicine; Cardiovascular Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea. Introduction: NF-kB promotes cell survival against external stress such as radiation. We examined whether NF-kB inhibition enhances the antiproliferative effects of radiation on vascular smooth muscle cells (VSMCs). Method and results: To evaluate the effects of NF-kB inhibition, NF-kB decoy oligonucleotides were transfected to VSMCs. The extent of cellular viability and proliferation was determined by MTT assay after gamma-ray irradiation using 137-Cs. Irradiation induced activation or nuclear translocation of NF-kB p65 in vascular smooth muscle cell which was significantly blocked by NF-kB decoy transfection. RT-PCR and western blotting showed reduced transcription and translation of ICAM, iNOS, and TNF-a, confirming the inactivation of NF-kB. NF-kB decoy potentiated antiproliferative effects of radiation so that the effect of 2 and 8-Gy radiation combined with NF-kB decoy transfection (48 hours after irradiation: 1.43 ± 0.21 and 1.50 ± 0.31, respectively) was similar to that of 16Gy radiation without NF-kB inhibition (1.63 ± 0.35). In addition, apoptosis was increased after NF-kB decoy transfection in irradiated VSMCs (apoptosis fraction:13.33 ± 2.08% vs. 26.29 ± 7.43%, for radiation only vs. radiation + NF-kB decoy transfection, p < .05). However, NF-kB inhibition did not show any additive effects on the cell cycle of irradiated VSMCs as measured by flow cytometry analysis. Conclusion: NF-kB inhibition reduces proliferation and survival of irradiated VSMCs and this seems to be through increased apoptosis rather than additive cell cycle arrest. Our data suggest the possibility of adjunctive gene therapy using NF-kB decoy to improve efficacy and to decrease the adverse effects of intracoronary radiation therapy.
P562 INFLUENCE OF ANGIOTENSIN II TYPE 1 RECEPTOR SIGNAL BLOCKADE ON THE BLOOD VESSEL PERMEABILITY IN MICE. Sano Hideto, Takakura Nobuyuki. Department of Stem Cell Biology, Cancer Research Institute, Kanazawa University, Kanazawa, Japan. It is known that the vascular endothelial growth factor (VEGF) is a major factor, which promotes blood vessel formation and vascular permeability. The promotion of blood vessel permeability by VEGF is one of the main causes of blood edema. Death from ischemic state or dyspnea is caused by the constriction of a blood vessel or a respiratory tract promoted by blood vessel edema. It is also known that the signals from Angiotensin (Ang) II-Ang II receptor system are involved in tissue edema. Angiotensin Converting Enzyme (ACE) transforms Ang I into Ang II. Blood pressure is adjusted through an Ang II mediated seventh membrane receptor. ACE inhibitors, Ang II type 1 receptor (ATR) antagonists are used as medical drugs, which controls Ang II-mediated hypertension. However, it was reported that ACE inhibitor has the side effects that induce blood vessel edema because of concernment from bradykinin. We considered the relation of the blood vessel permeability and the signals controlled by an ATR and VEGF receptor. In miles assay of a mouse, ATR blocker (Candesartan) markedly inhibited the VEGF-mediated blood vessel permeability. Moreover, in order to consider the relation between the blood vessel permeability of VEGF and an ATR in detail, miles assay was performed using the Ang II type 1a receptor (AT1a) deficient mice. Although expectedly the blood vessel permeability induced by VEGF in AT1a deficient mice were reduced compared with that in wild type mice, the blood vessel leakage by inflammation, which were promoted by mustard oil, were not altered. These results suggested that ATR blockade specifically inhibits the blood vessel permeability induced by VEGF. In order to clarify the mechanism, we are trying to analyze protein phosphorylation in the downstream of VEGF receptor and ATR, intracellular Ca2+ uptake, endothelial nitric oxide (NO) synthase activity and so on. NO is one of a pleiotropic mediator involved in the regulation of vascular tone including vascular relaxation and leakage. Interaction with Ca2 + calmodulin binding in endothelial cells needs to NO synthase activity. It is possible that the vascular permeability induced by NO through the VEGF signal pathway might be specifically inhibited by blocking of ATR cascade. By proving this hypothesis, antagonist of ATR might be utilized for VEGF gene therapy in which the edema is a side effect and for the drug delivery system to the cancer.
