Long-term serial cultivation of arterial and capillary endothelium from adult bovine brain

Summary Cerebrovascular endothelial cells from adult bovine brain were carried successfully in long-term, serial culture. Endothelial cells were obtained from the middle and anterior cerebral arteries and from capillaries isolated from grey matter of the cerebral cortex or caudate nucleus. Capillary cells were found to grow best in RPMI 1640 with 20% fetal bovine serum. They did not require tumor-conditioned medium or matrix-coated surfaces, although fibronectin was used to enhance the initial plating efficiency of the primary cultures. The same conditions were used to support satisfactory growth of arterial endothelial cells; however they did not grow as rapidly as the capillary cells. Retention of endothelial-specific characteristics were shown for capillary-derived cells carried up to Passage 28, arterial-derived cells up to Passage 11, and after frozen storage of both types of cultured cells. Cultures of both arterial and capillary cells stained positively for Factor VIII antigen, exhibited a nonthrombogenic surface, and produced prostacyclin in response to arachidonic acid. Arterial endothelial cells produced more prostacyclin than capillary endothelium. The capillary cells had a unique tendency to assume a ringlike morphology after subculture and sometimes formed capillarylike networks of cell cords in dense cultures. When cultured in a three-dimensional plasma clot, capillary and arterial endothelial cells, but none of the other cell types studied, organized into tubelike structures reminiscent of capillary formation in vivo. The availability of long-term cultures of cerebrovascular endothelial cells provides an opportunity to compare properties of arterial and capillary endothelium from the same tissue and to investigate such processes as angiogenesis and blood-brain barrier induction. This work was supported in part by Public Health Service grant NS-18586 from the National Institute of Neurological and Communicative Disorders and Stroke and by a grant from The Council for Tobacco Research—U.S.A., Inc. C. E. was supported by the Universidad Autonoma of Madrid and the Ministerio de Universidades e Investigacion of Spain.     

Culture of mouse brain capillary endothelial cell lines that express factor VIII, γ-glutamyl transpeptidase,and form junctional complexes in vitro

Summary The isolation and culture of cell lines from mouse brain capillary endothelium (MBE) is described. Cells migrating from collagenase-treated capillary fragments proliferated rapidly in the 1st wk of culture forming large epithelioid cobblestonelike colonies. The cells showed only marginal proliferation after 2 to 3 wk in culture, until peripheral cells migrated away from the colony which exhibited a marked degree of proliferation. These cells were trypsinized and subcultured to confluence. The cells can be maintained for well over 40 passages and seem to retain their endothelial morphology. The endothelial origin of these cells was demonstrated by positive immunoperoxidase reactivity with Factor VIII-related antigen, specific binding ofBandeiraea simplicifolia lectin and γ-glutamyl transpeptidase activity. Electron microscopic examination of the MBE cells showed junctional complexes including intermediate junctions, but no tight junctions. The overall ultrastructure indicates that a degree of dedifferentiation has occurred, the cells ultrastructurally resembling immature endothelium. An earlier investigation of cultured mouse brain endothelial cells reported a cell line that had lost many functional and structural characteristics. Our study demonstrates, as the previous one, that a certain degree of dedifferentiation needs to occur if MBE cells are to be maintained for long-term culture. However, the degree of dedifferentiation seems to be variable, depending in part on the culture conditions employed. This work was supported in part by grants from the National Health and Medical Research Council of Australia and the Telethon and Royal Perth Hospital Research Foundations.     

