ÄαÈÂå¶ûÊæ Zhang Blood Vessels And The Protection Of The Spontaneously Hypertensive Rat Vascular Endothelial

Background:Nebivolol is a highly selectiveβ1 adrenoceptor blocker with additional vasodilating properties. It has been shown that the nebivolol-induced vasorelaxation is nitric oxide (NO) dependent. However, the nature of the transduction pathway that mediates nebivolol's activation remains elusive. PI3K/Akt/eNOS is an important cell signal transduction pathway. The serine/tyrosine protein kinase Akt phosphorylates endothelial cell nitric oxide synthase (eNOS) and enhances the ability of eNOS to generate NO. Previous studies have shown that the release of NO from the endothelium may be ascribed to the modulation of different types of K+ channels. It has also been show that there was a difference in the NO release induced by nebivolol among different arteries.Purpose:The present study was designed to determine the relaxation of nebivolol on different arteries (aorta, carotid artery, renal artery, mesenteric artery and femoral artery); to observe the effects of NOS inhibitor, K+ channels inhibitors or PI3K/Akt inhibitors on the vasorelaxation induced by nebivolol in different rat arteries.Methods:Male Wistar rats (200-250g) were killed by cervical dislocation. Then thoracic aortas, carotid arteries, renal arteries, mesenteric arteries and femoral arteries were removed and placed in chilled (4℃) physiological salt solution (PSS) which was gassed with 100% O2. Aortas, carotid arteries were cleaned of connective tissue and fat, and cut into 3-4mm long rings. For the recording of tension the vessel rings were mounted, between 2 L-shaped stainless steel hooks, in 10ml organ baths filled with oxygenated PSS solution (bubbled with 100%O2 and maintained at 37℃) (pH 7.4). Each preparation was fixed, via a silk thread, to an isometric force transducer (A.D. Instruments, PowerLab, Australia) and force was recorded via PowerLab computer system (A.D. Instruments). Each vessel ring was subjected to an initial resting tension (aorta,2g; carotid artery, 1g). A 2h equilibration period was allowed before any experimental intervention, and during equilibration, the bath was flushed every 20 minutes with the fresh PSS. After equilibration, the rings were activated 2 times with 60mmol/L KCl. The integrity of endothelium was presumed by observation that the relaxation induced by 10-5mol/L acetylcholine (Ach) on 60 mmol/L KCl induced contraction was greater than 30%. When the contraction induced by 60 mmol/L KCl and the relaxation induced by 10-5 mol/L Ach were repeatable (the change was less than 10% between successive contractions or relaxation), the effects of the drugs to be tested were observed.The femoral artery, the third-order branches of the superior mesenteric artery and renal artery were isolated and cut into 2mm-long rings. The rings were mounted in separate 5ml tissue baths filled with PSS solution (bubbled with 100% O2 and maintained at 37℃) (pH 7.4) using 40μm steel wire in a small-vessel myograph (Multi Myograph System-610M, Danish Myo Technology A/S, Denmark) for tension recordings. The rings were normalized according to standard procedures and stretched to a state equal to 100mmHg,80mmHg, 100mmHg, respectively. The rings were equilibrated for at least for 1h, and during equilibration, the bath was flushed every 20 minutes with the fresh PSS. After equilibration, the rings were activated 2 times with 60mmol/L KC1. The integrity of endothelium was presumed by observation that the relaxation induced by 10"5 mol/L Ach on contraction induced by 60 mmol/L KCl was greater than 20%. When the contraction induced by 60 mmol/L KCl and the relaxation induced by 10-5 mol/L Ach were repeatable (the change was less than 10% between successive contractions or relaxation), the effects of the drugs to be tested were observed.Vessels were then washed extensively, and subsequently contracted with phenylephrine (Phe) (aorta and carotid artery were pre-contracted with 10"6 mol/L, mesenteric artery, femoral artery and renal artery were pre-contracted with 10"5 mol/L) or with 60 mmol/L KCl. Following attainment of plateau constriction in response to Phe or KCl, the logarithm cumulative concentration response curve (LCCRC) to nebivolol (10-7-10-5 mol/L) was performed. Then the vessels were contracted with Phe or 60mmol/L KCl again. Following attainment of plateau constriction, specific inhibitors of each type of K+channels:iberiotoxin (100 nmol/L), glibenclamide (0.1 mmol/L),4-aminopyridine (4-AP,1 mmol/L), BaCl2 (1 mmol/L), wortmannin (5×10"7 mol/L, a selective inhibitor of phosphatidylinositol 3-kinase (PI3K)), Akt inhibitor (lL-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate,10-5 