Discordant effects of nicotine on endothelial cell proliferation, migration, and the inward rectifier potassium current

The inward rectifier K+ current (K(ir)) determines the resting membrane potential of endothelial cells. Basic fibroblast growth factor (bFGF) has been shown to activate K(ir) and acts as angiogenic factor and vasodilator. In contrast, nicotine has been demonstrated to reduce endothelium-dependent vasorelaxation by increasing radical formation. Aim of the present study was to investigate whether nicotine modulates K(ir) and if this plays a role in bFGF-mediated proliferation, migration and nitric oxide (NO)-formation of endothelial cells. Using the patch-clamp technique in cultured endothelial cells of human umbilical cord veins (HUVEC), we found characteristic K(ir), which were blocked by extracellular barium (100 micromol/l). Perfusion with nicotine (1 nmol/l-10 micromol/l) revealed a dose-dependent reduction of K(ir). The simultaneous perfusion with bFGF (50 ng/ml) and nicotine (10 micromol/l) still significantly reduced K(ir) (n = 8; P < 0.01). Cell counts revealed that bFGF-mediated proliferation of HUVEC was significantly inhibited when using 1-10 micromol/l nicotine (n = 8, P < 0.01). The bFGF-induced endothelial cell migration--examined using the "Fences-Migration-Assay"--was significantly reduced by 10 mumol/l nicotine (n = 12; P < 0.05). NO-production was examined using a cGMP-Radioimmunoassay. The significant bFGF-induced increase of cGMP-levels was reduced by nicotine (n = 10; P < 0.05). Our data indicate that the modulation of K(ir) seems to be an essential pathway in the antagonistic effects of nicotine on bFGF-mediated endothelial cell growth, migration and NO-formation.     

Margatoxin inhibits VEGF-induced hyperpolarization, proliferation and nitric oxide production of human endothelial cells

BACKGROUND: Vascular endothelial growth factor (VEGF) induces proliferation of endothelial cells (EC) in vitro and angiogenesis in vivo. Furthermore, a role of VEGF in K(+) channel, nitric oxide (NO) and Ca(2+) signaling was reported. We examined whether the K(+) channel blocker margatoxin (MTX) influences VEGF-induced signaling in human EC. METHODS: Fluorescence imaging was used to analyze changes in the membrane potential (DiBAC), intracellular Ca(2+) (FURA-2) and NO (DAF) levels in cultured human EC derived from human umbilical vein EC (HUVEC). Proliferation of HUVEC was examined by cell counts (CC) and [(3)H]-thymidine incorporation (TI).RESULTS: VEGF (5--50 ng/ml) caused a dose-dependent hyperpolarization of EC, with a maximum at 30 ng/ml (n=30, p<0.05). This effect was completely blocked by MTX (5 micromol/l). VEGF caused an increase in transmembrane Ca(2+) influx (n=30, p<0.05) that was sensitive to MTX and the blocker of transmembrane Ca(2+) entry 2-aminoethoxydiphenyl borate (APB, 100 micromol/l). VEGF-induced NO production was significantly reduced by MTX, APB and a reduction in extracellular Ca(2+) (n=30, p<0.05). HUVEC proliferation, examined by CC and TI, was significantly increased by VEGF and inhibited by MTX (CC: -58%, TI --121%); APB (CC --99%, TI--187%); N-monomethyl-L-arginine (300 micromol/l: CC: -86%, TI --164%). CONCLUSIONS: VEGF caused an MTX-sensitive hyperpolarization which results in an increased transmembrane Ca(2+) entry that is responsible for the effects on endothelial proliferation and NO production.     

Nitroglycerin-mediated vasorelaxation is modulated by endothelial calcium-activated potassium channels