Bone Marrow�Cderived Endothelial Progenitor Cells and Endothelial Cells May Contribute to Endothelial Repair in the Kidney Immediately After Ischemia�CReperfusion

In ischemic acute kidney injury, renal blood flow is decreased. We have previously shown that reperfused, transplanted kidneys exhibited ischemic injury to vascular endothelium and that preservation of peritubular capillary endothelial integrity may be critical to recovery from ischemic injury. We hypothesized that bone marrow–derived (BMD) endothelial progenitor cells (EPCs) might play an important role in renal functional recovery after ischemia. We tested this hypothesis in recipients of cadaveric renal allografts before and for 2 weeks after transplantation. We found that the numbers of circulating CD34-positive EPCs and CD146-positive endothelial cells (ECs) decreased immediately after ischemia–reperfusion. In renal allograft tissues obtained 1 hr after reperfusion, CD34-positive cells were more frequently observed along the endothelial lining of peritubular capillaries compared with non-ischemic controls. Moreover, 0–17.5% of peritubular capillary ECs were of recipient origin. In contrast, only 0.1–0.7% of tubule cells were of recipient origin. Repeat graft biopsy samples obtained 35 and 73 days after transplant did not contain capillary ECs of recipient origin, whereas 1.4% and 12.1% of tubule cells, respectively, were of recipient origin. These findings suggest that BMD EPCs and ECs may contribute to endothelial repair immediately after ischemia–reperfusion. (J Histochem Cytochem 58:687–694, 2010)     

Role of transferrin in iron uptake by the brain: a comparative study

Summary The role of specific transferrin (Tf) and Tf receptor interaction on brain capillary endothelial cells in iron transport from the plasma to the brain was investigated by using Tf from several species of animals labeled with ⁵⁹Fe and ¹²⁵I, and 15-day and adult rats. The rate of iron transfer was much greater in the 15-day rats. It was greatest with Tf from the mammals, rat, rabbit and human, but much lower with chicken ovotransferrin and quokka (a marsupial), toad, lizard, crocodile, and fish Tf. The uptake of Tf by the brain showed a similar pattern, except for a very high uptake of ovotransferrin (ovo Tf). Iron uptake by the femurs (a source of bone marrow) was also high with Tf from the mammalian species and low with the other types of Tf, but showed little change with aging of the animals. It is concluded that iron transport into the brain is dependent on the function of Tf receptors, probably on capillary endothelial cells, and that these receptors show the same type of species specificity as the receptors on immature erythroid cells. Also, the decrease in iron uptake by the brain as rats age from 15 days to adulthood is specific for the brain and is not a general effect of the aging process.Abbreviations Tf transferrin - ovo Tf ovotransferrin     

Histochemical identification of cultured cells from human endometrium

Summary Histochemical techniques have been applied to the identification of cell types cultured from human endometrium. Previous work from this laboratory characterized two principtal cell types found in cultures of endometrium: a mature epithelial cell and another cell which was classified as the endometrial stromal cell based on light and electron microscopy. In this report we compare the histochemical staining of endometrial tissue in frozen sections to that of cultured cells. These results confirm the epithelial and stromal nature of the respective cell types. Several markers were found that could distinguish between cells of epithelial and stromal origin. The enzymes alkaline phosphatase, γ-glutamyltranspeptidase, peroxidase, and β-glucuronidase were localized in glandular and surface epithelia in frozen sections and in colonies of epithelial cells in culture. Stroma in frozen sections and cultured stromal cells contained leucine aminopeptidase and fibronectin. Epithelial sections and in culture could also be distinguished from cells of stromal origin by preferential binding of lotus and peanut lectin. Several other markers were found in both endometrial epithelium and stroma. J. M. S. was recipient of National Research Service Award CA09156 (National Cancer Institute); K. G. N. was recipient of National Research Service Award ES07017 (National Institute of Environmental Health Sciences); and D. G. K. was recipient of Research Career Development Award CA00431 from the National Cancer Institute, Bethesda, MD. Supported by Grant CA 31733 from the National Cancer Institute, Bethesda, MD.     

Primary culture of capillary endothelium from rat brain

To provide an in vitro system for studies of brain capillary function we developed a method for culture of brain capillary endothelial cells. Capillaries were isolated from rat brain and enzymatically treated to remove the basement membrane and contaminating pericytes. Subsequent Percoll gradient centrifugation resulted in a homogeneous population of capillary endothelial cells that attached to a collagen substrate and incorporated [3H]thymidine. Evidence for the endothelial nature of these cells was provided by the presence of Factor VIII antigen and angiotensin converting enzyme activity and by the failure of platelets to adhere to the cell surface. In addition, the cells were joined together by tight junctions. Thus, primary cultures of these cells retained both endothelial and blood-brain barrier features.     

