Interactive effects of insulin-like growth factor-1 and beta-estradiol on endothelial nitric oxide synthase activity in rat aortic endothelial cells

Insulin-like growth factor-1 (IGF-1) and beta-estradiol (E2) have vasodilatory effects, in part, through stimulation of vascular nitric oxide (NO) production. However, their interactive effects on endothelial nitric oxide synthase (eNOS) and NO production have not been previously studied in endothelial cells (EC). Employing rat aortic EC (RAEC), the effects of acute (20 and 30 minutes) and prolonged (4 hours) stimulation with 100 nmol/L IGF-1 and 1 nmol/L E2 (alone or in combination) were assessed with respect to protein levels and enzymatic activities for phosphatidyl inositol 3-kinase (PI3K) and serine/threonine kinase Akt (Akt), enzymes involved in eNOS activation. Exposure to IGF-1 for 30 minutes or E2 for 20 minutes increased insulin receptor substrate-1 (IRS-1) association with the regulatory (p85) subunit of PI3K, enhanced tyrosine phosphorylation of p85, and increased PI3K activity. Combined treatment had a greater effect on p85 phosphorylation and PI3K activity then either agonist alone. Moreover, IGF-1 and E2 enhanced Akt Ser(473) phosphorylation, with the effect of IGF-1 being much greater. Acute expose to both E2 (20 minutes) and IGF-1 (30 minutes) were associated with an increase in eNOS activity. Prolonged exposure (4 hours) to either IGF-1 or E2 increased expression of the p85 subunit as well as eNOS activity. Pretreatment with PI3K antagonist wortmannin (WT) prevented this increase in eNOS activity. The results suggest that IGF-1 and E2 may interact through PI3K/Akt-related pathways to increase eNOS activity.     

Altered insulin-mediated and insulin-like growth factor-1-mediated vasorelaxation in aortas of obese Zucker rats

OBJECTIVE: Insulin and insulin-like growth factor-1 (IGF-1) have vasorelaxant effects in vivo, which is dependent on nitric oxide (NO) production. The aim of this study was to investigate the vasorelaxant responses mediated by insulin and/or IGF-1 in aortas of obese Zucker rats. METHODS: The thoracic aortas of eight lean and eight obese Zucker rats (6 months old) were isolated for vasorelaxation analysis. Insulin-induced and IGF-1-induced vasorelaxant responses were evaluated by the isometric tension of aortic rings in the organ bathes. The roles of phosphatidylinositol 3-kinase (PI3K) and nitric oxide synthase (NOS) in vasorelaxant responses were examined by treating selective inhibitors, such as wortmannin (an inhibitor of PI3K) and N (omega)-nitro-L-arginine methyl ester (L-NAME, a NOS inhibitor). In addition, the vascular responses to sodium nitroprusside (SNP), a direct vasodilator of vascular smooth muscle, were examined. RESULTS: The insulin-induced vasorelaxation in aortas of obese rats was significantly decreased, whereas the IGF-1-induced vasorelaxation was significantly increased, compared with that in lean rats. After the pre-administration of wortmannin or L-NAME, the altered insulin-induced or IGF-1-induced vasorelaxation was abolished. There was no significant difference in the SNP-induced vasorelaxation between lean and obese rats. CONCLUSION: Our findings suggested that the decreased insulin-mediated vasorelaxation in obese rats appeared to be counteracted by the increased IGF-1-mediated vasorelaxation. Furthermore, the NO-dependent pathway was involved in the altered vasorelaxant responses. However, the SNP-induced vasorelaxation was not changed in obese rats.     

