Endomorphins exit the brain by a saturable efflux system at the basolateral surface of cerebral endothelial cells

Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are two highly selective mu-opiate receptor agonists. We recently demonstrated that EM-1 and EM-2 have a saturable transport system from brain-to-blood in vivo. Since the endothelial cells are the main component of the non-fenestrated microvessels of the blood-brain barrier (BBB), we examined whether these endogenous tetrapeptides have a saturable transport system in cultured cerebral endothelial cells. EM-1 and EM-2 binding and transport were studied in a transwell system in which primary mouse endothelial cells were co-cultured with rat glioma cells. We found that binding of both endomorphins was greater on the basolateral than the apical cell surface. Flux of EM-1 and EM-2 occurred predominantly in the basolateral to apical direction, each showing self-inhibition with an excess of the respective endomorphin. Transport was not influenced by the addition of the P-glycoprotein inhibitor, cyclosporin A. Neither the mu-opiate receptor agonist DAMGO nor the delta-opiate receptor agonist DPDPE had any effect on the transport. Thus, the results show that a saturable transport system for EM-1 and EM-2 occurs at the level of endothelial cells of the BBB, and unlike ß-endorphin and morphine, P-glycoprotein is not needed for the brain-to-blood transport. Cross-inhibition of the transport of each endomorphin by the other suggests a shared transport system that is different from mu- or delta-opiate receptors. As endormorphins are mainly produced in the CNS, the presence of the efflux system at the BBB could play an important role in pain modulation and neuroendocrine control.     

Chemically, mechanically, and hyperosmolarity-induced calcium responses of rat cortical capillary endothelial cells in culture

 The purpose of the present work was to characterize calcium responses of brain-capillary endothelial cells (BCEC), the cells forming the blood-brain barrier, to chemical, hyperosmolar and mechanical stimulation. Confluent BCEC cultures were grown from capillary fragments isolated from rat cerebral cortex. Intracellular free calcium ([Ca²⁺]_i) was measured using fura-2 and digital imaging. Our experiments show large endothelial calcium responses to substance P and ATP, up to a peak value of approximately 1000 and 600 nM, respectively, and these responses were observed in 2/3 of the cells. Calcium responses to bradykinin, histamine, and hyperosmolar sucrose or mannitol were smaller, attaining a peak in the range 180–340 nM, and were observed in a smaller fraction of the cells. No calcium responses were observed to high-potassium, l-glutamate, serotonin, carbachol, noradrenalin, and nitric-oxide donors. Consecutive superfusion of the cultures with ATP, bradykinin, and histamin showed that cells with a certain response pattern were spatially grouped; the response pattern itself varied widely between experiments. Mechanical stimulation of a single cell caused a calcium response in the stimulated cell in primary cultures and triggered an intercellularly propagating calcium wave in passaged cultures. Given the important effect of endothelial [Ca²⁺]_i on blood-brain barrier permeability and transport, we conclude that substance P and ATP are potential modulators of blood-brain barrier function. Hyperosmolarity-induced blood-brain barrier opening is probably not mediated through endothelial [Ca²⁺]_i. Received: 21 September 1998 / Accepted: 8 February 1999     

Repetitive/temporal hypoxia increased P-glycoprotein expression in cultured rat brain microvascular endothelial cells in vitro

The aim of the study was to investigate whether repetitive/temporal hypoxia up-regulated P-glycoprotein (P-gp) in cultured rat brain microvascular endothelial cells (rBMECs). Cultured rBMECs were used as in vitro blood brain barrier (BBB) model. Cells reached confluence were subjected to temporal hypoxic exposure. Under free-glucose cultured medium, the cells were covered by sterile paraffin oil for 15 min, inducing temporal hypoxic exposure. The hypoxic-exposure was carried out once every day up to 8 days, leading to the repetitive/temporal hypoxia in rBMECs. The cell viability was tested using CCK-8 kit, function and levels of P-gp in the cells were measured using rhodamine 123 uptake and western blot, respectively. It was found that 8-temporal hypoxic exposure induced 1.6-fold increase of P-gp level in cells, accompanied by decrease of cellular accumulation of rhodamine 123. Cellular accumulation of phenobarbital was also decreased. These findings indicated that repetitive/temporal hypoxia may be one of the factors resulting in P-gp overexpression in refractory epilepsy.     

