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nitrate with risk factor of coronary heart disease in patients with essential hypertension
Thursday October 2, 2003: Poster Session NO, NO synthase and oxidative stress atherothrombotic vascular disease, may be a mediator as well as a marker of atherosclerosis. On the other hand, oxidative stress, caused by inflammatory processes, could represent endogenous reason for antioxidant deficiency. However, the relationship between CRP and oxidative stress remains unclear. The objective of this study was to test the relationship between CRP, oxidative stress and endothelial cell function. Methods and Results: We conducted a cross-sectional analysis among 106 healthy men (40.0±0.89 yrs, 25.4±0.30kg/m2 ) enrolled in the antioxidant study. In unadjusted analysis, higher levels of CRP were seen with increasing body mass index (P=0.002), diastolic blood pressure (P=0.000), total homocysteine (P=0.041) and lymphocyte DNA damage, an oxidative stress marker (P=0.000). CRP levels showed an inverse relationship with HDL-cholesterol (P=0.009), β-carotene (P=0.039) and flow-mediated dilation (FMD, endothelium dependent) (P=0.049) of brachial artery. In multivariate analyses, we found independent relationships between CRP levels and lymphocyte DNA damage (β coefficient=0.501, P=0.000), FMD (β coefficient=-0.348, P=0.009), HDL-cholesterol (β coefficient= -0.312, P=0.017) and β-carotene (β coefficient= -0.306, P=0.019). Lymphocyte DNA damage was also accelerated by the increased level of CRP (β coefficient=0.508, P=0.002). Conclusions: Our present data suggest that serum levels of CRP are independently interrelated with oxidative stress marker in healthy men. 4P-1057
Endothelial dysfunction and oxidative stress in vasculature of growth hormone transgenic mice
I.J.L. Andersson 1 , M. Johansson 1 , L. Gan 1,2 , G. Bergström 1,2 . 1 Dept. of Physiology, 2 Dept. of Clinical Physiology, Sahlgrenska Academy, Göteborg University, Göteborg, Sweden Objectives: Mice over expressing bovine growth hormone (bGH) display several risk factors for development of atherosclerosis, such as hypertension, insulin resistance and high levels of LDL cholesterol. We examined conduit vessels from these mice with the aim of exploring the role of oxidative stress for endothelial function. Methods: Endothelial function of aorta and carotid artery from 9-11 weeks old female transgenic and control mice was assessed in a wire myograph by the response to acetylcholine before and after treatment with a superoxide dismutase mimetic (MnTBAP, 10−5 mol/L). The mRNA expression of several factors of importance for endothelial function were measured in aortic tissue by real time PCR. Results: Carotid artery from bGH showed impaired endothelium dependent relaxations to acetylcholine (3 × 10−8 - 6 × 10−6 mol/L) compared with carotid artery from control mice (maximum relaxation 46±7% vs. 69±8% P<0.05). This difference was abolished after treatment with MnTBAP. Endothelial function of aorta from bGH and control mice was intact (maximum relaxation 68±10% vs. 69±6%). Compared to control tissue, aortic tissue from bGH showed increased mRNA levels of extra cellular superoxide dismutase (ecSOD, + 61±20%, P<0.05) and endothelial nitric oxide synthase (eNOS, + 68±16%, P<0.05) and decreased levels of caveolin (-28±11%, P<0.05). Conclusion: These data suggest that an upregulation of eNOS and ecSOD counteracts oxidative stress in aorta of bGH mice, while the endothelial dysfunction in the carotid artery could be due to imbalance between the production of free radicals and antioxidant defence. It is possible that GH is involved in the regulation of eNOS and caveolin, and therefore exerts a protective role against endothelial dysfunction in bGH mice. 4P-1058
Cytoplasmic tyrosine kinase PYK2 is essential for eNOS activation, followed by angiogenesis and regulation of vascular tonus
Nitric oxide (NO) plays critical role in regulation of vascular tonus and also angiogenesis.Understanding the mechanism of eNOS activation is essential to develop therapy for vascular disease. Its activation is induced with phosphorylation at specific threonin residues by Akt or Ca2+ dependent CaMKinase. However, more upstream remains to be fully clarified. Meanwhile, we have generated the knock-out mice of cytoplasmic tyrosine kinase PYK2 which