OBJECTIVE: Recent in vitro data suggest, large conductance calcium-activated K+ channels (BKCa) modulate the vascular response to nitric oxide (NO). The in vivo implications and the characteristics of this interaction are not clear. This study firstly investigates whether modulation of BKCa affects the vascular response to nitroglycerin (NTG)-derived NO in vivo and in the isolated heart and secondly examines the influence of endothelial BKCa on NTG-mediated vasodilation in vitro. METHODS: The hypotensive effect of NTG was measured in conscious, chronically catheterized rats during i.v. infusions of iberiotoxin (IbTX, a selective inhibitor of BKCa) or placebo. Similarly, NTG-induced flow-changes in the isolated perfused rat heart were examined before and after IbTX treatment (0.1 microM). Concentration-relaxation curves to NTG in the presence of various K+ channel modulating agents were performed in vitro on porcine coronary arteries with and without intact endothelium. RESULTS: I.v. infusion of IbTX reduced the in vivo hypotensive effect of NTG by 55% (before IbTX: 32.0 +/- 3.0 mmHg, vs. after IbTX: 14.5 +/- 3.2 mmHg, P < 0.05) and nearly abolished NTG-induced increase in coronary flow in the isolated perfused heart (P < 0.05). In vitro, this effect depended on an intact endothelium (endothelium intact segments; NTG: pD2 = 5.8 +/- 0.1, Emax = 97.6 +/- 3.2% vs. NTG + IbTX: pD2 = 4.9 +/- 0.2, Emax = 49.7 +/- 6.2%, P < 0.05; endothelium denuded segments; NTG: pD2 = 6.9 +/- 0.1, Emax = 104.0 +/- 1.4% vs. NTG + IbTX: pD2 = 6.7 +/- 0.1, Emax = 100 +/- 1.2%, P > 0.05). CONCLUSION: The results suggest, that modulation of endothelial BKCa significantly affects NTG-induced vasorelaxation in vitro, in the isolated perfused heart and in vivo.     

Statins inhibit hypoxia-induced endothelial proliferation by preventing calcium-induced ROS formation

Pathological hypoxia plays an important role in many diseases, such as atherosclerosis, cancer, and rheumatoid arthritis. The aim of the present study was to examine the effects of different statins on hypoxia-induced endothelial cell signalling. Human umbilical cord vein endothelial cells (HUVEC) were treated with NaCN (CN, 2.5 mmol/l) to simulate a transient hypoxia. The CN-induced increase of endothelial cell numbers was significantly (n = 10, p < 0.01) reduced by the Ca(2+) chelator BAPTA (10 micromol/l), or the reactive oxygen species (ROS) scavenger N-acetylcysteine (ACC, 1 mmol/l), or the NAD(P)H-oxidase inhibitor diphenyleneiodonium (DPI, 5 micromol/l). In detail, cell numbers were (in percentage of control): 163.24 (CN), 90.06 (CN+ACC), 92.06 (CN+DPI). Intracellular-Ca(2+) and -ROS, analysed by fluorescence imaging, were significantly increased by CN. Interestingly, the CN-induced increase of ROS was in part Ca(2+)-dependent, whereas the Ca(2+) increase was not ROS-dependent. Simvastatin (5 micromol/l), fluvastatin (2.5 micromol/l), and cerivastatin (0.1 micromol/l) all reduced CN-induced proliferation, ROS generation and Ca(2+) increase. Cell viability was not reduced by the statins and the antiproliferative effect was completely reversed by mevalonate (500 micromol/l). In conclusion our study demonstrates that statins block hypoxia-associated endothelial proliferation by preventing the increase of Ca(2+) and ROS.     

Modulation of Ca2+-activated K+ channels in human vascular cells by insulin and basic fibroblast growth factor.

Insulin and basic fibroblast growth factor (bFGF) play an important role in the pathogenesis of atherosclerosis and have been shown to have vasodilatory effects. Since modulation of vascular ion channels determines membrane potential and thereby influences essential Ca2+-dependent intracellular pathways, we have investigated the effect of insulin and bFGF on Ca2+-activated K+ channels (BKCa) in human umbilical vein endothelial cells (HUVEC) and smooth muscle cells. The latter were obtained from either atherosclerotic plaques (SMCP) or from media segments (SMCM) of human coronary arteries. Using the patch-clamp technique, insulin (100 microU/ml) caused a significant increase in BKCa open-state probability in SMCP and HUVEC, whereas no significant changes were observed in SMCM. Basic FGF (30 ng/ml) revealed a significant increase in BKCa activity in HUVEC and a significant decrease in the BKCa open-state probability in SMCP, but caused no changes in SMCM. Thus, growth factors modulate vascular BKCa in a cell-type specific manner, which may be of importance concerning vasoactive and atherogenic effects of growth factors.     