Verapamil induces upregulation of P-glycoprotein expression on human monocyte derived dendritic cells

Overexpression of P-glycoprotein, a transmembrane drug efflux pump that mediates efflux of chemotherapeutic agents contributes to drug resistance in many leukaemia and other cancerous cells. Non-malignant cells including leukocytes also express P-glycoprotein, but physiologic functions for P-glycoprotein are poorly defined. Recently, P-glycoprotein expression has been described in human mononuclear phagocytes and Langerhans cells. It has been shown to play a role in phagocytic cell transmigration through endothelial-lined vessels in an ablumenal-lumenal direction, a process that mimics their migration into lymphatic vessels. Using the monoclonal antibody 4E3, and the P-glycoprotein antagonist, verapamil, the expression of P-glycoprotein on human monocyte-derived dendritic cells was evaluated. Dendritic cells used in this study were CD1a⁺, CD11c⁺, CD14^-, CD80⁺, CD83⁺, CD86⁺ and MHC-II^High. The expression of these markers increased significantly as the cells matured. P-glycoprotein expression was upregulated as the dendritic cells matured as well as in the presence of the “inflammatory stress” of the pathogenic bacteria Strept. pyogenes. Addition of verapamil or Strept. pyogenes to the culture medium during the final 24 hours significantly upregulated P-glycoprotein expression. Immortalized cell lines did not upregulate P-glycoprotein in the presence of verapamil. Evaluation of other normal cells showed that P-glycoprotein upregulation in the presence of verapamil was also a characteristic of macrophages. This novel observation of the upregulation of P-glycoprotein in the presence of verapamil appears to be a characteristic of activated myeloid derived antigen presenting cells and suggest that P-glycoprotein is essential for these cells as when it is blocked, they respond by increasing expression of this protein. In summary, this work describes that human dendritic cells generated from plastic-adherent monocytes rapidly upregulate expression of P-glycoprotein as they mature, and in the presence of inflammatory stress and the pharmacological agent verapamil, which blocks P-glycoprotein activity, suggesting that P-glycoprotein may play a role in activation as well as in migration of dendritic cells.     

Isolation and cultivation of porcine brain capillary endothelial cells as an in vitro model of the blood-brain barrier

Summary Capillary endothelial cells were isolated from the brains of Yucatan miniature swine and were cultivated to serve as an in vitro model for blood-brain barrier studies. The procedure included mechanical and enzymatic digestion of the brain tissue followed by separation of the capillary cells, based on size and density, from contaminating cell types. The purity of the cultures was further enhanced by manipulating the growth medium composition. The cells possess typical capillary endothelial cell morphology, the Factor VIII related antigen, and the ability to accumulate acetylated low-density lipoprotein. Cells from passages 4–6 were grown on polycarbonate membranes suspended between two chambers of media: analogous to the capillary lumen and the interstitium of the brain. A barrier was established within 4 days, as demonstrated by a resistance to the passage of albumin and an electrical current.     

Age-dependent decline of blood-brain barrier P-glycoprotein expression in the canine brain

The efflux transporter P-glycoprotein serves as a major molecular gatekeeper at the blood-brain barrier. It has been suggested that a reduction of P-glycoprotein activity with aging might enhance exposure of brain tissue to exogenous and endogenous compounds thereby contributing to the development of neurodegenerative diseases.Brain tissue from owner-kept dogs renders an excellent tool to study the impact of aging on the background of variable environmental and genetic influencing factors. Therefore, we determined expression rates of P-glycoprotein in canine post-mortem tissue from 23 non-laboratory dogs. P-glycoprotein expression in the parahippocampal cortex exhibited a negative correlation with age. Analysis of the area labeled for P-glycoprotein in dogs aged >100 months revealed a 72% drop in P-glycoprotein expression as compared to young adults aged 23-36 months. Respective data from the dentate hilus and dentate gyrus indicated an earlier drop with a reduction by 77 and 80% in dogs aged 37-99 months in comparison with younger individuals. In contrast to the decline observed with aging in dogs without plaques, P-glycoprotein expression rates rather tended to increase with further aging in dogs with plaque formation.In conclusion, the thorough analysis of P-glycoprotein expression rates in non-laboratory dogs revealed a significant decline with aging. The data strongly support the concept that age-dependent changes might predispose to neurodegenerative diseases. In the early pathogenesis of Alzheimer's disease which is modelled by diffuse plaques in the canine brain, an up-regulation of P-glycoprotein might act as a compensatory mechanism to enhance Abeta efflux from the brain. Future studies are necessary to further evaluate the correlation between Abeta deposits and P-glycoprotein expression in different phases of the disease.     