促红细胞生成素抑制血管紧张素Ⅱ诱导的大鼠心脏成纤维细胞中转化生长因子β1和胶原的表达

目的 探讨促红细胞生成素(EPO)对血管紧张素Ⅱ(AngⅡ)诱导的心脏成纤维细胞(CF)中转化生长因子(TGF)-β1蛋白表达和胶原生成的影响,以及磷脂酰肌醇-3-激酶(PD-K)/Akt信号途径和一氧化氮合酶(NOS)在其中的作用.方法 应用胰酶和胶原酶双酶法分离培养新生大鼠CF细胞,应用EPO、Ang Ⅱ、PI3-K抑制剂LY294002、NOS抑制剂L-NAME等不同因素干预.ELISA法检测CF中Ⅰ型和Ⅲ型胶原的浓度.化学酶法检测CF培养液中的NO浓度以及NOS总的活性及其亚型的活性.Western blot检测Akt、p-Akt、内皮型一氧化氮合酶(eNOS)、iNOS和TGF-β1蛋白的表达.结果 EPO剂量依赖性的抑制Ang Ⅱ诱导的CF培养液中Ⅰ型和Ⅲ型胶原表达以及提高NO的浓度.10 U/ml的EPO对Ⅰ型和Ⅲ型胶原浓度的抑制分别达到了28%和46%,同时NO浓度则提高了154%.EPO也显著抑制了Ang Ⅱ促CF中TGF-β1蛋白的表达,同时Akt的磷酸化水平显著提高,并促进eNOS蛋白的表达.应用LY294002使eNOS蛋白表达水平明显下降,培养液中的NO浓度也随之下降.L-NAME不能降低eNOS蛋白表达,但抑制了NO的生成.EPO抑制Ang Ⅱ诱导的CF中TGF-β1蛋白的表达以及Ⅰ型和Ⅲ型胶原合成作用均能被二者阻断.结论 EPO可抑制Ang Ⅱ诱导的新生大鼠CF中TGF-β1的表达以及Ⅰ型和Ⅲ型胶原表达,可能是通过激活PI3-K/Akt信号途径促使CF中eNOS表达,从而促进NO的表达来实现.     

Impairment of PI3-K/Akt pathway underlies attenuated endothelial function in aorta of type 2 diabetic mouse model

The phosphatidylinositol 3-kinase (PI3-K) pathway, which activates serine/threonine protein kinase Akt, enhances endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production. We investigated the involvement of the PI3-K/Akt pathway in the relaxation responses to acetylcholine (ACh) and clonidine in a new type 2 diabetic model (streptozotocin plus nicotinamide-induced diabetic mice). Plasma glucose and insulin levels were significantly elevated in our model, and intravenous glucose tolerance tests revealed clear abnormalities in glucose tolerance and insulin responsiveness. Although in our model the ACh-induced relaxation and NOx- (NO2-+NO3-)/cGMP production were unchanged, the clonidine-induced and insulin-induced relaxations and NOx-/cGMP production were all greatly attenuated. In control mice, the clonidine-induced and insulin-induced relaxations were each abolished by LY294002 and by Wortmannin (inhibitors of PI3-K), and also by Akt-inhibitor treatment. The ACh-induced relaxation was unaffected by such treatments in either group of mice. The expression level of total Akt protein was significantly decreased in the diabetic mice aorta, but those for the p85 and p110gamma subunits of PI3-K were not. The clonidine-induced Ser-473 phosphorylation of Akt through PI3-K was significantly decreased in our model; however, that induced by ACh was not. These results suggest that relaxation responses and NO production mediated via the PI3-K/Akt pathway are decreased in this type 2 diabetic model. This may be a major cause of endothelial dysfunction (and the resulting hypertension) in type 2 diabetes.     

Insulin-like growth factor signaling pathways in rat hepatic stellate cells: importance for deoxyribonucleic acid synthesis and hepatocyte growth factor production

It has been shown recently that insulin-like growth factor 1 (IGF-1) increases both DNA synthesis and hepatocyte growth factor (HGF) production in cultured hepatic stellate cells. In this study, we used selective blockers to investigate crucial signaling pathways for these effects of IGF-1 in cultured rat hepatic stellate cells. Both LY 294002 [a phosphatidylinositol 3-kinase (PI3-K) inhibitor], and rapamycin [a blocker of activation of the serine/threonine p70 S6 kinase (p70S6K), a molecule downstream from PI3-K] completely reversed the IGF-1-induced stimulation of DNA synthesis. Mitogen-activated protein kinase (MAPK) inhibition by PD 98059 had a less pronounced suppressory effect, although the used PD 98059 dose was fully effective in inhibiting MAPK phosphorylation. Both LY 294002 and PD 98059 lowered the IGF-1-induced increase of HGF in the medium by about 40%, but LY 294002 was 10 times more potent than PD 98059. Inhibition of p70S6K activation by rapamycin blocked IGF-1-induced DNA synthesis but not the increase in HGF. In conclusion, PI3-K (and, to some extent, MAPK) signaling pathways seem to be important for IGF-1-stimulated DNA synthesis and HGF production. DNA synthesis also seems to be dependent on rapamycin-sensitive activation of the PI3-K effector p70S6K.     

