Quantitative assessment of the normal cerebral microvasculature by endothelial barrier antigen (EBA) immunohistochemistry: application to focal cerebral ischemia

Cerebrovascular endothelium participates importantly in the pathophysiology of ischemic injury. Endothelial barrier antigen (EBA) is a protein located in the luminal plasma membrane of normal central and peripheral nervous-system endothelium. In this study, we assessed the sensitivity and specificity of EBA as a quantitative marker of normal endothelium and characterized alterations of EBA immunohistochemistry following focal cerebral ischemia. Anesesthetized, non-ischemic control rats (N=6) were studied. Other animals (N=5) received 90 min of middle cerebral artery occlusion (MCAo) followed by 3-day survival. Brains were prepared by perfusion-fixation and paraffin-embedding. For EBA immunohistochemistry, a monoclonal antibody (1:2000 dilution) was used. Adjacent sections were reacted for activated microglia by isolectin immunochemistry. Morphometric image-analysis was carried out in standardized microscopic fields. In control brains, pial and parenchymal blood vessels of all sizes were distinctly and selectively immunolabeled for EBA; background staining was absent. EBA-positive vascular profiles occupied 4.3+/-0.36% (mean+/-S.D.) of the microscopic field. The mean area of each identified profile was 51+/-13 micromter(2). The low coefficients of variation for both numbers of profiles (17%) and fractional areas (8%) denoted high inter-animal consistency. In brains with prior MCAo, numbers of EBA-immunoreactive vascular profiles in infarcted cortex and striatum were reduced by 39 and 46%, respectively, and their fractional areas were decreased by 63 and 76%, respectively, compared to contralateral hemisphere. Activated microglia were prominent in zones of frank infarction and in adjacent paramedian cortex; the latter region, however, showed normal-appearing EBA-immunostaining. EBA-immunohistochemistry provides a sensitive and specific index of normal cerebrovascular endothelial structures of all sizes. The technique lends itself well to quantitative morphometry and is applicable to perfusion-fixed paraffin-embedded material. EBA immunoreactivity declines in zones of ischemic infarction.     

内皮抑素治疗人喉癌裸鼠模型的实验研究

目的 研究内皮抑素对裸鼠喉鳞状细胞癌皮下移植模型的抑瘤作用,探讨其抑瘤机制及人喉癌生物治疗新方法。方法 建立人喉癌Hep-Ⅱ细胞株裸鼠皮下接种模型。应用内皮抑素治疗,观察肿瘤生长情况,对用药后肿瘤组织进行病理学检查,通用型二步法免疫组化检查及电镜观察。计数微血管密度(microvessel density,MVD),计算增殖细胞核抗原(proliferationg cell nuclear antigen,PCNA)和血管内皮生长因子(vascuoar endothelial growth factor,VEGF)阳性率。结果 在实验组与对照组之间,鼠净重、瘤重、瘤体积及瘤重/鼠净重,经t检验P值<0.05或<0.01,差异均有显著性意义,抑瘤率为45.9％。病理学检查及电镜观察表明,实验组应用内皮抑素治疗肿瘤生长受到抑制,细胞分裂少见,可见肿瘤细胞的坏死凋亡,新生血管明显减少。MVD、PCNA及VEGF表达在实验组明显低于对照组,经t检验P值分别为<0.01、<0.05、<0.05,差异均有显著性意义。结论 内皮抑素可显著抑制人喉癌裸鼠模型肿瘤的生长和发展,其可能机制为抑制肿瘤血管生成。     

玻璃体切割手术对角膜内皮细胞的影响

目的 观察玻璃体切割手术对角膜内皮细胞影响的相关因素。 方法 回顾分析21 3例玻璃体切割手术患者213只眼手术前及手术后1周、1、3个月时角膜内皮镜检查结果。其中单纯玻璃体切割手术78只眼，玻璃体切割手术联合白内障摘除手术135只眼；硅油填充34只眼，C³F⁸填充53只眼，无眼内填充物者126只眼。 结果 单纯玻璃体切割组、玻璃体切割联合白内障摘除后囊完整组手术前后不同时期角膜内皮细胞数差异无显著性的意义（P均>0.05）；无后囊且眼内填充C³F⁸或硅油组手术后较手术前角膜内皮细胞数明显减少，其差异有显著性的意义（P<0.05）。 结论 玻璃体切割联合白内障摘除且晶状体后囊不完整者，眼内填充C³F⁸或硅油对角膜内皮细胞有一定的损伤作用。（中华眼底病杂志，2004，20：101-103）     