Increased P-glycoprotein function and level after long-term exposure of four antiepileptic drugs to rat brain microvascular endothelial cells in vitro

To investigate whether long-term exposure to four typical antiepileptic drugs (AEDs): phenobarbital (PB), phenytoin (PHT), carbamazepine (CBZ) and valproic acid (VPA) can increase P-glycoprotein (P-gp) level and function in primary cultured rat brain microvascular endothelial cells (rBMECs) in vitro, the rBMECs were incubated in culture medium containing indicated drugs (PB, PHT, CBZ, VPA and rifampin) for 60 days in a gradient concentration manner. Age-matched cells were incubated in normal culture medium. After a 60-day exposure to the indicated drugs, P-gp function and level in cells were measured using rhodamine 123 (Rho123) accumulations and Western blot analysis, respectively. Lower Rho123 accumulation in drug-treated cells was found than that in age-matched cells. Cyclosporin A (CsA) and verapamil (Ver) increased Rho123 accumulation both in drug-treated cells and age-matched cells. The magnitude of increased Rho123 accumulation in drug-treated cells was larger than that in age-matched cells. Higher P-gp levels were found to be consistent with decrease of Rho123 accumulation in drug-treated cells. The results verified the hypothesis that long-term exposure to the four antiepileptic drugs can induce P-gp function and level in rBMECs.     

Role of N-linked glycosylation in expression of E-selectin on human endothelial cells

E-selectin is a cytokine-inducible membrane glycoprotein capable of mediating adhesion of leukocytes to endothelial cells. It is highly glycosylated, containing 11 sites for N-linked glycosylation. N-Glycosylation of E-selectin was analyzed by endoglycosidase treatment. Analysis of immunoprecipitated E-selectin from human umbilical vein endothelial cells (HUVEC) by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed that E-selectin was completely resistant to endoglycosidase H, but sensitive to peptide N-glycanase F digestion. This suggested that all N-linked oligosaccharide chains were of the complex type. The role of N-linked glycosylation in surface expression and secretion of E-selectin was studied using interleukin-1-stimulated HUVEC, cultured in the presence of the soluble glycosylation inhibitors tunicamycin or castanospermine. Cell surface expression was analyzed by indirect flow cytometry. N-Glycosylation was blocked by tunicamycin, and resulted in a significantly reduced surface expression of E-selectin, whereas castanospermine only marginally reduced E-selectin expression. The deglycosylated forms of E-selectin were also found to be fully capable of mediating adhesion of HT-29 cells in vitro. In conclusion, these studies show that E-selectin is heavily glycosylated with complex type N-linked oligosaccharides and that N-glycosylation is important for expression of E-selectin on human endothelial cells.     

Isolation and characterization of human smooth muscle cells from umbilical cord vein and their reconstitution in a vascular co-culture model with underlying endothelial cells