P563 ROLE OF ENOS IN MATERNAL CARDIOVASCULAR CHANGES DURING PREGNANCY. Shathiyah Kulandavelu, Dawei Qu, S. Lee Adamson. Samuel Lunenfeld Research Institute, Toronto, Ontario. Nitric oxide (NO) is believed to contribute to the marked systemic vasodilation of pregnancy leading to marked increases in cardiac output (CO), plasma volume (PV) and a decrease in blood pressure (BP). The mechanisms involved in mediating these changes are not fully understood, but NO has been shown to be increased in normal pregnancy and lack of NO has been implicated in pregnancy-induced hypertension. We hypothesized that vasodilation in mice lacking the gene encoding for eNOS will be blunted leading to a blunting of these other cardiovascular responses to pregnancy. Control C57B6 and eNOS / mice were studied before pregnancy and in late gestation (17.5d). CO was calculated from mean velocity and ascending aortic diameter (C57 n = 8; eNOS / n = 12), and left ventricular chamber dimensions (C57 n = 4; eNOS / n = 3) were measured in isofluraneanesthetized mice using ultrasound. PV (by dye dilution) and BP (by tailcuff) were measured in awake mice (C57 n = 6; eNOS / n = 6). In controls, CO increased 48% by 17.5d of gestation ( p < .001) due to a 41% increase in stroke volume (SV) ( p < .001). HR did not change
P557 THE EVALUATION OF THE ADENOSINE RECEPTORS IN THE ISOLATED PERFUSED MOUSE HIND LIMB. Marianne Brodmann, Ulrike Lischnig, Andreas Lueger, Gerhard Stark, Ernst Pilger. Div of Angiology, Dep of Internal Medicine, University Hospital Graz, Austria, Div of Pharmacotherapy, Dep of Internal Medicine, University Hospital Graz, Austria. Purpose: Adenosine exerts its action through different receptors. The distribution of adenosine receptors is very inhomogeneous throughout the organism. The adenosine mediated peripheral vasodilatation is said to be adenosine A2 receptor mediated. The purpose of this study was to evaluate if the vasodilatative effect in the peripheral vessels is adenosine A2 receptor mediated and which receptor subgroup (A2Aor A2B) plays the important role. Methods: After equilibration with Tyrode´s solution and precontraction with epinephrine 1mM, in the first series of experiments adenosine in dosages from 10-6 to 10-3 mM was administered under normoxemic conditions. In the second series of experiments the vascular bed was precontracted once again with epinephrine 1mM and afterwards CGS 21860, an adenosine A2Areceptor agonist, in dosages from 10-8 to 10-5 mM was administered under normoxemic conditions. In the last series of experiments after precontraction with epinephrine 1mM an adenosine A2B receptor agonist, NECA, was administered in dosages of 10-8 to 10-5, too. Results: With adenosine at the highest dosage of 10-3mM the vasoconstrictive effect of epinephrine could nearly be abolished. The reduction of peripheral vascular resistance was 87.8 + 15.1% (x + SEM, n = 6). Under the addition of CGS 21680 vasodilatation was also achieved, but only in a significant way at dosages of 10-6 and 10-5, but the effect was less expressed compared to adenosine 10-3mM [47.7 + 31.0%versus 87.8 + 15.1% (x + SEM, n = 6)]. When NECA, the adenosine A2B receptor agonist was added the vasodilatative effect was nearly the same as with adenosine alone at all dosages. At the highest dosage of 10-5 mM a reduction of peripheral vascular resistance of 90.1 + 19.9% could be achieved. Conclusion: The vasodilatative effect of adenosine in peripheral vessels seems to be adenosine A2 receptor mediated, whereby the A2B receptor seems to play the key role.
