Murine Brain Capillary Endothelial Cells Research Articles

Characterization of an iPSC-based barrier model for blood-brain barrier investigations using the SBAD0201 stem cell line

BackgroundBlood-brain barrier (BBB) models based on primary murine, bovine, and porcine brain capillary endothelial cell cultures have long been regarded as robust models with appropriate properties to examine the functional transport of small molecules. However, species differences sometimes complicate translating results from these models to human settings. During the last decade, brain capillary endothelial-like cells (BCECs) have been generated from stem cell sources to model the human BBB in vitro. The aim of the present study was to establish and characterize a human BBB model using human induced pluripotent stem cell (hiPSC)-derived BCECs from the hIPSC line SBAD0201.MethodsThe model was evaluated using transcriptomics, proteomics, immunocytochemistry, transendothelial electrical resistance (TEER) measurements, and, finally, transport assays to assess the functionality of selected transporters and receptor (GLUT-1, LAT-1, P-gp and LRP-1).ResultsThe resulting BBB model displayed an average TEER of 5474 ± 167 Ω·cm2 and cell monolayer formation with claudin-5, ZO-1, and occludin expression in the tight junction zones. The cell monolayers expressed the typical BBB markers VE-cadherin, VWF, and PECAM-1. Transcriptomics and quantitative targeted absolute proteomics analyses revealed that solute carrier (SLC) transporters were found in high abundance, while the expression of efflux transporters was relatively low. Transport assays using GLUT-1, LAT-1, and LRP-1 substrates and inhibitors confirmed the functional activities of these transporters and receptors in the model. A transport assay suggested that P-gp was not functionally expressed in the model, albeit antibody staining revealed that P-gp was localized at the luminal membrane.ConclusionsIn conclusion, the novel SBAD0201-derived BBB model formed tight monolayers and was proven useful for studies investigating GLUT-1, LAT-1, and LRP-1 mediated transport across the BBB. However, the model did not express functional P-gp and thus is not suitable for the performance of drug efflux P-gp reletated studies.

Highly cationic cell-penetrating peptides affect the barrier integrity and facilitates mannitol permeation in a human stem cell-based blood-brain barrier model

The blood-brain barrier (BBB) allows passive permeation of only a limited number of, primarily lipophilic, low-molecular weight drugs that obey the so-called “rule of CNS likeness”. Therefore, novel strategies to facilitate drug delivery across the BBB are needed. Cell-penetrating peptides (CPPs) enable delivery of various therapeutic cargoes into cells and may potentially serve as shuttles for delivery of brain-specific drugs across the BBB. The CPPs Tat47–57 and penetratin are prototypical cationic CPPs, whereas apidaecin and oncocin belong to the group of proline-rich cationic antimicrobial peptides displaying CPP-like properties.The aim of the present study was to investigate the potential of Tat47–57, penetratin, apidaecin, and oncocin for interaction with and permeation of the BBB in vitro. We also studied whether the CPPs facilitated permeation of the paracellular flux marker mannitol as well as the transcellular flux marker propranolol. The peptides were labelled with the fluorophore 6-TAMRA (T) for visualization and quantification purposes. CPP membrane-adherence, membrane-embedding, and cellular uptake as well as barrier-permeation were evaluated in murine brain capillary endothelial cells (bEND3) and human induced pluripotent stem cell-derived (Bioni-010c) brain capillary endothelial-like monolayers. The cationic and the proline-rich cationic CPPs were taken up into the Bioni-010c monolayers. T-Tat47–57, T-apidaecin, and T-oncocin also permeated Bioni-010c monolayers, whereas T-penetratin did not. However, both T-Tat47–57 and T-penetratin affected the barrier integrity to a degree that facilitated permeation of 14C-mannitol. These results may therefore pave the way for future CPP-mediated brain delivery of small drugs that do not obey the “rule of CNS likeness”.

Abstract 252: Tumor treating fields (TTFields) affect blood brain barrier (BBB) integrity in vitro and in vivo

Abstract Background: The blood-brain barrier (BBB) may impede application of drugs to the brain for treatment of malignant brain tumors, in particular glioblastoma multiforme (GBM). Alternating electric fields with intermediate frequency and low intensity, called Tumor Treating Fields (TTFields), are an established novel adjuvant treatment modality for GBM. Here, the effect of TTFields on BBB permeability is analyzed. Material and Methods: After TTFields treatment with a frequency of 100-300 kHz for up to 72 h, immortalized murine brain capillary endothelial cells (cerebEND) grown on cover slips and transwell inserts were stained for immunofluorescent assessment of the tight junction proteins Claudin-5 and ZO-1. Transendothelial electrical resistance (TEER) was applied to investigate BBB integrity. Moreover, BBB permeability was determined by fluorescein isothiocyanate (FITC) staining followed by flow cytometry. For in vivo analysis, the increase in vessel permeability was quantified by utilizing i.v. injected Evans Blue (EB) in rats during TTFields application to the brain (100 kHz, 72 h). Results: The BBB was disturbed by treatment with TTFields as tight junction proteins were delocalized from the cell boundaries to the cytoplasm with maximal effects at 100 kHz. TTFields application significantly reduced the BBB integrity by 65% and significantly increased the BBB permeability for 4 kDa large molecules. Initial recovery of the cell morphology was observed 48 h post-treatment and a complete recovery could be detected after 96 h, indicating a reversibility of the TTFields effect on the BBB. Average accumulation of EB in the rat brain was significantly increased by TTFields application to the rats head. Conclusion: In the future, TTFields could be utilized to deliver drugs generally unable to cross the BBB to the central nervous system as TTFields at a frequency of 100 kHz are potentially able to disrupt the BBB. The data presented on in vitro and in vivo application of TTFields to permeabilize the BBB may be a rationale for a phase I clinical trial and clinical application in the future. Citation Format: Almuth F. Kessler, Clara M. Schaeffer, Malgorzata Burek, Ursula Ruschig, Catherine Tempel-Brami, Tali Voloshin, Moshe Giladi, Ellaine Salvador, Ralf-Ingo Ernestus, Mario Löhr, Carola Förster, Carsten Hagemann. Tumor treating fields (TTFields) affect blood brain barrier (BBB) integrity in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 252.