Smooth muscle cells have been isolated from human umbilical cord veins, characterized and cultured for the development of an endothelialsmooth muscle cell co-culture system. After harvesting endothelial cells, the umbilical cords were trimmed of amnion, connective tissue and arteries, split into pieces, cut open longitudinally and placed with the luminal surface of the explant down onto a culture plate, without the use of proteolytic enzymes. Adherent primary cells were sequentially passaged and various cytological/biochemical characterizations were performed between passages 2 and 10. Cells stained positive for antibodies against smooth muscle actin, negative for antibodies against factor VIII and displayed typical hill and valley morphology when confluent. Cell proliferation was stimulated and supported in a concentration-dependent manner by both human serum and fetal bovine serum over the range 1%–20%. The use of human serum at concentrations >10% decreased the population doubling time during exponential growth by circa 50%. The cells were also characterized by high seeding efficiencies (>70%) and retained their diploid karyotype for up to 3 months in culture. Endothelial cells and smooth muscle cells prepared from umbilical veins were then seeded at varying densities onto either side of porous tissue culture inserts coated with fibronectin. Utilising the measurement of electrical resistance, the optimal seeding density of 5×10⁴ cells/cm² for each cell type gave maximal resistance across the cell bi-layer already after 24 hours, which remaining essentially unaltered for up to 4 days of culture and which was always substantially higher than the resistance of filters seeded only with endothelial cells on one side. This was not substantially affected either by increasing passage of the HUVSM cells cultured with a fixed passage of endothelial cells, or by varying the donor origin of the endothelial cells. In terms of functionality of the selective permeability of the model, the calcium ionophore ionomycin (25 M), added to the endothelial side of the bi-layer, caused a 30% reduction in the electrical resistance across the co-culture within 60 minutes, with control resistance being re-established within 1 hour of removal of the ionophore by washing. These results clearly indicate that smooth muscle cells and endothelial cells prepared from the same human blood vessel can be reconstituted into a functional vascular model suitable for the study of biochemical, physiological and toxicological phenomena in the human vascular wall.Abbreviations BCA bicinchoninic acid - BSA bovine serum albumin - DMEM DMEM medium supplemented with 4.5 g/l glucose, 100 IU/ml penicillin, 100 g/ml streptomycin and 1.25 g/ml fungizone - DMEM-1 DMEM supplemented with 20% FBS, 300 IU/ml penicillin, 300 g/ml streptomycin and 3.75 mg/ml fungizone - DMEM-2 DMEM supplemented with 20% FBS, 100 IU/ml of penicillin, 100 g/ml of streptomycin and 1.25 g/ml of fungizone - DMSO dimethyl sulfoxide - FBS fetal bovine serum - HPF human pulmonary fibroblasts - HS human serum - HUVE human umbilical vein endothelial - HUVSM human umbilical vein smooth muscle - M199 M199 medium supplemented with 20% HS, 100 IU/ml penicillin, 100 g/ml streptomycin, 1.25 g/ml fungizone and 2 mM L-glutamine - P passage number - PBS phosphate buffered saline - TCA trichloroacetic acid - vwF von Willebrand factor (factor VIII)     

葡萄糖对人脐静脉内皮细胞蛋白C受体表达的影响及吡格列酮的干预作用

目的:研究葡萄糖对人脐静脉内皮细胞蛋白C受体(EPCR)mRNA 表达的影响,以及吡格列酮的干预作用。方法:体外培养人脐静脉内皮细胞(HUVECs),分别以流式细胞术和RT-PCR技术确认HUVECs膜上EPCR的表达水平和 mRNA 水平的表达。再分别以含不同浓度D-葡萄糖(5、10、30、50 mmol/L)的培养基以及含吡格列酮(5、10、20 μmol/L)或不含吡格列酮的高糖(50 mmol/L)培养基孵育HUVECs 24 h,行剂量和时间依赖性实验,并采用实时定量PCR技术测定HUVECs细胞 EPCR mRNA 的表达。结果:随着培养基D-葡萄糖浓度的增加,HUVECs培养24 h后其EPCR mRNA 的表达逐渐下调。在采用吡格列酮干预后, 50 mmol/L高糖处理的HUVECs EPCR mRNA 表达的下调得到明显改善。结论:(1)EPCR 在HUVECs上高表达,高糖可通过下调EPCR mRNA 的表达而损伤内皮细胞功能。(2)吡格列酮可阻止高糖诱导的HUVECs EPCR mRNA表达的下调,从而保护内皮细胞功能。     

Modulation of transendothelial permeability and expression of ATP-binding cassette transporters in cultured brain capillary endothelial cells by astrocytic factors and cell-culture conditions

