Effect of human astrocytes on the characteristics of human brain-microvascular endothelial cells in the blood-brain barrier

A blood-brain barrier (BBB) model in vitro was established by cultivating human brain-microvascular endothelial cells (HBMECs) with the regulation of human astrocytes (HAs) (HBMEC/HA). Astrocyte-conditioned medium (ACM) was employed to constitute a confluent monolayer of HBMECs without directly conjugated HAs. HBMECs exhibited an orientated multiplication on the supporting membrane; while HAs grew in an overlapping fashion. In addition, HBMECs could propagate over the membrane pore, and the end-feet of HAs extended into the membrane pore to improve the integral feature of the BBB. HBMEC/HA demonstrated a high transendothelial electrical resistance (TEER) about 230 Ω cm2 and low permeability of propidium iodide (PI) about 4 × 10?? cm/s. The order in TEER was HBMEC/HA>HBMECs with 100% ACM>HBMECs with 50% ACM > HBMECs. The reverse order was valid for the permeability of PI and uptake of calcein-AM by HBMECs. The tranwell culture of HBMECs and HAs displays appropriate characteristics of the BBB and can be applied to estimate the delivery efficiency of therapeutic chemicals for the brain-related disease.     

Impact of arginine-modified solid lipid nanoparticles on the membrane charge of human brain-microvascular endothelial cells

Electrokinetic and electrostatic properties of human brain-microvascular endothelial cells (HBMECs) with the uptake of l-arginine (Arg)-modified solid lipid nanoparticles (RSLNs) were investigated. The exposure of these HBMECs to radiofrequency electromagnetic field (EMF) was also considered. As compared with the original culture of HBMECs, the uptake of the biomimetic RSLNs induced smaller absolute values of electrophoretic mobility, zeta potential, Donnan potential, and fixed charge density. In addition, an increase in the coverage fraction of Arg on the external layers of the RSLNs reduced the electrical characteristics of HBMECs. An increase in the power of EMF also decreased the charge of RSLNs-incorporating HBMECs. On the contrary, softness of HBMECs was enhanced by an increased coverage fraction of Arg and an increased power of EMF. Electrophysiology of HBMECs can be efficiently mediated by the novel RSLNs and exposure to EMF through fluctuation and redistribution of the membrane charge.     

Evaluation of surface charge density and surface potential by electrophoretic mobility for solid lipid nanoparticles and human brain-microvascular endothelial cells

Electrophoretic mobility, zeta potential, surface charge density, and surface potential of cacao butter-based solid lipid nanoparticles (SLN) and human brain-microvascular endothelial cells (HBMEC) were analyzed in this study. Electrophoretic mobility and zeta potential were determined experimentally. Surface charge density and surface potential were evaluated theoretically via incorporation of ion condensation theory with the relationship between surface charge density and surface potential. The results revealed that the lower the pH value, the weaker the electrostatic properties of the negatively charged SLN and HBMEC. A higher content of cacao butter or a slower stirring rate yielded a larger SLN and stronger surface electricity. On the contrary, storage led to instability of SLN suspension and weaker electrical behavior because of hydrolysis of ionogenic groups on the particle surfaces. Also, high H+ concentration resulted in excess adsorption of H+ onto HBMEC, rendering charge reversal and cell death. The largest normalized discrepancy between surface potential and zeta potential occurred at pH = 7. For a fixed biocolloidal species, the discrepancy was nearly invariant at high pH value. However, the discrepancy followed the order of electrical intensity for HBMEC system at low pH value because mammalian cells were sensitive to H+. The present study provided a practical method to obtain surface charge properties by capillary electrophoresis.     

Sequence-specific transport of oligonucleotides into human endothelial cells

This study is devoted to interactions of oligonucleotides containing specific DNA motifs with and transport into human endothelial cells. Those motifs are overrepresented in a pool of short DNA firmly bound to the surfaces of these cells. The data obtained by various methods suggest that the binding of oligonucleotides to the cell membrane and their accumulation in endothelial cells depend on both the presence of specific DNA motifs (CATGCAT, GATCCA, or TACGT) and their combination in the oligonucleotides under study. The oligonucleotides penetrate into endothelial cells via receptor-mediated endocytosis, which is confirmed by flow cytometry and fluorescence microscopy in the presence of potential inhibitors and competitors of intracellular transport.