Enhanced nitric oxide bioavailability in coronary arteries prevents the onset of heart failure in rats with myocardial infarction

AimThe endothelium, mainly via nitric oxide (NO) release, adjusts the coronary flow. Cardiac function is closely linked to blood flow; thus, we tested the hypothesis that NO modulation in coronary arteries could be differentially adjusted after myocardial infarction (MI) in the presence or absence of heart failure (HF).Methods and resultsFour weeks after coronary occlusion, the infarcted rats were subdivided into rats without (MI) or with HF signs according to haemodynamic parameters. The septal coronary arteries were subsequently used to perform functional and molecular experiments. Acetylcholine (ACh)-induced relaxation was decreased in the coronary arteries following HF, whereas it was enhanced in the arteries of the MI compared with those of SHAM-operated (SO) rats. The relaxation induced by the NO donor was similar among the groups. NO production, which was evaluated by 4,5-diaminofluorescein diacetate, was reduced in the coronary arteries of the HF group and increased in the arteries with MI after ACh-induced stimulation. HF coronary arteries exhibited oxidative stress, which was evaluated via ethidium bromide-positive nuclei, whereas it was decreased in MI. To evaluate the mechanisms involved in the enhanced ACh-induced relaxation in the arteries following MI, certain septal coronary arteries were pre-incubated with L-NAME (a nonselective NO synthase (NOS) inhibitor), 7-NI (a selective neuronal NOS (nNOS) inhibitor) or LY294002 (a PI3-kinase inhibitor). L-NAME and LY294002 reduced ACh-induced relaxation in the MI and SO rats; however, these effects were greater in the MI arteries. 7-NI reduced only the ACh-relaxation in MI. In addition, the eNOS, nNOS, Akt, and superoxide dismutase isoform protein expressions were greater in the coronary arteries of the MI than in those of the SO groups.ConclusionOur data suggested that endothelial function was closely related to cardiac function after coronary occlusion. The coronary arteries from the HF rats exhibited reduced NO bioavailability, whereas the MI rats exhibited increased NO bioavailability because of increased eNOS/nNOS/PI3-kinase/Akt pathway and a reduction in ROS generation. These results suggest that enhanced NO modulation can prevent the onset of HF.     

Effects of angiotensin II on nitric oxide generation in growing and resting rat aortic endothelial cells

OBJECTIVES: To assess the effects of angiotensin II (ang II) and its receptors on nitric oxide (NO) production and endothelial NO synthase (eNOS) activity and expression with respect to rat aortic endothelial cell (RAEC) growth. To also assess whether an intact endothelium is required for ang II activity. METHODS: RAEC were treated with different doses of ang II, Ca(2+) ionophore A23187, valsartan (an AT(1) receptor inhibitor) or PD-123319 (an AT(2) receptor inhibitor) alone or in combination for 24 h before measuring nitrite levels by Griess reaction as an index of NO production and eNOS activity by L-[3H]-arginine to L-[3H]-citrulline conversion assay. eNOS mRNA and protein expressions were determined by Northern and Western analyses, respectively. The requirement of endothelium for ang II-mediated relaxant/contractile effects was investigated by isometric tension studies. RESULTS: NO production and eNOS activity/expression were almost two-fold greater in proliferating RAEC. Ang II or Ca(2+) ionophore A23187 enhanced NO production in proliferating and confluent RAEC without altering the fold-difference in basal NO release. Both valsartan and PD-123319 significantly diminished NO production in RAEC treated with ang II but not Ca(2+) ionophore A23187 while NG-nitro-L-arginine (L-NNA, 10 micromol/l) equally decreased NO generation in response to both stimulators. L-NNA, valsartan and PD-123319 also abolished endothelium-dependent vasorelaxant responses to ACh and Ca(2+) ionophore A23187 in the presence of ang II. Sodium nitroprusside (SNP), a NO donor, increased endothelium-independent vasorelaxant responses that were augmented by valsartan but not L-NNA or PD-123319 in the presence of ang II. CONCLUSIONS: Ang II induces vascular NO production through endothelial AT(1) and AT(2)-receptors. This may be beneficial in counterbalancing its vasoconstrictor effect on vascular smooth muscle cells.     

Up-regulation of nNOS and associated increase in nitrergic vasodilation in superior mesenteric arteries in pre-hepatic portal hypertension

