Microvessel Endothelial Cell Culture Research Articles

Regulation of P‐glycoprotein by orphan nuclear receptors in human brain microvessel endothelial cells

In mammalian systems, pregnane X receptor (PXR) and constitutive androstane receptor (CAR) have been recognized as xenobiotic-sensors which can up-regulate the functional expression of drug transporters, such as P-glycoprotein (P-gp). In the brain, an increase in P-gp expression can further limit drug permeability across the blood-brain barrier (BBB) and potentially reduce CNS pharmacotherapy efficacy. At present, the involvement of human PXR (hPXR) and CAR (hCAR) in the regulation of P-gp expression at the human BBB is unknown. In this study, we investigate the role of hPXR and hCAR in the regulation of P-gp expression using a human cerebral microvessel endothelial cell culture system. We demonstrate that activation of hPXR and hCAR by their respective ligands leads to P-gp induction at both mRNA and protein levels, while pharmacological inhibitors of hPXR and hCAR prevent ligand-mediated P-gp induction. Ligand-induced nuclear translocation of hPXR is observed, although such effect could not be demonstrated for hCAR. Furthermore, down-regulation of hPXR and hCAR proteins using small-interfering RNA decreased P-gp expression. Our findings provide first evidence for P-gp regulation by hPXR and hCAR at the human BBB and suggest insights on how to achieve selective P-gp regulation at this site.

TRPV4 channel expression and function in blood‐brain barrier (BBB) microvessel endothelial cells

RT-PCR analysis revealed TRPV4 mRNA expression in microvessels isolated from mouse brain, in rat brain microvessel endothelial cell cultures, and in an immortalized mouse BBB endothelial cell line, bEnd3. The analysis was extended with bEnd3 cells grown on coverslips. Both immunoblotting and immunohistochemistry verified expression of TRPV4 protein in the bEnd3 cells and immunofluorescent imaging showed TRPV4 within the cytoplasm and at the cell membrane. To evaluate function, intracellular calcium levels were measured in bEnd3 cells on coverslips using fura 2 fluorescence imaging (37 °C). Cells were activated by application of: 1) the PKC-inactive phorbol ester, 4α-phorbol 12,13-decanoate (4α-PDD, 100 nM), a selective activator of TRPV4; 2) the PKC-active phorbol ester, phorbol 12-myristate 13-acetate (PMA, 100 nM); and 3) mechanical stimulation via hypotonic swelling (225 mOsm/kg). All stimuli activated Ca influx and were partially inhibited by the general TRPV channel blocker, ruthenium red (RR, 1 μM). Similar responses were demonstrated in TRPV4-transfected HEK 293 cells, but not in non-transfected cells. It is concluded that TRPV4 is expressed in BBB endothelial cells and that it may function as a mechanosensitive channel similar to that previously demonstrated for TRPV4 function in TRPV4-transfected HEK 293 cells.

Human cerebral microvessel endothelial cell culture as a model system to study the blood-brain interface in ischemic/hypoxic conditions.

(1) Cerebral ischemia and reperfusion induce several changes on the endothelial cells at the microcirculatory level. (2) Vasogenic brain edema due to compromised blood-brain barrier, transformation of the endothelial cell surface from an anticoagulant to a procoagulant surface are important factors in the pathogenesis of ischemic stroke. (3) Release of prostaglandins, endothelin-1, complement proteins, and matrix metalloproteinase-9 by microvascular endothelial cells are other components in the complex mechanism of brain ischemia/hypoxia. (4) Ultrastructural studies documented the opened paracellular avenues in the course of vasogenic edema in different experimental models (5) Tight junctions of endothelial cells have been characterized with freeze fracture electron microscopy, and the process of transvesiculation was analyzed using rapid freeze and freeze substitution procedure before electron microscopy studies (6) In endothelial cell-culture experiments, we used rodent and later human brains. (7) Endothelial cells co-cultured with astroglia resulted in an elaborate tight junctional complex. (8) This co-culture technique becomes the basis of in vitro blood-brain barrier studies On endothelial cells of human brain origin, different regulatory factors found to be responsible for the complex mechanism of ischemic stroke.

Investigation of substance P transport across the blood-brain barrier

The transport of substance P (SP) was investigated using the bovine brain microvessel endothelial cell culture model of the blood-brain barrier (BBB). The samples were derivatized precolumn with naphthalene dialdehyde, then analyzed by cyclodextrin-modified micellar electrokinetic chromatography with laser-induced fluorescence detection. SP crossed the BBB in both the apical-to-basolateral and basolateral-to-apical directions through an active transport mechanism. The transport of SP from the apical side was demonstrated to be via transcytosis. The N-terminal (SP 1–4) and C-terminal (SP 3–11) fragments were also found to permeate the BBB from the apical side.