     

Adhesion behaviors of human trophoblast cells by contact with endothelial cells

Although it is still not clear whether migratory trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by a mechanism of directional cell division/proliferation, this process involves the attachment and adhesion of trophoblasts to endothelial cells lining the blood vessel walls. This raises the possibility that the cell–cell contact with endothelial cells may regulate trophoblast cell adhesion behaviors according to the surrounding flow condition. To test this, the adhesion forces of early gestation human trophoblast cells (TCs) cultured on glass slides coated with type I rat collagen or cultured with human umbilical vein endothelial cells (HUVECs) were measured quantitatively using a micropipette aspiration technique. Then, the resistance of TCs co-cultured with HUVECs to flow-induced shear stress was assessed with a flow chamber technique. The results showed that the adhesion force of TCs to glass slides coated with collagen was positively correlated with the concentration of collagen. By contact with endothelial cells, the adhesion force and the resistance to shear stress for the TCs were significantly enhanced. The interdiction of integrin β1 interaction remarkably reduced the adhesion forces of TCs to endothelial cells, hence their resistance to shear stress. The results therefore suggest that the contacts of TCs with endothelial cells enhance the adhesion forces of human TCs, partially by regulating with the integrin β1 according to the flow condition (i.e., the shear stress) in such a way to prevent the TCs from being carried downstream by flowing blood.     

Targeting delivery of paclitaxel into tumor cells via somatostatin receptor endocytosis

BACKGROUND: The binding of somatostatin (SST) to endogenous G-protein-coupled receptors (SST receptors or SSTRs) is followed by internalization of SST, and, several reports have shown that a high density of SSTRs is present on most hormone-secreting tissue tumors. Facile synthesis of the long-acting SST analog, octreotide, has previously been described. Octreotide might be of practical value in developing tumor tracers and in serving as a carrier of cytotoxic antitumor drugs. RESULTS: Fluorescein-labeled octreotide was internalized into the cytosol of human breast MCF-7 carcinoma cells via binding to SSTRs. Octreotide-conjugated paclitaxel (taxol) was created by coupling taxol-succinate to the amino-terminal end of octreotide. This conjugate retains the biological activity of taxol in inducing formation of tubulin bundles, eventually causing apoptosis of MCF-7 cells. Cytotoxicity of octreotide-conjugated taxol is mainly mediated by SSTR, as shown by the observation that octreotide pretreatment can rescue the induced cell death. In comparison with free taxol, this conjugate shows much less toxicity in Chinese hamster ovary cells. CONCLUSIONS: Octreotide-conjugated taxol exerts the same antitumor effect of free taxol on stabilizing microtubule formation and inducing cell death. This conjugate triggers tumor cell apoptosis mediated by SSTRs and is exclusively toxic to SSTR-expressing cells. Octreotide-conjugated taxol is less toxic to low-SSTR-expressing cells compared with free taxol. Our results strongly indicated that octreotide-conjugated taxol demonstrates cell selectivity and may be used as a targeting agent for cancer therapy.     

A class of human proteins that deliver functional proteins into mammalian cells in vitro and in vivo

We discovered a class of naturally occurring human proteins with unusually high net positive charge that can potently deliver proteins in functional form into mammalian cells both in?vitro and also in murine retina, pancreas, and white adipose tissues in?vivo. These findings represent diverse macromolecule delivery agents for in?vivo applications, and also raise the possibility that some of these human proteins may penetrate cells as part of their native biological functions.     

