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3P-0665 Intra-individual variation of whole blood viscosity measured with scanning capillary viscometry
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Wednesday October 1, 2003: Poster Session Smooth muscle cells
circulates, thereby exerting a relatively low and turbulent shear stress on the vessel wall. Consequently, shear stress is thought to play a role in the initiation and development of atherosclerotic lesions. Recent studies have indicated that urokinase plasminogen activator (uPA) is involved in the migration and proliferation of vascular smooth muscle cells and that uPA expression is elevated in atherosclerotic human aorta. Thus, we examined whether turbulent shear stress affects the expression of uPA in cultured endothelial cells. Methods: Confluent monolayers of human coronary artery endothelial cells (HCAECs) were exposed to laminar or turbulent shear stress (1.5 dynes/cm2 ) for 1, 3, 6, 12, 24, and 48 hours, in a parallel-plate-type or cone-plate-type apparatus. Changes in the level of uPA mRNA were then determined using real-time PCR, and the amount of uPA protein released from the cells was measured using ELISA. Results: The HCAECs basally released uPA. When the HCAECs were exposed to turbulent shear stress for 1, 3, 6, 12, 24, and 48 hours, the release of uPA increased by 2-3-fold of the basal release. The uPA mRNA level began to increase after 6 hours and continued to increase with time, reaching a peak (around 2.8-fold of the control level) after 24 hours. When exposed to laminar shear stress for 12, 24, and 48 hours, uPA release decreased significantly below the basal level. The uPA mRNA levels also decreased after 6 and 12 hours, and returned to the control level after 24 and 48hors. Conclusion: Our results indicate that turbulent, but not laminar, shear stress upregulates the expression of the uPA gene and increases the release of uPA by HCAECs. Turbulent flow-induced uPA release may be involved in atherogenesis. 3P-0665
Intra-individual variation of whole blood viscosity measured with scanning capillary viscometry
A. Boss, K. Kensey, W. Hogenauer, R. Mannion. Rheologics, Inc., Exton, PA, USA Scanning capillary viscometry (SCV) makes it feasible to measure whole blood viscosity (WBV) in a clinical setting. Understanding the role elevated WBV plays in cardiovascular disease requires one to understand WBV measurement capabilities and limitations. The Rheolog™, a portable SCV device measures WBV across a wide range of blood shear rates. We evaluated 20 health males to determine diurnal variation, meal effect, and WBV variability within a 14-day study period. On full study days, viscosity was measured nine times during an 11-hour period, including before and after meals of varying nutritional content. Single, 8-hour fasting measurements were performed in the morning on four other study days. Diurnal variation: Pre-meal measurements were performed on all full study days. Mean WBV (1 sec−1 was 5.4% higher before breakfast compared to later in the day. The fasting time point also showed the largest variation, (SD = 3.47 at 1 sec−1 vs. 2.55 on average for the remainder of the day). Meal effect: At shear rates of 1, 2 and 5 sec−1 , WBV increased 1 hour post-meal compared to pre-meal values, whereas no significant change was observed at a shear rate of 300 sec−1 . However, the mean post-meal increase was small. At 1 sec−1 , the mean increase post-breakfast was 3.5%. It was not possible to discriminate the effect of meals with different lipid levels. Day to day variation: The mean fasting intra-individual SD was 3.45 at 1 sec−1 and 0.19 at 300 sec−1 . Conclusion: Day-to-day variation of WBV, particularly at shear rates of 5 sec−1 and lower, should be considered when evaluating WBV measurements. In normal subjects, there was no benefit to using fasting measurements instead of measurements taken at any other time of day, provided the measurements were performed at least 2 hours after a meal. 3P-0666
Three dimensional configuration of the aorta determines its susceptibility to aneurysm through concentrated wall shear distribution pattern
D. Mori, K. Tsubota, S. Wada, T. Yamaguchi. Dept. of Bioengineering and Robotics, Tohoku University, Sendai, Japan Our aim was to analyze the flow in various aortic arch models using computational fluid dynamics simulation in order to investigate the correlation between the aneurysms of the thoracic aorta, and the distortion of the arch from a fluid dynamical point of view. It was observed that the global feature of the secondary flow pattern, and the WSS distribution did not depend on the X, and Y angles measured by Yoshii et al. from qualitative view. The X and Y angles of Yoshii can be interpreted as parameters of the distortion of the aorta. However, the quantitative value of
the WSS was demonstrated to be apparently affected by the angles. This result suggest that the distortion of the aortic arch strongly influences the wall shear stress distribution in the site where the aneurysm preferentially develops.