Substance induces migration of capillary endothelial cells: A novel NK-1 selective receptor mediated activity

Substance P (SP) has been indicated as a main mediator of neurogenic inflammation, leading to vasodilation, increase in vascular permeability and modulation of immune cell function. Certain vascular effects produced by SP are endothelium mediated. We have studied the effect of SP and of selective NK-1, NK-2 and NK-3 receptor agonists on migration of cultured capillary endothelial cells of bovine origin. Our results indicate that SP (10⁻¹⁴–10⁻⁶ M) induces a concentration-dependent migration of endothelial cells with maximal activity at 10⁻¹⁰ M. This effect was mimicked by the selective NK-1 receptor agonist which showed a similar concentration-dependent curve, while selective NK-2 and NK-3 receptor agonists were ineffective. Our conclusions are that endothelial cells possess specific receptors for SP of the NK-1 type which affect mobilization of capillary endothelial cells.     

Human semen as a source of epithelial cells for culture

Summary When washed cells from human semen samples were plated out, epithelial cultures were obtained. The human ejaculates used as starting material contained, in addition to spermatazoa, 10³ to 10⁷ cells of other types, including granulocytes, macrophages lymphocytes, spermatocytes and epithelial cells. Although no fractionation of cell types was attempted, semen samples yielded epithelial cultures uncontaminated by fibroblasts. The cultured cells appeared characteristically epithelial with a polygonal shape, interdigitating cell membranes, and desmosomes. ABH blood-group antigenic determinants of the donor were expressed with variable frequency as a surface antigen on these cells. About half the trials gave some cell attachment. Most cultures remained as small, tight colonies, but a few reached confluency in about 5 weeks and could be subcultured successfully. Cell proliferation, as monitored by [³H]thymidine incorporation into nuclear macromolecules, ceased in less than 2 months. Aided by grants from the National Science Foundation (BMS-72-02219 A04 and PCM 76-81029 to E. A. K.), the Public Health Service (CA 12504 to O. J. M.), and the National Foundation-March of Dimes. The earlier portions of this study were carried out under a Program Project Grant from the National Institutes of Health (No. 5 PO GM 18153).     

Functional expression of the P-glycoproteinmdr in primary cultures of bovine cerebral capillary endothelial cells

The P-glycoproteinmdr is expressed not only in tumoral cells, but also in nontransformed cells, including the specialized endothelial cells of brain capillaries which build up the blood-brain barrier. Since all previously identified blood-brain barrier markers are rapidly lost when cerebral capillary endothelial cells are maintained in primary culture, we have investigated whether P-glycoprotein (P-gp) would follow the same rule, in order to address the influence of the cerebral environment on the specific P-gp expression in the brain endothelium. As compared to freshly isolated purified cerebral capillaries, P-glycoprotein was detected by immunochemistry at a high level in 5–7 day primary cultures. In our culture conditions, P-glycoprotein was immunodetected at a lower molecular weight than that found in freshly isolated capillaries. Enzymatic deglycosylation led to the same 130 kDa protein for both fresh and cultured samples, suggesting that P-gp post-translational modifications were altered in primary cultures. However, studies on the uptake and efflux of the P-gp substrate [³H]vinblastine, and on the effect of variousmdr reversing agents on the uptake and efflux, clearly indicated that the efflux pump function of the P-glycoprotein was maintained in primary cultures of bovine cerebral capillary endothelial cells. P-Glycoprotein may thus represent the first blood-brain barrier marker which is maintained in cerebral endothelial cells cultured in the absence of factors originating from the brain parenchyma.Abbreviations BBB blood-brain barrier - BCEC brain capillary endothelial cells - -GT -glutamyltranspeptidase - HBSS Hank's balanced salt solution - Mab monoclonal antibody - mdr multidrug resistance - P-gp P-glycoprotein     