is similar to focal adhesion kinase (FAK). We here showed that analysis of PYK2-/- mice revealed novel function of PYK2 in activation of eNOS-NO system. Method and Result: Stimulation of aorta with VEGF or acetylcholin and also stimulation of hindlimb muscle with hypoxia activates PYK2. In PYK2-/mice, these stimuli induced Akt1 activation in aorta and also cytoplasmic Ca2+ mobilization in single endothelial cell, followed by eNOS activation and NO production, was impaired. Subsequently, acetylcholin induced cGMP production in aorta, which production is mediated with eNOS-NO, and following its vasodilatation activity was reduced. Consistently, PYK2-/- mice exhibited 20% higher blood pressure than wild type mice. On the other hand, VEGF has been shown to induce angiogenesis. Indeed, PYK2-/- mice showed that angiogenesis in response to VEGF pellet placed on the cornea was reduced. Furthermore, VEGF induces angiogenesis in ischemic hindlimb. Indeed, PYK2-/-mice exhibited that angiogenesis in ichemic hindlmb, which was eNOS-NO system dependent, was impaired in PYK2-/- mice. Moreover, in PYK2 -/- mice, VEGF and subsequent eNOS-NO induced migration of endothelial cells, which is required for angiogenesis, were impaired. Conclusion: PYK2 is the critical activation for eNOS-NO system through activation of Akt1 and Ca2+ mobilization, leading to angiogenesis and regulation of vascular tonus. 4P-1059
Association of eNOS expression and activity and serum levels of nitrite/nitrate with risk factors of coronary heart disease in patients with essential hypertension
N. Gumanova, O. Litinskaya, A. Safaryan, D. Nebieridz, V. Metelskaya. National Research Center for Medicine, Moscow, Russia Endothelial dysfunction due to altered nitric oxide synthesis is a common denominator of hypertension and coronary heart disease (CHD). We evaluated the association of eNOS function with CHD risk factors in patients with essential hypertension types I and II. The following parameters were assayed: eNOS protein expression by Western, eNOS activity in intact bovine aortic endothelial cells (BAEC) after 24 h incubation with 10% patient serum, and serum levels of nitrite/nitrate. Serum level of nitrite/nitrate was significantly lower (89.6±6.81 µM; mean±SEM) in the groups with combination of metabolic risk factors which included hypertension, overweight and hyperlipidemia (n=70) versus 124.8±9.46 µM in the group without risk factors (n=20, p<0.02). The patient serum increased eNOS protein expression in BAEC by 36% in the groups with combination of metabolic risk factors (p<0.001). The eNOS activity was unchanged. This can be due to certain compensatory mechanisms in the endothelium which result in increased eNOS production associated with low serum levels of nitrite/nitrate in patients with combination of metabolic risk factors. Data show the strong association of altered nitric oxide synthesis with CHD risk factors. 4P-1060
Basal production of nitric oxide is increased in the microcirculation in type 2 diabetes despite endothelial dysfunction: association with oxidative stress and blood pressure
R. Woodman, D. Playford, K. Croft, G. Watts. Faculty of Medicine and Dentistry, University of Western Australia, Australia Background: Basal microcirculatory blood flow is determined by endothelial release of nitric oxide (NO) and non-NO vasoactive agents. Basal microcirculatory flow may be enhanced in Type 2 diabetes despite endothelial dysfunction. We assessed basal forearm blood flow (FABF) without and with NO blockade (FABF-NO) and examined changes in blood flow in association with CV risk factors and plasma F2-isoprostanes, a marker of oxidative stress. Methods: FABF was measured using venous occlusion strain-guage plethysmography in 43 Type 2 diabetic men and women without autonomic neuropathy (mean ± SE) (55.3 ± 1.1 yrs) and 15 age-matched controls (55.5 ± 2.9 yrs). NG-monomethyl-L-arginine (L-NMMA, 4mmol/min for 8 minutes) was infused into the brachial artery to study the inhibition of basal release of NO. Acetylcholine (Ach) (7.5, 15 and 30 µg/min) and sodium nitroprusside (SNP) (1.5, 3 and 10 µg/min) were infused separately to assess endothelium-dependent and endothelium-independent function. Results: Diabetic subjects had significantly higher serum triglycerides, insulin and systolic blood pressure (SBP), and lower HDL-cholesterol and vasodilatory responses to ACh and SNP (p<0.01 for each) than controls. After adjusting for age, sex, insulin and forearm circumference, FABF (p=0.01) and FABF-NO (p=0.001) were increased in diabetic subjects compared with controls whilst % inhibition of blood flow during L-NMMA was not different.
