Immuno-electron microscopic analysis of the distribution of ICAM-1 in human inflammatory tissue

An immuno-electron microscopic analysis was undertaken to determine ICAM-1 expression on vascular endothelium in human tonsils and in synovia from patients with rheumatoid arthritis. ICAM-1 was preferentially expressed on high endothelial venules (HEV) located in the parafollicular regions of the tonsils and on HEV located in the villous processes of the synovia. On both tissues, these areas contained the greatest number of perivascular lymphocytes. In contrast, ICAM-1 was only weakly expressed on the low endothelium lining capillaries, venules and sinusoids. In both tonsils and synovia, ICAM-1 was confined to the luminal and lateral surfaces of the endothelial cells and absent from the abluminal surfaces adjacent to the basement membrane. We propose that in inflammatory tissues, ICAM-1 mediates the interaction of circulating lymphocytes with the high endothelial cells, but may not have a major role in promoting their migration through the whole thickness of the blood vessel wall.     

Immuno-electron microscopic analysis of the distribution of ICAM-1 in human inflammatory tissue

An immuno-electron microscopic analysis was undertaken to determine ICAM-1 expression on vascular endothelium in human tonsils and in synovia from patients with rheumatoid arthritis. ICAM-1 was preferentially expressed on high endothelial venules (HEV) located in the parafollicular regions of the tonsils and on HEV located in the villous processes of the synovia. On both tissues, these areas contained the greatest number of perivascular lymphocytes. In contrast, ICAM-1 was only weakly expressed on the low endothelium lining capillaries, venules and sinusoids. In both tonsils and synovia, ICAM-1 was confined to the luminal and lateral surfaces of the endothelial cells and absent from the abluminal surfaces adjacent to the basement membrane. We propose that in inflammatory tissues, ICAM-1 mediates the interaction of circulating lymphocytes with the high endothelial cells, but may not have a major role in promoting their migration through the whole thickness of the blood vessel wall.     

Caveolin-1 immuno-expression in human gastric cancer: histopathogenetic hypotheses

The immunohistochemical expression of caveolin-1 (cav-1) was evaluated in a series of gastric carcinomas (GC) and in the adjacent normal gastric mucosa. Cav-1 immuno-expression was found in most GC (94%) with a significantly higher amount in the Lauren intestinal type in comparison to the diffuse-type carcinomas. Interestingly, gastric intestinal metaplasia as well as the cells at the base and neck of gastric pits within all fundic mucosal fragments showed an evident cav-1 immuno-staining, suggesting a histogenetic derivation of these lesions from the trans-differentiation of chief cells or from a cryptic progenitor population at the base of fundic glands, as recently hypothesized by other authors. The absence of significant correlations between cav-1 immuno-expression and the other clinico-pathological parameters, such as the stage of disease or the patients overall survival, indicates that the role of cav-1 in GC is neither stage-specific nor related to prognosis.     

Expression of divalent metal transporter 1 (DMT1) isoforms in first trimester human placenta and embryonic tissues

BACKGROUND: Divalent metal transporter 1 (DMT1) is a transmembrane glycoprotein which mediates the proton-coupled transport of a variety of divalent metal ions. Two isoforms, which differ by the presence (DMT1-IRE) or absence (DMT1-nonIRE) of an iron-responsive element (IRE) in their 3' untranslated region, are implicated in apical iron transport and endosomal iron transport respectively. Although the expression pattern of DMT1 isoforms is tissue specific in adult, data regarding its expression in embryonic tissues are lacking. METHODS: Semiquantitative RT-PCR and immunohistochemistry were used to study the mRNA and protein expression of both DMT1 isoforms in embryonic tissues between 8 and 14 weeks gestational age. RESULTS: DMT1-IRE and DMT1-nonIRE expressions were ubiquitous in embryonic tissues examined. In the lung, statistically significant correlations were found between the levels of DMT1 isoform expression and gestational age. In the placenta, DMT1-IRE was the predominantly expressed isoform. Both isoform proteins were localized in embryonic epithelial cellular membrane. CONCLUSION: Both DMT1 isoforms are ubiquitously expressed in embryonic tissues in the first trimester. Predominant DMT1-IRE isoform expression in placenta suggests an iron-regulatory mechanism reminiscent of that in the adult duodenum. Epithelial distributions of both DMT1 isoforms are associated with the absorptive or excretory functions of the expressed tissues.     