Confluent cell monolayers of brain capillary endothelial cells (BCEC) are used widely as an in vitro cell culture model of the blood–brain barrier. The present study describes the influence of cell-culture conditions on tight junctions, filamentous-actin cytoskeleton, and expression of ATP-binding cassette (ABC) transporters in primary cell cultures of porcine BCEC. Astrocyte as well as C6 glioma-conditioned cell culture medium was used in combination with retinoic acid, dexamethasone, cyclic adenosine monophosphate (cAMP) analogs, or 1,25-dihydroxyvitamin D₃. It was shown that C6-conditioned medium led to a reorganization of filamentous actin and to an improved staining of zonula occludens-associated protein-1 (ZO-1). Further optimization of these culture conditions was achieved with cAMP analogs and dexamethasone. Retinoic acid, as well as 1,25-dihydroxyvitamin D₃, did not improve cellular tight junctions as judged by filamentous actin, ZO-1 rearrangement, and transcellular electrical resistance (TER) measurements. However, these morphological changes did not influence the paracellular permeability of the extracellular marker sucrose. Expression of ABC transporters such as P-glycoprotein, multidrug resistance-associated protein-1( MRP1), and MRP2 were compared by measuring messenger RNA (mRNA) levels in whole-brain tissue, isolated brain capillaries, and cultured cells. In freshly isolated BCEC, mRNA levels of MRP2 and P-glycoprotein dropped by two- to sevenfold, respectively, whereas MRP1 mRNA levels were slightly increased. During cell culture, mRNA levels of MRP1 and MRP2 decreased by up to fivefold, while P-glycoprotein levels remained constant. These results were unaltered by different cell-culture conditions. In conclusion, the present study suggests that paracellular permeability, as well as mRNA expression of the studied ABC transporters in primary cultures, of porcine BCEC are insensitive toward changes in cell-culture conditions.Abbreviations ACEM Astrocyte-conditioned endothelial medium - BCEC Brain capillary endothelial cells - C6CEM C6-conditioned endothelial medium - TER Transcellular electrical resistance Due to an error in the citation line, this revised PDF (published in December 2003) deviates from the printed version, and is the correct and authoritative version of the paper.     

MiR-16 modulate temozolomide resistance by regulating BCL-2 in human glioma cells

Temozolomide (TMZ) with radiotherapy is the current standard of care for newly diagnosed glioma. However, glioma patients who are treated with the drug often develop resistance to it and some other drugs. Recently studies have shown that microRNAs (miRNAs) play an important role in drug resistance. In present study, we first examined the sensitivity to temozolomide in six glioma cell lines, and established a resistant variant, U251MG/TR cells from TMZ-sensitive glioma cell line, U251MG. We then performed a comprehensive analysis of miRNA expressions in U251MG/TR and parental cells using cancer microRNA PCR Array. Among the downregulated microRNAs was miR-16, members of miR-15/16 family, whose expression was further validated by qRT-PCR in U251MG/TR and U251MG cells. The selective microRNA, miR-16 mimics or inhibitor was respectively transfected into U251MG/TR cells and AM38 cell. We found that treatment with the mimics of miR-16 greatly decreased the sensitivity of U251MG/TR cells to temozolomide, while sensitivity to these drugs was increased by treatment with the miR-16 inhibitor. In addition, the downregulation of miR-16 in temozolomide-sensitive AM38 cells was concurrent with the upregulation of Bcl-2 protein. Conversely, overexpression of miR-16 in temozolomide-resistant cells inhibited Bcl-2 expression and decreased temozolomide resistance. In conclusion, MiR-16 mediated temozolomide-resistance in glioma cells by modulation of apoptosis via targeting Bcl-2, which suggesting that miR-16 and Bcl-2 would be potential therapeutic targets for glioma therapy.     

