- Email: [email protected]
3P-0644 New 3-hydroxy-3 methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitor, pitavastatin has biphasic effect on angiogenesis
Wednesday October 1, 2003: Poster Session Vascular biology vessels (p<0.0001, One-factor ANOVA). There was no significant relationship between VEGF-D expression and lesion types or the number of blood vessels. Conclusion: These data suggest that VEGF-C produced by foamy macrophages may act as an endogenous regulator for neovascularization, including the both lymphatic and vascular vessels, in the atherosclerotic intima, which may play an important role in coronary plaque progression. 3P-0643
Pulsatile pressure and shear stress regulate the differentiation of marrow stromal cells into smooth muscle cells
T. Yasu 1 , N. Kobayashi 1 , H. Ueba 1 , M. Sata 2 , S. Hashimoto 1 , M. Saito 1 , M. Kuroki 1 , M. Kawakami 1 . 1 Omiya Medical Center, Jichi Medical School, Amanuma, Saitama; 2 Tokyo University, Japan Aim: Smooth-muscle-cell (SMC) precursors derived from marrow stromal cells have attracted an increasing attention as a contributing factor to neointimal formation in injured arteries. The present study investigates effects of pulsatile pressure and shear stress on the differentiation of marrow stromal cells into SMC populations. Methods: Marrow stromal cells were isolated from rat bone marrow by adherent cell culture and density-gradient separation. After 7, 14, and 21 days of preceding static culture, marrow stromal cells were exposed to three kinds of mechanical forces induced by pulsatile fluid flow over 24 hr: 1) flow dominance [0.6/1.2 L/min, 10 mmHg]; 2) pulsatile pressure dominance [0.1 L/min, 120/60 mmHg]; and 3) the combination of pulsatile flow and pressure [0.6/1.2 L/min, 120/60 mmHg]. We examined the change of cell orientation and the expression of smooth-muscle myosin heavy chain (SM-MHC) in static control and three kinds of stress-exposed cells using immunofluorescent staining and Western blot analysis. Results: In immunofluorescence study, the incidence of SM-MHC positive cells was higher in the pressure-dominance and the combined stresses than that of control at 14(P<0.05) and 21 days of prior culture (P<0.01). Relative SM-MHC induction in Western blot analysis was: 29.0 ± 1.4 (mean ± SE) in flow-dominant(P<0.05), 42.0 ± 7.4 in pressure-dominant (P<0.01), and 44.0 ± 11.0 in the combined stresses (P<0.01)compared to the control at 14 days of preceding culture. Conclusion: The present study demonstrates that pulsatile fluid-flow and hydro-pressure promote the expression of SM-MHC on marrow stromal cells. These findings are concordant with the thesis that marrow-derived mesenchymal progenitors adhere to and differentiate into SMC populations in the surface of injured arteries. 3P-0644
New 3-hydroxy-3 methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitor, pitavastatin has biphasic effect on angiogenesis
Background and Purpose: 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor (statin) reduces serum cholesterol level strongly and prevents coronary events. In addition to the cholesterol lowering effect, the direct effects of statins to vascular wall, including angiogenesis, were demonstrated by the recent studies. In the present study, we initially investigated the effect of pitavastatin, which is a new member of statins and has stronger cholesterol lowering effect than conventional statins, on proliferation, migration and viability of endothelial cells, and then on angiogenesis. Method: Migration of adult human epidermal microvessel endothelial cells (HMVECs) were examined by scratch wound assay and chemotaxis chamber. Cell proliferation was estimated by metabolic activity (WST-1). Cell viability was evaluated by trypan blue dye exclusion test. The effect of pitavastatin on angiogenesis was examined by quail chorioallantoic membrane (CAM) assay. Results: High concentration (>0.1 µmol/l) of pitavastatin dose-dependently inhibited bFGF-induced migration and proliferation of HMVECs, and increased trypan blue positive cells. In contrast, pitavastatin at low concentration (<0.01 µmol/l) did not affect HMVEC proliferation and migration, but significantly reduced trypan blue positive cells. High dose of pitavastatin (0.5 µmol/embryo) significantly reduced (-25.9% versus bFGF only) angiogenesis. In contrast, low dose of pitavastatin (0.15 µmol/embryo) tended to increase bFGF-induced angiogenesis (+11.9% versus bFGF only). Conclusions: Pitavastatin has biphasic effect on endothelial cells and angiogenesis. These results suggest that pitavastatin could be applied for angiogenic diseases such as malignancy and inflammation at high dose, and
