Isolation of endothelial cells from human placental microvessels: effect of different proteolytic enzymes on releasing endothelial cells from villous tissue

Approaches for the isolation of human placental microvascular endothelial cells (HPMEC) using proteolytic enzymes have been described recently. However, the isolation procedure and enzyme composition most suitable for optimal disaggregation of placental tissue and isolation of HPMEC has not yet been established. We tested different proteolytic enzymes and enzyme mixtures for their capabilities of releasing endothelial cells from human term placental villous tissue. Best results were obtained with a mixture of collagenase/dispase/deoxyribonuclease I (0.28%/0.25%/0.01%). By adding a discontinuous Percoll gradient centrifugation step to the enzymatic dispersion, about 1 x 10(6) cells/g tissue with more than 30% von Willebrand factor (vWf)-positive cells were obtained. However, the total cell number and number of vWf-positive cells were highly dependent on the lot of collagenase used. A perfusion step prior to mincing of villous tissue did not increase the amount of vWf-positive cells. We conclude that the methods described in this study are suitable to isolate high yields of HPMEC and that the composition of the collagenase preparation is crucial to the successful release of endothelial cells from placental tissue. To obtain pure HPMEC, further separation steps, e.g., cell sorting with antibodies against endothelial specific cell surface antigens are necessary.     

Isolation and characterization of lymphatic microvascular endothelial cells from human tonsils

Human lymphatic endothelial cells (LECs) have isolated prevalently from human derma and tumors. As specialized lymphatic organs within the oropharynx, palatine tonsils are easily obtained and rich in lymphatic venules. Using a two-step purification method based on the sorting of endothelial cells with Ulex Europaeus Agglutinin 1 (UEA-1)-coated beads, followed by purification with monoclonal antibody D2-40, we successfully purified LECs from human palatine tonsils. The LECs were expanded on flasks coated with collagen type 1 and fibronectin for up to 8-10 passages and then analyzed for phenotypic and functional properties. Cultured cells retained the phenotypic pattern of the lymphatic endothelium of palatine tonsils and expressed functional VEGFR-3 molecules. In fact, stimulation with VEGFR-3 ligand, the vascular endothelium grow factor C, induced a marked increase in cell proliferation. Similarly to blood endothelial cells (BECs), LECs were able to form tube-like structure when seeded in Cultrex basement membrane extract. Comparative studies performed on LECs derived from palatine tonsils and iliac lymphatic vessels (ILVs), obtained with the same procedures, showed substantial discrepancies in the expression of various lymphatic markers. This points to the existence of micro- and macrovessel-derived LECs with different phenotypes, possibly involving different biological activities and functions. Palatine tonsil- and ILV-derived LECs may, therefore, represent new models for investigating function and biochemical properties of these lymphatic endothelia.     

Isolation and characterization of microvascular endothelial cells from chicken fat pads

Summary Microvascular endothelial cells from abdominal fat pads of 6-wk-old broiler chickens were isolated to provide anin vitro system to study their physiological functions. The isolation procedure produced clumps of 10–30 cells, which attached to culture vessels in 4 h and attained confluency in 2 wk. At confluency, cells had a cobblestone appearance but were not contact inhibited and detached from the bottom of the culture vessel 2 wk after reaching confluency. The cells internalized acetylated low density lipoproteins, a characteristic of endothelial cells. This property was used to obtain pure endothelial cell cultures using the cell sorter. When cultured over Matrigel, a reconstituted matrix, the cells aligned themselves into chordlike structures and formed branching microvessels. Cells plated on type I collagen-coated culture flasks occasionally formed chordlike structures and proliferated at a faster rate than cells plated on Matrigel. Cells cultured on laminin-coated plates were slender and had long cytoplasmic extensions however, cells cultured on uncoated plastic had fibroblastic morphology. These properties are similar to those described for microvessel endothelial cells isolated from tissues of other species.     