BACKGROUND/AIMS: Splanchnic arterial vasodilation in portal hypertension has been attributed largely to vascular NO overproduction. Three NO-synthase (NOS) isoforms have been identified of which e(ndothelial)-NOS has been found up-regulated and i(nducible)-NOS not expressed in the splanchnic circulation in portal hypertension. So far, n(euronal)-NOS has not been investigated and hence, the current study evaluates nNOS-expression and nNOS-mediated vasorelaxation in a model of portal vein-ligated rats (PVL). METHODS: Mesenteric vasculature of PVL and sham rats was evaluated for nNOS-protein (immunohistochemically and Western blotting). In vitro perfused de-endothelialized mesenteric arterial vasculature was pre-constricted with norepinephrine (EC(80)) and tested for nNOS-mediated vasorelaxation by periarterial nerve stimulation (PNS, 2-12 Hz, 45V) before and after incubation with the NOS-inhibitor L-NAME (10(-4)M). RESULTS: nNOS was localized to the adventitia of the mesenteric arterial tree showing more intense staining and increased protein expression in PVL as compared to sham rats. PNS induced a frequency-dependent vasorelaxation, which was more pronounced in PVL rats. L-NAME abolished this difference in nerval-mediated vasorelaxation, the effect being significantly greater in PVL than in sham animals. CONCLUSIONS: Perivascular nNOS-protein expression is enhanced in mesenteric arteries in portal hypertension mediating an increased nerval NO-mediated vasorelaxation. This nNOS-derived NO overproduction may play an important role in the pathogenesis of arterial vasodilation in portal hypertension.     

促红细胞生成素对乳鼠心肌细胞肥大及P13K/Akt-eNOS信号转导通路的影响

目的 探讨促红细胞生成素(EPO)对血管紧张素Ⅱ(AngⅡ)诱导的肥大心肌细胞的影响,以及磷脂酰肌醇3激酶(PI3K)/丝氨酸苏氨酸激酶(Akt)-内皮型一氧化氮合酶(eNOS)信号转导通路在其中的作用.方法 分离乳鼠心肌细胞,利用AngⅡ诱导建立心肌细胞肥大模型,以心肌细胞表面积和心钠素(ANF)mRNA表达作为心肌细胞肥大观察指标.观察不同浓度EPO对肥大心肌细胞的影响,并利用PI3K抑制剂LY294002和一氧化氮合酶抑制剂L-NAME对其相关机制进行探讨,I司时对细胞培养液中一氧化氮(NO)浓度进行检测,蛋白免疫印迹法检测磷酸化Akt(p-Akt)、Akt、磷酸化eNOS(p-eNOS)和eNOS蛋白表达情况.结果 20 U/ml EPO能抑制由AngⅡ诱导的心肌细胞肥大,表现为心肌细胞表面积和ANF mRNA表达均减少(P＜0.05).EPO能激活Akt,促进eNOS及p-eNOS表达增加(均P＜0.05),并使NO合成增加(P＜0.01).LY294002和L-NAME能逆转EPO的抗心肌细胞肥大作用,减少NO产最(P＜0.05).蛋白免疫印迹法榆测显示,LY294002能够抑制EPO对p-Akt、p-eNOS和eNOS蛋白表达的促进作用,而L-NAME能抑制eNOS的磷酸化(均P＜0.05).结论 EPO能够抑制AngⅡ诱导的心肌细胞肥大,该作用可能是通过激活P13K/Akt信号转导通路,促进eNOS表达与活化,从而促进NO的合成来实现的.     

Phosphatidylinositol 3-kinase and focal adhesion kinase are early signals in the growth factor-like responses to thrombospondin-1 seen in human vascular smooth muscle.

Thrombospondin-1 (TSP-1) is a matricellular protein that is expressed in negligible amounts in normal blood vessels but is markedly upregulated in vascular injury. Although TSP-1 can act as a pleiotropic regulator for human vascular smooth muscle cells (HVSMCs), the intracellular signaling pathways stimulated by this protein remain obscure. In cultured HVSMCs derived from saphenous vein, TSP-1 induces tyrosine phosphorylation of a number of cellular proteins, with a complex temporal pattern of activation. Immunoprecipitation techniques have identified the early tyrosine-phosphorylated signals as being the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-K) and focal adhesion kinase (FAK). Tyrosine phosphorylation of the p85 subunit of PI 3-K showed a biphasic response to TSP-1 stimulation, which corresponded to a biphasic activation of the lipid kinase. Treatment with both wortmannin and LY294002 inhibited PI 3-K activity of HVSMCs but did not affect tyrosine phosphorylation of the p85 regulatory subunit. TSP-1-stimulated FAK phosphorylation, however, was substantially reduced by these inhibitors, as was the TSP-1-induced chemotaxis of these cells. These results suggest that activation of PI 3-K is an early signal induced by TSP-1 and is critical for chemotaxis. Activation of this kinase precedes and may occur upstream from FAK phosphorylation, although the nature of the interaction between these 2 enzymes remains obscure.     