SMOOTH MUSCLE CELLS 3P-0667
Mechanism of inhibitory effect of statins on vascular smooth muscle cell growth: Comparison with atorvastatin and pravastatin
M. Igarashi 1 , A. Hirata 1 , Y. Kadomoto 1 , N. Sugae 2 , H. Yamaguchi 2 , Y. Jimbu 2 , T. Kato 2 , M. Tominaga 1 . 1 Department of Laboratory Medicine, Yamagata University School of Medicine, Yamagata; 2 Third Department of Internal Medicine, Yamagata University School of Medicine, Japan The aim of this study was to characterize the mechanism(s) of inhibitory effect of statins such as atorvastatin and pravastatin on cultured rat vascular smooth muscle cell (VSMC) growth. VSMCs were harvested from the aortae of male Sprague-Dawley rats by the medial explant technique. After being starved, the cells were cultured in Dulbecco modified Eagle medium (DMEM) containing 5% fetal bovine serum (FBS) or 5 ng/ml of platelet-derived growth factor (PDGF)-BB either with various concentrations of atorvastatin (final concentrations: 1, 5, and 10 mM), or of pravastatin (final concentrations: 1, 10, and 20 mM). The measurement of DNA synthesis was measured by the addition of [3 H]-thymidine and the growth assay was performed by the method of Lowry et al. The levels of phosphorylated extracellular signal-regulated protein kinase (ERK) 1/2, and proliferative cell nuclear antigen (PCNA) were evaluated by the immunoblot analysis. Pravastatin, when solubilized with water, did not affect any change of these parameters. However, both atorvastatin and pravastatin solubilized with DMSO dose-dependently decreased the values of DNA synthesis, total cellular protein concentration and cell number, and the levels of phosphorylated ERK 1/2, PCNA. In contrast, these agents did not induce apoptosis in the condition of 5% FBS. These results indicated that both atorvastatin and pravastatin could inhibit the VSMC growth by the same mechanism(s). 3P-0669
Identification of estrogen-regulated genes in vascular smooth muscle cells
T. Watanabe 1 , M. Akishita 2 , T. Nakaoka 3 , Y. Miyahara 1 , H. Aburatani 4 , M. Yoshizumi 5 , K. Kozaki 1 , Y. Ouchi 1 . 1 Graduate School of Medicine, University of Tokyo, Tokyo; 2 Kyorin University School of Medicine; 3 Institute of Medical Science, University of Tokyo; 4 Research Center for Advanced Science and Technology, University of Tokyo; 5 Graduate School of Biomedical Science, Hiroshima University, Japan Background: Estrogen has diverse effects on the vasculature, such as vasodilation, endothelial growth, and inhibition of vascular smooth muscle cell (VSMC) proliferation and migration. However, little is known about the genes that are regulated by estrogen in the vascular wall. Methods and Results: Wistar rats were ovariectomized (OVX group) or sham-operated (Sham group), and 2 weeks after the operation, were subjected to subcutaneous implantation of placebo pellets (OVX+V group) or estradiol pellets (OVX+E group). Endothelium-denuded aortic tissue was analyzed 2 weeks after implantation. With the use of high-density oligonucleotide microarray analysis, the expression of approximately 7000 genes was analyzed. Of all the genes with different expression levels between the OVX+E group and the OVX+V group, those that have been reported to be expressed in the vasculature, were focused. Finally, four genes, caveolin-1, two LIM proteins (enigma and SmLIM) and Id3a, were identified. Microarray as well as real-time polymerase chain reaction showed that the expression levels of these genes were significantly higher in the OVX+E group than in the OVX+V group. To clarify whether estrogen directly upregulates these genes in the vascular wall, Northern blot analysis was performed using cultured rat VSMC. Addition of 100 nmol/L estradiol for 24 hours increased the mRNA levels of all four genes. Conclusions: We have identified four genes that are under the control of estrogen and possibly contribute to vascular function.