Increased caveolin-1, a cause for the declined adipogenic potential of senescent human mesenchymal stem cells

Mesenchymal stem cell (MSC) has drawn much attention in the aspect of tissue renewal and wound healing because of its multipotency. We initially observed that bone marrow-derived human MSCs (hMSCs) divided poorly and took flat and enlarged morphology after expanded in culture over a certain number of cell passage, which resembled characteristic features of senescent cells, well-studied in human diploid fibroblasts (HDFs). More interestingly, adipogenic differentiation potential of hMSCs sharply declined as they approached the end of their proliferative life span. In this study, altered hMSCs were verified to be senescent by their senescence-associated beta-galactosidase (SA-beta-gal) activity and the increased expression of cell cycle regulating proteins (p16(INK4a), p21(Waf1) and p53). Similar as in HDFs, basal phosphorylation level of ERK was also significantly increased in senescent hMSCs, implying altered signal paths commonly shared by the senescent cells. Insulin, a major component of adipogenesis inducing medium, did not phosphorylate ERK 1/2 more in senescent hMSCs after its addition whereas it did in young cells. In senescent hMSCs, we also found a significant increase of caveolin-1 expression, previously reported as a cause for the attenuated response to growth factors in senescent HDFs. When we overexpressed caveolin-1 in young hMSC, not only insulin signaling but also adipogenic differentiation was significantly suppressed with down-regulated PPARgamma2. These data indicate that loss of adipogenic differentiation potential in senescent hMSC is mediated by the over-expression of caveolin-1.     

小凹蛋白与内皮型一氧化氮合成酶在人肝硬化组织中的表达及意义

目的 检测人肝硬化组织中小凹蛋白(caveolin-1)、内皮型一氧化氮合成酶(eNOS)的细胞定位及蛋白表达水平的变化,探讨caveolin-1的表达对eNOS的影响.方法 免疫组织化学染色检测30例肝硬化患者活检后肝组织和30例肝外伤、肝血管瘤患者正常肝组织中caveolin-1和eNOS的细胞定位.Western印迹检测caveolin-1和eNOS的蛋白表达水平变化.结果 caveolin-1和eNOS均主要分布于肝窦内皮细胞中,caveolin-1在肝硬化组和对照组表达阳性率有差异,分别为87%和40%(P＜0.05).eNOS在肝硬化组和对照组表达阳性率有差异,分别为33%和66%(P＜0.05).caveolin-1在肝硬化组织中表达较正常肝组织中明显增强.eNOS在肝硬化组织中呈低水平表达,较正常肝组织中表达明显减少.结论 肝硬化肝窦内皮细胞的损伤降低了eNOS的表达.caveolin-1的过表达促进eNOS-caveolin-1复合物的形成,加剧了门静脉高压症的发生.     

小凹蛋白与内皮型一氧化氮合成酶在人肝硬化组织中的表达及意义

目的检测人肝硬化组织中小凹蛋白(caveolin-1)、内皮型一氧化氮合成酶(eNOS)的细胞定位及蛋白表达水平的变化,探讨caveolin-1的表达对eNOS的影响。方法免疫组织化学染色检测30例肝硬化患者活检后肝组织和30例肝外伤、肝血管瘤患者正常肝组织中caveolin-1和eNOS的细胞定位。Western印迹检测caveolin-1和eNOS的蛋白表达水平变化。结果caveolin-1和eNOS均主要分布于肝窦内皮细胞中,caveolin-1在肝硬化组和对照组表达阳性率有差异,分别为87%和40%(P<0.05)。eNOS在肝硬化组和对照组表达阳性率有差异,分别为33%和66%(P<0.05)。caveolin-1在肝硬化组织中表达较正常肝组织中明显增强。eNOS在肝硬化组织中呈低水平表达,较正常肝组织中表达明显减少。结论肝硬化肝窦内皮细胞的损伤降低了eNOS的表达。caveolin-1的过表达促进eNOS-caveolin-1复合物的形成,加剧了门静脉高压症的发生。     