mol/L, a selective inhibitor of Akt) and NG-nitro-L-arginine methyl ester (L-NAME,100μmol/L), an inhibitor of NO synthase were added to the bathing medium respectively. When the plateau constriction was attained, nebivolol (10-7-10'5 mol/L) was added cumulatively. So the effects of NOS inhibitor, K+channels inhibitors or PI3K/Akt inhibitors on the vasorelaxation induced by nebivolol in different rat arteries were studied.Results:Nebivolol (10-7-10-5 mol/L) concentration-dependently relaxed pre-contractions induced by KC1 and PE in different rat arteries. The Emax of the relaxation of nebivolol was different according to the contractors. The rank order of vasodilator efficacy as measured in relation to the maximal vasodilation induced by nebivolol (10"7-10-5 mol/L) was:mesenteric artery (96.7±12.8%)≈aorta (94.2±5.4%)>femoral artery (84.8±12.1%)≈renal artery (80.3±3.4%)>carotid artery (74.9±10.1%) pre-contracted with Phe. In arteries contracted with 60mmol/L KCl, the maximal vasodilation was similar, expect in mesenteric artery (90.57±15.2%):femoral artery (75.7±17.7%), aorta (73.1±11.3%), renal artry (71.6±6.2%), carotid artery (70.9±7.1%). The vasodilator effect of nebivolol, in concentrations of 3 and 10μmol/L, was significantly blocked by 100μmol/L of the NOS inhibitor L-NAME in all arteries The exposure of the vessel rings to a selective inhibitor of phosphatidylinositol 3-kinase (PI3K) wortmannin (5×10-7 mol/L) or a selective inhibitor of Akt (1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate,10-5 mol/L) did not influence nebivolol-induced vasorelaxation. Similarly, K+channels blockers:iberiotoxin (100 nmol/L), glibenclamide (0.1 mmol/L),4-aminopyridine (1 mmol/L), or BaCl2(1 mmol/L) had no influence on the relaxation of nebivolol in arteries precontracted by Phe or KCl.Conclusion:Nebivolol produced a concentration-dependent vasodilation in different rat arteries pre-contracted by Phe or KCl. In the isolated rat aorta, carotid artery, femoral artery, mesenteric artery and renal artery, neither K+channels nor PI3K/Akt/eNOS pathway was involved in the relaxation induced by nebivolol. Background:Hypertension is a major risk factor for cardiovascular disease. Experiments show that there is endothelial dysfunction in animal and human hypertension, which in turn accelerates the damage of target organs. Endothelia dysfunction, which may manifest as vasomotor imbalance and changes in vaso-active substances and so on, is the initiator factors and carrier of "endothelial-high blood pressure-cardiovascular events" chain. Vascular endothelial damage enables the vascular remodeling. The remodeling can be an early manifestation of vascular structural changes and the basis for a number of cardiovascular events. It has been became a new target for hypertension treatment to improve the endothelia function. Nebivolol is a highly selectiveβ1-adrenergic receptor antagonist that can blockβ1-adrenergic receptor and promote endothelium-dependent vasodilation by increasing bioavailability of nitric oxide (NO). Compared with other traditionalβ-adrenergic receptor antagonists, nebivolol, without having the metabolic defects, is a bestβ-adrenergic receptor antagonist at present. Studies show that nebivolol can ameliorate the vascular reaction of aorta and improve the plasma NO concentration of SHR. But the data is few about the effect of nebivolol on the reaction of arteriole, the vaso-activesubstance in artery and the reconstruction of vascular.Purpose:The aim of the study was to assess the protective effect of nebivolol on endothelial dysfunction in spontaneously hypertensive rats (SHR) by observing the reconstruction of arteries, vascular endothelial damage, levels of vaso-activesubstance and the vascular responses of different arteries.Methods:SHR and age-matched Wister-Kyoto (WKY) rats were randomly divided into 4 groups:(1) SHR+Nebivolol group(n=6):SHR treated with nebivolol(8 mg/kg/day, i.g.); (2) SHR+Atenolol group (n=6):SHR treated with Atenolol (80 mg/kg/day, i.g.); (3) SHR control group (n=6):SHR treated with vehicle once daily, i.g.; (4) WKY control group (n=6):WKY treated with vehicle once daily, i.g. Therapy continued for 8 weeks, body weight and tail systolic blood pressure were measured weekly. At the end of this study, animals were anesthetized with 3% sodium pentobarbital (30 mg/kg, i.p.). Blood was draw by abdominal aortic puncture, then the aorta, carotid artery, renal, mesentery and femoral artery were moved rapidly. Renal and a part of aorta were fixed with 4% paraformaldehyde to observe the vascular morphological changes, including vessel lumen diameter (L), media thickness (M) and M/L. Expression of FVM in aorta was performed by