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
THURSDAY
M. Okigaki 1 , K. Amano 1 , T. Yamashita 2 , S. Kawashima 2 , M. Yokoyama 2 , D. Jin 3 , S. Takai 3 , M. Miyazaki 3 , H. Matsubara 4 , T. Iwasaka 1 . 1 Department of Medicine II and Cardiovascular Center, Kansai Medical University; 2 Division of Cardiovascular and Respiratory Medicine, Kobe University; 3 Department of Phamacology, Osaka Medical College; 4 Department of Cardiology and Vascular Regenerative Medicine, Kyoto Prefectural University School of Medicine, Japan
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Endothelial dysfunction and oxidative stress in vasculature of growth hormone transgenic mice
I.J.L. Andersson 1 , M. Johansson 1 , L. Gan 1,2 , G. Bergström 1,2 . 1 Dept. of Physiology, 2 Dept. of Clinical Physiology, Sahlgrenska Academy, Göteborg University, Göteborg, Sweden Objectives: Mice over expressing bovine growth hormone (bGH) display several risk factors for development of atherosclerosis, such as hypertension, insulin resistance and high levels of LDL cholesterol. We examined conduit vessels from these mice with the aim of exploring the role of oxidative stress for endothelial function. Methods: Endothelial function of aorta and carotid artery from 9-11 weeks old female transgenic and control mice was assessed in a wire myograph by the response to acetylcholine before and after treatment with a superoxide dismutase mimetic (MnTBAP, 10−5 mol/L). The mRNA expression of several factors of importance for endothelial function were measured in aortic tissue by real time PCR. Results: Carotid artery from bGH showed impaired endothelium dependent relaxations to acetylcholine (3 × 10−8 - 6 × 10−6 mol/L) compared with carotid artery from control mice (maximum relaxation 46±7% vs. 69±8% P<0.05). This difference was abolished after treatment with MnTBAP. Endothelial function of aorta from bGH and control mice was intact (maximum relaxation 68±10% vs. 69±6%). Compared to control tissue, aortic tissue from bGH showed increased mRNA levels of extra cellular superoxide dismutase (ecSOD, + 61±20%, P<0.05) and endothelial nitric oxide synthase (eNOS, + 68±16%, P<0.05) and decreased levels of caveolin (-28±11%, P<0.05). Conclusion: These data suggest that an upregulation of eNOS and ecSOD counteracts oxidative stress in aorta of bGH mice, while the endothelial dysfunction in the carotid artery could be due to imbalance between the production of free radicals and antioxidant defence. It is possible that GH is involved in the regulation of eNOS and caveolin, and therefore exerts a protective role against endothelial dysfunction in bGH mice. 4P-1058
Cytoplasmic tyrosine kinase PYK2 is essential for eNOS activation, followed by angiogenesis and regulation of vascular tonus
Nitric oxide (NO) plays critical role in regulation of vascular tonus and also angiogenesis.Understanding the mechanism of eNOS activation is essential to develop therapy for vascular disease. Its activation is induced with phosphorylation at specific threonin residues by Akt or Ca2+ dependent CaMKinase. However, more upstream remains to be fully clarified. Meanwhile, we have generated the knock-out mice of cytoplasmic tyrosine kinase PYK2 which
is similar to focal adhesion kinase (FAK). We here showed that analysis of PYK2-/- mice revealed novel function of PYK2 in activation of eNOS-NO system. Method and Result: Stimulation of aorta with VEGF or acetylcholin and also stimulation of hindlimb muscle with hypoxia activates PYK2. In PYK2-/mice, these stimuli induced Akt1 activation in aorta and also cytoplasmic Ca2+ mobilization in single endothelial cell, followed by eNOS activation and NO production, was impaired. Subsequently, acetylcholin induced cGMP production in aorta, which production is mediated with eNOS-NO, and following its vasodilatation activity was reduced. Consistently, PYK2-/- mice exhibited 20% higher blood pressure than wild type mice. On the other hand, VEGF has been shown to induce angiogenesis. Indeed, PYK2-/- mice showed that angiogenesis in response to VEGF pellet placed on the cornea was reduced. Furthermore, VEGF induces angiogenesis in ischemic hindlimb. Indeed, PYK2-/-mice exhibited that angiogenesis in ichemic hindlmb, which was eNOS-NO system dependent, was impaired in PYK2-/- mice. Moreover, in PYK2 -/- mice, VEGF and subsequent eNOS-NO induced migration of endothelial cells, which is required for angiogenesis, were impaired. Conclusion: PYK2 is the critical activation for eNOS-NO system through activation of Akt1 and Ca2+ mobilization, leading to angiogenesis and regulation of vascular tonus. 4P-1059