雷帕霉素洗脱支架置入动物体内后的支架内皮化进程

目的研究雷帕霉素洗脱支架(CypherTM)对血管内皮再生的影响。方法杂种犬6只,经麻醉后行血管造影,选择动脉血管直径为2.25 mm的直血管段在远端及近端分别置入直径为2.25 mm的CypherTM支架及Bx son ic支架。术后仍继续给予抗血小板治疗,分别在术后1月、3月时行再次血管造影后取标本行扫描电镜检查。结果术后1月、3月血管造影CypherTM支架及Bx son ic支架均无管腔丢失。1月时扫描电镜显示CypherTM支架内皮覆盖不完整,有一定的血小板黏附、聚集;Bx son ic支架内皮覆盖完整,未见血小板黏附聚集。3月时CypherTM支架及Bx son ic支架内皮覆盖均较完整,无血小板黏附、聚集。结论CypherTM支架内皮再生较裸金属支架缓慢,有一定的血小板激活。3个月时才能完全实现内皮化。置入CypherTM支架的患者应进行更为严格的抗血小板治疗。     

EGFP Kallikrein重组真核表达载体的构建及其在兔内皮祖细胞中的表达

目的：构建携带人组织激肽释放酶（tHK）基因，以绿色荧光蛋白(GFP)为报告基因的重组真核表达载体,并导入内皮祖细胞(EPCs)中表达，验证tHK基因修饰EPCs用于血管病治疗的可行性。方法：PCR扩增tHK目的基因，将该全长基因定向克隆至真核表达载体pEGFP N3上，构建重组质粒载体。并利用脂质体转染体外培养的EPCs，在活细胞状态下用荧光显微镜直接观察tHK EGFP融合蛋白在细胞中的表达；用RT PCR 方法验证tHK在mRNA水平的表达；用ELISA方法验证tHK蛋白水平的表达。结果：酶切和测序证实pEGFP Kallikrein重组质粒构建正确，重组质粒载体在EPCs 中获得了表达,表达的融合蛋白具有tHK 和EGFP 的双重活性。结论：通过基因克隆方法成功构建了pEGFP Kallikrein重组质粒载体，并且在EPCs 中稳定表达，为进一步研究tHK基因修饰EPCs双重治疗血管病的技术策略奠定了基础。     

I型三磷酸鸟苷环化水解酶在2型糖尿病大鼠主动脉表达的改变

目的:探讨血管内皮功能异常状态下,I型三磷酸鸟苷环化水解酶(GTPCH I)mRNA在2型糖尿病及硫辛酸干预大鼠主动脉中的表达。方法:选择雄性Wistar大鼠35只,随机选择10只作为正常对照组(NC组),25只采用高脂饲料喂养8周后,小剂量链脲佐菌素注射诱导建立2型糖尿病大鼠模型(成模21只),其中10只给予硫辛酸50 mg/kg腹腔注射,作为硫辛酸干预组(LA组),其余11只作为2型糖尿病组(T2DM组)。所有大鼠饲养16周后均处死测定其主动脉舒张功能,并用半定量RT-PCR检测主动脉GTPCH ImRNA表达。结果:T2DM组和LA组主动脉的内皮依赖性血管舒张(EDVR)明显减弱,而LA组较T2DM组有明显改善(P<0.05)。主动脉GTPCH I mRNA的表达在T2DM组下降70.28%,LA组下降26.88%,两组差异具有统计学意义(P<0.05)。结论:T2DM大鼠离体主动脉EDVR减弱时,主动脉GTPCH I mRNA表达显著下降。硫辛酸可改善EDVR,此作用可能与改善GTPCH I mRNA表达有关。     

槲皮素与芦丁对离体大鼠主动脉环的舒张作用及机制

目的:比较研究槲皮素与芦丁对离体大鼠胸主动脉环的作用及其可能的途径。方法:采用累积加药法,检测槲皮素和芦丁对去氧肾上腺素(pheny lephrine,PE)预收缩的胸主动脉环张力的影响。结果:槲皮素对离体大鼠内皮完整和去内皮的胸主动脉环均有浓度依赖性的舒张作用,而芦丁对PE预收缩血管的舒张作用是内皮依赖性的。槲皮素和芦丁对内皮完整的胸主动脉环的最大舒张反应分别为(77.20±6.11)%和(44.28±7.48)%,但两者对内皮完整的胸主动脉环最大舒张的半数有效浓度无明显差异。用一氧化氮合酶抑制剂L-NAM E(0.1 mm o l/L)预处理后,可阻断芦丁诱导的舒张血管作用,但不能阻断槲皮素引起的舒张血管作用;用鸟苷酸环化酶抑制剂亚甲蓝(10μm o l/L)预处理后,两者的血管舒张作用均被阻断。用环氧合酶抑制剂吲哚美辛(10μm o l/L)预处理后可减弱槲皮素诱导的舒张血管作用,但不能阻断芦丁引起的舒张血管作用。结论:槲皮素的舒血管作用强于芦丁,槲皮素可能是通过鸟苷酸环化酶和环氧合酶途径产生非内皮依赖性的血管舒张作用,而芦丁可能是通过NO-鸟苷酸环化酶途径产生内皮依赖性的血管舒张作用。     