S195
P559 INCREASED CONTRACTILITY OF ANG II IN THE AORTA OF CARDIOMYOPATHIC HAMSTERS IS MEDIATED BY AN INCREASED ANG II-BINDING CAPACITY AND RELEASE OF ET-1. Maria J. Crespo, Pablo I. Altieri, Nelson Escobales. Department of Physiology. University of Puerto Rico, School of Medicine, GPO Box 365067, San Juan, Puerto Rico 00936-5067, Department of Physiology. University of Puerto Rico, School of Medicine, GPO Box 365067, San Juan, Puerto Rico 00936. Heart failure (HF) is a multifactorial and progressive disease that has been associated with multiple systemic and vascular alterations. Previous reports from our laboratory showed that in 2-month-old Bio-To2 Syrian cardiomyopathic hamsters (SCH) that have not yet developed the clinical manifestations of HF, the vascular contractility induced by 0.1 microM angiotensin II (Ang. II) was approximately 44% greater than in control animals (1.3 ± 0.1g vs. 0.9 ± 0.1 g respectively, n = 9, P < .05 ). This finding was observed concomitantly with an increased aortic ACE activity. To further evaluate the mechanisms underlying Ang II-enhanced vascular contraction, concentration-response curves for Ang II (0.01 nM- 10 microM) were constructed in the presence and absence of prazosin (alpha-1 blocker), NS-398 (selective COX-2 blocker) and BQ-123 (ETAreceptor antagonist) in aortic rings from 2-month-old SCH. Age-matched golden hamsters were used as controls (CTL). The binding capacity and affinity of the AT1 receptor were also evaluated in aortic homogenates using 125I-Ang II. Our results indicate that incubation with neither 10 microM prazosin or 10 microM NS-398 modify EC50 or Emax values for Ang II indicating that norepinephrine and prostaglandins are not involved in the contractile action of Ang II. However, 10 microM BQ123 reduced by 40% the contraction induced by 1.0 microM Ang II (from 1.05 ± 0.04 to 0.6475 ± 0.06 g/mg tissue, n = 5, P < .05), suggesting that the action of Ang II is mediated in part, by ET-1 release. The ET-1 dependent contraction decreases to 29% at 0.1 microM Ang II concentration. Although dissociation constants for labeled Ang II were found to be similar in the aorta of SCH and CTL animals (Kd : CTL = 2 nM and SCH = 8 nM), 125IAng II binding capacity was about 10-fold greater in SCH than in CTL (Bmax: CTL = 140 and SCH = 1270 fmol/mg protein). Altogether these results suggest that in 2-month-old SCH the action of Ang II is mediated both, by an increased binding capacity for the hormone and ET-1 release. Supported by NIH Grant 2 SO6 GM08224 MBRS-SCORE
P560 MOLECULAR IDENTIFICATION OF HETEROTETRAMERIC K+ CHANNELS IN HUMAN AORTIC ENDOTHELIAL CELLS: A DOMINANT CONTRIBUTION OF KIR2.1 AND KIR2.2. Yun Fang, Victor Romanenko, Congzhu Shi, Gernot Schram, Stanley Nattel, Peter F. Davies, Irena Levitan. Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, USA, Montreal Heart Institute, Montreal, PQ, Canada. Numerous studies have shown that endothelial K+ channels play an important role in endothelium-dependent regulation of vascular tone. Indeed, K+ channels were shown to regulate Ca2+ signaling and NO release, and are implicated in the regulation of endothelial cell proliferation. In addition, endothelial K+ channels were shown to be one of the fastest endothelial responses to flow suggesting an active role in endothelium mechanotransduction. Recently, we have shown that endothelial K+ channels are significantly suppressed by enriching the cells with cholesterol, an effect that may play a major role in the development of hypercholesterolemia-induced endothelial injury. To investigate this hypothesis further, however, it is essential to identify the molecular nature of the endothelial K+ channels. Several complementary strategies were applied, therefore, to determine the molecular identity of K+ channels in human aortic endothelial
S196