乳腺癌组织中Caveolin-1、MMP-2的表达及意义

目的 探讨Caveolin-1及MMP-2蛋白在乳腺癌及正常乳腺组织中的表达及两者的相关性.方法 采用EnVision法免疫组化检测86例乳腺癌及20例正常乳腺组织中Caveolin-1及MMP-2蛋白的表达.结果 乳腺癌中Caveolin-1的阳性率(52.3%)低于正常乳腺组织(95.0%)(P<0.05),而MMP-2的阳性率高于正常乳腺组织(P<0.05).Caveolin-1在乳腺癌的表达与淋巴结转移、临床分期相关(P<0.05),而与患者年龄、组织学分级及肿瘤大小无关(P>0.05).MMP-2在乳腺癌的表达与淋巴结转移、组织学分级及临床分期相关(P<0.05),而与患者年龄及肿瘤大小无关(P>0.05).在乳腺癌中Caveolin-1和MMP-2的表达呈负相关(r=-0.472,P<0.05).结论 Caveolin-1低表达与MMP-2过表达可能是乳腺组织恶性转变以及乳腺癌发生浸润转移的重要生物学标志,联合检测Caveolin-1和MMP-2对预测乳腺癌的浸润转移有重要意义.     

Rb1延缓人脐静脉内皮细胞早熟性衰老与caveolin-1表达的关系

目的:血管内皮细胞衰老是动脉粥样硬化发生的病理生理机制之一。本研究旨在探讨人参皂苷Rb1延缓人脐静脉内皮细胞(HUVECs)早熟性衰老与窖蛋白1(caveolin-1)表达的关系,为延缓HUVECs衰老提供新的靶点。方法:建立60μmol/L过氧化氢(H_2O_2)诱导的HUVECs早熟性衰老模型,根据细胞形态学的变化、衰老相关β-半乳糖苷酶(SA-β-Gal)染色阳性率和细胞周期评估内皮细胞衰老,采用Western blot和激光共聚焦显微成像的方法检测caveolin-1的变化,观察人参皂苷Rb1对HUVECs衰老的作用及其相关的分子机制。结果:60μmol/L H_2O_2可成功地诱导内皮细胞衰老,早熟性衰老的HUVECs体积变大,SA-β-Gal活性明显增加,细胞发生G_1期阻滞,细胞增殖受抑制,caveolin-1表达增多。与H_2O_2处理组相比,人参皂苷Rb1预处理延缓HUVECs早熟性衰老,SA-β-Gal染色阳性细胞百分比降低,G_0/G_1期细胞比例下降,caveolin-1表达减少。结论:人参皂苷Rb1可通过抑制caveolin-1的表达延缓H_2O_2诱导的HUVECs早熟性衰老。     

Expression of caveolin-1 is correlated with Akt-1 in colorectal cancer tissues

Caveolin is a major component of caveolae which is a plasma membrane microdomain. The emerging role of caveolin in tumorigenesis was based mainly on in vitro experiments with cancer cell lines. We performed semi-quantitative RT-PCR for caveolin, Akt and EGFR to understand the role of caveolins in colorectal tumor biology. Cancer tissue samples and the neighboring normal colon mucosa were obtained from 95 colorectal cancer patients who underwent operations at Ewha Womans University Mokdong Hospital. With these fresh tissues, semi-quantitative RT-PCR was performed by coamplification of the gene for caveolin-1, EGFR and Akt-1 with beta-actin. The average age was 60.21+/-13.33 years old, and sex ratio was 1.44:1. Caveolin-1 is more expressed in tumors than normal mucosa (P=0.025). The expression of caveolin-1 and Akt-1 had a definitive positive relationship (P=0.002). But, the expression of caveolin-1 and EGFR was not significantly related. We could not find correlations between caveolin-1 expression and clinical factors. In conclusion, caveolin-1 is more expressed in cancer tissues than normal colon and related with Akt-1, not with EGFR expression in colorectal cancer tissues, which suggests that signaling for caveolin-1 affects Akt-1 activation, but this reaction is not initiated by EGFR stimulation in colon cancer.     