immunohistochemistry analysis. Levels of NO (nitric acid reducdase assay), endothelin-1 (ET-1, radioimmunoassay), angiotensin II (AngⅡ, radioimmunoassay) in plasma, aorta and von Willebrand factor (vWF, ELISA) in plasma were measured. The contraction of different arteries (aorta, carotid artery, renal artery, femoral artery and mesentery artery) to Phe, KCl, ET-1, AngⅡand relaxation to acetylcholine (Ach) and sodium nitroprusside (SNP) were also studied in vitro.Results:(1) During the study, the body weight and systolic blood pressure were higher in SHR than in age-matched WKY. Nebivolol and atenolol had no effect on the body weight of SHR. Compared with atenolol, treatment with nebivolol induced an acute and significant reduction in systolic blood pressure; this reached a maximum in the fourth week.(2) Morphometry study of aorta:There was no difference in L among different groups, but M and M/L increased in SHR than in WKY. Treatment with nebivolol reduced M and M/L in SHR while Atenolol had no effect.(3) Morphometry study of interlobular artery in renal:The L decreased, but M and M/L of interlobular artery increased in SHR. Treatment with nebivolol raised L and lessened M and M/L in SHR, but atenolol had no effect.(4) Thoracic aorta immunohistochemistry assay to identify FⅧin endothelia:The expression of FⅧwas weakly positive in SHR than in WKY, the brown endothelium was not intact and continuous. Treated with nebivolol, the expression of FⅧincreased and the brown endothelium was clear and intact, however atenolol had no effect.(5) Level of vWF in plasma:The level of vWF increased in SHR than in WKY. Nebivolol reduced the level of vWF in SHR, but atenolol had no influence.(6) Levels of NO, ET-1 and NO/ET-1 in plasma and aorta:The levels of NO, NO/ET-1 in plasma and aorta were lowered and ET-1 in aorta was increased in SHR than in WKY; but no difference was found in plasma ET-1 between SHR and WKY. Nebivolol increased the level of NO and NO/ET-1 in plasma and aorta, but had no influence on level of ET-1 in aorta and plasma. Atenolol had no effect on theses changes in SHR.(7) Levels of Ang II and NO/AngⅡin plasma and aorta:The level of Ang II was increased and NO/Ang II was reduced in SHR plasma and aorta than in WKY. Nebivolol had no effect on Ang II in plasma, but reduced it in aorta. So the NO/Ang II in plasma and aorta were increased after nebivolol treatment. Atenolol had no effect on the changes in SHR.(8) Vascular reaction of different arteries:The contractions to Phe, KCl in different arteries; to ET-1 in carotid artery; to AngⅡin aorta and carotid artery were increased in SHR than in WKY. The relaxation to Ach decreased in SHR than in WKY, but there was no difference of relaxation to SNP among groups. Nebivolol reduced the contraction of different arteries to Phe, KCl, AngⅡand ET-1 in SHR, but there was difference in the reduction based on the kind of arteries. It was marked to KCl in carotid artery and femoral artery, to Phe in aorta, renal artery, femoral artery, and to ET-1 and Ang II in aorta. Nebivolol increased the relaxation of different arteries to Ach, especially in renal artery, but had no effect on the relaxation of different arteries to SNP.Atenolol just decreased the contraction of different arteries to KCl apart from renal artery and mesenteric artery; to Phe besides mesenteric artery; to ET-1, AngⅡin aorta and carotid artery. Atenolol increased the relaxation of renal artery to Ach in SHR, but had no effect on the relaxation to SNP.Conclusion:The antihypertensive effect of nebivolol in SHR was accompany by protect on endothelial function:retarding vascular remodeling; reducing the injury of aorta endothelia cell; modulate the unbalance of vasoactive substances in aorta and plasma; improving the vascular reaction to different vasomotors, resuming the balance of relaxation and contraction. Objectives:Endothelial dysfunction plays an important role in the development of cardiovascular diseases. Nitric oxideis is recognized as a major mediator in regulating vascular construction and function. Aasymmetric dimethylarginine (ADMA), an endogenous NOS inhibitor that compatetively inhibits nitric oxide synthase (NOS) activity and reduces NO level, has been considered a new predictor of endothelial dysfunction. In a variety of pathophysiological conditions, plasm ADMA has been found rising significantly, and then may induce endothelial dysfunction. With the understanding of endothelial dysfunction and pathological roles of ADMA in cardiovascular disease, it may become a new therapeutic avuenues to improve endothelial function by reducing ADMA level. Nebivolol can protect vascular endothlia function. But data is few about the relationship