Lysophosphatidylcholine-induced modulation of Ca(2+)-activated K(+)channels contributes to ROS-dependent proliferation of cultured human endothelial cells

Proliferation of endothelial cells plays a crucial role in the process of atherosclerotic plaque destabilization. The major component of oxidized low-density lipoprotein lysophosphatidylcholine (LPC) has been shown to promote endothelial proliferation by increasing the production of reactive oxygen species (ROS). Since K(+) channels are known to control the cell cycle, we investigated the role of Ca(2+)-activated K(+) channels (BK(Ca)) in the regulation of LPC-induced endothelial proliferation and ROS generation. A significant increase of cell growth induced by LPC (20 micromol/l; cell counts (CCs): +87%, thymidin incorporation: +89%; n = 12, P < 0.01) was observed, which was inhibited by the BK(Ca) inhibitor iberiotoxin (IBX; 100 nmol/l), by the NAD(P)H-oxidase inhibitor diphenyleneiodonium (5 micromol/l) and by transfection with antisense (AS) oligonucleotides against NAD(P)H oxidase, whereas N(G)-monomethyl-l-arginine (l-NMMA) further increased LPC-induced cell growth. Using the patch-clamp technique a significant increase of BK(Ca) open-state probability (control: 0.004 +/- 0.002; LPC: 0.104 +/- 0.035; n = 21, P < 0.05) by LPC was observed. Using dichlorofluorescein fluorescence microscopy a significant increase of ROS induced by LPC was reported, that was blocked by IBX and Ca(2+) antagonists. Intracellular Ca(2+) measurements revealed a capacitative Ca(2+) influx caused by LPC. Bioactivity of nitric oxide (NO) was measured using a [(3)H]-cGMP radioimmunoassay. LPC significantly decreased acetylcholine-induced NO synthesis. LPC significantly increased cGMP levels in endothelial cells transfected with AS, which was blocked by IBX. In conclusion, our results demonstrate that LPC activates BK(Ca) thereby increasing ROS production which induces endothelial proliferation. In addition LPC-induced BK(Ca)-activation contributes to increased cGMP levels, if ROS production is prevented by AS.     

Modulation of endothelial Ca(2+)-activated K(+) channels by oxidized LDL and its contribution to endothelial proliferation

OBJECTIVE: Oxidized low-density lipoprotein (oxLDL) plays an important role in causing endothelial dysfunction and initiating atherosclerosis. Some of the endothelial functions have been shown to be modulated by changes in cellular electrophysiological properties. Therefore, we analysed the effect of oxLDL on endothelial Ca(2+)-activated K(+) channels (BK(Ca)) and its contribution to oxLDL-mediated changes of proliferation and syntheses of nitric oxide (NO). METHODS: The patch-clamp technique was used to study the behavior of BK(Ca) in human endothelial cells of umbilical cord veins (HUVEC). Changes of intracellular Ca(2+) were measured by means of Fura-2 imaging. Cell counts and [3H]-thymidine incorporation were used to analyse endothelial proliferation. Synthesis of NO was measured by means of [3H]-cGMP radioimmunoassay. RESULTS: oxLDL (10 microg/ml) caused a significant increase of BK(Ca) activity, whereas preincubation of HUVEC with an antibody against the lectin-like-oxLDL-receptor-1 (LOX-1) abolished BK(Ca) activation. Fura-2 measurements revealed a biphasic increase of intracellular Ca(2+) after application of the atherogenic lipid. Endothelial proliferation was significantly increased by oxLDL. The highly selective BK(Ca) inhibitor iberiotoxin (100 nmol/l IBX) blocked this proliferative response. Acetylcholine-induced NO synthesis was significantly decreased by IBX. Interestingly, oxLDL significantly decreased acetylcholine-induced NO synthesis if the production of superoxide was not blocked by antisense oligonucleotides against the NAD(P)H-oxidase. CONCLUSIONS: Our data demonstrate that oxLDL activates BK(Ca), which plays an important role in oxLDL-mediated endothelial proliferation. Acetylcholine-induced NO synthesis is modulated by BK(Ca), whereas the reduction of acetylcholine-induced NO-synthesis by oxLDL is related to an increase in superoxide production.     

Thiamine corrects delayed replication and decreases production of lactate and advanced glycation end-products in bovine retinal and human umbilical vein endothelial cells cultured under high glucose conditions