that low dose of pitavastatin may be available as therapeutic drug for ischemic diseases such as arteriosclerosis obliterance. 3P-0645
Direct evidence of angiopoietin-1 released from pericytes upon hypoxia on the induction of endothelial cell migration and tube formation
Y. Park, I. Jo. Division of Cardiovascular Research, Department of Biomedical Sciences, National Institute of Health, Seoul, Republic of Korea The angiopoietin/Tie2 system has been known to plays a critical role in angiogenesis by controlling several signaling pathways involved in cell migration, proliferation and survival. Furthermore, this angiogenic process appears to be regulated by co-ordinated actions of the endothelial cells (EC) with the pericytes around the endothelial cells. Previously, we reported that hypoxia, well-known angiogenic factor, rapidly up-regulated angiopoietin-1 (Ang-1) mRNA, but not Ang-2, from bovine retinal pericytes (BRP) (Microvascular Res. 65:125-131, 2003). In this study, we investigated the potential role of Ang-1 released from BRP upon hypoxia on bovine aortic EC (BAEC). BRP were incubated for indicated times in either normoxic (NCM) or hypoxic conditioned medium (HCM). After incubation, each medium was collected and added into BAEC to examine their migration and tube formation. Ang-1 protein in hypoxic pericyte was detected by western blot analysis using Ang-1 antibody. Ang-1 protein was significantly up-regulated by hypoxia in a timedependent manner, while only basal and constant level of Ang-1 proteinwas found in normoxic pericyte. EC migration was significnatly observed to occur using HCM in a dose-dependent manner and the maximal induction was found to be at 20% HCM. Concurrently, BAEC tube formation on Matrigel was also significantly induced by 20% HCM. Both BAEC migration and tube formation were completely blocked by anti Ang-1 antibody. These results, together with our more findings of partial attenuation of HCM-induced EC migration and tube formation by antibodies against Ang-2 and Tie2, suggest a critical role of Ang-1 released from hypoxic BRP on EC migration and tube formation. In conclusion, our data suggest that hypoxia induces Ang-1 secretion from pericyte in a paracrine manner, which in turn may stimulates the migration and tube formation of EC, Ang-1 deficient cells. 3P-0646
Smoking cessation rapidly increases endothelial progenitor cells (EPCs) in peripheral blood in chronic smokers
T. Kondo, M. Hayashi, K. Kinoshita, K. Takeshita, K. Kobayashi, N. Inoue, T. Murohara. Department of Cardiology, Nagoya University Graduate School of Medicine, Japan Objective: Endothelial progenitor cells (EPCs) circulate in adult peripheral blood and contribute to neovascularization and collateral formation. Smoking is a strong risk factor for cardiovascular disease and thromboantiitis obliterans (Buerger’s disease). Reduced EPCs may have been involved in the process of these diseases. However, it is not clear that smoking cessation restores the level of circulating EPCs. Methos and Results: In this study we examined the EPCs level using flow cytometry by counting CD34-positeive cells, AC133-positive cells, and CD34/AC133-double positive cells. The EPCs level was tend to be lower in smokers (N=15) than in non-smokers (N=9) (p=0.101 for CD34(+), p=0.022 for AC133(+), p=0.060 for CD34(+)AC133(+) cells respectively). Smokers quitted smoking with nicotine patch (N=8) or without nicotine patch (N=7). After 1 month, all ex-smokers resume smoking. The EPCs level rapidly increased after smoking cessation (p=0.0012 for CD34(+), p=0.007 for AC133(+), p=0.008 for CD34(+)AC133(+) cells respectively) and decreased when they resumed smoking (p=0.0106 for CD34(+), p=0.0171 for AC133(+), p=0.0031 for CD34(+)AC133(+) cells respectively). The degree of EPCs change didn’t differ between the subjects with or without nicotine patch. Conclusion: The EPCs level was lower in chronic smokers than nonsmokers. Short term smoking cessation (1 month) rapidly and significantly increased the level of EPCs and re-start smoking significantly decrease EPCs. The possible mechanisms by which smoking impair the EPCs level in peripheral blood may be the decreased EPCs production form bone marrow or increased degradation of EPCs.