A polarized localization of amino acid/carnitine transporter B(0,+) (ATB(0,+)) in the blood-brain barrier

Brain capillary endothelial cells control the uptake and efflux from the brain of many hydrophilic compounds due to highly specialized transporters often localized in a polarized way. Localization of Na⁺- and Cl⁻-dependent amino acid and carnitine transporter B^0,+ (ATB^0,+) was studied in a co-culture of bovine brain capillary endothelial cells (BBCEC) grown on filters above astrocytes (an in vitro blood-brain barrier model). Immunoblotting and three-dimensional immunocytochemistry analysis with anti-B^0,+antibodies demonstrated the presence of this transporter and its prevalent co-localization with P-glycoprotein i.e. at the apical side. The sensitivity of leucine uptake through the apical membrane to 2-aminobicyclo-[2.2.1]-heptane-2-carboxylic acid (BCH), d-serine as well as sodium and chloride replacement confirm the functioning of ATB^0,+ and suggests an important physiological role of ATB^0,+ in controlling the delivery of amino acids and carnitine to the brain.     

Comparison of coulter volumes with radiometrically determined intracellular water volumes for cultured cells

Summary During methotrexate-induced differentiation of cultured human choriocarcinoma (BeWo) cells, proliferation is inhibited, morphologic and biochemical changes occur, and giant, often multinucleated, cells form. We have used the increase in cell volume as a marker of the mature syncytiotrophoblastlike phenotype. Uninduced and differentiated BeWo cells are not spherical, and theoretical considerations suggested that deviations in shape could result in significant errors in Coulter volume. To determine if the values obtained by electrical pulse sizing reflected the actual mass of BeWo cells, we have evaluated the relationship between Coulter volumes and intracellular water volumes obtained using a shape-independent estimate for eight cell types. A close correlation (r ²=0.97) was found, indicating that cell volume changes in populations of irregularly shaped cells can be accurately measured using a Coulter instrument. Supported by an operating grant from the National Cancer Institute of Canada. N.S.B. was a recipient of a studentship award from the Alberta Heritage Foundation for Medical Research. C.E.C. is a Senior Research Scientist of the National Cancer Institute of Canada. The McEachern Laboratory is a research facility of the Faculty of Medicine, University of Alberta, and the Cross Cancer Institute, Edmonton, Alberta.     

Effects of chelators on iron uptake and release by the brain in the rat

The iron chelators desferrioxamine (DFO), pyridoxal isonicotinoyl hydrazone (PIH), 2,2-bipyridine, diethylenetriamine penta-acetic acid (DTPA) and 1,2 dimethyl-3-hydroxy pyrid-4-one (CP20) were analysed for their ability to change⁵⁹Fe uptake and release from the brain of 15- and 63-day rats either during or after intravenous injection of⁵⁹Fe-¹²⁵I-transferrin. DTPA was the only chelator unable to significantly reduce iron uptake into the brain of 15-day rats. This indicates that iron is not released from transferrin at the luminal surface of brain capillary endothelial cells. CP20 was able to reduce iron uptake in the brain by 85% compared to 28% with DFO. Only CP20 was able to significantly reduce brain iron uptake in 63 day rats. Once⁵⁹Fe had entered the brain no chelator used was able to mediate its release. All of the chelators except CP20 had similar effects on femur iron uptake as they did on brain uptake, suggesting similar iron uptake mechanisms. It is concluded that during the passage of transferrin-bound iron into the brain the iron is released from transferrin within endothelial cells after endocytosis of transferrin.     