Summary This study aimed at verifying whether thiamine, a co-enzyme which decreases intracellular glycolysis metabolites by allowing pyruvate and glyceraldheyde 3-phosphate to enter the Krebs cycle and the pentose-phosphate shunt, respectively, corrects delayed replication caused by high glucose concentrations in cultured human umbilical vein (HUVEC) and bovine retinal endothelial cells (BREC). After incubation in physiological (5.6 mmol/l) or high (28.0 mmol/l) glucose with or without 150 μmol/l thiamine, cells were counted and proliferation assessed by mitochondrial dehydrogenase activity. Lactate was measured in both cell types as an index of glycolytic activity and fluorescent advanced glycosylation end-products (AGE) concentration was determined in the HUVEC lysate. Both cell counts and proliferation assays in either of the cell types confirmed the impairment to cell replication induced by high glucose. When thiamine was added to cells kept under high glucose conditions, the number of surviving cells was significantly increased and the reduced cell proliferation appeared to be corrected. Lactate assays confirmed the increased production of this metabolite by BREC and HUVEC in high glucose, which was reduced by thiamine. Fluorescent AGE determination showed that thiamine may prevent non-enzymatic glycation in HUVEC. Thiamine restores cell replication, decreases the glycolytic flux and prevents fluorescent AGE formation in endothelial cells cultured in high glucose, suggesting that abnormal levels of glycolytic metabolite(s) may damage cells. [Diabetologia (1996) 39: 1263–1268] Received: 18 March 1996 and in revised form: 12 June 1996     

Divergent biological actions of coronary endothelial nitric oxide during progression of cardiac hypertrophy

Coronary endothelial NO synthase expression and NO bioactivity were investigated at sequential stages during the progression of left ventricular hypertrophy. Male guinea pigs underwent abdominal aortic banding or sham operation. Left ventricular contractile function was quantified in isolated ejecting hearts. Coronary endothelial and vasodilator function were assessed in isolated isovolumic hearts in response to boluses of bradykinin (0.001 to 10 micromol/L), substance P (0.01 to 100 micromol/L), diethylamine NONOate (DEA-NO) (0.1 to 1000 micromol/L), N(G)-monomethyl-L-arginine monoacetate (L-NMMA) (10 mmol/L), and adenosine (10 mmol/L). At a stage of compensated left ventricular hypertrophy (3 weeks), left ventricular endothelial NO synthase protein expression was unaltered (Western blot and immunocytochemistry). Vasoconstriction in response to L-NMMA was increased in banded animals compared with sham-operated animals (13.8+/-2.1% versus 6.2+/-1.3%, n=10; P<0.05), but agonist- and DEA-NO-induced vasodilation was similar in the 2 groups. At a stage of decompensated left ventricular hypertrophy (8 to 10 weeks), left ventricular endothelial NO synthase protein expression was significantly lower in banded animals (on Western analysis: banded animals, 7.8+/-0.4 densitometric units; sham-operated animals, 12.2+/-1.7 densitometric units; n=5; P<0.05). At this time point, vasoconstriction in response to L-NMMA was similar in the 2 groups, but vasodilatation in response to bradykinin (30.9+/-2.4% versus 39.7+/-2.2%, n=10; P<0.05), DEA-NO (26.2+/-1.8% versus 34.6+/-1.8%, n=10; P<0.05), and adenosine (24.3+/-2.0% versus 35.7+/-2.0%, n=10; P<0.01) was attenuated in banded animals. These findings indicate that there is an increase in the basal activity of NO (without a significant change in endothelial NO synthase expression) in early compensated left ventricular hypertrophy, followed by a decrease in both endothelial NO synthase expression and NO bioactivity during the transition to myocardial failure.     

HMG-CoA reductase inhibition improves endothelial cell function and inhibits smooth muscle cell proliferation in human saphenous veins

OBJECTIVES: This study examined effects of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitor cerivastatin on human saphenous vein (SV), endothelial cells (EC) and smooth muscle cells (SMC). BACKGROUND: Venous bypass graft failure involves EC dysfunction and SMC proliferation. Substances that improve EC function and inhibit SMC proliferation would be of clinical relevance. METHODS: Both EC and SMC were isolated from SV. Endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production were analyzed by immunoblotting and porphyrinic microsensor. The SMC proliferation was assayed by 3H-thymidine incorporation. Protein kinases and cell cycle regulators were analyzed by immunoblotting. RESULTS: Cerivastatin (10(-9) to 10(-6) mol/liter) enhanced eNOS protein expression and NO release (about two-fold) in EC in response to Ca2+ ionophore (10(-6) mol/liter). This was fully abrogated by the HMG-CoA product mevanolate (2 x 10(-4) mol/liter). In SMC, platelet-derived growth factor (5 ng/ml) enhanced 3H-thymidine incorporation (298 +/- 23%, n = 4), activated cyclin-dependent kinase (Cdk2), phosphorylated Rb and down-regulated p27Kip1 (but not p21CiP1). Cerivastatin reduced the 3H-thymidine incorporation (164 +/- 11%, p < 0.01), inhibited Cdk2 activation and Rb phosphorylation, but did not prevent p27Kip1 down-regulation, nor p42mapk and p70S6K activation. Mevalonate abrogated the effects of cerivastatin on Cdk2 and Rb but only partially rescued the 3H-thymidine incorporation (from 164 +/- 11% to 211 +/- 13%, n = 4, p < 0.01). CONCLUSIONS: In humans, SVEC inhibition of HMG-CoA/mevalonate pathway contributes to the enhanced eNOS expression and NO release by cerivastatin, whereas in SMC, inhibition of this pathway only partially explains cerivastatin-induced cell growth arrest. Inhibition of mechanisms other than p42mapk and p70S6K or Cdk2 are also involved. These effects of cerivastatin could be important in treating venous bypass graft disease.     