Lack of transport of erythropoietin across the human placenta as studied by an in vitro perfusion system

The transfer of human recombinant erythropoietin (rhEPO) from the maternal to the fetal side was investigated using the technique of in vitro perfusion of an isolated cotyledon of human placenta, with recirculation of the perfusate (130 ml) in separate closed maternal and fetal circuits. rhEPO (221–512 U), together with [¹⁴C]BSA (bovine serum albumin, 44.8 kBq or 2,688,000 dpm), was added to the maternal circuit only. Despite a considerably lower molecular weight of EPO mol. wt.=30,400 Da) compared to BSA (mol. wt.= 69,000 Da), no difference was found in their transfer across the placenta from the maternal to the fetal side, which was very low for both macromolecules. The total transfer of rhEPO derived from the concentration measured in the samples taken from the fetal circuit at the end of 4–5 h of perfusion, was in the range of 0.04% of the amount initially added to the maternal compartment. A similar amount of transfer was determined for [¹⁴C]BSA (0.04–0.07%,n=12). In conclusion, by direct determination in a dually in vitro perfused human placental cotyledon, no significant transfer of rhEPO from the maternal to the fetal side could be shown.     

Histologic distribution and biochemical properties of alpha 1-microglobulin in human placenta

PROBLEM: The embryo is protected from immunologic rejection by the mother, possibly accomplished by immunosuppressive molecules located in the placenta. We investigated the distribution and biochemical properties in placenta of the immunosuppressive plasma protein alpha 1-microglobulin. METHOD OF STUDY: Placental alpha 1-microglobulin was investigated by immunohistochemistry and, after extraction, by electrophoresis, immunoblotting and radioimmunoassay. RESULTS: alpha 1-Microglobulin staining was observed in the intervillous fibrin and in syncytiotrophoblasts, especially at sites with syncytial injury. Strongly stained single cells in the intervillous spaces and variably stained intravillous histiocytes were noted. Solubilization of the placenta-matrix fraction and placenta membrane fraction released predominantly the free form of alpha 1-microglobulin, but, additionally, an apparently truncated form from the placenta-membrane fraction. The soluble fraction of placenta contained two novel alpha 1-microglobulin complexes. CONCLUSIONS: The biochemical analysis indicates the presence in placenta of alpha 1-microglobulin forms not found in blood. The histochemical analysis supports the possibility that alpha 1-microglobulin may function as a local immunoregulator in the placenta.     

Caveolin-1 promotes tumor cell proliferation and vasculogenic mimicry formation in human glioma

Vasculogenic mimicry (VM) plays an important role in human glioma progression and resistance to antiangiogenic therapy as a compensatory neovascularization mechanism in malignant tumors. Caveolin-1 (Cav-1) has been found to contribute to VM formation. However, it remains largely unknown whether Cav-1 expression correlates with VM in glioma. In this study, we examined CAV-1 expression levels and VM in human glioma cell lines and in 94 human gliomas with different grades of malignancy, and present Cox proportional hazards regression. The molecular role of Cav-1 in glioma cells was investigated using quantitative polymerase chain reaction (qRT-PCR) assays, western blotting, CCK-8 assays, and tubule formation assays. Cav-1 expression and VM formation were positively correlated with each other and both were closely associated with glioma development and progression as evidenced by the presence of cystic tumor, shortened survival time, and advanced-stage glioma in glioma patients with Cav-1 overexpression/increased VM formation. Cav-1 promoted U251 glioma cell proliferation and VM formation in a Matrigel-based 3D culture model. VM-associated factors including hypoxia-inducible factor 1α (HIF-1α) and p-Akt was significantly elevated by Cav-1 overexpression but suppressed by siCav-1 in U251 cells. Collectively, our study identified Cav-1 as an important regulator of glioma cell proliferation and VM formation, contributing to glioma development and progression.     