Overexpression of endothelial NO synthase induces angiogenesis in a co-culture model

OBJECTIVE: Angiogenesis is a complex multistep process that involves endothelial cell (EC) migration, proliferation and differentiation into vascular tubes. NO has been reported to be a downstream mediator in the angiogenic response to a variety of growth factors, but the mechanisms by which NO promotes neovessel formation is not clear. We hypothesized that NO directly contributes to EC migration and capillary tube formation. METHODS: Since previous studies have noted important biological differences between NO produced pharmacologically by NO-donor compounds compared to that from NO synthase (NOS), we used a cell-based gene transfer approach to increase NO production in a co-culture model of in vitro angiogenesis. Rat smooth muscle cells (SMCs) were transfected with plasmids containing VEGF(121), VEGF(165) (SMC(VEGF)), endothelial NOS (SMC(eNOS)) or the empty vector (SMC(Cont)). Expression of the eNOS in SMC(eNOS) was confirmed by Northern analysis, NADPH-diaphorase activity, and nitrite/nitrate levels, whereas VEGF production was confirmed using ELISA. Calf pulmonary artery ECs (CPAECs) were cultured on the fibrin matrix with (co-culture) or without underlying SMCs (monoculture). RESULTS: Co-culture of ECs with SMC(Cont) had no effect on EC differentiation compared with EC in monoculture (differentiation index, DI=2.8+/-3.4 vs. 2.1+/-2.8, respectively, NS). In contrast, co-culture with SMC(eNOS) resulted in the formation of extensive capillary-like structures within 48 h (DI=17.2+/-5.9, P<0.001 versus SMC(Cont)), which was significantly inhibited using a NOS inhibitor, L-NAME (3 mM) (DI=4.5+/-3.04, P<0.001 versus SMC(eNOS)). Similarly, SMC(VEGF121) induced an angiogenic response (DI=14.2+/-3.8), which was also significantly inhibited by L-NAME (DI=5.9+/-1.8, P<0.05). In using the Boyden chamber model, SMC(eNOS), but not SMC(Cont) increased EC migration to a similar extent as SMC(VEGF121), and both were significantly inhibited with L-NAME. CONCLUSIONS: These data support an important paracrine role for endogenously produced NO in EC migration and differentiation in vitro, and suggest that the cell-based eNOS gene transfer may be a useful approach to increase new blood vessel formation in vivo.     

Oxygen radical-mediated reduction in basal and agonist-evoked NO release in isolated rat heart

Oxygen free radicals (OFR) play a primary role in ischemia-reperfusion-mediated vascular dysfunction and this is paralleled by a loss of endothelial nitric oxide synthase (eNOS) activity. The authors tested whether a direct exposure to OFR may affect vascular relaxation by altering nitric oxide (NO) release. Effects of electrolysis(EL)-generated OFR on basal and agonist-evoked NO release were monitored in isolated rat hearts by oxyhemoglobin assay. Electrolysis-induced changes were compared with those obtained after 30 min perfusion with NOS and cyclooxygenase (COX) inhibitors NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) and indomethacin (INDO, 1 m M). Electrolysis-generated hydroxyl radical (.OH) formed by.O2-and H2O2 via the Fenton reaction as revealed by Electron Paramagnetic Resonance (EPR). After EL, basal NO release declined by 60% and coronary perfusion pressure (CPP) increased by approximately 70%. L-NAME/INDO perfusion similarly lowered NO release (-63%) but increased CPP less than EL (56+/-3%P<0.03 v post-EL). In presence of excess substrates and cofactors eNOS activity was not affected by EL. Both acetylcholine (ACh; 1 microM) and bradykinin (BK; 10 n M) had minimal effect in reversing EL-induced vasoconstriction, whereas both partially reversed L -NAME/INDO-mediated constriction. Sodium nitroprusside (SNP, 1 microM) completely reversed L-NAME/INDO constriction and partly countered that after EL (-38+/-2.5, P<0.001). Acetylcholine-evoked NO release was nearly abolished by both treatments whereas BK still elicited partial NO release after eNOS/cyclooxygenase inhibition (P<0.001) but not after EL. In conclusion, OFR severely impair NO-mediated coronary vasorelaxation affecting both basal and agonist-evoked NO release but not eNOS activity. However, EL also significantly blunts NOS/COX-independent vasodilation suggesting alteration of other vasodilatative pathways.     