波动性高糖对人脐静脉血管内皮细胞的损伤及其机制的研究

目的 观察波动性高糖在体外诱导人脐静脉血管内皮细胞(human umbilical vein endothelial cell,HUVEC)的损伤作用,并探讨其机制.方法 以HUVEC为研究对象,体外培养至第三代,实验分为:A组为正常组(正常培养的细胞),B组给予恒定加入5.5mmol/L 葡萄糖(Glu),C组给予恒定加入7.8mmol/L Glu,D组给予每24h轮换加入5.5mmol/L/7.8mmol/L Glu,E组给予恒定加入14.5mmol/L Glu,F组给予每24h轮换加入5.5mmol/L/14.5mmol/L Glu,G组给予恒定加入20mmol/L Glu,H组给予每24h轮换加入5.5mmol/L/20mmol/L Glu各组分别与HUVEC孵育5d,测定各组细胞液中的细胞活力(MTT)指标.测定正常对照组、恒定性高糖组(E组和G组)及波动性高糖组(F组和H组)细胞上清液中丙二醛(MDA)的含量、超氧化物歧化酶活力(SOD)、一氧化氮(NO)的含量、细胞间黏附分子-1(ICAM-1)含量及细胞凋亡率,并在显微镜下观察细胞形态变化.结果 培养液中SOD、NO的含量在波动性高糖组与恒定性高糖组均与对照组比较,差异有统计学意义(P＜0.05);培养液中MDA及ICAM-1的含量在波动性高糖组与恒定性高糖组均与对照组比较,差异有统计学意义(P＜0.05).波动性高糖与恒定性高糖均可导致HUVECs出现凋亡的形态学变化,波动性高糖组的凋亡率与恒定性高糖组比较,差异有统计学意义(P＜0.05).结论 (1)波动性高糖和恒定性高糖对人脐静脉血管内皮细胞均有损伤,且波动性高糖对其损伤更大.(2)波动性高糖体外诱导人脐静脉血管内皮细胞损伤的机制可能与其氧化应激水平更高,炎症反应加重及细胞凋亡率增加有关.     

Nitric oxide inhibits proliferation of human endothelial cells via a mechanism independent of cGMP.

Endothelial cell-derived nitric oxide (NO) has been suggested to inhibit smooth muscle cell proliferation, resulting in the reduction of intimal hyperplasia during atherogenesis. The present study investigates the role of NO from exogenous and endogenous sources on the proliferation of human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (CAEC). Three different NO-generating compounds [sodium nitroprusside (SNP), S-nitroso-glutathione (GSNO) and S-nitroso-acetylpenicillamine (SNAP)] were found to inhibit endothelial cell proliferation measured with three independent methods (cell counting, [3H]thymidine incorporation, DNA histograms) with significant inhibition occurring at concentrations > or = 100 microM. Growth-inhibiting effects were observed after long-term treatment (18-96 h) as well as after short stimulation with NO donors (10 min with a subsequent NO donor-free culture period of 18 h) and were comparable in culture medium (20% serum, growth factor supplementation) and serum-deficient medium (1% serum). The NO donor effects were mediated by the release of NO as they were prevented by NO scavenging. Superoxide dismutase (SOD) was found not to interfere with these effects suggesting that peroxynitrite formation was unlikely to be involved. 1H-[l,2,4]Oxadiazolo[4,3,-alpha]quinoxalin-1-one (ODQ), a specific inhibitor of the soluble guanylate cyclase, was observed not to alter the antiproliferative effects of NO donors although it completely prevented NO-mediated increase of cyclic guanosine 3',5'-monophosphate (cGMP), suggesting that the NO-induced growth inhibition was not mediated by cGMP. Furthermore, inhibition of endogenous NO production by N-nitro-L-arginine methylester (L-NAME) did not affect endothelial cell growth regardless of using serum plus growth factor supplement, growth factor supplement alone, or thrombin to stimulate proliferation. We suggest that constitutively synthesized NO may not regulate endothelial cell proliferation whereas the growth-inhibiting NO effects may occur when an inducible NO synthase associated with a persistently high NO production is expressed in the atherosclerotic vessel wall.     