Isolation and culture of rat microvascular endothelial cells

The purpose of this study is to identify the separation techniques that result in pure cultures of rat microvascular endothelial cells (MECs). A multistep process is used to optimize the separation of the cells from rat epididymal fat pads, obtaining as pure a culture as possible within a relatively short processing time. The process initially employs the digestion, filtration, and density gradient separation steps. We further describe the use of an attachment phase that allows the differential adherence of contaminating cell types. Immunomagnetic purification is the final step in the process and is performed using anti-PECAM-1 (CD31) monoclonal antibody-labeled DynaBeads.     

Cultivation of microvascular endothelial cells from human preputial skin

Summary A procedure is described for the isolation and cultivation of microvascular endothelium from human skin. Neonatal foreskins are pooled, washed, minced, and dissociated by a mixture of collagenase and dispase. Microvascular endothelium, liberated in the form of intact capillary fragments, is incompletely separated from fibroblasts and epidermal cells by sieving through nylon mesh, followed by velocity sedimentation on 5% bovine serum albumin. The endothelium-enriched fraction has been maintained in primary culture for up to 3 weeks. The resulting epithelioid colonies have been characterized morphologically by both light and transmission electron microscopy and manifest all of the structural features that distinguish other, large-vessel endothelia in culture. In addition, immunohistochemical studies using an indirect fluorescent antibody technique demonstrate that these cells contain the endothelium-specific product, Factor VIII antigen. This work was supported by National Institutes of Health Grants AM18904 and AM20571, the RGK Foundation, the Charlotte and Sidney Lifschultz Foundation, the Juvenile Diabetes Foundation, and the South Carolina Geenral Medical Faculty Research Appropriation.     

Culture of murine brain microvascular endothelial cells that maintain expression and cytoskeletal association of tight junction-associated proteins

A readily obtainable in vitro paradigm of the blood-brain barrier (BBB) would offer considerable benefits. Toward this end, in this study, we describe a novel method for purifying murine brain microvascular endothelial cells (BMEC) for culture. The method uses limited collagenase-dispase digestion of enriched brain microvessels, followed by immunoisolation of digested, microvascular fragments by magnetic beads coated with antibody to platelet-endothelial cell adhesion molecule-1. When plated onto collagen IV-coated surfaces, these fragments elaborated confluent monolayers of BMEC that expressed, as judged by immunocytochemistry, the adherens junction-associated proteins, VE-cadherin and beta-catenin, as well as the tight junction (TJ)-associated proteins, claudin-5, occludin, and zonula occludin-1 (ZO-1), in concentrated fashion along intercellular borders. In contrast, cultures of an immortalized and transformed line of murine brain capillary-derived endothelial cells, bEND.3, displayed diffuse cytoplasmic localization of occludin and ZO-1. This difference in occludin and ZO-1 staining between the two endothelial cell types was also reflected in the extent of association of these proteins with the detergent-resistant cytoskeletal framework (CSK). Although both occludin and ZO-1 largely partitioned with the CSK fraction in BMEC, they were found predominantly in the soluble fraction of bEND.3 cells, and claudin-5 was found associated equally with both fractions in BMEC and bEND.3 cells. Moreover, detergent-extracted cultures of the BMEC retained pronounced immunostaining of occludin and ZO-1, but not claudin-5, along intercellular borders. Because both occludin and ZO-1 are thought to be functionally coupled to the detergent-resistant CSK and high expression of TJs is considered a seminal characteristic of the BBB, these results impart that this method of purifying murine BMEC provides a suitable platform to investigate BBB properties in vitro.     