Novel nitric oxide synthase--dependent mechanism of vasorelaxation in small arteries from hypertensive rats

To determine the mechanism(s) involved in vasorelaxation of small arteries from hypertensive rats, normotensive (NORM), angiotensin II-infused (ANG), high-salt (HS), ANG high-salt (ANG/HS), placebo, and deoxycorticosterone acetate-salt rats were studied. Third-order mesenteric arteries from ANG or ANG/HS displayed decreased sensitivity to acetylcholine (ACh)-induced vasorelaxation compared with NORM or HS, respectively. Maximal relaxations were comparable between groups. Blockade of Ca(2+)-activated K(+) channels had no effect on ANG versus blunting relaxation in NORM (log EC(50): -6.8+/-0.1 versus -7.2+/-0.1 mol/L). NO synthase (NOS) inhibition abolished ACh-mediated relaxation in small arteries from ANG, ANG/HS, and deoxycorticosterone acetate-salt versus blunting relaxation in NORM, HS, and placebo (% maximal relaxation: ANG: 2.7+/-1.8; ANG/HS: 7.2+/-3.2; NORM: 91+/-3.1; HS: 82.1+/-13.3; deoxycorticosterone acetate-salt: 35.2+/-17.7; placebo: 79.3+/-10.3), indicating that NOS is the primary vasorelaxation pathway in these arteries from hypertensive rats. We hypothesized that NO/cGMP signaling and NOS-dependent H(2)O(2) maintains vasorelaxation in small arteries from ANG. ACh increased NOS-dependent cGMP production, indicating that NO/cGMP signaling is present in small arteries from ANG (55.7+/-6.9 versus 30.5+/-5.1 pmol/mg), and ACh stimulated NOS-dependent H(2)O(2) production (ACh: 2.8+/-0.2 micromol/mg; N(omega)-nitro-l-arginine methyl ester hydrochloride+ACh: 1.8+/-0.1 micromol/mg) in small arteries from ANG. H(2)O(2) induced vasorelaxation and catalase blunted ACh-mediated vasorelaxation. In conclusion, Ca(2+)-activated K(+) channel-mediated relaxation is dysfunctional in small mesenteric arteries from hypertensive rats, and the NOS pathway compensates to maintain vasorelaxation in these arteries through NOS-mediated cGMP and H(2)O(2) production.     

Catechin Averts Experimental Diabetes Mellitus-Induced Vascular Endothelial Structural and Functional Abnormalities

Diabetes mellitus is associated with an induction of vascular endothelial dysfunction (VED), an initial event that could lead to the pathogenesis of atherosclerosis and hypertension. Previous studies showed that catechin, a key component of green tea, possesses vascular beneficial effects. We investigated the effect of catechin hydrate in diabetes mellitus-induced experimental vascular endothelial abnormalities (VEA). Streptozotocin (50 mg/kg, i.p., once) administration to rats produced diabetes mellitus, which subsequently induced VEA in 8 weeks by markedly attenuating acetylcholine-induced endothelium-dependent relaxation in the isolated aortic ring preparation, decreasing aortic and serum nitrite/nitrate concentrations and impairing aortic endothelial integrity. These abnormalities in diabetic rats were accompanied with elevated aortic superoxide anion generation and serum lipid peroxidation in addition to hyperglycemia. Catechin hydrate treatment (50 mg/kg/day p.o., 3 weeks) markedly prevented diabetes mellitus-induced VEA and vascular oxidative stress. Intriguingly, in vitro incubation of L-NAME (100 μM), an inhibitor of nitric oxide synthase, or Wortmannin (100 nM), a selective inhibitor of phosphatidylinositol 3-kinase (PI3K), markedly prevented catechin hydrate-induced improvement in acetylcholine-provoked endothelium-dependent relaxation in the diabetic rat aorta. Moreover, catechin hydrate treatment considerably reduced the elevated level of serum glucose in diabetic rats. In conclusion, catechin hydrate treatment prevents diabetes mellitus-induced VED through the activation of endothelial PI3K signal and subsequent activation of eNOS and generation of nitric oxide. In addition, reduction in high glucose, vascular oxidative stress, and lipid peroxidation might additionally contribute to catechin hydrate-associated prevention of diabetic VEA.     

Nitric oxide synthase inhibition increases aortic stiffness measured by pulse wave velocity in rats

OBJECTIVE: The present study was to examine whether endogenous nitric oxide (NO) plays a role in the regulation of vascular stiffness. METHODS: Pulse wave velocity (PWV) was determined as the time delay between the foot of pressure waves recorded simultaneously at the aortic arch and abdominal aorta (just above the bifurcation) in anesthetized Sprague-Dawley rats. A decrease in vascular compliance results in an increase in PWV. RESULTS: A bolus injection of a NO synthase inhibitor, L-NAME (30 mg/kg), significantly increased PWV, accompanied by an increase in blood pressure. Since changes in blood pressure are known to affect PWV, phenylephrine (PE) was administered to mimic the blood pressure changes induced by L-NAME, thus compensating for the pressure-dependent component of the PWV changes. At each given level of mean arterial pressure (MAP), PWV was significantly higher with L-NAME than with PE treatment, suggesting that acute withdrawal of endogenous NO reduces aortic compliance independent of changes in MAP. In rats chronically treated with L-NAME (0.5 g/l in drinking water) for 3 weeks, PWV was even higher than those acutely treated with L-NAME (at MAP=150 mmHg). This additional increase in vascular stiffness may be due to the remodeling of the vascular wall as a result of chronic NOS inhibition and hypertension. CONCLUSION: These data demonstrate that NO modulates vascular compliance independent of blood pressure changes and that an intact endogenous NO system is required to maintain normal vascular compliance.     