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
Wednesday October 1, 2003: Poster Session Smooth muscle cells
circulates, thereby exerting a relatively low and turbulent shear stress on the vessel wall. Consequently, shear stress is thought to play a role in the initiation and development of atherosclerotic lesions. Recent studies have indicated that urokinase plasminogen activator (uPA) is involved in the migration and proliferation of vascular smooth muscle cells and that uPA expression is elevated in atherosclerotic human aorta. Thus, we examined whether turbulent shear stress affects the expression of uPA in cultured endothelial cells. Methods: Confluent monolayers of human coronary artery endothelial cells (HCAECs) were exposed to laminar or turbulent shear stress (1.5 dynes/cm2 ) for 1, 3, 6, 12, 24, and 48 hours, in a parallel-plate-type or cone-plate-type apparatus. Changes in the level of uPA mRNA were then determined using real-time PCR, and the amount of uPA protein released from the cells was measured using ELISA. Results: The HCAECs basally released uPA. When the HCAECs were exposed to turbulent shear stress for 1, 3, 6, 12, 24, and 48 hours, the release of uPA increased by 2-3-fold of the basal release. The uPA mRNA level began to increase after 6 hours and continued to increase with time, reaching a peak (around 2.8-fold of the control level) after 24 hours. When exposed to laminar shear stress for 12, 24, and 48 hours, uPA release decreased significantly below the basal level. The uPA mRNA levels also decreased after 6 and 12 hours, and returned to the control level after 24 and 48hors. Conclusion: Our results indicate that turbulent, but not laminar, shear stress upregulates the expression of the uPA gene and increases the release of uPA by HCAECs. Turbulent flow-induced uPA release may be involved in atherogenesis. 3P-0665
Intra-individual variation of whole blood viscosity measured with scanning capillary viscometry
A. Boss, K. Kensey, W. Hogenauer, R. Mannion. Rheologics, Inc., Exton, PA, USA Scanning capillary viscometry (SCV) makes it feasible to measure whole blood viscosity (WBV) in a clinical setting. Understanding the role elevated WBV plays in cardiovascular disease requires one to understand WBV measurement capabilities and limitations. The Rheolog™, a portable SCV device measures WBV across a wide range of blood shear rates. We evaluated 20 health males to determine diurnal variation, meal effect, and WBV variability within a 14-day study period. On full study days, viscosity was measured nine times during an 11-hour period, including before and after meals of varying nutritional content. Single, 8-hour fasting measurements were performed in the morning on four other study days. Diurnal variation: Pre-meal measurements were performed on all full study days. Mean WBV (1 sec−1 was 5.4% higher before breakfast compared to later in the day. The fasting time point also showed the largest variation, (SD = 3.47 at 1 sec−1 vs. 2.55 on average for the remainder of the day). Meal effect: At shear rates of 1, 2 and 5 sec−1 , WBV increased 1 hour post-meal compared to pre-meal values, whereas no significant change was observed at a shear rate of 300 sec−1 . However, the mean post-meal increase was small. At 1 sec−1 , the mean increase post-breakfast was 3.5%. It was not possible to discriminate the effect of meals with different lipid levels. Day to day variation: The mean fasting intra-individual SD was 3.45 at 1 sec−1 and 0.19 at 300 sec−1 . Conclusion: Day-to-day variation of WBV, particularly at shear rates of 5 sec−1 and lower, should be considered when evaluating WBV measurements. In normal subjects, there was no benefit to using fasting measurements instead of measurements taken at any other time of day, provided the measurements were performed at least 2 hours after a meal. 3P-0666
Three dimensional configuration of the aorta determines its susceptibility to aneurysm through concentrated wall shear distribution pattern
D. Mori, K. Tsubota, S. Wada, T. Yamaguchi. Dept. of Bioengineering and Robotics, Tohoku University, Sendai, Japan Our aim was to analyze the flow in various aortic arch models using computational fluid dynamics simulation in order to investigate the correlation between the aneurysms of the thoracic aorta, and the distortion of the arch from a fluid dynamical point of view. It was observed that the global feature of the secondary flow pattern, and the WSS distribution did not depend on the X, and Y angles measured by Yoshii et al. from qualitative view. The X and Y angles of Yoshii can be interpreted as parameters of the distortion of the aorta. However, the quantitative value of
the WSS was demonstrated to be apparently affected by the angles. This result suggest that the distortion of the aortic arch strongly influences the wall shear stress distribution in the site where the aneurysm preferentially develops.