HIV-Tat protein induces P-glycoprotein expression in brain microvascular endothelial cells

Among the different factors which can contribute to CNS alterations associated with HIV infection, Tat protein is considered to play a critical role. Evidence indicates that Tat can contribute to brain vascular pathology through induction of endothelial cell activation. In the present study, we hypothesized that Tat can affect expression of P-glycoprotein (P-gp) in brain microvascular endothelial cells (BMEC). P-gp is an ATP-dependent cellular efflux transporter which is involved in the removal of specific non-polar molecules, including drugs used for highly active antiretroviral therapy (HAART). Treatment of BMEC with Tat(1-72) resulted in P-gp overexpression both at mRNA and protein levels. These alterations were confirmed in vivo in brain vessels of mice injected with Tat(1-72) into the hippocampus. Furthermore, pre-treatment of BMEC with SN50, a specific NF-kappaB inhibitor, protected against Tat(1-72)-stimulated expression of mdr1a gene, i.e. the gene which encodes for P-gp in rodents. Tat(1-72)-mediated changes in P-gp expression were correlated with increased rhodamine 123 efflux, indicating the up-regulation of transporter functions of P-gp. These results suggest that Tat-induced overexpression of P-gp in brain microvessels may have significant implications for the development of resistance to HAART and may be a contributing factor for low efficacy of HAART in the CNS.     

Isolation and characterisation of human pulmonary microvascular endothelial cells from patients with severe emphysema

Background

Loss of the pulmonary microvasculature in the pathogenesis of emphysema has been put forward as a credible alternative to the classical inflammatory cell driven proteolysis hypothesis. Mechanistic studies in this area have to date employed animal models, immortalised cell lines, primary endothelial cells isolated from large pulmonary arteries and non-pulmonary tissues and normal human pulmonary microvascular endothelial cells. Although these studies have increased our understanding of endothelial cell function, their relevance to mechanisms in emphysema is questionable. Here we report a successful technique to isolate and characterise primary cultures of pulmonary microvascular endothelial cells from individuals with severe emphysema.

Methods

A lobe of emphysematous lung tissue removed at the time of lung transplantation surgery was obtained from 14 patients with severe end-stage disease. The pleura, large airways and large blood vessels were excised and contaminating macrophages and neutrophils flushed from the peripheral lung tissue before digestion with collagenase. Endothelial cells were purified from the cell mixture via selection with CD31 and UEA-1 magnetic beads and characterised by confocal microscopy and flow cytometry.

Results

Successful isolation was achieved from 10 (71%) of 14 emphysematous lungs. Endothelial cells exhibited a classical cobblestone morphology with high expression of endothelial cell markers (CD31) and low expression of mesenchymal markers (CD90, αSMA and fibronectin). E-selectin (CD62E) was inducible in a proportion of the endothelial cells following stimulation with TNFα, confirming that these cells were of microvascular origin.

Conclusions

Emphysematous lungs removed at the time of transplantation can yield large numbers of pulmonary microvasculature endothelial cells of high purity. These cells provide a valuable research tool to investigate cellular mechanisms in the pulmonary microvasculature relevant to the pathogenesis of emphysema.     

Immortalized mouse brain endothelial cells are ultrastructurally similar to endothelial cells and respond to astrocyte-conditioned medium

Summary Studies of brain microvessel endothelial cell physiology and blood-brain barrier properties are often hampered by the requirement of repeatedly producing and characterizing primary endothelial cell cultures. The use of viral oncogenes to produce several immortalized brain microvessel cell lines has been reported. The resulting cell lines express many properties of the blood-brain barrier phenotype but do not completely mimic primary endothelial cells in culture. As immortalized brain microvessel endothelial cell lines have not yet been produced from mice, we transformed mouse brain endothelial cells with the adenovirus E1A gene using a retroviral vector (DOL). Eight of 11 clones produced exhibited an endothelial-like cobblestone morphology and were characterized as endothelial with a panel of antibodies, lectins, and ultrastructural criteria. These cells are endothelial in origin and share ultrastructural features with primary cultures of endothelial cells. Examination of freeze fracture and transmission electron micrographs show adherens junctions exist between the transformed cells, and culture in astrocyte-conditioned medium induces the formation of gap junctions. This is one indication that responses to astrocyte-derived factors are retained by the transformed cell lines.     