Endothelium-dependent relaxation by acetylcholine is impaired in hypertriglyceridemic humans with normal levels of plasma LDL cholesterol

OBJECTIVES: Patients with high triglyceride (of which very low density lipoproteins [VLDL] are the main carriers), but with normal low density lipoprotein (LDL) cholesterol levels, were examined for in vivo endothelium function status. BACKGROUND: Very low density lipoproteins inhibit endothelium-dependent, but not -independent, vasorelaxation in vitro. METHODS: Three groups were studied: 1) healthy volunteers (n = 10; triglyceride 1.24+/-0.14 mmol/liter, LDL cholesterol 2.99+/-0.24 mmol/liter); 2) hypertriglyceridemic (n = 11; triglyceride 6.97+/-1.19 mmol/liter, LDL cholesterol 2.17+/-0.2 mmol/liter, p < 0.05); and 3) hypercholesterolemic (n = 10; triglyceride 2.25+/-0.29 mmol/liter, LDL cholesterol 5.61+/-0.54 mmol/liter; p < 0.05) patients. Vasoactive responses to acetylcholine, sodium nitroprusside, noradrenaline, N(G)-monomethyl-L-arginine and postischemic hyperemia were determined using forearm venous occlusion plethysmography. RESULTS: Responses to acetylcholine (37 microg/min) were significantly dampened both in hypercholesterolemic (% increase in forearm blood flow: 268.2+/-62) and hypertriglyceridemic patients (232.6+/-45.2) when compared with controls (547.8+/-108.9; ANOVA p < 0.05). Responses to sodium nitroprusside (at 1.6 microg/min: controls vs. hypercholesterolemics vs. hypertriglyceridemic: 168.7+/- 25.1 vs. 140.6+/-38.9 vs. 178.5+/-54.5% increase), noradrenaline, N(G)-monomethyl-L-arginine and postischemic hyperemic responses were not different among the groups examined. CONCLUSIONS: Acetylcholine responses are impaired in patients with pathophysiologic levels of plasma triglycerides but normal plasma levels of LDL cholesterol. The impairment observed was comparable to that obtained in hypercholesterolemic patients. We conclude that impaired responses to acetylcholine normally associated with hypercholesterolemia also occur in hypertriglyceridemia. These findings identify a potential mechanism by which high plasma triglyceride levels may be atherogenic independent of LDL cholesterol levels.     

miR-206对血管内皮细胞增殖的影响及其机制

目的研究miR-206对血管内皮细胞增殖的影响及其机制,为临床动脉硬化疾病提供新的诊疗方向。方法收集临床病理首次确诊为糖尿病下肢动脉粥样硬化病变的患者病变处动脉内皮组织和正常内皮组织,qPCR检测miR-206的表达;而后通过基础实验研究将HUVEC分为未转染组、miR-206-mimics组和miR-206-inhibitor组,观察转染效率;三组细胞均采用CCK8检测细胞增殖情况,Transwell进行迁移能力评估,流式细胞仪检测细胞周期情况,Western blot检测Cx43表达情况。结果糖尿病动脉硬化患者下肢动脉(均为胫腓动脉)病变处内皮细胞miR-206的mRNA表达较正常组织内皮细胞明显增加(P0.05);HUVEC转染miR-206-mimics后,miR-206表达显著升高(P0.05),转染miR-206-inhibitor后,miR-206表达明显降低(P0.05);在HUVEC转染miR-206-mimics 24 h后,细胞增殖率相比于未转染组和miR-206-inhibitor组要明显降低(P0.05);在HUVEC转染miR-206-mimics后,细胞迁移能力明显降低(P0.05),细胞周期在G2期发生阻滞,而HUVEC转染miR-206-inhibitor后细胞迁移能力提高(P0.05);miR-206-mimics组细胞Cx43表达明显降低,而miR-206-inhibitor组细胞Cx43表达明显增加。结论 miR-206可以抑制血管内皮细胞的增殖,将内皮细胞阻滞在有丝分裂G2期,并且抑制内皮细胞迁移,其对内皮细胞增殖的影响可能是通过调节Cx43表达来实现的。     