重组人促红细胞生成素对人脐静脉内皮细胞CyclinD1表达的影响

目的探讨重组人促红细胞生成素(rHuEPO)对人脐静脉内皮细胞(HUVEC)细胞周期蛋白CyclinD1的影响。方法采用细胞培养及免疫组织化学方法检测不同剂量(10、20、40U/mL)rHuEPO作用下HUVEC中CyclinD1表达的变化。结果对组照及10、20、40U/mlrHuEPO实验组内皮细胞CyclinD1表达阳性百分率(%)分别为26±3、41±5、69±3、65±4,与对照组相比,rHuEPO作用后HUVEC中CyclinD1表达明显增强(P﹤0.05),且随rHuEPO剂量增加作用增强,呈剂量效应关系。结论rHuEPO能明显促进体外培养内皮细胞CyclinD1表达,提示rHuEPO具促内皮细胞增殖作用。     

辛伐他汀对香烟烟雾提取物诱导的人脐静脉内皮细胞表达sEPCR和mEPCR的影响

目的: 研究辛伐他汀对香烟烟雾提取物(CSE)诱导的人脐静脉内皮细胞(HUVECs) 表达可溶性内皮细胞蛋白C受体(sEPCR)和膜联内皮细胞蛋白C受体(mEPCR)的干预作用。方法: 体外培养的4~6代HUVECs 随机分为对照组、5%CSE组、不同浓度辛伐他汀组及辛伐他汀干预组,辛伐他汀组分别加入50、100、200 μmol/L辛伐他汀液孵育24 h,辛伐他汀干预组先以50、100、200 μmol/L辛伐他汀预处理细胞2 h,再与5%CSE孵育24 h。收集各组细胞及上清液,ELISA法检测上清液中sEPCR蛋白含量,实时定量PCR法检测各组细胞中mEPCR mRNA的表达。结果: (1)5%CSE组sEPCR蛋白含量高于对照组,mEPCR mRNA表达低于对照组,差异均有统计学意义(均P<0.05);(2)100 μmol/L与200 μmol/L辛伐他汀组sEPCR蛋白含量均高于对照组,低于5%CSE组,其mEPCR mRNA表达均低于对照组,高于5%CSE组,差异均有统计学意义(均P<0.05);(3)各辛伐他汀干预组sEPCR蛋白含量均低于5%CSE组,但高于对照组及相应浓度的辛伐他汀组;相反,各辛伐他汀干预组mEPCR mRNA表达均高于5%CSE组,低于对照组及相应浓度的辛伐他汀组,差异均有统计学意义(均P<0.05)。结论: 在体外,辛伐他汀通过上调HUVECs mEPCR mRNA的表达,降低sEPCR的分泌,对CSE介导的内皮细胞凝血功能障碍可能具有一定的改善作用。     

不同浓度哇巴因对人血管内皮细胞的作用

目的：研究不同浓度哇巴因对血管内皮细胞(HUVEC)的作用及生理浓度哇巴因激活的血管内皮细胞早期反应基因。方法： MTT法观察哇巴因对血管内皮细胞增殖的影响，台盼蓝染色及乳酸脱氢酶活性测定细胞活力。免疫组织化学染色法测定细胞核增殖抗原（PCNA）表达情况。以包含8 464条人类基因的DNA芯片检测血管内皮细胞受生理浓度哇巴因活化后的基因表达谱，并从中筛选出早期反应基因。 结果： 生理浓度（0.3-0.9 nmol/L）的哇巴因能刺激细胞增殖，且这种增殖不与剂量呈正相关，刺激增殖的最适浓度为0.3 nmol/L，最大效应作用时间为1-2 h。0.9-1.8 nmol/L的哇巴因抑制内皮细胞的增殖，引起细胞水肿和凋亡。但10 nmol/L的高浓度哇巴因却能够引起细胞的增殖。血管内皮细胞受哇巴因作用2 h后，基因表达谱研究显示340条基因出现表达差异，其中上调的共有145 条，多数与细胞代谢和转录调控相关，显著上调的6条。 结论： 哇巴因在参与维持血管内皮细胞的正常增殖及高血压引起的血管重塑中起重要作用。     

Proliferation of glomerular capillary endothelial cells under the influence of hypophyseal intermediate lobe materials

Summary Bovine hypophyseal intermediate lobe tissue and colloid have a particular affinity for adult cells of mesodermal origin. Intermediate lobe materials utilized as an erythropoietic stimulant induces the proliferation of glomerular capillary endothelial cells.These cells meet the requirements of erythroid precursors for they demonstrate that they belong to a class capable of proliferating, differentiating and undergoing multiple divisions resulting in the formation of colonies of cells belonging to the erythroid series.The present observations show that intermediate lobe materials simulate erythropoietin(s) and that endothelial cells, when properly stimulated, differentiate along the erythroid cell line.     