Selective inhibition of amino-terminal methionine processing by TNP-470 and ovalicin in endothelial cells

BACKGROUND: The angiogenesis inhibitors TNP-470 and ovalicin potently suppress endothelial cell growth. Both drugs also specifically inhibit methionine aminopeptidase 2 (MetAP2) in vitro. Inhibition of MetAP2 and changes in initiator methionine removal in drug-treated endothelial cells have not been demonstrated, however. RESUTLS: Concentrations of TNP-470 sufficient to inactivate MetAP2 in intact endothelial cells were comparable to those that inhibited cell proliferation, suggesting that MetAP2 inhibition by TNP-470 underlies the ability of the drug to inhibit cell growth. Both drug-sensitive and drug-insensitive cell lines express MetAP1 and MetAP2, indicating that drug sensitivity in mammalian cells is not simply due to the absence of compensating MetAP activity. With a single exception, detectable protein N-myristoylation is unaffected in sensitive endothelial cells treated with TNP-470, so MetAP1 activity can generally compensate when MetAP2 is inactive. Analysis of total protein extracts from cells pulse-labeled with [(35)S]-methionine following TNP-470 treatment revealed changes in the migration of several newly synthesized proteins. Two of these proteins were identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cyclophilin A. Purification and amino-terminal sequencing of GAPDH from TNP-470-treated cells revealed partial retention of its initiator methionine, indicating that methionine removal from some, but not all, proteins is affected by MetAP2 inactivation. CONCLUSIONS: Amino-terminal processing defects occur in cells treated with TNP-470, indicating that inhibition of MetAP2 by the drug occurs in intact cells. This work renders plausible a mechanism for growth inhibition by TNP-470 as a consequence of initiator methionine retention, leading to the inactivation of as yet unidentified proteins essential for endothelial cell growth.     

New insights into human farnesyl pyrophosphate synthase inhibition by second-generation bisphosphonate drugs

Pamidronate, alendronate, APHBP and neridronate are a group of drugs, known as second-generation bisphosphonates (2G-BPs), commonly used in the treatment of bone-resorption disorders, and recently their use has been related to some collateral side effects. The therapeutic activity of 2G-BPs is related to the inhibition of the human Farnesyl Pyrophosphate Synthase (hFPPS). Available inhibitory activity values show that 2G-BPs act time-dependently, showing big differences in their initial inhibitory activities but similar final IC₅₀ values. However, there is a lack of information explaining this similar final inhibitory potency. Although different residues have been identified in the stabilization of the R₂ side chain of 2G-BPs into the active site, similar free binding energies were obtained that highlighted a similar stability of the ternary complexes, which in turns justified the similar IC₅₀ values reported. Free binding energy calculations also demonstrated that the union of 2G-BPs to the active site were 38 to 54 kcal mol^?1 energetically more favourable than the union of the natural substrate, which is the basis of the inhibition potency of the hFPPS activity.     

The identification of proliferation and tumour-induced proteins in human endothelial cells: a possible target for tumour therapy

The continued growth and spread of tumours is dependent on the proliferation of the endothelial cells of their vasculature. The presence of proliferation- or tumour-induced surface proteins on these endothelial cells would offer a suitable epitope for monoclonal antibody therapy of tumours. Using cultured human umbilical and capillary endothelial cells, we have stimulated them with simple mitogens and tumour conditioned media and examined the proteins induced by [35S]methionine incorporation and 125I-surface-labelling. Two-dimensional polyacrylamide gel electrophoresis revealed the induction of proliferation and tumour-related antigens on the surface of the endothelial cells. Subsequent monoclonal antibody studies suggest that tumour specific surface proteins are present on most tumour endothelium.     

Effect of surface functionalization of MCM-41-type mesoporous silica nanoparticles on the endocytosis by human cancer cells

We have synthesized a series of MCM-41-type mesoporous silica nanoparticles (MSN). The surface of the MSNs are functionalized with 3-aminopropyl (AP), 3-guanidinopropyl (GP), 3-[N-(2-guanidinoethyl)guanidino]propyl (GEGP), and N-folate-3-aminopropyl (FAP). In contrast to the zeta-potential of -18.4 mV for FITC-MSN, the values of zeta-potential for AP-, GP-, GEGP-, and FAP-functionalized FITC-MSNs in 100 mM PBS buffer (pH 7.4) increased positively from -11.3, -10.6, -4.0, to +4.9 mV, respectively. The uptake efficiency, endocytosis mechanism, and biocompatibility of these organically functionalized MSNs were investigated with human cervical cancer cells (HeLa). Flow cytometry results suggested that the endocytosis of MSN could be manipulated by different surface functionalization. The immunocytochemistry study indicated that the uptake of these MSNs by HeLa cells was surface functional group dependent and involved several different mechanisms of endocytosis. Confocal fluorescence micrographs showed that the different surface functionalities of MSNs could also affect their ability to escape endosomal entrapment, which is a key factor in designing effective intracellular delivery vehicles.     