Dietary supplementation with marine omega-3 fatty acids improve systemic large artery endothelial function in subjects with hypercholesterolemia

OBJECTIVE: This work was undertaken to determine whether dietary supplementation with marine omega-3 fatty acids improve systemic large artery endothelial function in subjects with hypercholesterolemia. BACKGROUND: Marine omega-3 fatty acids improve vascular function, but the underlying mechanism(s) are unclear. We studied the effects of marine omega-3 fatty acids on large artery endothelial function in subjects with hypercholesterolemia. METHODS: Hypercholesterolemic subjects with no other known cause for endothelial dysfunction were recruited to a prospective, placebo-controlled, randomized, double-blind, parallel-group study. Treatment with omega-3 fatty acids at a dose of 4 g/day (n = 15/group) was compared with placebo, at the beginning (day 0) and end (day 120) of a four-month treatment period. Endothelial function was assessed pre- and posttreatment by noninvasive ultrasonic vessel wall tracking of brachial artery flow-mediated dilation (FMD). RESULTS: Treatment with marine omega-3 fatty acids resulted in a significant improvement in FMD (0.05 +/- 0.12 to 0.12 +/- 0.07 mm, p < 0.05) and a significant reduction in triglycerides (2.07 +/- 1.13 to 1.73 +/- 0.95 mmol/liter, p < 0.05), whereas treatment with placebo resulted in no change in FMD (0.03 +/- 0.10 to 0.04 +/- 0.10 mm) or triglycerides (2.29 +/- 2.09 to 2.05 +/- 1.36 mmol/liter) (both p < 0.05 treated compared with control). Responses to sublingual glyceryl trinitrate were unchanged. CONCLUSIONS: Marine omega-3 fatty acids improve large artery endothelium-dependent dilation in subjects with hypercholesterolemia without affecting endothelium-independent dilation.     

Evidence for the stimulatory effect of resveratrol on Ca(2+)-activated K+ current in vascular endothelial cells

OBJECTIVE: Resveratrol, a natural phytoalexin compound, is present in grapes and wine, and it can produce vasorelaxation. However, little is known of its mechanisms of action on ionic currents in endothelial cells. METHODS: The effect of resveratrol on Ca(2+)-activated K+ currents in an endothelial cell line (HUV-EC-C) originally derived from human umbilical vein was investigated with the aid of the patch-clamp technique. RESULTS: In the whole-cell configuration, resveratrol reversibly increased the amplitude of K+ outward currents. The increase in outward current caused by resveratrol was greatly inhibited by iberiotoxin (200 nM) or paxilline (1 microM), but not by glibenclamide (10 microM), tamoxifen (10 microM), or beta-bungarotoxin (200 nM). Thus, this outward current is believed to be Ca(2+)-activated K+ current (I K(Ca)). In the inside-out configuration, bath application of resveratrol (30 microM) caused no change in the single-channel conductance, but increased the activity of large-conductance Ca(2+)-activated K+ (BKCa) channels. Resveratrol enhanced the channel activity in a concentration-dependent manner. The EC50 value for resveratrol-induced channel activity was 20 microM. The resveratrol-stimulated increase in the channel activity was independent of internal Ca2+. Resveratrol (30 microM) also shifted the activation curve of BKCa channels to less positive membrane potentials. The change in the kinetic behavior of BKCa channels caused by resveratrol in these cells in due to an increase in mean open time and a decrease in mean closed time. In a pancreatic islet endothelial cell line (MS1), resveratrol (30 microM) also increased the activity of intermediate-conductance KCa channels. CONCLUSIONS: These results provide evidence that in addition to the presence of antioxidative activity, resveratrol can also stimulate KCa channels in endothelial cells. The direct stimulation of these KCa channels by resveratrol may be responsible for its effect on the functional activities of endothelial cells.     