Ultrastructural localization of lectin binding sites in the developing brain microvasculature

 The temporo-spatial patterning of lectin-binding sites was examined by lectin histochemistry and quantitative methods in the microvasculature of the optic tectum of 9-, 14-, 20-day-old embryos and 30-day-old chickens. Horseradish peroxidase and colloidal-gold-labelled lectins were used for detection of β-d-galactose (RCA-I, Ricinus communis agglutinin-I) and of N-acetylglucosamine and sialic residues (WGA, Wheat germ agglutinin) at light and electron microscopical levels. At the light microscopical level, RCA-I and WGA binding sites were detectable in the early embryonic capillaries in a diffuse staining pattern; in later embryonic stages and in adult animals, RCA-I labelling became located on the abluminal surface of the vessels, while WGA staining was detected on the luminal surface. Ultrastructurally, gold labelling for RCA-I was seen intracytoplasmically in endothelial cells in 9-day-old embryos. In 14-to 20-day-old embryos and in chickens, binding sites for RCA I were detected in endothelial tight junctions and basement membranes. In contrast, labelling of the gold-coupled WGA lectin was distributed almost exclusively on the luminal endothelial surface already in early embryos. The results indicate that the endothelial cells of the optic tectum acquire functional polarity early in their development and that glycoconjugates containing β-d-galactose residues are involved in the biochemical composition of the tight junctions and basement membrane, which are considered to be key structures in blood-brain barrier (BBB) differentiation. Accepted: 9 October 1997     

Alterations in the activity and expression of endothelial NO synthase in aged human endothelial cells

This study was to investigate factors underlying the age-related decrease in NO production in vascular endothelial cells. The age-related changes in NO production, the activity and expression level of eNOS, and eNOS binding proteins, were studied in HUVECs.NO production in HUVECs significantly decreased in an age-dependent manner. The potentiation of NO production by l-Arg was significantly suppressed by L-NIO (eNOS-specific inhibitor) in young HUVECs and was suppressed by 1400W (iNOS-specific inhibitor) in aged HUVECs. The aged HUVECs had lower eNOS protein levels than young cells. eNOS phosphorylation at Ser-1177 (active) decreased gradually from PDL 23 through 40, and eNOS phosphorylation at Thr-495 (inactive) increased in aged cells. Changes of intracellular eNOS binding proteins, such as caveolin-1, pAkt, and Hsp90, as well as interaction between eNOS and eNOS binding proteins, indicated decreasing enzyme activity in aged HUVECs.Aging might decrease the activity as well as expression level of eNOS in HUVECs. And the decrease in eNOS activity probably implicated to the alterations in the regulatory binding proteins. For further study, it needs to be confirmed that the age-related change in the intracellular distribution of eNOS and the relative contribution of eNOS and iNOS on vascular dysfunction in aged endothelial cells.     

人外周血内皮祖细胞的分离、培养和扩增

目的:改进从人外周血中分离、培养和体外扩增血管内皮祖细胞(EPC)的方法。方法:采用不同淋巴细胞分离液,用密度梯度离心法从外周血分离EPC;用CD34免疫磁性活化细胞分选系统(MACS)分离CD34 细胞;分别培养在包被和不包被有人纤维连接蛋白(HFN)的培养板内;采用细胞免疫化学法检测内皮细胞表面标志CD31、CD34和vWF的表达。结果:从成人外周血可分离获得EPC;不同的分离条件可影响获得EPC的数量和质量,HFN对EPC的生长有促进作用。结论:进一步改进从人外周血分离获取EPC并行体外扩增的方法,为EPC的研究奠定了基础。