Association of eNOS expression and activity and serum levels of nitrite/nitrate with risk factors of coronary heart disease in patients with essential hypertension
N. Gumanova, O. Litinskaya, A. Safaryan, D. Nebieridz, V. Metelskaya. National Research Center for Medicine, Moscow, Russia Endothelial dysfunction due to altered nitric oxide synthesis is a common denominator of hypertension and coronary heart disease (CHD). We evaluated the association of eNOS function with CHD risk factors in patients with essential hypertension types I and II. The following parameters were assayed: eNOS protein expression by Western, eNOS activity in intact bovine aortic endothelial cells (BAEC) after 24 h incubation with 10% patient serum, and serum levels of nitrite/nitrate. Serum level of nitrite/nitrate was significantly lower (89.6±6.81 µM; mean±SEM) in the groups with combination of metabolic risk factors which included hypertension, overweight and hyperlipidemia (n=70) versus 124.8±9.46 µM in the group without risk factors (n=20, p<0.02). The patient serum increased eNOS protein expression in BAEC by 36% in the groups with combination of metabolic risk factors (p<0.001). The eNOS activity was unchanged. This can be due to certain compensatory mechanisms in the endothelium which result in increased eNOS production associated with low serum levels of nitrite/nitrate in patients with combination of metabolic risk factors. Data show the strong association of altered nitric oxide synthesis with CHD risk factors. 4P-1060
Basal production of nitric oxide is increased in the microcirculation in type 2 diabetes despite endothelial dysfunction: association with oxidative stress and blood pressure
R. Woodman, D. Playford, K. Croft, G. Watts. Faculty of Medicine and Dentistry, University of Western Australia, Australia Background: Basal microcirculatory blood flow is determined by endothelial release of nitric oxide (NO) and non-NO vasoactive agents. Basal microcirculatory flow may be enhanced in Type 2 diabetes despite endothelial dysfunction. We assessed basal forearm blood flow (FABF) without and with NO blockade (FABF-NO) and examined changes in blood flow in association with CV risk factors and plasma F2-isoprostanes, a marker of oxidative stress. Methods: FABF was measured using venous occlusion strain-guage plethysmography in 43 Type 2 diabetic men and women without autonomic neuropathy (mean ± SE) (55.3 ± 1.1 yrs) and 15 age-matched controls (55.5 ± 2.9 yrs). NG-monomethyl-L-arginine (L-NMMA, 4mmol/min for 8 minutes) was infused into the brachial artery to study the inhibition of basal release of NO. Acetylcholine (Ach) (7.5, 15 and 30 µg/min) and sodium nitroprusside (SNP) (1.5, 3 and 10 µg/min) were infused separately to assess endothelium-dependent and endothelium-independent function. Results: Diabetic subjects had significantly higher serum triglycerides, insulin and systolic blood pressure (SBP), and lower HDL-cholesterol and vasodilatory responses to ACh and SNP (p<0.01 for each) than controls. After adjusting for age, sex, insulin and forearm circumference, FABF (p=0.01) and FABF-NO (p=0.001) were increased in diabetic subjects compared with controls whilst % inhibition of blood flow during L-NMMA was not different.
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
THURSDAY
M. Okigaki 1 , K. Amano 1 , T. Yamashita 2 , S. Kawashima 2 , M. Yokoyama 2 , D. Jin 3 , S. Takai 3 , M. Miyazaki 3 , H. Matsubara 4 , T. Iwasaka 1 . 1 Department of Medicine II and Cardiovascular Center, Kansai Medical University; 2 Division of Cardiovascular and Respiratory Medicine, Kobe University; 3 Department of Phamacology, Osaka Medical College; 4 Department of Cardiology and Vascular Regenerative Medicine, Kyoto Prefectural University School of Medicine, Japan
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