年龄增加对人血管内皮依赖性舒张作用的实验研究及机制探讨

目的探讨年龄增加对人血管内皮依赖性舒张作用的影响及其初步机制。方法采用血管环张力测定法,在15例不同年龄段（51~83岁）胃癌患者手术切除的胃网膜动脉上,测定0.001~10 μmol/L乙酰胆碱（ACh）诱发的血管内皮依赖性舒张作用和内皮依赖性超级化因子（EDHF）样血管舒张作用的变化;采用实时荧光定量PCR（qRT-PCR）方法观察不同年龄人血管中的内皮型NO合酶（eNOS）、环氧合酶（COX）、胱硫醚γ裂解酶（CSE）以及C-型利钠肽（CNP）基因表达的改变。结果相同剂量下,ACh诱导出人动脉内皮依赖性的舒张作用会随着年龄的增加而减弱（P<0.05）,其EDHF样血管舒张作用也明显降低（P<0.05）;qRT-PCR发现随着年龄的增加,其血管eNOS、CSE和CNP mRNA表达降低(P<0.05),而COX mRNA表达的年龄趋势不明显。结论随年龄的增加,血管内皮依赖性舒张和EDHF样舒张作用降低,可能与内皮合成的NO和CNP等舒血管物质减少有关。     

Phosphodiesterase Inhibitor Effect on Small Artery Function in Preeclampsia

Objective.?To determine if the phosphodiesterase type 5 inhibitor, sildenafil citrate improves endothelial-dependent relaxation of small arteries from women with preeclampsia.?Methods.?Myometrial and omental biopsies were taken from women participating in a randomized placebo-controlled trial using sildenafil citrate in women with preeclampsia. Vasoconstriction and endothelial-dependent relaxation of small arteries was measured utilizing wire myography.?Results.?Vasoconstriction and endothelial-dependent relaxation of myometrial and omental arteries were not altered in women taking sildenafil.?Conclusion.?Acute effects may have been lost as sildenafil administration occurred many hours prior to myography. Plasma sildenafil levels may have been lower than required for vascular response.     

The Effects of Preeclampsia and Oxygen Environment on Endothelial Release of Matrix Metalloproteinase‐2

Background: There is evidence of altered vascular endothelial function in women with preeclampsia as well as in the endothelial cells from umbilical vessels of preeclamptic pregnancies. Matrix metalloproteinase (MMP)‐2 is elevated in the plasma of preeclamptic women and is a mediator of vascular reactivity; however, whether MMP‐2 release is altered in preeclamptic endothelial cells is unknown. We hypothesize that MMP‐2 release is enhanced in endothelial cells from preeclamptic compared with uncomplicated pregnancies and that this phenomenon may be mediated by an oxygen‐dependent mechanism. Our specific hypothesis is that cells from normal pregnancies will demonstrate enhanced MMP‐2 release at low oxygen (< 0.5%, 2%) compared to high oxygen (20%), thus mimicking the behavior of preeclamptic cells. Methods: Human umbilical vein endothelial cells (HUVECs) from preeclamptic pregnancies (n = 4) and normal pregnancies (n = 4) were incubated for 12 hr in standard culture conditions (20% oxygen). In a separate series of experiments, HUVECs from normal pregnancies (n = 6) were incubated for 12 hr at < 0.5%, 2%, and 20% oxygen. Supernatants were analyzed for MMP‐2 and tissue inhibitors of metalloproteinases (TIMP)‐1 and ‐2. Results: The HUVECs from women with preeclampsia demonstrated significantly enhanced release of MMP‐2 (p < 0.05), TIMP‐1 (p < 0.001), and TIMP‐2 (p = 0.01) compared to normal cells. MMP‐2 release from HUVECs from uncomplicated pregnancies was significantly elevated at 2% oxygen compared to < 0.5% and 20% oxygen (p < 0.05). TIMP‐1 and ‐2 secretion was not altered with varying oxygen. Conclusions: Preeclamptic endothelial cells demonstrate significantly enhanced MMP‐2, TIMP‐1 and TIMP‐2 release compared to normal cells. Our data show that there are significant effects of oxygen tension on MMP‐2 release from normal cells; however, the magnitude of the enhanced release is small when compared to the differences in MMP‐2 release in cells from preeclamptic and normal pregnancies. Furthermore, TIMP‐1 and ‐2 release is not affected by changes in oxygen. It is unlikely that oxygen is a key mediator of the enhanced MMP‐2, TIMP‐1 and TIMP‐2 release observed in preeclamptic cells.