Fish Oil Blunted Nicotine-Induced Vascular Endothelial Abnormalities Possibly via Activation of PPARγ-eNOS-NO Signals

Nicotine exposure is associated with an induction of vascular endothelial dysfunction (VED), a hallmark of various cardiovascular disorders. The present study investigated the effect of fish oil in nicotine-induced experimental VED. VED was assessed by employing isolated aortic ring preparation, estimating aortic and serum nitrite/nitrate, aortic superoxide anion generation, and serum TBARS, and carrying out electron microscopic and histological studies of thoracic aorta. Nicotine (2 mg/kg/day, i.p., 4 weeks) administration produced VED in rats by attenuating acetylcholine-induced endothelium-dependent relaxation in the isolated aortic ring preparation, decreasing aortic and serum nitrite/nitrate concentration, impairing endothelial integrity, and inducing vascular oxidative stress. Treatment with fish oil (2 mL/kg/day p.o., 4 weeks) markedly prevented nicotine-induced endothelial functional and structural abnormalities and oxidative stress. However, administration of GW9662, a selective inhibitor of PPARγ, to a significant degree attenuated fish oil-associated anti-oxidant action and vascular endothelial functional and structural improvements. Intriguingly, in vitro incubation of L-NAME (100 μM), an inhibitor of nitric oxide synthase (NOS), markedly attenuated fish oil-induced improvement in endothelium-dependent relaxation in the aorta of nicotine-administered rats. Nicotine administration altered the lipid profile by increasing serum total cholesterol, which was significantly prevented by fish oil treatment. The vascular protective potential of fish oil in preventing nicotine-induced VED may pertain to its additional properties (besides its lipid-lowering effect) such as activation of PPARγ and subsequent possible activation of endothelial NOS and generation of nitric oxide, and consequent reduction in oxidative stress.     

Activation of endothelial nitric oxide synthase by cilostazol via a cAMP/protein kinase A- and phosphatidylinositol 3-kinase/Akt-dependent mechanism

We investigated the effect of cilostazol on nitric oxide (NO) production in human aortic endothelial cells (HAEC). Cilostazol increased NO production in a concentration-dependent manner, and NO production was also increased by other cyclic-AMP (cAMP)-elevating agents (forskolin, cilostamide, and rolipram). Cilostazol increased intracellular cAMP level, and that effect was enhanced in the presence of forskolin. In Western blot analysis, cilostazol increased phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser(1177) and of Akt at Ser(473) and dephosphorylation of eNOS at Thr(495). Cilostazol's regulation of eNOS phosphorylation was reversed by protein kinase A inhibitor peptide (PKAI) and by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Moreover, the cilostazol-induced increase in NO production was inhibited by PKAI, LY294002, and N(G)-nitro-l-arginine methyl ester hydrochloride (l-NAME), a NOS inhibitor. In an in vitro model of angiogenesis, cilostazol-enhanced endothelial tube formation, an effect that was completely attenuated by inhibitors of PKA, PI3K, and NOS. These results suggest that cilostazol induces NO production by eNOS activation via a cAMP/PKA- and PI3K/Akt-dependent mechanism and that this effect is involved in capillary-like tube formation in HAEC.     

Melatonin restores endothelium-dependent relaxation in aortic rings of pancreatectomized rats

In rats turned hyperglycemic by a subtotal pancreatectomy, a decreased relaxation response of aortic rings to acetylcholine (ACh) was found; this effect was amplified by preincubation in a high glucose medium (44 mmol/L). The relaxation response to ACh did not occur in endothelium-denuded rings or after the aortic rings were exposed to l-nitro-arginine methyl ester [L-NAME, a nitric oxide (NO) synthase inhibitor]. Incubation with the NO donor sodium nitroprusside (SNP) restored the impaired relaxation response seen in endothelium-denuded or L-NAME-treated aortic rings. Pancreatectomy decreased the vasorelaxation of aortic rings caused by SNP. Only in pancreatectomized rats, incubation in a high glucose medium impaired the relaxation effect of SNP. To assess whether melatonin preincubation reversed the impaired relaxation response to ACh (intact endothelium aortic rings) or to SNP (endothelium-denuded or L-NAME-treated rings) in hyperglycemic rats, cumulative dose-response curves were performed in the presence of 10(-5) mol/L melatonin. Melatonin preincubation did not modify ACh-induced relaxation of aortic rings in a normal glucose concentration but was highly effective in preventing the impairment of relaxation caused by a high glucose solution. Melatonin was also effective in restoring the impaired SNP-induced vasorelaxation seen in endothelium-denuded or L-NAME-treated aortic rings from hyperglycemic rats. The results further support the improvement by melatonin of the endothelial-mediated relaxation in blood vessels of diabetic rats.     