Delivery of bioactive,gel-isolated proteins into live cells

The delivery of proteins into live cells is a promising strategy for the targeted modulation of protein-protein interactions and the manipulation of specific cellular functions. Cellular delivery can be facilitated by complexing the protein of interest with carrier molecules. Recently, an amphipatic peptide was identified, Pep-1 (KETWWETWWTE WSQPKKKRKV), which crosses the plasma membrane of many cell types to carry and deliver proteins as large as antibodies. Pep-1 effectively delivers proteins in solution; but Pep-1 is not suitable for delivering sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) isolated proteins because Pep-1 complexes with cargo proteins are destroyed by SDS. Here, we report cellular delivery of SDS-PAGE-isolated proteins, without causing cellular damage, by using a nonionic detergent, Triton X-100, as carrier. To determine the specificity of our method, we separated antibodies against different intracellular targets by nonreducing SDS-PAGE. Following electrophoresis, the antibody bands were detected by zinc-imidazole reverse staining, excised, in-gel refolded with Triton X-100, and eluted in detergent-free phosphate-buffered saline. When overlaid on cultured NIH 3T3 cells, the antibodies penetrated the cells localizing to their corresponding intracellular targets. These results are proof-of-principle for the delivery of gel-isolated bioactive proteins into cultured cells and suggest new ways for experimental protein therapy and for studying protein-protein interactions using gel-isolated protein.     

Low molecular weight heparin enhances prostacyclin production by cultured human endothelial cells.

Confluent cultures of vascular endothelial cells derived from the human umbilical vein were incubated in a serum-free medium in the presence of low molecular weight heparin (LMWH) with molecular weights of 4000-6000 dalton (Da), or of unfractionated heparin (UFH) with average molecular weight 12,000 Da, and prostacyclin production was determined by radioimmunoassay for 6-keto-prostaglandin F1 alpha, the stable metabolite of prostacyclin. LMWH at 1 U/ml as anti-factor Xa activity significantly increased prostacyclin production after 6h or longer; however, UFH at 1 USP U/ml did not induce such a significant change. The LMWH-induced increase in prostacyclin production occurred at 0.1 U/ml and above after 6 h of treatment. Since prostacyclin is both a potent inhibitor of platelet aggregation and a vasodilator, it was suggested that the increased endothelial cell prostacyclin production induced by LMWH may be a component of the anticoagulant activity of the drug.     

Effects of flavonoid derivatives on human microvascular endothelial cells

Some natural compounds, including flavonoids, are active in vasculature re-growth during hair follicle disruption, but their effects have not been yet evaluated directly on microvascular endothelial cells. Skin vascularisation regulates the physiological blood supply required for hair growth and its dysregulation is the basis of several human diseases. Follicle-derived vascular endothelial growth factor (VEGF) release from follicular keratinocytes promotes perifollicular vascularisation and increases follicle and hair size, while blockade of VEGF-mediated angiogenesis leads to impaired hair growth. Here, we tested three flavonoids, namely visnadin (VSD), hesperidin (HSP) and baicalin (BC), on cultured human microvascular endothelial cells (HMEC), comparing their effects with minoxidil (MXD), a synthetic drug broadly used in the treatment of androgenetic alopecia. The response to these compounds was assayed in terms of endothelial survival, proliferation, tubulogenesis and proangiogenic signalling. We show that BC promotes HMEC proliferation, while both VSD and MXD enhance tubulogenesis. Interestingly, only HSP increases VEGFR-2 phosphorylation.     