Subcellular localization of leukotriene receptors in human endothelial cells

Leukotriene B₄ (LTB₄), a potent chemotactic and immune-modulating mediator, signals via two receptors, BLT₁ and BLT₂. Recently, we reported that BLT₁ is the predominating BLT expressed on human umbilical vein endothelial cells (HUVEC), and that BLT₁ mediated functions are enhanced by LTB₄ and lipopolysaccharide (LPS), but not by TNFα. Here, we demonstrate that BLT₁ is found on the outer cell membrane of HUVECs but also in intracellular granules, co-localized with monocyte chemotactic protein-1 and P-selectin, but not with interleukin-8 and von Willebrand factor. Upon stimulation with LTB₄ or LPS, more BLT₁ protein is found, now evenly distributed over the cytoplasm and in the cell nucleus, but less on the cell surface. An MAP kinase inhibitor prevented this enhancement and translocation, suggesting this signaling pathway to be crucial. Thus, BLT₁, a G-protein-coupled 7-transmembrane receptor, is located in various subcellular compartments in endothelial cells, which may have implications for cellular LT dependent responses and target accessibility for BLT₁ antagonists.     

Chlamydia pneumoniae infection alters the junctional complex proteins of human brain microvascular endothelial cells

Chlamydia pneumoniae has been identified and associated with multiple sclerosis (MS) and Alzheimer's disease (AD) pathogenesis, although the relationship of this organism in these diseases remains controversial. We have hypothesized that one potential avenue of infection is through the junctional complexes between the blood-brain barrier (BBB) endothelia. C. pneumoniae is characteristically a respiratory pathogen, but has been implicated in atherosclerosis, coronary artery disease, and neuroinflammatory conditions. C. pneumoniae infection may lead to endothelial damage, junctional alterations, and BBB breakdown. Therefore, in this study, C. pneumoniae infection of human brain microvascular endothelial cells (HBMECs) resulted in increased expression of the zonula adherens proteins beta-catenin, N-cadherin, and VE-cadherin, and decreased expression of the tight junctional protein occludin, as determined by immunocytochemistry and Western blot analyses. These events may underlie a mechanism for the regulation of paracellular permeability while maintaining barrier integrity during C. pneumoniae infection associated with neuropathologies such as MS and AD.     

Isolation and characterization of goat retinal microvascular endothelial cells

In this report, we demonstrate a method for the isolation of pure homogeneous endothelial cell population from goat’s eye without using multi-step procedure and sophisticated instrument facilities. Microvascular endothelial cell from goat’s retina were isolated using enzymatic method and cultured in Iscove’s Modified Dulbecco’s Medium supplemented with 10% fetal bovine serum. Five to 7 d after plating, a monolayer of endothelial cells was formed. These cells were identified as endothelial cells by morphology and confirmed by positive immunocytochemistry for vWF, CD31, VE-cadherin, CD146, VCAM-1, and ICAM-1, a specific marker for endothelial cells. We have compared both the mechanical and non-mechanical enzymatic methods in isolating pure endothelial cells. Cells plated on 4% gelatin-coated dishes resulted in tubular morphology, a characteristic of endothelial cells. This method is simpler and cost-effective when compared with other previously reported methods. These endothelial cells will be more helpful to identify the role of various factors in angiogenic-related disease such as diabetic retinopathy.     