Vascular insulin/insulin-like growth factor-1 resistance in female obese Zucker rats

Because insulin resistance/diabetes may cause inordinate vascular complications in females, we have investigated the effects of insulin and insulin-like growth factor (IGF-1) on vascular reactivity in 12-week-old female Zucker obese (Ob) rats, a rodent model of insulin resistance and its lean (Ln) age-matched counterpart. Endothelium intact aortic rings from Ob animals and their Ln littermates (12 weeks of age) were subjected to contractile concentration responses to phenylephrine (PE) followed by relaxation to isoproterenol (Iso), with and without preincubation for 2 hours with cholera toxin (CTX; 1 microg/mL) or pertussis toxin (PTX; 2 microg/mL) and before and after incubation with either insulin or IGF-1 (100 nmol/L) for 1 hour. Systolic blood pressure was higher (138 +/- 3 v. 109 +/- 4 mm Hg; P <.0001) in the 12-week-old Ob rats. Contractile responses to PE were similar in both groups; however, both insulin and IGF-1 induced a paradoxical increase (P <.001) in contraction in Ob vasculature (929 +/- 92 v. 679 +/- 25 mg, respectively). CTX alone decreased contraction in the Ob (P <.02) and PTX in the Ln (P <.02), but there were no interactions between either IGF-1 or insulin and the toxins. Marked impairment of relaxation to Iso was seen in aortic rings of these female Ob rats (ED(50) = 2.6 micromol/L v. 418 nmol/L, P =.0002), an effect exacerbated by preincubation with either insulin or IGF-1 (P =.0001). Again, no role for G-proteins could be demonstrated. Insulin-dependent glucose uptake was severely impaired (P <.05) in aortic segments of the Ob insulin-resistant rats. Insulin receptor binding, tyrosine kinase activity (TKA), and abundance of several G-protein alpha subunits (inhibitory and stimulatory) in solubilized arterial membrane preparations (assessed by Western blot) were comparable in the 2 groups. These results indicate that resistance to the vascular actions of insulin/IGF-1 in female Ob rats is a postreceptor event that parallels glucose uptake resistance and is independent of G-proteins.     

Contribution of nitric oxide to the pathogenesis of cirrhotic cardiomyopathy in bile duct-ligated rats

BACKGROUND & AIMS: Decreased cardiac contractility and beta-adrenergic responsiveness have been observed in cirrhosis, but the etiology remains unclear. We aimed to test the role of nitric oxide (NO), a negative inotropic agent, in the pathogenesis of cirrhotic cardiomyopathy in a rat model. METHODS: Cirrhosis was induced by bile duct ligation. Four weeks after ligation or sham operation, cardiac levels of tumor necrosis factor (TNF)-alpha, guanosine 3,5'-cyclic monophosphate (cGMP), inducible NOS (NOS2), and endothelial constitutive NOS (NOS3) messenger RNA (mRNA) and protein were determined. Serum nitrite/nitrate level was measured. Cardiac contractile function was evaluated in isolated left ventricular papillary muscles in the absence and presence of the NOS inhibitor nitro-L-arginine methyl ester (L-NAME). RESULTS: Cardiac TNF-alpha, NOS2 mRNA and protein, cGMP, and serum interleukin (IL)-1beta and nitrite/nitrate levels were significantly higher in cirrhotic rats than sham controls. No significant differences in NOS3 mRNA or protein were found between cirrhotic and sham control rats. Baseline isoproterenol-stimulated papillary muscle contractile force was significantly lower in the cirrhotic group; with L-NAME incubation, contractile force increased significantly in cirrhotic rats but was unaffected in the controls. In normal papillary muscles, IL-1beta attenuated the contractility, but coincubation with L-NAME again reversed this attenuation. Incubation with the exogenous NO donor S-nitroso-N-acetyl-penicillamine also blunted papillary muscle contractility. CONCLUSIONS: These results suggest that cytokine-induced stimulation of NOS2 plays a significant role in the pathogenesis of cirrhotic cardiomyopathy.