Phenotypic diversity in cultured cerebral microvascular endothelial cells

Summary Diversity exists in both the structure and function of the endothelial cells (EC) that comprise the microvasculature of different organs. Studies of EC have been aided by our ability to first isolate and subsequently establish cultures from microvascularized tissue. After the isolation of microvessel endothelial cells (MEC) derived from rat cerebrum, we observed morphologic differences in colonies of cells that grew in primary cultures. The morphologies ranged from a cobblestone phenotype considered typical of EC in culture to elongated and stellate cell appearances. Serially passaged cell lines were established based on two parameters: initially by growth and, second, on differences in primary colony morphology using selective weeding techniques. Each culture was examined for the presence of EC-characteristic markers which include Factor-VIII-related antigen, angiotensin-I-converting enzyme activity, collagen type IV synthesis, and PGI₂ production. Variable expression of each of these characteristics among the established EC lines was observed. Growth curves established for each of the EC cultures demonstrated differences in both population doubling rates and cell densities at confluence. The endocytic capacity of each EC line was also evaluated. Our ability to isolate and establish a number of morphologically distinct EC cultures indicates that diversity exists within the EC that comprise the cerebral microvasculature. Diversity in the established cell lines suggests either the EC that line the brain microvasculature exist as a mosaic or that morphologically distinct cultures may originate from different microanatomical origins (arteriolar, true capillary, or venular) or may have resulted from cells at different points in their in vitro life spans at the time of isolation. This research was supported by grants HLO3227 and HLO1514 from the National Institutes of Health, Bethesda, MD.     

Rac is Essential in the Transformation of Endothelial Cells by Polyoma Middle T

Expression of the Polyoma Middle T (PyMT) antigen in endothelial cells results in single-step transformation to hemangioma producing malignant cells. To study the mechanism of PyMT transformation, we used the PyMT induced mouse brain endothelial cell line, bEND.3, expressing constitutively active and dominant negative mutants of the small GTPase Rac. The bEND.3 cell phenotype of tumorigenesis, loss of normal growth control and formation of cysts rather than capillary tubes in fibrin gels was reversed by expression of dominant negative Rac. The mechanism of N17 Rac action in blocking the endothelial cell transformant, PyMT, did not involve effects of Rac on the actin cytoskeleton since this component of the bEND.3 cell phenotype was not affected. Furthermore, the PyMT induced activation of the plasminogen activator (PA)/plasmin system was not affected by Rac inhibition. Inhibition of the downstream effectors of Rae, phosphatidylinositol 3-kinase (P13-K) and p70^S6k, which are known to be constitutively activated by PyMT transformation, inhibited bEND. cell proliferation and cyst formation in fibrin gels even in cells expressing V12 constitutively active Rac, but they did not restore capillary tube formation. These results demonstrate that middle T antigen induced endothelial cell transformation requires signal transduction by Rac. The downstream Rac effectors, P13-K and p70^S6k, mediate PyMT/Rac effects on cell proliferation and cyst formation, but other unknown effectors of PyMT are required for the cytoskeletal changes and activation of the PA/plasmin system.     

Exogenous expression of claudin-5 induces barrier properties in cultured rat brain capillary endothelial cells

Claudins are thought to be major components of tight junctions (TJs), and claudin-5 and -12 are localized at TJs of the blood-brain barrier (BBB). Claudin-5-deficient mice exhibit size-selective (<800 Da) opening of the BBB. The purpose of this study was to clarify the expression levels of claudin-5 and -12 in rat brain capillary endothelial cells, and to examine the ability of claudin-5 to form TJs in cultured rat brain capillary endothelial cells (TR-BBB). Expression of claudin-5 mRNA in rat brain capillary fraction was 751-fold greater than that of claudin-12. The level of claudin-5 mRNA in the rat brain capillary fraction (per total mRNA) was 35.6-fold greater than that in whole brain, while the level of claudin-12 mRNA was only 13.9% of that in whole brain, suggesting that expression of claudin-12 mRNA is not restricted to brain capillaries. Transfection of TR-BBB cells with the claudin-5 gene afforded TR-BBB/CLD5 cells, which showed no change in expression of claudin-12 or ZO-1, while the expressed claudin-5 was detected at the cell-cell boundaries. The permeability surface product of [(14)C]inulin at a TR-BBB/CLD5 cell monolayer was significantly smaller (P < 0.01) than that for the parental TR-BBB cells, and the values of the permeability coefficient (Pe) were 1.14 x 10(-3) and 11.6 x 10(-3) cm/min, respectively. These results indicate that claudin-5, but not claudin-12, is predominantly expressed in brain capillaries, and plays a key role in the appearance of barrier properties of brain capillary endothelial cells.     