Caveolin-1在TGF-β1诱导人支气管上皮细胞间质化中的作用

目的:探究小窝蛋白1(caveolin-1)在转化生长因子β1(TGF-β1)诱导的人支气管上皮细胞(16HBE细胞)上皮-间充质转化(EMT)中的作用。方法:以16HBE细胞株为研究对象,免疫荧光、RT-q PCR和Western blot实验检测16HBE细胞EMT过程中caveolin-1的mRNA和蛋白表达;Western blot检测siRNA干扰caveolin-1对16HBE细胞EMT的影响。结果:Caveolin-1广泛存在于16HBE细胞膜上,TGF-β1刺激后,caveolin-1的mRNA和蛋白表达减少(P0.05)。与TGF-β1组比较,caveolin-1 siRNA和TGF-β1共同作用促进了细胞形态的转化,抑制了E-钙黏蛋白的蛋白表达而促进了α-SMA的蛋白表达(P0.05)。TGF-β1刺激16HBE细胞后,AKT和Smad3在30 min磷酸化水平最高,与0 min对照组比较显著增加(P0.05);用siRNA干扰caveolin-1基因后再用TGF-β1刺激16HBE细胞30 min,下游信号蛋白分子AKT和Smad3的磷酸化水平增高,与TGF-β1组比较显著增加(P0.05)。结论:TGF-β1能下调16HBE细胞的caveolin-1表达水平;caveolin-1可能参与了TGF-β1诱导的16HBE细胞EMT过程中TGF-β1/Smad通路和PI3K-AKT通路的活化。     

窖蛋白-1在肺癌中的表达及意义

目的 探讨窖蛋白-1（caveolin-1）在不同类型肺癌组织中的表达及其与微血管密度（MVD）和临床病理因素之间的关系。方法 对154例原发性肺癌、相应癌旁正常肺组织及36例淋巴结转移癌行caveolin-1免疫组织化学染色;对154例原发性肺癌行CD34免疫组织化学（SP法）染色并进行微血管密度计数;Western印迹法检测其中50例新鲜肺癌组织及其癌旁正常肺组织中caveolin-1的表达情况。结果 caveolin-1为膜／质表达蛋白,在正常支气管上皮细胞和肺泡上皮细胞中的阳性率为100％。在肺癌组织中的阳性率为59．1％（91／154）,低于癌旁正常肺组织,P＜0．01;Western印迹结果进一步证实caveolin-1在肺鳞癌、肺腺癌组织中的表达均显著低于癌旁正常肺组织,P＜0．01。caveolin-1在小细胞肺癌（SCLC）和非小细胞肺癌（NSCLC）中的阳性率分别为7．1％和64．3％,二者间差异有统计学意义,P＜0．01。NSCLC中,有淋巴结转移组caveolin-1表达高于无淋巴结转移组（P=0．005）;Ⅲ、Ⅳ期组caveolin-1表达显著高于Ⅰ、Ⅱ期组（P=0．042）,caveolin-1表达与NSCLC的其他临床病理因素及MVD值无关（P＞0．05）。结论caveolin-1其作为一种肿瘤抑制因子的同时,可能还具有促进NSCLC进展和转移的活性。     

Distribution of caveolin in the muscle spindles of human skeletal muscle

The aim of the present study was to demonstrate the location of the different members of the caveolin (cav) family in human muscle spindles. Twenty spindles of three human muscles (vastus medialis, ischiocavernosus, bulbospongiosus) from 12 cadavers were immunohistochemically stained for cav‐1, cav‐2, and cav‐3, and the equatorial and polar regions evaluated. All layers of the outer and inner spindle capsule and all blood vessels within the spindle stained for cav‐1 and cav‐2. In the muscle spindle, intrafusal muscle fibres stained selectively for cav‐3, but with a patchy appearance. Caveolinopathies may therefore also include changes in muscle spindle function.     

Distinctive expression profiles of Caveolin-1 and Notch-1 protein in patients with nasal polyps or sinonasal inverted papillomas

Background

Nasal polyposis (NP) and sinonasal inverted papillomas (SIP) are considered benign lesions capable of recurrence or malignant transformation although not with the same prevalence. Since fluctuations of Caveolin-1 and Notch-1 proteins expression have been reported in many pathologies, the current study aimed to investigate their involvement in the epithelial transformation observed in SIPs compared to NP.

Apical and basolateral localisation of GLUT2 transporters in human lung epithelial cells

Glucose concentrations of normal human airway surface liquid are ~12.5 times lower than blood glucose concentrations indicating that glucose uptake by epithelial cells may play a role in maintaining lung glucose homeostasis. We have therefore investigated potential glucose uptake mechanisms in non-polarised and polarised H441 human airway epithelial cells and bronchial biopsies. We detected mRNA and protein for glucose transporter type 2 (GLUT2) and glucose transporter type 4 (GLUT4) in non-polarised cells but GLUT4 was not detected in the plasma membrane. In polarised cells, GLUT2 protein was detected in both apical and basolateral membranes. Furthermore, GLUT2 protein was localised to epithelial cells of human bronchial mucosa biopsies. In non-polarised H441 cells, uptake of d-glucose and deoxyglucose was similar. Uptake of both was inhibited by phloretin indicating that glucose uptake was via GLUT-mediated transport. Phloretin-sensitive transport remained the predominant route for glucose uptake across apical and basolateral membranes of polarised cells and was maximal at 5–10 mM glucose. We could not conclusively demonstrate sodium/glucose transporter-mediated transport in non-polarised or polarised cells. Our study provides the first evidence that glucose transport in human airway epithelial cells in vitro and in vivo utilises GLUT2 transporters. We speculate that these transporters could contribute to glucose uptake/homeostasis in the human airway.     