Expression of Membrane-Bound and Soluble Cell Adhesion Molecules by Human Brain Microvessel Endothelial Cells

Expression of membrane-bound (mb) and soluble (s) forms of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) induced by tumor necrosis factor-α (TNF-α) has been measured by enzyme-linked immunosorbent assay in cultured human brain microvessel endothelial cells. Both the mb and the s forms of VCAM-1 and ICAM-1 were upregulated by TNF-α; however, the stimulation of the s forms was delayed in time. When piracetam, a neuroprotective drug, was added to the tissue culture medium simultaneously with TNF-α, the expression of mbVCAM-1 and ICAM-1 was lowered. Differential upregulation of mb and s forms of adhesion molecules and a novel effect of piracetam have been demonstrated in human brain microvessel endothelial cell cultures.

Brain microvessel endothelial cell culture systems.

The cellular basis of the blood-brain barrier (BBB) was defined as the endothelial lining of the brain microvessels in the late 1960s (Reese and Karnovsky, 1967; Brightman and Reese, 1969). This unique endothelial lining was recognized as the sole restrictive permeability barrier to the passage of nutrients, drugs, and metabolic waste products between the blood and the central nervous system. Consequently, investigations on the BBB have focused on developing an understanding of the transport and functional properties of this specialized endothelium (Bradbury, 1993).

Human brain microvessel endothelial cell culture as a model system to study vascular factors of ischemic brain.

Cerebral ischemia is caused by reduced blood supply at the microcirculatory level. In the microvessels, the main elements of the reperfusion injury following brain ischemia are the transformation of endothelial cell-surface from anticoagulant to procoagulant property, leukocyte adhesion, sludge or clot formation. There is a paucity of information on how hemostatic factors, cytokines, lipoprotein(a) (Lp(a)) and endothelin-1 (ET-1), being responsible for ischemic/reperfusion injury, interact with human brain microvessel endothelium (HBEC). There are no data furthermore about the expression of complement proteins of HBEC influenced by cytokines or fibrinolytic factors. Previously we established optimal conditions for culturing HBEC. Cell contraction induced by thrombin, plasmin, miniplasmin was recorded. The reassembly of F-actin was observed after thrombin treatment. ICAM-1 upregulation was measured following TNF-alpha, IL-1-alpha and thrombin incubation. Plasmin and miniplasmin downregulated the ICAM-1 in our cell culture system. Lp(a) modulated the thromboresistant cell-surface by reduction of t-PA and u-PA, but PAI-1 remained unchanged. Lp(a) modulated the ET-1 production by early increasing and late decreasing, in a bimodal manner. The increased secretion of ET-1 by cytokines (TNF-alpha, IL-1-alpha) was reduced in the presence of Lp(a). Gradual increase of complement proteins (factor H, factor B, C4) was induced by cytokines. Plasmin and miniplasmin augmented a rapid increase of C4. Some factors of complex relationship between regulators and modulators of endothelial adhesion molecules have been demonstrated in a human cell culture system prepared from brain microvessel endothelium. A unified concept of sequential events of ischemia/reperfusion in the brain has not yet developed.

Uptake of surfactant-coated poly(methyl methacrylate)-nanoparticles by bovine brain microvessel endothelial cell monolayers

The objective of this study was to determine the influence of coating of radiolabeled poly(methylmethacrylate) nanoparticles (PMMA nanoparticles) with surfactants on their uptake by bovine microvessel endothelial cell cultures (BMEC cultures). For this purpose, BMEC cultures were grown in 24-well culture plates, where they formed confluent monolayers 10–12 days after seeding. The nanoparticle suspensions were then incubated with the cell cultures at 37°C, and the radioactivity within the cell cultures and the supernatant was measured after 30 min, 2 h and 6 h. The rates of uptake of the coated nanoparticles and of a control group of uncoated nanoparticles were compared.

The interphase microtubule cytoskeleton of five different phenotypes of microvessel endothelial cell cultures derived from bovine corpus luteum

The interphase microtubule cytoskeleton of five different microvessel endothelial cell cultures, recently established from bovine corpus luteum, was analysed using anti-tubulin immunofluorescence. An antibody against acetylated microtubules detected four cell types each of which possessed a single cilia. The length of the cilia were up to 10 μm for cell types 1 and 2. Ciliary stubs had a length of up to 0.37 μm in cell types 4 and 5. Cilia were missing in cell type 3. Long and short cilia were located in the perinuclear region from where cytoplasmic microtubules radiated. Cell type 3 displayed straight microtubules rather than the wavy path seen in the other cell types. The amount of tyrosinated microtubules visualized by a specific antibody was consistently higher than that of posttranslationally acetylated microtubules. The latter were more apparent in cell types 4 and 5 than in the other cell types. We conclude: Differences in the cytoplasmic microtubule inventory of each microvessel endothelial cell type points at individual functions maintained in culture.