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
WEDNESDAY
M. Katsumoto, T. Shingu, R. Kuwashima, S. Nomura, Y. Umeda, K. Chayama. Department of Medicine and Molecular Science Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan
203
Pulsatile pressure and shear stress regulate the differentiation of marrow stromal cells into smooth muscle cells
T. Yasu 1 , N. Kobayashi 1 , H. Ueba 1 , M. Sata 2 , S. Hashimoto 1 , M. Saito 1 , M. Kuroki 1 , M. Kawakami 1 . 1 Omiya Medical Center, Jichi Medical School, Amanuma, Saitama; 2 Tokyo University, Japan Aim: Smooth-muscle-cell (SMC) precursors derived from marrow stromal cells have attracted an increasing attention as a contributing factor to neointimal formation in injured arteries. The present study investigates effects of pulsatile pressure and shear stress on the differentiation of marrow stromal cells into SMC populations. Methods: Marrow stromal cells were isolated from rat bone marrow by adherent cell culture and density-gradient separation. After 7, 14, and 21 days of preceding static culture, marrow stromal cells were exposed to three kinds of mechanical forces induced by pulsatile fluid flow over 24 hr: 1) flow dominance [0.6/1.2 L/min, 10 mmHg]; 2) pulsatile pressure dominance [0.1 L/min, 120/60 mmHg]; and 3) the combination of pulsatile flow and pressure [0.6/1.2 L/min, 120/60 mmHg]. We examined the change of cell orientation and the expression of smooth-muscle myosin heavy chain (SM-MHC) in static control and three kinds of stress-exposed cells using immunofluorescent staining and Western blot analysis. Results: In immunofluorescence study, the incidence of SM-MHC positive cells was higher in the pressure-dominance and the combined stresses than that of control at 14(P<0.05) and 21 days of prior culture (P<0.01). Relative SM-MHC induction in Western blot analysis was: 29.0 ± 1.4 (mean ± SE) in flow-dominant(P<0.05), 42.0 ± 7.4 in pressure-dominant (P<0.01), and 44.0 ± 11.0 in the combined stresses (P<0.01)compared to the control at 14 days of preceding culture. Conclusion: The present study demonstrates that pulsatile fluid-flow and hydro-pressure promote the expression of SM-MHC on marrow stromal cells. These findings are concordant with the thesis that marrow-derived mesenchymal progenitors adhere to and differentiate into SMC populations in the surface of injured arteries. 3P-0644
New 3-hydroxy-3 methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitor, pitavastatin has biphasic effect on angiogenesis
Background and Purpose: 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor (statin) reduces serum cholesterol level strongly and prevents coronary events. In addition to the cholesterol lowering effect, the direct effects of statins to vascular wall, including angiogenesis, were demonstrated by the recent studies. In the present study, we initially investigated the effect of pitavastatin, which is a new member of statins and has stronger cholesterol lowering effect than conventional statins, on proliferation, migration and viability of endothelial cells, and then on angiogenesis. Method: Migration of adult human epidermal microvessel endothelial cells (HMVECs) were examined by scratch wound assay and chemotaxis chamber. Cell proliferation was estimated by metabolic activity (WST-1). Cell viability was evaluated by trypan blue dye exclusion test. The effect of pitavastatin on angiogenesis was examined by quail chorioallantoic membrane (CAM) assay. Results: High concentration (>0.1 µmol/l) of pitavastatin dose-dependently inhibited bFGF-induced migration and proliferation of HMVECs, and increased trypan blue positive cells. In contrast, pitavastatin at low concentration (<0.01 µmol/l) did not affect HMVEC proliferation and migration, but significantly reduced trypan blue positive cells. High dose of pitavastatin (0.5 µmol/embryo) significantly reduced (-25.9% versus bFGF only) angiogenesis. In contrast, low dose of pitavastatin (0.15 µmol/embryo) tended to increase bFGF-induced angiogenesis (+11.9% versus bFGF only). Conclusions: Pitavastatin has biphasic effect on endothelial cells and angiogenesis. These results suggest that pitavastatin could be applied for angiogenic diseases such as malignancy and inflammation at high dose, and
that low dose of pitavastatin may be available as therapeutic drug for ischemic diseases such as arteriosclerosis obliterance. 3P-0645