Regulation of cell growth by fatty acid-CoA ligase 4 in human hepatocellular carcinoma cells

Fatty acid-CoA ligase 4 (FACL4) is a central enzyme controlling the unesterified free arachidonic acid (AA) level in cells and the free AA is known to induce apoptosis. We have recently reported that expression of FACL4 is upregulated in about 40% of human hepatocellular carcinoma (HCC) and 50% of HCC cell lines, suggesting that FACL4 may be involved in liver carcinogenesis. In this study, we investigated whether HCC cell growth is regulated by FACL4. Immunoblot analysis showed that SNU 398 cells express very low or no detectable level of FACL4. We, therefore, transfected the SNU 398 cells with FACL4 expression vector, and clones expressing FACL4 were pooled and analyzed. We found that forced expression of FACL4 in SNU 398 promotes the growth of cells. In addition, we observed that triacsin C, a FACL4 inhibitor, inhibits the growth of Hep 3B cell line which expresses high level of endogenous FACL4. We also found that the triacsin C-mediated growth inhibition in Hep 3B cells results from the induction of apoptosis with evidence of Bcl-2 reduction. Altogether, our data show that FACL4 affects HCC cell growth and suggest that modulation of FACL4 expression/activity is an approach for treatment of HCC.     

Shear stress stimulates phosphorylation of protein kinase A substrate proteins including endothelial nitric oxide synthase in endothelial cells

Fluid shear stress plays a critical role in vascular health and disease. While protein kinase A (PKA) has been implicated in shear-stimulated signaling events in endothelial cells, it remains unclear whether and how PKA is stimulated in response to shear stress. This issue was addressed in the present study by monitoring the phosphorylation of endogenous substrates of PKA. Shear stress stimulated the phosphorylation of cAMP responsive element binding protein (CREB) in a PKA-dependent manner. Western blot analysis using the antibody reactive against the consensus motif of PKA substrates detected two proteins, P135 and P50, whose phosphorylation was increased by shear stress. The phosphorylation of P135 was blocked by a PKA inhibitor, H89, but not by a phosphoinositide 3-kinase inhibitor, wortmannin. Expression of a constitutively active PKA subunit stimulated P135 phosphorylation, supporting the potential of P135 as a PKA substrate. P135 was identified as endothelial nitric oxide synthase (eNOS) by immunoprecipitation study. PKA appeared to mediate shear stress-stimulated eNOS activation. Shear stress stimulated intracellular translocation of PKA activity from 'soluble' to 'particulate' fractions without involving cellular cAMP increase. Taken together, this study suggests that shear stress stimulates PKA-dependent phosphorylation of target proteins including eNOS, probably by enhancing intracellular site-specific interactions between protein kinase and substrates.     

Gene delivery into brain capillary endothelial cells using Antp-modified DNA-loaded nanoparticles

Our aim is to test the characteristics and expression activity in brain capillary endothelial cells (BCECs) of DNA-loaded nanoparticles before and after the modification with Antennapedia peptide (Antp). Chitosan (CH), polyethylenimine (PEI) and polyamidoamine (PAMAM) were chosen to prepare the nanoparticles; Lipofectamine 2,000 served as control. The incorporation efficiency was determined by agarose gel electrophoresis and PicoGreen assay. Transmission electron microscopy was performed to observe the diameter. The expression efficiency was qualified with fluorescence microscopy and quantified with the luciferase assay system. Heparin and DNase I were employed to investigate the stability. The results indicated that the gene products of DNA/CH nanoparticles and DNA/Lipofectamine 2,000 complexes whether modified or unmodified with Antp could not be observed within BCECs. The DNA/PEI nanoparticles and DNA/PAMAM nanoparticles could be internalized into BCECs to a certain extent, and the expression efficiency was enhanced significantly with the modification of Antp. The diameter increased from 100 to 200 nm after modification with Antp. The nanoparticles could protect themselves from the displacement of anionic substances and the digestion of DNase I. These observations demonstrated that Antp, polymer and DNA could form stable nanoparticles, and the modification of Antp can enhance the expression efficiency of DNA-loaded nanoparticles in BCECs.