Isolation of a storage and secretory organelle containing Von Willebrand protein from cultured human endothelial cells

Von Willebrand protein was synthesized and secreted by human endothelial cells in culture. Ca²⁺ ionophore A23187 and phorbol myristate acetate stimulated the release of Von Willebrand protein from the cultured cells. Stimulated release was accompanied by the disappearance of rod-like structures from the cultured endothelial cells immunostained for Von Willebrand protein, suggesting the existence of a storage organelle for Von Willebrand protein in these cells (Loesberg, C., Gonsalves, M.D., Zandbergen, J., Willems, C., Van Aken, W.G., Stel, H.V., Van Mourik, J.A. and De Groot, P.G. (1983) Biochim. Biophys. Acta 763, 160–168). Cultured human endothelial cells were fractionated on a density gradient of colloidal silica. Von Willebrand protein was found in two organelle populations: a buoyant one sedimenting with a variety of cell organelle marker enzymes, including those of the Golgi apparatus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum and plasma membrane fragments (peak density of this fraction: 1.08 g·ml^?1), and a dense one with a peak density of 1.12 g·ml^?1. The dense organelles containing Von Willebrand protein were apparently free of other organelles. Stimulating Von Willebrand protein release with phorbol myristate acetate or Ca²⁺ ionophore A23187 resulted in a decrease or even complete disappearance of Von Willebrand protein from the high-density organelle fraction, implying a role of this organelle in the stimulus-induced release of Von Willebrand protein. The Von Willebrand protein content of the buoyant fraction was lowered to some extent or did not change upon incubation of the cells with ionophore A23187 and phorbol myristate acetate. Restoration of Von Willebrand protein content of the dense organelle fraction after stimulation occurred within 2 days; this was accompanied by recurrence of immunostaining of rod-shaped structures in cells and an increase in cellular Von Willebrand protein. The excretion of restored Von Willebrand protein could be stimulated again.     

Isolation and characterization of microvascular endothelial cells from developing corpus luteum.

We have isolated and characterized microvascular endothelial cells from the developing rabbit corpus luteum. The isolated cells express Factor VIII-related antigen and angiotensin-converting enzyme, internalize acetylated low-density lipoprotein, and form capillary-like tubules in collagen gel cultures. Of the mitogens tested, only basic fibroblast growth factor stimulated the proliferation of these cells. Transforming growth factor-beta 1 and tumor necrosis factor-alpha strongly inhibited the proliferation of these endothelial cells. Platelet-derived growth factor, epidermal growth factor, insulin-like growth factor-1, histamine, prostaglandins, sex steroids, and interleukin-6 (interferon-beta 2) had no effect on the proliferation of these microvascular endothelial cells from the corpus luteum, whereas interleukin-1 alpha and 1 beta were mildly inhibitory. Endothelial cells are an essential component of corpus luteum physiology. Therefore, the availability of these cells will allow us to investigate the potential interactions between endothelial cells and luteal cells in vitro.     

HIV-1 envelope protein gp140 binding studies to human brain microvascular endothelial cells

Human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein gp140 interacts with its specific receptors on the surface of the target cells leading to cellular activation through various signaling pathways. The effect of blocking the chemokine repertoire in human brain microvascular endothelial cells in HIV dementia (HAD) disease has not been reported. Characterizing the nature of HIV-1 envelope protein gp140 (T-tropic, HXBc2) receptor binding conditions to HBMEC is critical to gain insight into the HIV dementia, and eventually to rationally design the agents to block envelope protein receptor interactions. HIV-1 gp140 oligomers were purified and separated to monomers, dimers, and trimers. The binding conditions of gp140 to HBMEC chemokine receptor, CXCR4, were optimized with an aim of understanding the structural interactions in HAD. Analysis of the interaction between HIV-1 gp140 and CXCR4 of HBMEC by saturation binding, cross-competition analysis with radiolabeled SDF and gp140, revealed a strong interaction, specificity between HIV-1 gp140 and CXCR4. Our binding data demonstrate that HIV-1 envelope protein gp140 enters cells by protein receptor mediated interactions that are regulated by the conformational state of the gp140 at physiological environment (pH and temperature). The CXCR4 antibody 12G5 inhibited SDF-1 binding to HBMEC indicating the specificity of gp140 binding to HBMEC. Scatchard analysis revealed the presence of approximately 70250 gp140 binding sites per cell with a K(d) of 4.5 nM. Cross-competition experiments using labeled SDF-1 and gp140 revealed that both unlabeled SDF-1 and gp140 are capable of displacing their radiolabeled counterparts. The binding assay conditions and radioligand binding assay are highly valuable to identify and design better HIV inhibitors for HAD.     