Direct evidence of angiopoietin-1 released from pericytes upon hypoxia on the induction of endothelial cell migration and tube formation
Y. Park, I. Jo. Division of Cardiovascular Research, Department of Biomedical Sciences, National Institute of Health, Seoul, Republic of Korea The angiopoietin/Tie2 system has been known to plays a critical role in angiogenesis by controlling several signaling pathways involved in cell migration, proliferation and survival. Furthermore, this angiogenic process appears to be regulated by co-ordinated actions of the endothelial cells (EC) with the pericytes around the endothelial cells. Previously, we reported that hypoxia, well-known angiogenic factor, rapidly up-regulated angiopoietin-1 (Ang-1) mRNA, but not Ang-2, from bovine retinal pericytes (BRP) (Microvascular Res. 65:125-131, 2003). In this study, we investigated the potential role of Ang-1 released from BRP upon hypoxia on bovine aortic EC (BAEC). BRP were incubated for indicated times in either normoxic (NCM) or hypoxic conditioned medium (HCM). After incubation, each medium was collected and added into BAEC to examine their migration and tube formation. Ang-1 protein in hypoxic pericyte was detected by western blot analysis using Ang-1 antibody. Ang-1 protein was significantly up-regulated by hypoxia in a timedependent manner, while only basal and constant level of Ang-1 proteinwas found in normoxic pericyte. EC migration was significnatly observed to occur using HCM in a dose-dependent manner and the maximal induction was found to be at 20% HCM. Concurrently, BAEC tube formation on Matrigel was also significantly induced by 20% HCM. Both BAEC migration and tube formation were completely blocked by anti Ang-1 antibody. These results, together with our more findings of partial attenuation of HCM-induced EC migration and tube formation by antibodies against Ang-2 and Tie2, suggest a critical role of Ang-1 released from hypoxic BRP on EC migration and tube formation. In conclusion, our data suggest that hypoxia induces Ang-1 secretion from pericyte in a paracrine manner, which in turn may stimulates the migration and tube formation of EC, Ang-1 deficient cells. 3P-0646
Smoking cessation rapidly increases endothelial progenitor cells (EPCs) in peripheral blood in chronic smokers
T. Kondo, M. Hayashi, K. Kinoshita, K. Takeshita, K. Kobayashi, N. Inoue, T. Murohara. Department of Cardiology, Nagoya University Graduate School of Medicine, Japan Objective: Endothelial progenitor cells (EPCs) circulate in adult peripheral blood and contribute to neovascularization and collateral formation. Smoking is a strong risk factor for cardiovascular disease and thromboantiitis obliterans (Buerger’s disease). Reduced EPCs may have been involved in the process of these diseases. However, it is not clear that smoking cessation restores the level of circulating EPCs. Methos and Results: In this study we examined the EPCs level using flow cytometry by counting CD34-positeive cells, AC133-positive cells, and CD34/AC133-double positive cells. The EPCs level was tend to be lower in smokers (N=15) than in non-smokers (N=9) (p=0.101 for CD34(+), p=0.022 for AC133(+), p=0.060 for CD34(+)AC133(+) cells respectively). Smokers quitted smoking with nicotine patch (N=8) or without nicotine patch (N=7). After 1 month, all ex-smokers resume smoking. The EPCs level rapidly increased after smoking cessation (p=0.0012 for CD34(+), p=0.007 for AC133(+), p=0.008 for CD34(+)AC133(+) cells respectively) and decreased when they resumed smoking (p=0.0106 for CD34(+), p=0.0171 for AC133(+), p=0.0031 for CD34(+)AC133(+) cells respectively). The degree of EPCs change didn’t differ between the subjects with or without nicotine patch. Conclusion: The EPCs level was lower in chronic smokers than nonsmokers. Short term smoking cessation (1 month) rapidly and significantly increased the level of EPCs and re-start smoking significantly decrease EPCs. The possible mechanisms by which smoking impair the EPCs level in peripheral blood may be the decreased EPCs production form bone marrow or increased degradation of EPCs.
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
WEDNESDAY
M. Katsumoto, T. Shingu, R. Kuwashima, S. Nomura, Y. Umeda, K. Chayama. Department of Medicine and Molecular Science Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan
203