Cerebral microvessels and derived cells in tissue culture

Summary An endothelial cell line has been established from a primary culture of cerebral microvessels isolated from Swiss-Webster mice. The microvessels were isolated by a mechanical dispersion and filtration technique. The cells that emerged from these microvessels, maintained in organoid cultures, proliferated and formed plaques of a single or mixed cell type. The endothelial cell line, designated ME-2, was isolated from one such morphologically homogeneous cell plaque, using both cloning ring techniques and C₆ glioma-conditioned medium. An endothelial specific antiserum was made in rabbits and was used immunocytochemically to confirm the cell type of origin of the ME-2 cell line. Not only did the cell type specific antiserum react exclusively with endothelial cells in vivo, but in the brain the antiserum localized preferentially to the luminal membrane of the endothelium. The ME-2 endothelial cells have retained several of their unique properties such as cytomorphology, growth characteristics, and cell type specific surface antigens throughout the life of the line (in one case 40 passages before senescence). This work was supported in part by an Arteriosclerosis Specialized Center of Research grant from the National Heart, Lung and Blood Institute, National Institutes of Health, Grant HL-14230, and Grant 584-127703 from the Veterans Adminsitration. This paper is dedicated to the memory of Steve Frommes, Electron Microscopist and Photographer.     

Long-term culture of human endothelial cells

Summary Human umbilical vein endothelial cells can be grown in vitro for 28 passages (CPDL 58) in Medium 199 supplemented with newborn bovine serum and a partially purified growth factor derived from bovine brain. Newborn bovine serum is superior to fetal bovine serum for the proliferation of human umbilical vein endothelial cells seeded at low density in the presence of the growth factor. The endothelial cells, which can be passaged every 7 to 10 d at a 1-to-5 split ratio, retain their morphological and biochemical characteristics. The proliferation of cells seeded at low density (10³/cm²) is proportional to the concentration of the growth factor present in the medium. The growth factor, which has an isoelectric point between 5.0 and 5.5, can support cell proliferation at reduced serum concentrations; half-maximal growth is achieved in medium containing the growth factor and 3% serum. The brain endothelial cell growth factor does not stimulate DNA synthesis significantly in cultures of human skin fibroblasts. This research was supported by grants from the U.S. Public Health Service (AG 01732, HL 16387, and HL 07080), the Cystic Fibrosis Foundation, and the New York and American Heart Associations. Victor B. Hatcher is an Established Fellow of the New York Heart Association and a recipient of the Ann Weinberg Cystic Fibrosis Research Scholarship Award.     

Increased expression of β-catenin in brain microvessels of a segmentally trisomic (Ts65Dn) mouse model of Down syndrome

We examined the distribution of β-catenin and endogenous blood serum albumin at the ultrastructural level in blood microvessels (capillaries) from brains of control and trisomic Ts65Dn mice. Morphological examination revealed an increased immunolabeling for β-catenin in endothelial substructures of the capillary network, such as intercellular junctions, cytoplasm, and nuclei. These immunosignals were significantly increased in all endothelial substructures from trisomic mice. These changes, however, did not affect the blood–brain barrier function of the entire microvascular network, because the increased uptake of albumin by endothelial cells and the eventual escape of this protein (microleakage) into the perivascular neuropil were noted only in a few capillary profiles. Nevertheless, these findings suggest the involvement of some segments of the microvascular network in the brain pathology associated with DS.