Mfn2基因突变体诱导大鼠颈动脉球囊损伤后血管平滑肌细胞凋亡

目的:探讨线粒体融合素2(Mfn2)基因蛋白激酶A(PKA)磷酸化位点突变对大鼠颈动脉球囊损伤后血管平滑肌细胞凋亡的影响.方法:建立大鼠颈动脉球囊损伤模型,感染组分别以携带全长Mfn2基因的重组腺病毒(Adv-Mfn2)、携带不同Mfn2基因PKA磷酸化位点突变体的重组腺病毒(Adv-Mfn2-S442A,Adv-Mfn2-S442D)和仅含有半乳糖苷酶基因的对照腺病毒(Adv-LacZ)感染损伤的颈总动脉段,以注射磷酸盐缓冲液(PBS)作为未感染对照组,同时以假手术组作为正常对照组,分别于术后5 d、14 d取材,应用免疫组化检测外源基因的表达,原位末端转移酶标记法(TUNEL)检测凋亡细胞,苏木精-伊红染色行组织形态学分析.结果:术后5 d,免疫组化证实Adv-Mfn2、Adv-Mfn2-S442A、Adv-Mfn2-S442D 3组Mfn2蛋白表达水平明显增加,TUNEL染色显示Adv-Mfn2、Adv-Mfn2-S442A组凋亡细胞数较对照组、Adv-LacZ组和Adv-Mfn2-S442D组明显增加(P<0.01);与Adv-Mfn2组相比,Adv-Mfn2-S442A 组凋亡细胞数更多(P<0.01).术后14 d,苏木精-伊红染色显示Adv-Mfn2组、Adv-Mfn2-S442A组内膜/中膜面积比明显低于对照组、Adv-LacZ组和Adv-Mfn2-S442D组(P<0.01);且Adv-Mfn2-S442A组内膜/中膜面积比值较Adv-Mfn2组更低(P<0.01).结论:高表达Mfn2基因可促进大鼠颈动脉球囊损伤后血管平滑肌细胞凋亡,且Mfn2-S442A促凋亡作用更强,而Mfn2-S442D无此作用.     

重组诱导型一氧化氮合酶在V79细胞中的表达及其对血管平滑肌细胞增殖的影响

目的探讨成纤维细胞基因转染后产生的一氧化氮对血管平滑肌细胞增殖的影响. 方法将诱导型一氧化氮合酶基因通过真核表达载体转入V79成纤维细胞中,用Griess法测定转染细胞生成的一氧化氮的量.将含有一氧化氮的细胞转染上清液加到培养的血管平滑肌细胞中,观察对平滑肌细胞生长状态的影响,用流式细胞仪分析细胞周期的变化.     

重组人诱导型一氧化氮合酶转染平滑肌细胞及对其增殖的影响

目的探讨血管平滑肌细胞基因转染后对血管平滑肌细胞增殖的影响. 方法将诱导型一氧化氮合酶基因通过真核表达载体转入血管平滑肌细胞中,用Griess法测定转染细胞生成的一氧化氮的量;观察对平滑肌细胞生长状态的影响,用流式细胞仪分析细胞周期的变化.     

血管紧张素原对动脉粥样硬化的作用及机制

目的探讨血管紧张素原(AGT)对动脉粥样硬化(AS)的作用机制。方法构建AS小鼠模型,RT-PCR检测AS小鼠斑块组织和正常小鼠主动脉血管组织中AGT的表达水平。以人巨噬细胞RAW264.7和人主动脉平滑肌细胞(HA-VSMC)为研究对象,转染siRNA AGT、siRNA control,实时荧光定量PCR(RT-PCR)检测转染后的AGT水平。MTT检测细胞转染后细胞的增殖情况,流式细胞仪检测转染后的细胞凋亡情况。Western印迹检测转染后细胞中Caspase-3、Bcl-2、Bax蛋白表达水平。结果 AS小鼠斑块组织中AGT的表达水平高于正常小鼠主动脉组织(P<0.01)。siRNA AGT可以有效抑制巨噬细胞RAW264.7和人主动脉平滑肌细胞HA-VSMC中AGT的转录表达。转染siRNA AGT后的巨噬细胞RAW264.7存活率与siRNA control组比较差异显著(P<0.01),转染siRNA AGT后的主动脉平滑肌细胞HA-VSMC存活率与siRNA control组差异显著(P<0.01),抑制AGT的表达可以抑制巨噬细胞和平滑肌细胞的增殖。转染siRNA AGT后的巨噬细胞RAW264.7凋亡率显著高于siRNA control组(P<0.01);转染siRNA AGT后的主动脉平滑肌细胞HA-VSMC凋亡率显著高于siRNA control组(P<0.01)。转染siRNA AGT后的巨噬细胞RAW264.7和主动脉平滑肌细胞HA-VSMC中Caspase-3、Bax蛋白表达量显著高于siRNA control组,Bcl-2蛋白表达量显著低于siRNA control组(均P<0.01)。结论AGT在鼠AS斑块组织中过表达,抑制AGT可以抑制AS相关细胞增殖,促进细胞凋亡,作用机制与凋亡相关蛋白Caspase-3、Bcl-2、Bax有关。     

TFPI基因转染对血管平滑肌细胞中细胞凋亡抑制蛋白的调控

目的研究组织因子途径抑制物(TFPI)基因转染对大鼠血管平滑肌细胞中细胞凋亡抑制蛋白(IAP)表达的影响,探讨TFPI诱导细胞凋亡的可能机制。方法将含有人TFPI基因的重组腺病毒或含β-半乳糖苷酶(LacZ)基因的重组腺病毒或DMEM在体外分别转染大鼠血管平滑肌细胞,用ELISA方法测定转染后细胞中TFPI蛋白的表达水平,用RT-PCR方法测定基因转染后不同时间点细胞中c-IAP1mRNA的表达,用Western-blot方法检测基因转染后不同时间点细胞中survivin的表达。结果基因转染后1天在血管平滑肌细胞中即可检测到TFPI蛋白表达,峰值出现在转染后第3天;基因转染后3天和7天,TFPI组c-IAP1 mRNA的表达与对照组相比明显减少(P0.05);基因转染后3、5、7天,TFPI组survivin的表达与对照组相比明显减少(P0.05),且具有明显的时间依赖性。结论 TFPI可能通过抑制IAP的表达来发挥诱导平滑肌细胞凋亡的作用,从而抑制冠状动脉介入治疗后再狭窄发生。     

内皮型一氧化氮合酶基因体内转染对大鼠颈总动脉损伤后血管狭窄程度的影响

目的 将内皮型一氧化氮合酶基因转染至球囊损伤后的大鼠颈总动脉中，观察其对血管狭窄程度的影响。方法 将大鼠分为正常对照组、空白对照组、pcDNA3．1（-）组和内皮型一氧化氮合酶组。除正常对照组外均行颈总动脉内膜球囊损伤术，术中采用脂质体栽体FuGENE6分别介导真核表达栽体pcDNA3．1-内皮型一氧化氮合酶和pcDNA3，1（-）转染至损伤后的内皮型一氧化氮合酶组和pcDNA3．1（-）组大鼠血管中。于术后2用处死大鼠，以逆转录-聚合酶链反应法检测转染后体外培养的血管平滑肌细胞中内皮型一氧化氮合酶mRNA的表达。并于术后1月和2月处死大鼠，检测颈总动脉血管狭窄的程度。结果 2周后转染pcDNA3．1-内皮型一氧化氮合酶基因的血管平滑肌细胞中可以检测出内皮型一氧化氮合酶mRNA的表达，1月和2月后转染pcDNA3．1-内皮型一氧化氮合酶基因的颈总动脉狭窄程度明显小于空白对照组和pcDNA3．1（-）组（P〈0．01）。结论 内皮型一氧化氮合酶基因体内转染可以在血管平滑肌细胞中成功表达，且可以明显改善球囊损伤后颈总动脉狭窄的程度。     

CXCR4基因修饰骨髓间充质干细胞移植对损伤血管内皮化的影响

目的:探讨基质细胞衍生因子受体4(CXCR4)基因修饰的骨髓间充质干细胞(BMSCs)移植对大鼠颈动脉球囊成形术后内皮修复的影响。方法:运用密度梯度离心法、贴壁培养并扩增BMSCs,用携带增强绿色荧光蛋白(EGFP)的慢病毒转染BMSCs,获得CXCR4修饰的BMSCs(EGFP-CXCR4-BMSCs)。建立大鼠颈动脉球囊损伤模型,随机分成PBS移植对照组(对照组)、空病毒转染BMSCs组(EGFP-BMSCs组)及CXCR4转染BMSCs移植组(CXCR4-BMSCs组)。MTT分别检测慢病毒转染细胞后第1、2、3、4、5d的细胞活力,计算细胞增殖率;细胞移植后14d取血管组织行免疫荧光检测GFP表达,28d行免疫组织化学染色检测血管内膜血小板-内皮细胞黏附分子(CD31)及血管内皮生长因子(VEGF)的表达;苏木精-伊红(HE)染色检测血管形态学变化;Western blot检测血管局部内皮型一氧化氮合酶(eNOS)的表达。结果:BMSCs转染EGFP空病毒载体及CXCR4后,与对照组相比,各时间点细胞增殖率无统计学差异。CXCR4-BMSCs组损伤血管处有较多的阳性GFP表达。CXCR4-BMSCs与EGFP-BMSCs组血管新生内膜面积、新生内膜/中膜面积均较对照组减轻(均P0.05),CXCR4-BMSCs组尤为明显(P0.05)。CXCR4-BMSCs与EGFP-BMSCs组血管内膜较对照组相比均有CD31及VEGF的表达(均P0.05),损伤血管eNOS表达量较对照组均明显增高(均P0.05),以CXCR4-BMSCs组较为明显。结论:CXCR4基因修饰不影响BMSCs的增殖,CXCR4-BMSCs较单纯EGFP-BMSCs移植更能促进动脉损伤后早期再内皮化,降低血管成形术后再狭窄。     

重组腺病毒AdCat对血管平滑肌细胞

目的探讨腺病毒载体介导的过氧化氢酶基因转染对体外培养的人血管平滑肌细胞增殖与凋亡的影响.方法用含过氧化氢酶基因的重组腺病毒(AdCat)转染人血管平滑肌细胞,采用Western blot 方法检测血管平滑肌细胞过氧化氢酶的表达,应用细胞计数方法观察血管平滑肌细胞的增殖活性,应用流式细胞术和Hoechst 33258 染色等方法对血管平滑肌细胞的凋亡进行研究.结果Western blot显示AdCat转染后血管平滑肌细胞过氧化氢酶表达明显增多;细胞计数显示AdCat组明显抑制细胞增殖,与阴性对照组、空白对照组相比差异有统计学意义(P<0.01);流式细胞术显示AdCat组与阴性对照组、空白对照组比较凋亡率明显增加(P<0.01).经Hoechst 33258染色后,AdCat组凋亡细胞明显多于空白对照组.结论重组腺病毒AdCat转染导致血管平滑肌细胞过氧化氢酶过度表达,抑制血管平滑肌细胞的增殖,促进血管平滑肌细胞的凋亡.     

多沙唑嗪对血管狭窄和血清一氧化氮的影响

目的探讨多沙唑嗪对兔腹主动脉球囊损伤后血管狭窄的影响,及其与血清一氧化氮、血管内膜中膜平滑肌细胞增生的关系。方法23只新西兰兔随机分为正常对照组、球囊损伤组、多沙唑嗪组。正常对照组不予任何方式处理。另两组行腹主动脉球囊损伤术,同时多沙唑嗪组应用多沙唑嗪控释片4mg/d,观察各组血清一氧化氮以及血管损伤处血管狭窄、血管内膜中膜增殖细胞核抗原(PCNA)的变化。结果应用多沙唑嗪4周后多沙唑嗪组血清一氧化氮含量增高,血管损伤处血管内膜、外膜增生减轻,新生内膜面积减少,管腔面积增加,内弹力层和外弹力层包围面积增加,血管内膜中膜PCNA阳性平均灰度降低。结论多沙唑嗪可以抑制球囊损伤后兔腹主动脉血管的狭窄,抑制血管内膜中膜平滑肌细胞增殖,增加血清一氧化氮含量。     

骨髓间充质干细胞局部移植对兔腹主动脉成形术后再狭窄和平滑肌细胞增殖的影响

目的探讨兔腹主动脉血管成形术后,骨髓间充质干细胞(BMSCs)移植对兔腹主动脉再狭窄和平滑肌细胞增殖的影响及机制。方法将60只新西兰大白兔随机分为对照组、损伤组、移植组各20只。所有动物均穿刺股动脉,对照组不进行球囊扩张损伤腹主动脉,损伤组和移植组送入球囊扩张损伤腹主动脉,损伤组注射等量的磷酸盐缓冲液(PBS),移植组用经4,6-联脒-2-苯基吲哚(DAPI)荧光标记的BMSCs以1×107/kg的细胞数经血管注射到损伤血管的局部。术后4 w取腹主动脉行免疫组织化学染色检测平滑肌肌动蛋白(α-SM-actin)、增殖细胞核抗原(PCNA)在血管内膜上的表达情况,测定PCNA阳性细胞核数量,计算PCNA增殖指数。HE染色行血管形态计量分析包括内膜中膜厚度、内膜中膜面积,衡量血管狭窄情况。结果损伤组新生血管内膜层α-SM-actin表达明显强于对照组(P<0.05),移植组新生血管内膜α-SM-actin表达,与损伤组比较明显增强(P<0.05)。对照组血管内膜无PCNA阳性表达,损伤组PCNA阳性细胞数较对照组明显增多(P<0.05)。移植组PCNA阳性细胞数较损伤组明显减少(P<0.05)。损伤组腹主动脉明显狭窄,内膜厚度、内膜面积、中膜厚度、中膜面积、血管狭窄程度等指标明显高于移植组和对照组(P<0.05),移植组腹主动脉有所狭窄,但血管形态学指标与对照组比较差异无统计学意义(P>0.05)。结论兔腹主动脉球囊损伤后导致血管平滑肌细胞增殖及动脉管腔狭窄。BMSCs血管局部移植可抑制血管平滑肌细胞的增殖,减轻动动脉粥样硬化及狭窄程度。     

Manipulating glutathione-S-transferases may prevent the development of tolerance to nitroglycerin

Tolerance to clinically important organic nitrates such as nitroglycerin (NTG) has been experimentally related to endothelial dysfunction and vascular oxidative stress. Anti-oxidant enzymes such as the glutathione-S-transferases (GSTs) could potentially play a protective role in NTG tolerance. Our previous work showed that an α-class glutathione-S-transferase (GSTA4-4) defends against oxidative damage in the vascular wall; therefore, we asked whether overexpression of GSTA4-4 in endothelial cells and smooth muscle cells might alter the development of tolerance to NTG. Stable transfections of mouse pancreatic islet endothelial cells (MS1) with cDNA of mGSTA4-4, and human fetal aortic vascular msmooth muscle cells (FLTR) with cDNA of hGSTA4-4 were established. TT cytotoxicity, apoptosis, nitric oxide (NO) synthases, both endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) and cyclic guanosine mono-phosphate (cGMP) were measured. Endothelial cells overexpressing mGSTA4-4, and smooth muscle cells overexpressing hGSTA4-4 were more resistant to cytotoxic injury by NTG, assessed at 24 h (p<0.05). In ooth endothelial and smooth muscle cells, NTG-induced apoptosis was inhibited by GST overexpression. Following dosing in a relevant tolerance-inducing NTG protocol, we found that GSTA4-4-overexpressing cells demonstrated significant downregulation of NOS enzymes; NO release, unchanged by the tolerance protocol in both wild-type and vector-transfected cells, was augmented in GST-overexpressing cells (p<0.01); cGMP levels in control cells fell, whereas it rose in GSTA4-4 overexpressing cells (p<0.05). Our results demonstrate that overexpression of GST isozymes can protect endothelial cells and smooth muscle cells against oxidative stress associated with NTG, and markedly alter cellular responses to repeated doses, or tolerance. By manipulating GSTs, physiological tolerance to NTG may be diminished or eliminated.     

Glutathione-S-transferase A4-4 modulates oxidative stress in endothelium: possible role in human atherosclerosis

The role of alpha-class mammalian glutathione S-transferases (GSTs) in the protection of many cell types, including vascular smooth muscle cells, against oxidant damage has been demonstrated, but the role of GSTs in the endothelial cell is not well studied. In order to examine the role of GSTs in the endothelial cell, a stable transfection of mouse pancreatic islet endothelial cells (MS1) with cDNA of mGSTA4-4, mouse isozyme of GSTs with activity in vascular wall, was established. Transfected cells demonstrated significantly higher GSTs enzyme activity and expressed significantly increased resistance to the cytotoxicity of allylamine, acrolein, 4-hydroxy-2-nonenal (4-HNE), and H(2)O(2) (P < 0.05). A significantly higher rate of proliferation and lower baseline level of intracellular malondialdehyde (MDA) and 4-HNE were present when compared to wild-type or vector-transfected MS1 endothelial cells (P < 0.05). Transfection protected MS1 endothelial cells from 4-HNE and H(2)O(2) induced apoptosis by inhibiting phosphorylation of c-Jun N-terminal kinases (p-JNK) and consequent activation of p53 and Bax. In early human fibrous atherosclerotic plaques, immunohistochemical studies demonstrated marked induction of hGSTA4-4 in endothelial cells overlying plaque, and in proliferating plaque vascular smooth muscle cells. Our results indicate that endothelial cell mGSTA4-4 can play a key role in protecting blood vessels against oxidative stress and, thus, is likely to be a critical defense mechanism against oxidants that act as atherogens.     

Increased superoxide production in nitrate tolerance is associated with NAD(P)H oxidase and aldehyde dehydrogenase 2 downregulation

Chronic administration of nitroglycerin (NTG) induces nitrate tolerance. Among possible underlying mechanisms, increased vascular production of reactive oxygen species (ROS) has emerged as a principal mechanism. Using cell culture and animal models of nitrate tolerance, we aimed to assess the impact of nitrates on NAD(P)H oxidases and aldehyde dehydrogenase 2 (ALDH2) expression. Rats and vascular smooth muscle cells were treated with NTG. Vascular reactivity was assessed by isometric tension studies. Superoxide was detected by dihydroethidium staining. Gene expression was measured by real-time polymerase chain reaction. NAD(P)H oxidase activity was measured using lucigenin-enhanced chemiluminescence. ALDH activity was measured biochemically, and NO consumption electrochemically. Nitrate tolerance was induced in rats by treatment with NTG for 3 days, and detected as impaired endothelium-dependent and -independent relaxation of aortic segments. Although superoxide production was increased in all aortic layers, expression of nox1, nox2 and nox4 was significantly decreased. Similarly, in vascular smooth muscle cells exposed to NTG for 6-24 h, NAD(P)H oxidase activity was increased, in spite of nox1 downregulation. In addition, expression and activity of ALDH-2 was decreased in nitrate-tolerant rings. Furthermore, exogenous addition of ALDH decreased superoxide generation in vitro and attenuated NO consumption in vascular smooth muscle cell homogenates. Our data suggest that in nitrate tolerance, activation of nox enzymes more than compensates for their downregulation, resulting in a net increase in superoxide and NO consumption. Furthermore, reduced ALDH-2 activity and expression leads to decreased NTG bioconversion. Therefore, both mechanisms reduce NO availability and impair vasorelaxation.     

Effects of long-term nitroglycerin treatment on endothelial nitric oxide synthase (NOS III) gene expression, NOS III-mediated superoxide production, and vascular NO bioavailability

Long-term nitroglycerin (NTG) treatment has been shown to be associated with cross-tolerance to endothelium-dependent vasodilators. It may involve increased production of reactive oxygen species (such as superoxide, O(2)(.-)) that rapidly inactivate the nitric oxide (NO) released from the endothelial cells. It remains to be elucidated, however, whether long-term treatment with NTG alters the activity and expression of the endothelial NO synthase (NOS III) and whether this enzyme can contribute to O(2)(.-) formation. We studied the influence of long-term NTG treatment on the expression of NOS III as assessed by RNase protection assay and Western blot. Tolerance was measured ex vivo in organ chamber experiments with rat aortic rings. O(2)(.-) and NO formation were quantified using lucigenin- and Cypridina luciferin analog-enhanced chemiluminescence as well as electron spin resonance (ESR) spectroscopy. Treatment of Wistar rats with NTG (Alzet osmotic minipumps, NTG concentration 10 microg x kg(-1) x min(-1)) for 3 days caused marked tolerance, cross-tolerance to the endothelium-dependent vasodilator acetylcholine, and a significant increase in O(2)(.-)-induced chemiluminescence. Tolerance was associated with a significant increase in NOS III mRNA to 236+/-28% and NOS III protein to 239+/-17%. In control vessels, the NOS inhibitor N(G)-nitro-L-arginine (L-NNA) increased the O(2)(.-)-mediated chemiluminescence, indicating that basal production of endothelium-derived NO depresses the baseline chemiluminescence signal. In the setting of tolerance, however, L-NNA decreased steady-state O(2)(.-) levels, indicating the involvement of NOS III in O(2)(.-) formation. Likewise, A23187-induced, NOS III-mediated O(2)(.-) production was more pronounced in tolerant than in control vessels. Vascular NO bioavailability as assessed with ESR spectroscopy using iron-thiocarbamate as a trap for NO was significantly reduced in tolerant vessels. Pretreatment of tolerant tissue in vitro with the protein kinase C (PKC) inhibitors reduced basal and stimulated NOS III-mediated O(2)(.-) production and partially reversed vascular tolerance. These findings suggest that NTG treatment increases the expression of a dysfunctional NOS III gene, leading to increased formation of O(2)(.-) and decreased vascular NO bioavailability. Normalization of NOS III-mediated O(2)(. -) production and improvement of tolerance with PKC inhibition suggests an important role for PKC isoforms in mediating vascular dysfunction caused by long-term NTG treatment.     

硝普钠对培养的猪主动脉平滑肌细胞增殖的影响

内皮细胞损伤和血管平滑肌细胞增殖在动脉粥样硬化发生过程中起关键作用，本工作作用离体冠状动脉实验证明硝普钠呈剂量依赖性松驰冠状动脉，且与内皮去除与否无关。用噻唑蓝比色法观察到硝普钠也能剂量依赖性抑制主动脉平滑肌细胞增殖。结果表明，ＮＯ对平滑肌细胞增殖具有调节作用。提示在临床上对于动脉粥样硬化及冠心病病人，长期使用硝普钠等与ＮＯ合成及释放有关的硝基化合物不仅可以缓解冠状动脉痉挛，而且还可抑制平滑肌细胞     

抑瘤素M在高血糖致人主动脉平滑肌细胞增殖中的作用

目的 研究高血糖对人巨噬细胞中抑瘤素M表达的影响以及抑瘤素M对人主动脉平滑肌细胞增殖的影响.方法 采用佛波酯孵育人单核/巨噬细胞系THP-1细胞,诱导其分化为巨噬细胞.应用人细胞因子抗体芯片筛选出受高血糖(15 mmol/L)调控的人巨噬细胞源性细胞因子,并经Western blot和酶联免疫吸附试验(ELISA)技术验证.运用逆转录-聚合酶链反应(RT-PCR)明确人主动脉内皮细胞、平滑肌细胞及巨噬细胞中是否存在抑瘤素M受体GP130-OSMRβ二聚体及GP130-LIFR二聚体表达.运用细胞增殖实验(XTT法)研究抑瘤素M对人主动脉平滑肌细胞增殖的影响.采用单因素方差分析进行统计学分析.结果 佛波酯孵育48 h可诱导THP-1细胞分化为巨噬细胞.细胞因子芯片结果显示高血糖可上调抑瘤素M在巨噬细胞中的表达,并为Western blot及ELISA所证实.RT-PCR显示人主动脉内皮细胞、平滑肌细胞及巨噬细胞均可表达GP130-OSMRβ二聚体及GP130-LIFR二聚体.XTT法证实抑瘤素M可促进血管平滑肌细胞增殖,与0μg/L抑瘤素M(吸光度为0.468±0.032)相比,5μg/L(吸光度为0.503±0.026)、10μg/L(吸光度为0.520±0.027)、20μg/L抑瘤素M(吸光度为0.513±0.026)人主动脉平滑肌细胞增殖均明显增加(F=10.40,P<0.05).结论 高血糖可促进巨噬细胞中抑瘤素M的表达和分泌.抑瘤素M通过与人主动脉平滑肌细胞中的受体结合而促进血管平滑肌细胞的增殖,进而部分参与糖尿病大血管病变的发生发展.     

Inducible nitric oxide synthase and atherosclerosis

Nitric oxide (NO) synthase induction in vascular smooth muscle cells may play a role in local vascular injury associated with atherosclerosis or postangioplasty restenosis by inhibiting smooth muscle cell proliferation and contraction, as well as by preventing leukocyte and platelet adhesion. The expression of inducible NO synthase is increased in balloon-injured arteries of experimental animals or in human atherosclerotic lesions. Replacement therapy with NO donors or NO synthase gene transfer may improve the clinical course of atherosclerosis or restenosis.     

Endothelial feedback and the myoendothelial projection

The endothelium plays a critical role in controlling resistance artery diameter, and thus blood flow and blood pressure. Circulating chemical mediators and physical forces act directly on the endothelium to release diffusible relaxing factors, such as NO, and elicit hyperpolarization of the endothelial cell membrane potential, which spreads to the underlying smooth muscle cells via gap junctions (EDH). It has long been known that arterial vasoconstriction in response to agonists is limited by the endothelium, but the question of how contraction of smooth muscle cells leads to activation of the endothelium (myoendothelial feedback) has, until recently, received little attention. Initial studies proposed the permissive movement of Ca(2+) ions from smooth muscle to endothelial cells to elicit release of NO. However, more recent evidence supports the notion that flux of IP(3) leading to localized Ca(2+) events within spatially restricted myoendothelial projections and activation of EDH may underlie myoendothelial feedback. In this perspective, we review recent data which supports the functional role of myoendothelial projections in smooth muscle to endothelial communication. We also discuss the functional evidence supporting the notion that EDH, as opposed to NO, is the primary mediator of myoendothelial feedback in resistance arteries.     

Agonists of toll-like receptors 2 and 4 activate airway smooth muscle via mononuclear leukocytes

RATIONALE: Toll-like receptors 2 and 4 (TLR2, TLR4) enable cellular responses to bacterial lipoproteins, LPS, and endogenous mediators of cell damage. They have an established role in the activation of leukocytes, endothelial cells, and some smooth muscle cell types, but their roles in airway smooth muscle are uncertain. OBJECTIVES: To determine the roles of TLRs in activation of airway smooth muscle. METHODS: Airway smooth muscle cells were cultured with TLR agonists, in the presence or absence of mononuclear leukocytes. MEASUREMENTS AND MAIN RESULTS: We observed expression of TLR2 and TLR4 mRNAs, which could be upregulated by treatment with proinflammatory cytokines in primary human airway smooth muscle, but no important functional responses to agonists of these TLRs were seen. Coincubation of airway smooth muscle with peripheral blood mononuclear cells, at concentrations as low as 250 mononuclear cells/ml, resulted in a marked cooperative response to TLR stimuli, and synergistic production of cytokines, including chemokines (interleukin [IL-]-8) and IL-6. This cooperative response was greater when monocytes were enriched and was transferable using supernatants from LPS-stimulated peripheral blood mononuclear cells. Activation of cocultures required IL-1 generation from mononuclear cells, and was blocked by IL-1 receptor antagonist, though IL-1 generation alone was not sufficient to account for the magnitude of mononuclear cell-dependent coculture activation. CONCLUSIONS: These data indicate that potent amplification of inflammation induced by TLR agonists, such as LPS, may be achieved by cooperativity between airway smooth muscle and leukocytes involved in immune surveillance or inflammation.     

Acetylcholine-induced endothelium-dependent vascular smooth muscle relaxation in nitroglycerin-tolerant isolated rat aorta

Summary Nitroglycerin (NTG) tolerance is recognized clinically, and its pharmacological mechanism has been thought to be due to a decrease in the accumulation of cyclic GMP (cGMP) which is a second messenger of NTG. Endothelium-derived relaxing factor (EDRF) also relaxes vascular smooth muscle through the activation of soluble guanylate cyclase and the production of cGMP. The purpose of this study was to investigate acetylcholine (ACh)-induced endothelium-dependent relaxation and cGMP response in NTG-tolerant isolated rat aorta. Ring strips prepared from the thoracic aorta of male Wistar rats were mounted in tissue baths and contracted with 10^–6 M norepinephrine. NTG and ACh relaxation responses were compared before and after 1 h treatment with 5×10^–4 M NTG. The chronological changes in tissue cGMP levels by 10^–6 M NTG and ACh were compared between a control group (untreated) and NTG-tolerant group (treated with 5×10^–4 M NTG for 1 h). The NTG dose-response curve shifted markedly to the right, but the ACh dose-response curve shifted to the left after the induction of NTG tolerance. In the control group, both NTG and ACh elevated the tissue cGMP levels, but in the NTG-tolerant group only ACh elevated cGMP significantly. However, in the NTG-tolerant group, the cGMP increase induced by ACh was smaller than that in the control group.These results suggest that NTG tolerance does not decrease, but rather augments ACh-induced endothelium-dependent vascular smooth muscle relaxation in isolated rat aorta.