Poster Abstracts / Cardiovascular Pathology 13 (2004) S139–S200
cells (HAECs). First, using RT-PCR analysis we have identified four types of inwardly-rectifying K+ channels belonging to a family of Kir2.x inward rectifiers: Kir2.1, 2.2, 2.3, and 2.4. The expression of all four channels was confirmed by sequencing. The relative transcript abundance of the different Kir2.x channels was determined using LightcyclerTM technology. The levels of 2.1, 2.2 and 2.4 mRNAs were found to be roughly similar whereas the level of Kir2.3 mRNA was significantly lower. Second, we performed functional analyses of the endothelial Kir current by testing the sensitivity of the current to the blocking effect of Ba2+ and to pH, and by characterizing the single channel basis of the current. The sensitivity of the whole cell current to Ba2+ was most similar to that of Kir2.1 whereas the contribution of Kir2.4 could be virtually excluded. We also observed that the current was not sensitive to pH, thereby, excluding significant contributions from Kir2.3 and Kir2.4 channels to the overall current. Consistent with these observations, the distribution of the unitary conductances suggested that Kir2.1 and Kir2.2 channels account for most, if not all, of the current. To probe whether the cells express heterotetrameric Kir channels, HAECs were transfected with dominant negative mutants of Kir2.1, 2.2,2.3 and 2.4 channels. The endogenous current was strongly suppressed (80-85%) by all four dominant negative mutants indicating that endogenous Kir subunits are capable of forming heterotetramers. On the basis of these observations, we suggest that endogenous Kir current in endothelial cells is formed by Kir2.1, Kir2.2 and their heterotetramers.
P561 NF-KB INHIBITION POTENTIATES ANTIPROLIFERATIVE EFFECTS OF RADIATION ON VASCULAR SMOOTH MUSCLE CELLS. Kyung-Woo Park, Hyun-Jai Cho, Seil Oh, Jin-Shik Park, Joong-Wha Chung, Young-Seok Cho, Hyo-Soo Kim, Myoung-Mook Lee, Young-Bae Park. Department of Internal Medicine, Seoul National University College of Medicine; Cardiovascular Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea. Introduction: NF-kB promotes cell survival against external stress such as radiation. We examined whether NF-kB inhibition enhances the antiproliferative effects of radiation on vascular smooth muscle cells (VSMCs). Method and results: To evaluate the effects of NF-kB inhibition, NF-kB decoy oligonucleotides were transfected to VSMCs. The extent of cellular viability and proliferation was determined by MTT assay after gamma-ray irradiation using 137-Cs. Irradiation induced activation or nuclear translocation of NF-kB p65 in vascular smooth muscle cell which was significantly blocked by NF-kB decoy transfection. RT-PCR and western blotting showed reduced transcription and translation of ICAM, iNOS, and TNF-a, confirming the inactivation of NF-kB. NF-kB decoy potentiated antiproliferative effects of radiation so that the effect of 2 and 8-Gy radiation combined with NF-kB decoy transfection (48 hours after irradiation: 1.43 ± 0.21 and 1.50 ± 0.31, respectively) was similar to that of 16Gy radiation without NF-kB inhibition (1.63 ± 0.35). In addition, apoptosis was increased after NF-kB decoy transfection in irradiated VSMCs (apoptosis fraction:13.33 ± 2.08% vs. 26.29 ± 7.43%, for radiation only vs. radiation + NF-kB decoy transfection, p < .05). However, NF-kB inhibition did not show any additive effects on the cell cycle of irradiated VSMCs as measured by flow cytometry analysis. Conclusion: NF-kB inhibition reduces proliferation and survival of irradiated VSMCs and this seems to be through increased apoptosis rather than additive cell cycle arrest. Our data suggest the possibility of adjunctive gene therapy using NF-kB decoy to improve efficacy and to decrease the adverse effects of intracoronary radiation therapy.