of protective effect of nebivolol on vascular and ADMA.Aim:To investigate the relationship of protective effect of nebivolol on vascular and ADMA.Methods:1. Effect of nebivolol on ADMA system of SHR. SHR and age-matched Wister-Kyoto (WKY) rats were randomly divided into 4 groups:(1) SHR+Nebivolol group (n=6):SHR treated with nebivolol(8 mg/kg/day, i.g.); (2) SHR+Atenolol group (n=6):SHR treated with Atenolol(80 mg/kg/day, i.g.); (3) SHR control group (n=6):SHR treated with vehicle once daily, i.g.; (4) WKY control group (n=6):WKY treated with vehicle once daily, i.g. After treatment for 8 weeks, rats were anesthetized with 3% sodium pentobarbital (30 mg/kg, i.p.). Blood was draw by abdominal aortic puncture, then the aorta, mesentery artery were moved rapidly and kept in-70℃. The levels of ADMA (ELISA), NO (nitric acid reducdase assay) and NOS activity (chemical method) in plasma were measured. The mRNA expression of eNOS, DDAH-2, PRMT-1 in aorta and mesenteric artery were measured by RT-PCR. The protein expression of DDAH-2 and PRMT-1 in aorta and mesenteric artery were measured by Western blot. ROS level in aorta were also measured.2. Effect of nebivolol on HUVECs cultured with AngⅡ. HUVECs were cultured with Ang II 1μmol/L for 24h in the absence or presence of nebivolol (5,10 or 20μmol/L) or atenolol (20μmol/L) for 1 h. The supernaent in the conditioned medium was collected by centrifugation for determination of ADMA (ELISA), NO (nitric acid reducdase assay) and NOS activity (chemical method). The expression of eNOS, DDAH-2, PRMT-1 mRNA (RT-PCR), the protein expression of DDAH-2 and PRMT-1 (Western blot), activity of DDAH in HUVECs were also measured.3. Effect of nebivolol on HUVECs cultured with ADMA. HUVECs were cultured with ADMA 16μmol/L for 24h in the absence or presence of nebivolol (5,10 or 20μmol/L) or atenolol (20μmol/L) for 1 h. The supernaent in the conditioned medium was collected by centrifugation for determination of NO and NOS activity. The expression of eNOS mRNA (RT-PCR), ROS in HUVECs were also measured.4. Effect of nebivolol on influence of ADMA on vascular reponse of rat aorta. Male Wistar rats were killed by cervical dislocation. Then thoracic aortas were removed quickly. Aortas was cleaned of connective tissue and fat, and cut into 3-4mm long rings. Each preparation was fixed, via a silk thread, to an isometric force transducer and force was recorded via PowerLab computer-7 -4 system (A.D. Instruments). The effects of ADMA (10-10 mol/L) on aorta resting tone, aorta pre-contracted by Phe (10-6mol/L), relaxation induced by Ach (10-10-10-5 mol/L) and the effect of nebivolol (10-5,10-6,10-5mol/L) on the function of ADMA on rat aorta were observed in virto.Rusults:1. Compared with WKY, the level of plasma ADMA was elevated significantly, plsma NO, activity of NOS were decreased in SHR. In aorta and mesenteric artery of SHR, the mRNA expression of eNOS, mRNA/protein expression of DDAH-2 reduced, but mRNA/protein expression of PRMT-1 increased. ROS level in aorta also increased in SHR than in WKY. Nebivolol treatment increased plasma NO and activity of NOS, decreased ADMA leve. Nebivlolol up regulated mRNA expression of eNOS and mRNA/protein expression of DDAH-2; down regulated mRNA/protein expression of PRMT-1 in aorta and mesenteric artery. Nebivolol reduced ROS level of SHR aorta. Atenolol had no effect on the changes in SHR.2. AngⅡ(1μmol/L) for 24h significantly decreased NO level and NOS activity, increased the mRNA/protein expression of PRMT-1, decreased DDAH activity, mRNA expression of eNOS and mRNA/expression of DDAH-2, resulting in elevation of ADMA level. Pretreatment with nebivolol (5,10 or 20μmol/L) concentration-dependently attenuated the above effects by AngⅡ. Atenolol (20μmol/L) had no influence on the effects induced by AngⅡ.3. ADMA (16μmol/L) for 24h significantly markedly decreased NO level, NOS activity and eNOS mRNA; and increased intracellular ROS generation. Pretreatment with nebivolol (5,10 or 20μmol/L) concentration-dependently attenuated the above effects by ADMA. Atenolol (20μmol/L) had no influence on the effects induced by ADMA.4. ADMA (10-7-10-4 mol/L) had no effect on the restiong tone of rat aorta, but ADMA concentration-dependently increased the tone of aorta pre-contracted by Phe (10 mol/L) and inhibited Ach (10-10-10-5 mol/L) induced relaxation in vitro. Nebivolol (10-5,10-6,10-5 mol/L) dose-dependently decreased the elevation of ADMA on pre-contracted rat aorta and the inhibition of ADMA 10-5 mol/L on relaxation induced by Ach.Conclusion:Nebivolol reduced ADMA by increasing DDAH activity/expression and reducing PRMT-1 expression. Nebivolol also can inhibit cell injury and contraction induced by ADMA.