SMOOTH MUSCLE CELLS 3P-0667
Mechanism of inhibitory effect of statins on vascular smooth muscle cell growth: Comparison with atorvastatin and pravastatin
M. Igarashi 1 , A. Hirata 1 , Y. Kadomoto 1 , N. Sugae 2 , H. Yamaguchi 2 , Y. Jimbu 2 , T. Kato 2 , M. Tominaga 1 . 1 Department of Laboratory Medicine, Yamagata University School of Medicine, Yamagata; 2 Third Department of Internal Medicine, Yamagata University School of Medicine, Japan The aim of this study was to characterize the mechanism(s) of inhibitory effect of statins such as atorvastatin and pravastatin on cultured rat vascular smooth muscle cell (VSMC) growth. VSMCs were harvested from the aortae of male Sprague-Dawley rats by the medial explant technique. After being starved, the cells were cultured in Dulbecco modified Eagle medium (DMEM) containing 5% fetal bovine serum (FBS) or 5 ng/ml of platelet-derived growth factor (PDGF)-BB either with various concentrations of atorvastatin (final concentrations: 1, 5, and 10 mM), or of pravastatin (final concentrations: 1, 10, and 20 mM). The measurement of DNA synthesis was measured by the addition of [3 H]-thymidine and the growth assay was performed by the method of Lowry et al. The levels of phosphorylated extracellular signal-regulated protein kinase (ERK) 1/2, and proliferative cell nuclear antigen (PCNA) were evaluated by the immunoblot analysis. Pravastatin, when solubilized with water, did not affect any change of these parameters. However, both atorvastatin and pravastatin solubilized with DMSO dose-dependently decreased the values of DNA synthesis, total cellular protein concentration and cell number, and the levels of phosphorylated ERK 1/2, PCNA. In contrast, these agents did not induce apoptosis in the condition of 5% FBS. These results indicated that both atorvastatin and pravastatin could inhibit the VSMC growth by the same mechanism(s). 3P-0669
Identification of estrogen-regulated genes in vascular smooth muscle cells
T. Watanabe 1 , M. Akishita 2 , T. Nakaoka 3 , Y. Miyahara 1 , H. Aburatani 4 , M. Yoshizumi 5 , K. Kozaki 1 , Y. Ouchi 1 . 1 Graduate School of Medicine, University of Tokyo, Tokyo; 2 Kyorin University School of Medicine; 3 Institute of Medical Science, University of Tokyo; 4 Research Center for Advanced Science and Technology, University of Tokyo; 5 Graduate School of Biomedical Science, Hiroshima University, Japan Background: Estrogen has diverse effects on the vasculature, such as vasodilation, endothelial growth, and inhibition of vascular smooth muscle cell (VSMC) proliferation and migration. However, little is known about the genes that are regulated by estrogen in the vascular wall. Methods and Results: Wistar rats were ovariectomized (OVX group) or sham-operated (Sham group), and 2 weeks after the operation, were subjected to subcutaneous implantation of placebo pellets (OVX+V group) or estradiol pellets (OVX+E group). Endothelium-denuded aortic tissue was analyzed 2 weeks after implantation. With the use of high-density oligonucleotide microarray analysis, the expression of approximately 7000 genes was analyzed. Of all the genes with different expression levels between the OVX+E group and the OVX+V group, those that have been reported to be expressed in the vasculature, were focused. Finally, four genes, caveolin-1, two LIM proteins (enigma and SmLIM) and Id3a, were identified. Microarray as well as real-time polymerase chain reaction showed that the expression levels of these genes were significantly higher in the OVX+E group than in the OVX+V group. To clarify whether estrogen directly upregulates these genes in the vascular wall, Northern blot analysis was performed using cultured rat VSMC. Addition of 100 nmol/L estradiol for 24 hours increased the mRNA levels of all four genes. Conclusions: We have identified four genes that are under the control of estrogen and possibly contribute to vascular function.
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan