内皮型一氧化氮合酶、内皮素-1、蛋白激酶C、核因子-κB在门静脉高压血管内皮细胞的表达及意义

目的 探讨门静脉局部内皮源性血管活性物质异常与内皮细胞应力信号转导通路激活的关系.方法 用免疫组织化学和免疫荧光激光扫描共聚焦显微镜技术检测18例门静脉高压症、10例外伤性脾破裂患者脾静脉和15例门静脉高压、10例对照组大鼠门静脉内皮细胞内皮型一氧化氮合酶(eNOS)、内皮素-1(ET-1)、蛋白激酶C(PKC)、核因子-κB(NF-κB)的表达,探讨门静脉高压时其血管内皮细胞eNOS、ET-1表达改变与信息分子PKC、NF-κB的关系.结果 实验组内皮细胞PKC表达呈阳性或强阳性,对照组则呈阴性或弱阳性表达.其平均吸光度分别为实验组0.068 2±0.009 2(人)/0.059 3±0.005 7(鼠),对照组0.025 7±0.008 2(人)/0.022 4±0.006 5(鼠),差异有统计学意义(P＜0.01).eNOS、ET-1与NF-κB在门静脉高压患者脾静脉和实验组大鼠模型门静脉内皮的荧光信号强度均显著高于对照组ET-1107.359±30.147比56.441±18.874,P＜0.01(人);91.017±32.517比51.065±19.018,P＜0.01(鼠).eNOS95.610±28.333比57.872±27.374,P＜0.01(人);88.712±28.159比50.897±16.546,P＜0.01(鼠).NF-κB90.098±30.964(人)/90.348±32.852(鼠)比48.358±19.326(人)/48.070±19.065(鼠),P＜0.01(人)/P＜0.01(鼠).脾/门静脉内皮细胞ET-1、eNOS的表达与PKC、NF-κB的表达呈正相关(P＜0.05).结论 门静脉血中ET-1和NO的增高是由于门静脉系统内皮细胞合成增多所致,内皮细胞合成NO的增多与其eNOS表达上调有关.门静脉高压时其血管内皮细胞的机械应力信号通路被激活,内皮细胞ET-1和eNOS表达上调与该通路激活有关.     

内皮型一氧化氮合酶、内皮素-1、蛋白激酶C、核因子-кB在门静脉高压血管内皮细胞的表达及意义

目的探讨门静脉局部内皮源性血管活性物质异常与内皮细胞应力信号转导通路激活的关系。方法用免疫组织化学和免疫荧光激光扫描共聚焦显微镜技术检测18例门静脉高压症、10例外伤性脾破裂患者脾静脉和15例门静脉高压、10例对照组大鼠门静脉内皮细胞内皮型一氧化氮合酶(eNOS)、内皮素-1(ET-1)、蛋白激酶C(PKC)、核因子-_κB(NF-_κB)的表达,探讨门静脉高压时其血管内皮细胞eNOS、ET-1表达改变与信息分子PKC、NF-_κB的关系。结果实验组内皮细胞PKC表达呈阳性或强阳性,对照组则呈阴性或弱阳性表达。其平均吸光度分别为实验组0．068 2±0．009 2(人)/0．059 3±0．005 7(鼠),对照组0．025 7±0．008 2(人)/0．022 4±0．006 5 (鼠),差异有统计学意义(P＜0．01)。eNOS、ET-1与NF-_κB在门静脉高压患者脾静脉和实验组大鼠模型门静脉内皮的荧光信号强度均显著高于对照组：ET-1：107．359±30．147比56．441±18．874．P＜0．01(人)；91．017±32．517比51．065±19．018,P＜0．01(鼠)。eNOS：95．610±28．333比57．872±27．374,P＜0．01(人)；88．712±28．159比50．897±16．546,P＜0．01(鼠)。NF-_κB：90．098±30．964(人)/90．348±32．852(鼠)比48．358±19．326(人)/48．070±19．065(鼠),P＜0．01 (人)/P＜0．01(鼠)。脾/门静脉内皮细胞ET-1、eNOS的表达与PKC、NF-_κB的表达呈正相关(P＜0．05)。结论门静脉血中ET-1和NO的增高是由于门静脉系统内皮细胞合成增多所致,内皮细胞合成NO的增多与其eNOS表达上调有关。门静脉高压时其血管内皮细胞的机械应力信号通路被激活,内皮细胞ET-1和eNOS表达上调与该通路激活有关。     

肝硬化患者血浆刺激内皮产生一氧化氮的实验研究

目的 研究肝硬化患者血浆能否刺激血管内皮细胞产生一氧化氮。方法 分别用正常人血浆和肝硬化患者血浆刺激人脐静脉血管内皮细胞,以硝酸还原酶法测定细胞培养上清NO浓度,逆转录-聚合酶链反应(RT-PCR)测定血管内皮细胞NO(eNOs)mRNA水平。结果肝硬化患者血浆组NO浓度显著高于正常人血浆组(P≤0.01);门静脉高压症患者血浆处理组eNOS mR-NA水平较正常人血浆组增高,且有时间依赖性,正常人血浆处理样品的各个时间点其RNA水平没有明显差异。结论 肝硬化患者血浆本身可刺激血管内皮细胞产生NO。     

胃癌血管内皮生长因子和一氧化氮合酶的表达与胃癌血管生成及临床病理的关系

目的研究血管内皮生长因子(VEGF)、诱导型一氧化氮合酶(iNOS)和内皮型一氧化氮合酶(eNOS)在人胃癌中表达的相关性;探讨3者与人胃癌血管生成和临床病理特征的关系;研究一氧化氮(NO)和VEGF的相互作用及NO在VEGF促肿瘤生长中的作用机制。方法应用免疫组织化学方法检测34例人胃癌手术切除标本VEGF、iNOS和eNOS的表达,第Ⅷ因子相关抗原(FⅧRAg)血管内皮细胞特异性染色计数肿瘤微血管密度(MVD)。结果(1)31例胃癌组织表达VEGF,25例表达iNOS,28例表达eNOS;(2)VEGF与iNOS的表达正相关,与eNOS的表达无相关;(3)VEGF、iNOS的表达与胃癌MVD呈正相关,表达与不表达eNOS其胃癌MVD的差异无显著性意义;(4)VEGF的表达与胃癌淋巴结转移和肿瘤浸润深度呈正相关,与肿瘤分化程度无关;iNOS表达与胃癌的浸润深度呈正相关,与胃癌分化程度及有无淋巴结转移无关;eNOS表达与胃癌浸润深度、分化程度及有无淋巴结转移均无关。结论(1)iNOS对VEGF的生成和发挥作用的过程有重要影响;(2)MVD随着VEGF和iNOS表达的增强而增加,说明两者对胃癌血管生成具有促进作用。     

蛋白激酶Cα在门静脉高压患者肝内外血管表达的研究

目的：研究蛋白激酶Cα（PKCα)在门静脉高压患者肝内外血管的表达，探讨其在门静脉高压形成机制中的作用。方法：对34例门静脉高压患者的肝内外血管与30例对照组织行PKCα免疫组织化学染色。用逆转录－聚合酶链反应（RT－PCR）测定门静脉高压患者脾静脉血管平滑肌细胞（VSMC）中PKCαmRNA的表达。结果：正常肝内外血管PKCα免疫组织化学染色呈阴性或弱阳性，门静脉高压患者的肝内血管和脾静脉呈强阳性，差异有非常显著性（P＜0．01）。RTPCR表明门静脉高压患者脾静脉VSMC中PKCαmRNA的表达是正常人的2．81倍。结论：PKCα过度表达可能是门静脉高压肝内外血管结构改变的重要原因，并可能改变其因管舒缩活性物质的合成及敏感性。     

人胃癌VEGF和NOS的表达与肿瘤血管生成的关系

目的　研究血管内皮生长因子 (VEGF)、诱导型一氧化氮合酶 (iNOS)及内皮型一氧化氮合酶 (eNOS)在人胃癌表达的相关性及与胃癌血管生成的关系 ,探讨NO和VEGF的相互作用及NO在VEGF促肿瘤生长中的作用机理。方法　应用免疫组化方法检测 34例胃癌组织中VEGF、iNOS和eNOS的表达及分布 ,用血管内皮细胞第Ⅷ相关抗原 (FⅧRAg)行免疫特异性染色计数肿瘤微血管密度 (MVD)。 结果　 34例胃癌组织中 ,表达iNOS者占 73.5 % ,表达eNOS者占 82 .4 % ,表达VEGF者占 91.2 % ;VEGF与iNOS的表达具有相关性 (P<0 .0 0 5 ) ,VEGF与eNOS的表达则无相关性 (P>0 .0 5 ) ;表达VEGF的胃癌其MVD明显高于不表达VEGF的胃癌 (P<0 .0 2 5 ) ,表达iNOS的胃癌其MVD明显高于不表达iNOS的胃癌 (P<0 .0 5 ) ,表达eNOS的胃癌MVD与不表达eNOS的胃癌差异无显著性意义 (P>0 .0 5 )。结论　胃癌组织中MVD随着VEGF和iNOS表达的增加而增加 ,提示两者对胃癌的血管生成起促进作用 ;VEGF与iNOS的表达具有相关性 ,提示iNOS在VEGF的生成和发挥作用过程中起重要作用。     

黄芪当归合剂对单侧输尿管梗阻大鼠肾脏血管活性物质变化的影响

目的 本研究通过观察黄芪当归合剂(A&A)对单侧输尿管梗阻(UUO)模型肾组织血管紧张素Ⅱ(Ang-Ⅱ)、内皮素-1(ET-1)、一氧化氮(NO)水平及一氧化氮合酶(NOS)的影响，以进一步揭示A&A抑制肾纤维化机制。方法 Wistar大鼠随机分为假手术(Sham)、UUO和UAA(UUO+A&A)组，造模后0、3、7、10 d分析各组肾脏中NO、Ang-Ⅱ、ET-1水平和NOS活性及3种NOS的表达。结果 (1)造模后UUO组的Ang-Ⅱ和ET-1水平持续增高；A&A仅在第3天时降低Ang-Ⅱ水平(P < 0.05)。(2)造模后UUO组的NO浓度在第10天时才明显增高；而UAA组中NO浓度逐渐增高，第3天时明显高于UUO组(P < 0.05)。(3)UUO组内皮型eNOS在髓质血管内表达增高，且UAA组与UUO组间没有显著性差异。第3天时UAA组神经型nNOS表达低于UUO组(P < 0.05)。UAA组cNOS的活性明显高于Sham和UUO组。诱导型iNOS表达和活性3组间差异均无统计学意义。结论 梗阻性肾病中，A&A在早期降低肾内Ang-Ⅱ水平、且持续增强eNOS活性使NO产生增加，从而可能降低血管张力、改善肾脏的缺血及缺氧状态，减轻肾间质纤维化。     

五味子乙素通过NOS/NO系统预防BaP所致HTR8-SVneo细胞氧化损伤

目的研究五味子乙素(Sch B)预防环境污染物苯并(a)芘(BaP)致人绒毛膜滋养层细胞HTR8-SVneo氧化损伤的机制。方法以HTR8-SVneo细胞为载体,构建BaP氧化应激模型。实验分为3组,BaP组、Sch B组和空白对照组。MTS法检测细胞存活率,还原酶法检测细胞培养液中一氧化氮(NO)及总一氧化氮合酶(TNOS)含量,用RT-PCR法检测细胞内诱导型一氧化氮合酶(iNOS)和内皮型一氧化氮合酶(eNOS)mRNA水平。结果 BaP组细胞存活率(72.2±0.9)%较对照组显著下降(P0.05);而0.1、0.5或2μmol/L Sch B+20μmol/L BaP各组的细胞存活率分别为(78.2±1.5)%、(86.5±0.6)%、(93.4±1.0)%,显著高于BaP组(P0.05)。BaP组细胞培养液中NO、TNOS含量、iNOS、eNOS mRNA表达量和eNOS蛋白表达量均显著高于对照组(P0.05);而与BaP组相比,Sch B组中NO、TNOS含量,iNOS、eNOS mRNA表达量和eNOS蛋白表达量均随着Sch B剂量的增加逐渐下降(P0.05)。结论 Sch B可有效预防BaP所致的HTR8-SVneo细胞氧化损伤,其机制可能与NOS/NO系统的平衡有关。     

异丙酚对脂多糖诱导人脐静脉内皮细胞一氧化氮合酶及NF—kB表达的影响

一氧化氮合酶（NOS）在血管内皮细胞中主要有两种亚型：内皮型一氧化氮合酶（eNOS）和诱生型一氧化氮合酶（iNOS），前者在生理状态下正常表达，后者则在炎性刺激等病理情况下被大量诱生。由iNOS所产生的过量NO可对血管内皮细胞造成损伤。NF-kB是一种与急性炎症密切相关的核转录因子，多种炎症介质如iNOS等基因的启动子和增强子均含有kB位点。前期研究证实异丙酚能减少脓毒症动物体内一氧化氮（NO）的产生，对内毒素（LPS）所致急性肺损伤具有保护作用。本研究拟观察异丙酚对人脐静脉内皮细胞eNOS、iNOS及NF-KB表达的影响，探讨异丙酚抗炎作用的机制。     

异丙酚对脂多糖诱导人脐静脉内皮细胞一氧化氮合酶及NF-κB表达的影响

一氧化氮合酶(NOS)在血管内皮细胞中主要有两种亚型:内皮型一氧化氮合酶(eNOS)和诱生型一氧化氮合酶 (iNOS),前者在生理状态下正常表达,后者则在炎性刺激等病理情况下被大量诱生。由iNOS所产生的过量NO可对血管内皮细胞造成损伤。NF-κB是一种与急性炎症密切相关的核转录因子,多种炎症介质如iNOS等基因的启动子和增强子均含有κB位点。前期研究证实异丙酚能减少脓毒症动物体内一氧化氮(NO)的产生,对内毒素(LPS)所致急性肺损伤具有保护作用。本研究拟观察异丙酚对人脐静脉内皮细胞eNOS、iNOS及NF-κB表达的影响,探讨异丙酚抗炎作用的机制。     

异丙酚对高血压大鼠胸主动脉内皮型一氧化氮合酶和诱导型一氧化氮合酶表达的影响

目的 评价异丙酚对高血压大鼠胸主动脉内皮型一氧化氮合酶(eNOS)和诱导型一氧化氮合酶(iNOS)表达的影响.方法 SD大鼠,雌雄各半,体重240～ 280 g,采用皮下注射去氧皮质酮的方法制备高血压模型,采用随机数字表法,将64只造模成功的大鼠随机分为4组(n＝16):高血压组(H组)、小剂量异丙酚组(P1组)、中剂量异丙酚组(P2组)和大剂量异丙酚组(P3组).P1组、P2组和P3组分别静脉输注异丙酚20、30、40 mg·kg-1·h-13 h,H组给予等容量生理盐水.分别于给药前、给药1h、3h时记录平均动脉压(MAP).给药3h时处死大鼠,摘眼球法采集血样,硝酸还原酶法测定血清一氧化氮(N0)浓度,取胸主动脉,采用RT-PCR和Western blot法测定eNOS mRNA、iNOS mRNA及其蛋白表达水平.结果 与H组比较,P1组、P2组和P3组给药3h时MAP降低,血清NO浓度升高,主动脉eNOS mRNA及其蛋白表达上调,主动脉iNOS mRNA及其蛋白表达下调,且呈剂量依赖性(P<0.05或0.01).结论 异丙酚降低高血压大鼠血压的机制与下调iNOS表达,上调血管内皮细胞eNOS表达,促进NO释放有关.     

Effect of endothelial cell-based iNOS gene transfer on cavernosal eNOS expression and mouse erectile responses

Inducible nitric oxide synthase (iNOS) gene transfer is reported to augment erectile responses in rats, although it is also shown to impair vasorelaxation in cerebral arteries. We investigated the effect of endothelial cell-based iNOS gene transfer on endothelial NOS (eNOS) expression and mouse erectile responses. Human coronary artery endothelial cells (EC) transduced with empty vector (control) or iNOS were grown in culture and transplanted into the corpus cavernosum of severe combined immunodeficient mice. Endothelial NOS expression was compared in control and iNOS-transduced cells grown in the presence or absence of a selective iNOS inhibitor, L-N6- (1-iminoethyl) lysine hydrochloride (L-NIL). At 3-5 days after cell transplantation, we recorded intracorporal pressure (ICP) responses to cavernosal nerve stimulation and measured cavernosal total NO and eNOS protein expression. In this study, EC transduced with iNOS produced significantly more NO than controls but exhibited a twofold downregulation of eNOS protein and mRNA. This effect was reversed by L-NIL. In vivo, the cell-based gene transfer of iNOS led to significantly increased ICP responses, compared to mice transplanted with control ECs. Consistent with the in vitro data, cavernosal lysates had significantly reduced eNOS expression. In conclusion, EC gene transfer of iNOS downregulates EC expression of eNOS by an NOS-dependent mechanism. In the cavernosum of mice transplanted with Inos-transduced EC, nerve-stimulated erectile responses were augmented by the short-term gene transfer. However, our findings suggest that iNOS gene transfer may have deleterious effects on endothelial function if used as a treatment for erectile dysfunction.     

Impaired tubulointerstitial expression of endothelin-1 and nitric oxide isoforms in donor kidney biopsies with postischemic acute renal failure

BACKGROUND: About 30% of cadaveric renal allografts, but almost never living-donor kidneys, develop postischemic acute renal-transplant failure (ARF). We therefore quantified the expression of essential reperfusion regulators in different compartments of cadaveric and living-donor kidney biopsies. METHODS: Specimens were obtained from donor kidneys at the end of the cold ischemia time before implantation and categorized into three groups according to donor source and early posttransplant function. Ten living-donor biopsies (LIV) were compared with nine cadaveric kidney biopsies (CAD) with primary posttransplant function (CAD-PF) and to nine with ARF (CAD-ARF). Laser capture microdissection was used to isolate glomeruli from tubulointerstitium. The gene expression of intercellular adhesion molecule (ICAM)-1, interleukin (IL)-1beta, endothelin (ET)-1, inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) was quantified in glomeruli and tubulointerstitium by real-time polymerase chain reaction (TaqMan). RESULTS: Tubulointerstitial areas of all CAD kidneys revealed significantly lower mRNA levels of all investigated genes compared with LIV. Tubulointerstitial ET-1, iNOS, and eNOS in CAD-ARF averaged only half of the expression in CAD-PF kidneys. ICAM-1 and IL-1beta mRNA concentrations were equal in CAD-PF and CAD-ARF. Glomerular expression of the investigated genes was equal in CAD and LIV kidneys with the exception of ICAM-1 and ET-1, which were two times higher in CAD-PF compared with LIV and CAD-ARF. CONCLUSION: These data suggest that CAD compared with LIV kidneys have an impaired expression of immune and vasoregulatory genes in the tubulointerstitium, which may represent reduced cellular vitality and capacity to adaptation. The observed further reduction of ET-1, iNOS, and eNOS expression in CAD-ARF might contribute to reperfusion injury and delayed allograft function.     

Parathyroid hormone stimulates the endothelial nitric oxide synthase through protein kinase A and C pathways.

BACKGROUND: Parathyroid hormone (PTH), the major systemic calcium regulating hormone has been implicated in the development of hypertension and the occurrence of uraemic vascular changes. As nitric oxide synthase (NOS) is involved in the production of nitric oxide, and acute PTH effect is characterized by vasodilation, the effect of PTH on the endothelial NOS (eNOS) system was measured in cultured human umbilical cord vein endothelial cells (HUVEC) and the pathways possibly involved were studied. METHODS: The presence of the PTH receptor-1 (PTHR1) on the HUVEC membrane was examined by RT-PCR, immunocytochemistry and western blot. HUVEC were stimulated with 10(-12) to 10(-10) mol/l PTH. The eNOS mRNA expression was established by RT-PCR and the eNOS protein levels were determined by western blot. The eNOS activity was measured by the conversion of [(14)C]arginine to [(14)C]citrulline. RESULTS: PTHR1 has been found to be expressed in HUVEC and its expression is depressed by increasing concentrations of PTH. PTH induced a significant increase in eNOS mRNA (10(-11) mol/l: 1.87 +/- 0.16, P = 0.012; 10(-10) mol/l: 1.96 +/- 0.28, P = 0.007, fold of control), and protein expression. The eNOS activity was also significantly stimulated (10(-11) mol/l: 1139 +/- 203; 10(-10) mol/l: 1323 +/- 216 vs control: 621 +/- 154 cpm/150 mug protein, P < 0.01). The addition of calphostin C (PKC inhibitor) or Rp-cAMP (PKA inhibitor) reduced the eNOS mRNA, protein expression and activity of PTH-stimulated HUVEC. The combined treatment of calphostin C and Rp-cAMP abolished the eNOS protein expression and activity. CONCLUSION: PTH induces an increased activity of the eNOS system; probably both PKA and PKC pathways are involved in this activation. Such data may explain the vasodilation observed after acute treatment with PTH.     

High glucose increases inducible NO production in cultured rat mesangial cells. Possible role in fibronectin production.

BACKGROUND/AIM: Increased nitric oxide (NO) generation and action have been suggested to be associated with glomerular hyperfiltration and increased vascular permeability early in diabetes. However, previous studies have primarily focused on the constitutive nitric oxide synthase (cNOS) pathway present in endothelial cells, and the role of the inducible NOS (iNOS) pathway in diabetic nephropathy has remained unclear. This study examined whether high glucose modulates NO synthesis by the iNOS pathway in rat mesangial cells. In addition, the effect of inhibition of the iNOS pathway on fibronectin production was determined to examine the role of the iNOS pathway in high glucose-induced extracellular expansion by mesangial cells. METHODS: NO synthesis by the iNOS pathway was evaluated by nitrite and iNOS mRNA and protein productions. The effects of protein kinase C (PKC) inhibitor and aldose reductase inhibitor on the iNOS mRNA expression and aminoguanidine, a relatively specific inhibitor of the iNOS on fibronectin protein production were examined. RESULTS: High 30 mM glucose concentration led to significant increases in nitrite production of rat mesangial cells upon stimulation with lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) compared with control 5.6 mM glucose concentration. Mesangial iNOS mRNA expression and protein production also increased significantly in response to high glucose. The addition of calphostin C, a PKC inhibitor, and 6-bromo-1,3-dioxo-1H-benz[d,e]isoquinoline-2(3H)-acetic acid, an aldose reductase inhibitor, significantly suppressed the enhancement of iNOS mRNA expression in high glucose concentration. High glucose also significantly increased fibronectin protein production of mesangial cells upon stimulation with LPS plus IFN-gamma compared to control glucose. Aminoguanidine reversed this high glucose-induced fibronectin production at dose inhibiting iNOS mRNA expression. CONCLUSIONS: These results indicate that high glucose enhances cytokine-induced NO production by rat mesangial cells, and that the activation of PKC and aldose reductase pathway may play a role in this enhancement. In addition, high glucose-induced NO production by the iNOS pathway may promote extracellular matrix accumulation by mesangial cells under certain condition.     

Induction of endothelin-1 expression by glucose: an effect of protein kinase C activation

Enhanced actions or levels of endothelin-1 (ET-1), a potent vasoconstrictor, have been associated with decreased blood flow in the retina and peripheral nerves of diabetic animals and may be related to the development of pathologies in these tissues. Hyperglycemia has been postulated to increase ET-1 secretion in endothelial cells. We have characterized the mechanism by which elevation of glucose is increasing ET-1 mRNA expression in capillary bovine retinal endothelial cells (BREC) and bovine retinal pericytes (BRPC). Elevation of glucose, but not mannitol, from 5.5 to 25 mmol/l for 3 days increased membranous protein kinase C (PKC) activities and ET-1 mRNA in parallel levels by 2-fold in BREC and BRPC. These effects were reversed by decreasing glucose levels to 5.5 mmol/l for an additional 2 days. Glucose-induced ET-1 overexpression was inhibited by a general PKC inhibitor, GF109203X, and a mitogen-activated protein kinase kinase inhibitor, PD98059, but not by wortmannin, a phosphatidylinositol 3-kinase inhibitor. By immunoblot analysis, PKC-beta2 and -delta isoforms in BREC were significantly increased relative to other isoforms in the membranous fractions when glucose level was increased. Overexpression of PKC-beta1 and -delta isoforms but not PKC-zeta isoform by adenovirus vectors containing the respective cDNA enhanced in parallel PKC activities, proteins, and basal and glucose-induced ET-1 mRNA expression by at least 2-fold. These results showed that enhanced ET-1 expression induced by hyperglycemia in diabetes is partly due to activation of PKC-beta and -delta isoforms, suggesting that inhibition of these PKC isoforms may prevent early changes in diabetic retinopathy and neuropathy.     

Regulation of endothelin synthesis by extracellular matrix in human endothelial cells

BACKGROUND: Vascular diseases are characterized by the presence of structural changes and the progressive loss of endothelial function. Although the biochemical basis of these structural changes have started to be outlined, it seems that accumulation of normal extracellular matrix proteins as well as the appearance of interstitial collagens, mainly collagen type I, characterize this process. On the other hand, a role for endothelial vasoactive factors has been proposed in the genesis of endothelial dysfunction, and it is generally accepted that changes in extracellular matrix composition may modify cell behavior. METHODS: Experiments were designed to test the influence of the supporting matrix on endothelin-1 (ET-1) synthesis by endothelial cells. Northern blot experiments were performed to analyze the prepro-endothelin-1 (prepro-ET-1) mRNA expression. ET-1 production was measured by ELISA. RESULTS: Cells grown on collagen type I (Col I) showed an increase of prepro-ET-1 mRNA level when compared with cells cultured on collagen type IV (Col IV). According to these results, the release of ET-1 to culture medium was also higher in Col I-grown cells than in those cultured on Col IV. Treatment of cells with a peptide that interferes with Col I integrins (D6Y), or with protein tyrosine kinase inhibitors such as genistein and herbimycin, completely abolished the effect of Col I. Moreover, experiments with antibodies against integrins suggest that these cell surface receptors could be involved in the modulation of ET-1 system by extracellular matrix. CONCLUSIONS: These results suggest that the presence of an abnormal extracellular matrix could stimulate endothelin synthesis by human endothelial cells, through integrin activation.     

红细胞生成素对慢性肾衰竭大鼠肾小球内皮细胞功能的影响

目的 探讨红细胞生成素（EPO）对慢性肾衰竭（CRF）大鼠肾小球内皮细胞功能的影响。 方法 采用分阶段5/6肾切除术制备大鼠慢性肾衰竭动物模型。实验动物按数字随机法分为4组：假手术组（对照组）、慢性肾衰竭组（模型组）及EPO干预的两个剂量组（小剂量组EPO用量30 U/kg,大剂量组EPO用量50 U/kg）。慢性肾衰竭大鼠皮下注射EPO 6周后处死。检测各组大鼠血肌酐（Scr）、血尿素氮（BUN）、尿蛋白、血红蛋白（Hb）和血压的变化,并观察肾组织病理改变。免疫组化法检测肾小球CD34、CD31表达;RT-PCR检测肾组织内皮素1（ET-1）、内皮细胞一氧化氮合酶（eNOS）和血管内皮细胞生长因子（VEGF） mRNA的表达。 结果 与模型组比较,EPO治疗能显著增加大鼠肾小球CD34、CD31的表达（均P < 0.05）;下调肾组织ET-1 mRNA的表达（P < 0.05）;上调肾组织eNOS和 VEGF mRNA的表达（均P < 0.05）。此外,EPO治疗还能使大鼠Scr、BUN、尿蛋白和血压水平显著降低（均P < 0.05）,Hb水平显著增高（P < 0.05）,肾组织病理损害明显减轻。 结论 EPO能减轻慢性肾衰竭大鼠肾脏的病理损害,改善肾功能。这种作用可能与其促进肾小球内皮细胞的修复和改善内皮功能有关。     

Enhanced expression of nitric oxide synthase in the early stage after increased pulmonary blood flow in rats.

OBJECTIVE: Evidence that vasodilator nitric oxide mediates normal pulmonary vascular tone has led to the hypothesis that endothelial injury induced by congenital heart disease with increased pulmonary blood flow disrupts these regulatory mechanisms and its associated altered vascular reactivity. Therefore, we hypothesized that increased pulmonary blood flow results in altered expression of endothelial nitric oxide synthase (eNOS). METHODS: We created an arteriovenous shunt in female Wistar (5-week-old) and measured the change of pulmonary blood flow and pressure immediately after and 1 month after the shunt operation. The protein levels of eNOS in the lung tissues of rats were assessed. RESULTS: The shunt immediately resulted in a significant increase in pulmonary blood flow (16.5 +/- 11.8% , pulmonary artery pressure (2.3 +/- 0.7 mm Hg), and blood O(2) saturation (16.1 +/- 11.8%) in the pulmonary artery. After 4 weeks, there was a significant increase in pulmonary blood flow (30.7 +/- 1.6%), pulmonary artery pressures (4.3 +/- 1.1 mm Hg), and blood O(2) content (43.3 +/- 17.5%). Western blot analysis demonstrated that eNOS protein was increased in the shunt lung 72 h after surgery and recovered to the control level 1 week later. CONCLUSION: This simple shunt model can induce early upregulation of eNOS expression with increased pulmonary blood flow and pulmonary artery pressure in rats.