Src family kinases and nitric oxide production are required for hepatocyte growth factor-stimulated endothelial cell growth

Hepatocyte growth factor (HGF) is a potent mitogen for vascular endothelial cells (EC); however, signal transduction pathways for HGF-stimulated EC growth remain unclear. In the present study we investigated the role of Src family kinases and nitric oxide (NO) in HGF-stimulated EC growth. Human umbilical vein endothelial cells (HUVEC) were stimulated with HGF and NO was measured by an NOx analyzing HPLC system. Activation of ERK1/2 and p38 MAPK was assessed by Western blot. NO production in HUVEC increased 1.8-fold by HGF. A Src family kinases inhibitor PP1 inhibited HGF-stimulated NO production by 71%. HUVEC growth increased 1.9-fold in cell number by HGF. PP1 and Nitro-L-arginine methylester (L-NAME) inhibited HGF-stimulated HUVEC growth by 51 and by 71%. ERK1/2 and p38 MAPK were phosphorylated by HGF and a MEK inhibitor PD98059 and a p38 MAPK inhibitor SB203580 inhibited HGF-stimulated HUVEC growth by 66% and by 58%; however, HGF-induced phosphorylation of ERK1/2 and p38 MAPK was not inhibited by L-NAME, indicating that NO is not an upstream activator of ERK1/2 and p38 MAPK. These findings demonstrated that Src family kinases regulate HGF-stimulated NO production in HUVEC and that HGF stimulates HUVEC growth through NO-dependent and NO-independent pathways.     

Plasma markers of NO synthase activity in women after ovarian hyperstimulation: influence of estradiol on ADMA

The beneficial effects of estrogen on vasculature are partially explained by an estrogen-induced increase in nitric oxide (NO) synthesis. Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of NO synthase. In the present study, the effect of 17beta-estradiol (E2) on ADMA and NO synthesis was investigated both in vivo and in vitro. Plasma NO and ADMA levels were measured in healthy women at a low menstrual estrogenic stage (E2 < 100 pg/ml) and in women who were undergoing ovarian hyperstimulation (E2 > 2000 pg/ml) before in vitro fertilization embryo transfer. Primary human umbilical vein endothelial cell (HUVEC) cultures were incubated with and without 100 nM E2 for 24 hours and the NO and ADMA levels of the media were measured. A nitric oxide analyzer was used for the detection of NO metabolites. ADMA and L-arginine were measured by high-performance liquid chromatography after precolumn derivatization with o-phthaldialdehyde. The IVF patients with high plasma E2 concentrations had significantly lower (48%, n = 12) plasma ADMA and higher (56%, n = 14) NO levels than the women at a low estrogenic stage. The incubation of HUVEC cultures with estradiol resulted in a significant decrease (47%, n = 10) in ADMA and an increase (46%, n = 10) in NO concentration in the culture media. Estradiol, by reducing ADMA, may therefore facilitate NO synthesis in endothelial cells. The protective effects of estradiol on vasculature may partly be related to a reduction in ADMA levels.     

Intracellular pH, Na+- and K+-activities at the onset of St. Thomas' cardioplegia: a study with ionselective microelectrodes

Cellular edema and cardiac arrhythmias are often closely related to intracellular ionic alterations and, moreover, are an actual problem of clinical cardioplegia and ischemia of the heart. In order to investigate whether the clinically widely used cardioplegic solution of the St. Thomas' Hospital may predispose for these complications, membrane potential (EM), intracellular pH (pHi), and intracellular sodium and potassium activity (aiNa- and aiK) in sheep heart Purkinje fibres were directly measured by means of neutral-carrier ion-selective microelectrodes during equilibration of the fibres' extracellular space with the cardioplegic solution. The experimental temperature was 35 degrees C throughout. During control conditions under superfusion of a HEPES-buffered Tyrode solution EM was -74.4 +/- 5.1 mV (n = 39), pHi was 7.18 +/- 0.05 (n = 16), and aiNa and aiK were 7.6 +/- 1.4 mmol/l (n = 15) and 118.9 +/- 4.6 mmol/l (n = 15) respectively. Superfusing the Purkinje strand for 10 minutes with the cardioplegic St. Thomas' solution led to a depolarization to -57.3 +/- 4.7 mV (n = 21), a slight aiNa decrease to 6.7 +/- 1.6 mmol/l (n = 15; p less than 0.01; cardioplegic solution without procaine) and an increase of aiK to 127.1 +/- 4.4 mmol/l (n = 6; p less than 0.01). pHi needed 15 minutes to reach a steady state value of 7.25 +/- 0.05 (n = 9). The alterations on post-cardioplegic reperfusion with Tyrode solution were reversible within 15 minutes. Simulation of the clinical situation of ice-cold application of the solution by inhibiting the cellular Na/K pump via 0.1 mmol/l dihydroouabaine added to the St. Thomas' solution resulted in a marked increase of aiNa to 10.7 +/- 1.5 mmol/l (n = 6; p less than 0.01; no procaine) during cardioplegic superfusion.(ABSTRACT TRUNCATED AT 250 WORDS)