小窝蛋白1脚手架区多肽减轻LPS诱导的小鼠急性肺损伤

目的:探究人工合成的小窝蛋白1(caveolin-1,Cav-1)脚手架区多肽cavtratin对血红素加氧酶1(heme oxygenase-1,HO-1)活性及脂多糖(lipopolysaccharide,LPS)诱导的小鼠急性肺损伤的作用。方法:成年雄性BALB/c小鼠随机分为6组,每组8~10只,实验分为对照(control)组、触足肽内化序列(Antennapedia internalization sequence,AP)组、LPS组、LPS+血晶素(hemin)组、LPS+hemin+cavtratin组和LPS+hemin+cavtratin+锌原卟啉(zinc protoporphyrin IX,Zn PP)组。小鼠气管滴注LPS 24 h后,苏木素-伊红染色观察肺组织病理形态变化;检测肺组织湿/干重比、肺泡灌洗液中细胞数和血清中乳酸脱氢酶活性。免疫荧光观察HO-1和Cav-1的结合情况并检测HO-1活性;实时荧光定量PCR检测炎症因子(IL-1β、IL-6、TNF-α、MCP-1和i NOS)的mRNA水平。结果:组织免疫荧光以及HO-1活性检测发现,与LPS组比较,LPS+hemin+cavtratin组HO-1与细胞膜Cav-1的结合减少,HO-1的活性增高(P0.05);与LPS组比较,LPS+hemin+cavtratin组肺组织受损程度明显减轻,肺湿/干重比、肺泡灌洗液细胞数和血清中乳酸脱氢酶活性显著降低(P0.05);与LPS组比较,LPS+hemin+cavtratin组炎症因子的mRNA表达降低(P0.05);HO-1活性抑制剂Zn PP可以消除cavtratin的保护作用。结论:Cavtratin可以通过减少HO-1与细胞膜Cav-1的结合使得HO-1活性增加,进而减轻LPS诱导的小鼠急性肺损伤。     

低氧诱导的小窝蛋白-1上调参与人肺腺癌细胞A549迁移和侵袭

 目的:探讨低氧诱导剂二氯化钴（CoCl2）对小窝蛋白-1（Cav-1）生成的调节作用及后者对人肺腺癌A549细胞迁移、侵袭的影响。方法:检测肺癌患者伴恶性胸水（MPE）和结核性胸膜炎患者胸水（TBPE）中Cav-1和缺氧诱导因子(HIF)-1α浓度,比较两者相关性;以CoCl2和（或）HIF-1α抑制剂YC-1作用于A549细胞ELISA法检测细胞上清Cav-1和HIF-1α浓度;分别采用细胞划痕实验及Transwell小室侵袭实验研究CoCl2刺激表达的Cav-1对A549细胞迁移和侵袭的影响。结果:MPE中Cav-1和HIF-1α浓度明显高于TBPE,两组患者胸水中Cav-1与HIF-1α均呈正相关。CoCl2浓度和时间依赖性诱导A549细胞Cav-1和HIF-1α生成,200 μmol/L或24 h达到峰值;浓度>200 μmol/L或作用时间超过24 h则呈现浓度或时间依赖性抑制。HIF-1α抑制剂YC-1浓度依赖性抑制HIF-1α和Cav-1生成。CoCl2浓度依赖性增强A549细胞迁移和侵袭,200 μmol/L作用最强;YC-1对上述过程产生抑制效应。结论: 肺癌患者胸腔积液中Cav-1浓度升高,低氧诱导Cav-1生成的变化可能参与了A549细胞迁移和侵袭,HIF-1α可能对Cav-1生成发挥影响。