P562 INFLUENCE OF ANGIOTENSIN II TYPE 1 RECEPTOR SIGNAL BLOCKADE ON THE BLOOD VESSEL PERMEABILITY IN MICE. Sano Hideto, Takakura Nobuyuki. Department of Stem Cell Biology, Cancer Research Institute, Kanazawa University, Kanazawa, Japan. It is known that the vascular endothelial growth factor (VEGF) is a major factor, which promotes blood vessel formation and vascular permeability. The promotion of blood vessel permeability by VEGF is one of the main causes of blood edema. Death from ischemic state or dyspnea is caused by the constriction of a blood vessel or a respiratory tract promoted by blood vessel edema. It is also known that the signals from Angiotensin (Ang) II-Ang II receptor system are involved in tissue edema. Angiotensin Converting Enzyme (ACE) transforms Ang I into Ang II. Blood pressure is adjusted through an Ang II mediated seventh membrane receptor. ACE inhibitors, Ang II type 1 receptor (ATR) antagonists are used as medical drugs, which controls Ang II-mediated hypertension. However, it was reported that ACE inhibitor has the side effects that induce blood vessel edema because of concernment from bradykinin. We considered the relation of the blood vessel permeability and the signals controlled by an ATR and VEGF receptor. In miles assay of a mouse, ATR blocker (Candesartan) markedly inhibited the VEGF-mediated blood vessel permeability. Moreover, in order to consider the relation between the blood vessel permeability of VEGF and an ATR in detail, miles assay was performed using the Ang II type 1a receptor (AT1a) deficient mice. Although expectedly the blood vessel permeability induced by VEGF in AT1a deficient mice were reduced compared with that in wild type mice, the blood vessel leakage by inflammation, which were promoted by mustard oil, were not altered. These results suggested that ATR blockade specifically inhibits the blood vessel permeability induced by VEGF. In order to clarify the mechanism, we are trying to analyze protein phosphorylation in the downstream of VEGF receptor and ATR, intracellular Ca2+ uptake, endothelial nitric oxide (NO) synthase activity and so on. NO is one of a pleiotropic mediator involved in the regulation of vascular tone including vascular relaxation and leakage. Interaction with Ca2 + calmodulin binding in endothelial cells needs to NO synthase activity. It is possible that the vascular permeability induced by NO through the VEGF signal pathway might be specifically inhibited by blocking of ATR cascade. By proving this hypothesis, antagonist of ATR might be utilized for VEGF gene therapy in which the edema is a side effect and for the drug delivery system to the cancer.
P563 ROLE OF ENOS IN MATERNAL CARDIOVASCULAR CHANGES DURING PREGNANCY. Shathiyah Kulandavelu, Dawei Qu, S. Lee Adamson. Samuel Lunenfeld Research Institute, Toronto, Ontario. Nitric oxide (NO) is believed to contribute to the marked systemic vasodilation of pregnancy leading to marked increases in cardiac output (CO), plasma volume (PV) and a decrease in blood pressure (BP). The mechanisms involved in mediating these changes are not fully understood, but NO has been shown to be increased in normal pregnancy and lack of NO has been implicated in pregnancy-induced hypertension. We hypothesized that vasodilation in mice lacking the gene encoding for eNOS will be blunted leading to a blunting of these other cardiovascular responses to pregnancy. Control C57B6 and eNOS / mice were studied before pregnancy and in late gestation (17.5d). CO was calculated from mean velocity and ascending aortic diameter (C57 n = 8; eNOS / n = 12), and left ventricular chamber dimensions (C57 n = 4; eNOS / n = 3) were measured in isofluraneanesthetized mice using ultrasound. PV (by dye dilution) and BP (by tailcuff) were measured in awake mice (C57 n = 6; eNOS / n = 6). In controls, CO increased 48% by 17.5d of gestation ( p < .001) due to a 41% increase in stroke volume (SV) ( p < .001). HR did not change