Immortalization of brain capillary endothelial cells with maintenance of structural characteristics of the blood-brain barrier endothelium

Summary Early passage bovine brain capillary endothelial cells were immortalized by transfection with the plasmid pSV3 neo. Cells from one clone, SV-BEC, expressed nuclear SV 40 large T antigen, displayed a contact-inhibited and anchorage-dependent proliferation, and a high sensitivity to the addition of exogenous basic fibroblast growth factor. SV-BEC cells are morphologically unaltered and express typical markers of endothelial cells: Factor VIII-related antigen, angiotensin-converting enzyme andGriffonia simplicifolia agglutinin binding site. Endothelium like immunoreactivity was detected in the conditioned medium from these cells. Moreover, SV-BECs present numerous intercellular tight junctions characteristic of the blood-brain barrier and possess functionalβ1- andβ2-adrenergic receptors, as observed on isolated bovine brain capillaries.     

A new astrocytic cell line which is able to induce a blood-brain barrier property in cultured brain capillary endothelial cells

Astrocytes, a member of the glial cell family in the central nervous system, are assumed to play a crucial role in the formation of the blood-brain barrier (BBB) in vertebrates. It was shown that astrocytes induce BBB-properties in brain capillary endothelial cells (BCEC) in vitro. We now established an astroglial cell line of non-tumoral origin. The cloned cell line (A7) shows a highly increased proliferation rate and expresses the astrocytic marker glial fibrillary acidic protein. Furthermore, the clone A7 expresses S-100-protein and vimentin, which are also expressed by primary cultured astrocytes. This cell line therefore shows general astrocytic features. In addition, we were able to show that A7 cells re-induce the BBB-related marker enzyme alkaline phosphatase in BCEC, when these two cell types are co-cultured. Thus we have a cell line which can be readily cultured in large quantities, shows common astrocyte properties and is able to influence BCEC with respect to a BBB-related feature. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biological characteristics of cultured cells derived from various types of human brain tumors

We placed in culture brain tumors from 45 cases (7 cases of astrocytoma, 2 from oligodendrogliomas, 2 glioblastomas, 2 ependymomas, 13 meningiomas, 6 pituitary adenomas, 5 neurinomas, a malignant lymphoma, a choroid plexus papilloma, and 6 metastatic tumors) and succeeded in making a primary culture from 33, and maintained 17 in vitro over a considerable period of time (greater than three months). In the early period of the primary cultures, the astrocytoma cells had cytoplasmic processes which contacted each other, the oligodendroglioma cells were small and spindle-shaped, the glioblastoma cells were neoplastic with pleopmorphic features and possessed cytoplasmic processes, the ependymoma cells formed a rosette-like cell arrangement, the meningioma cells were spindle- or round-shaped cells and characterized as forming psammoma bodies, the pituitary adenoma cells were round- or oval-shaped cells and produced growth hormone (GH), adenocorticoid tropic hormone (ACTH), prolactin, or other hypophyseal hormones, the choroid plexus papilloma cells were round-or polygonal and showed a papillary cell arrangement, the neurinoma cells were spindle- or fibrous-shaped cells, and the malignant lymphoma cells were round and formed cell aggregates floating in the culture medium.     

Heterogeneity of microvascular endothelial cells isolated from human term placenta and macrovascular umbilical vein endothelial cells

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand factor, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and fibroblasts. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial growth factors (VEGF121, VEGF165) and basic fibroblast growth factor (FGF-2) induced proliferation of all endothelial cell types. Placental growth factors PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- 20% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and 15 +/- 20%) and HPEC-A2 (15 +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.