Aβ1–42 oligomer induces alteration of tight junction scaffold proteins via RAGE-mediated autophagy in bEnd.3 cells

Compelling evidences have shown that amyloid-β (Aβ) peptide is one of the major pathogenic factors resulting in blood-brain barrier (BBB) disruption in Alzheimer's disease (AD). However, the mechanism underlying BBB breakdown remains elusive. In our present study, we employed murine brain capillary endothelial cells (bEnd.3) as an in vitro BBB model to investigate the role of autophagy in Aβ1–42 oligo induced BBB disruption. We first identified Aβ1–42 oligo cytotoxicity to bEnd.3 cells as observed in the reduced cell viability and downregulation of ZO-1, Occludin and Claudin-5. Based on the observation that both downregulated expression of p-mTOR/m-TOR and upregulated ratio of LC3-II/β-actin were induced by Aβ1–42 oligo, we then applied 3-MA, an inhibitor of autophagy, to test the role of autophagy in Aβ1–42 oligo induced Tight junction (TJ) proteins damage. Results have shown that 3-MA partially reversed Aβ1–42 oligo induced downregulation of ZO-1, Occludin and Claudin-5, which was further determined by LC3 siRNA. We also used rapamycin to activate autophagy and found that TJ proteins damage induced by Aβ1–42 was deteriorated even further. Given that the receptor of advanced glycation end-products (RAGE) is a pivotal receptor that mediates Aβ toxicity, RAGE siRNA was utilized to identify the involvement of RAGE in Aβ1–42 oligo induced autophagy. The results demonstrated a suppressed autophagy with increased p-mTOR/m-TOR and decreased LC3-II/β-actin as well as increased ZO-1, Occludin and Claudin-5 in transfected cells after Aβ1–42 oligo treatment, as compared to the non-transfected group. In summary, these results suggested that Aβ1–42 oligo induced TJ proteins disruption via a RAGE-dependent autophagy pathway.

Synthesis and deposition of basement membrane proteins by primary brain capillary endothelial cells in a murine model of the blood-brain barrier.

The brain vascular basement membrane is important for both blood-brain barrier (BBB) development, stability, and barrier integrity and the contribution hereto from brain capillary endothelial cells (BCECs), pericytes, and astrocytes of the BBB is probably significant. The aim of this study was to analyse four different invitro models of the murine BBB for expression and possible secretion of major basement membrane proteins from murine BCECs (mBCECs). mBCECs, pericytes and glial cells (mainly astrocytes and microglia) were prepared from brains of C57BL/6 mice. The mBCECs were grown as monoculture, in co-culture with pericytes or mixed glial cells, or as a triple-culture with both pericytes and mixed glial cells. The integrity of the BBB models was validated by measures of transendothelial electrical resistance (TEER) and passive permeability to mannitol. The expression of basement membrane proteins was analysed using RT-qPCR, mass spectrometry and immunocytochemistry. Co-culturing mBCECs with pericytes, mixed glial cells, or both significantly increased the TEER compared to the monoculture, and a low passive permeability was correlated with high TEER. The mBCECs expressed all major basement membrane proteins such as laminin-411, laminin-511, collagen [α1(IV)]2 α2(IV), agrin, perlecan, and nidogen 1 and 2 invitro. Increased expression of the laminin α5 subunit correlated with the addition of BBB-inducing factors (hydrocortisone, Ro 20-1724, and pCPT-cAMP), whereas increased expression of collagen IV α1 primarily correlated with increased levels of cAMP. In conclusion, BCECs cultured invitro coherently form a BBB and express basement membrane proteins as a feature of maturation. Cover Image for this issue: doi: 10.1111/jnc.13789.

Endogenous Agmatine Inhibits Cerebral Vascular Matrix Metalloproteinases Expression by Regulating Activating Transcription Factor 3 and Endothelial Nitric Oxide Synthesis

Earlier investigations from our laboratory demonstrated that the expression of matrix metalloproteinases (MMPs) was down-regulated by exogenously administered agmatine against ischemia-like injuries in the murine brain capillary endothelial (bEnd.3) cells. In our present study, we intended to investigate the mechanism involved in the inhibition of MMPs in bEnd.3 cells infected with retroviral containing human arginine decarboxylase (hADC) gene which can synthesize agmatine endogenously (ADCDeltabEnd.3 cells). The ADCDeltabEnd.3 cells were subjected to oxygen glucose deprivation (OGD, 6 hrs) with reperfusion (18 hrs). High performance liquid chromatography (HPLC) analysis revealed the high levels of agmatine in the ADCDeltabEnd.3 cells compared to other experimental groups. The results demonstrated significant decrease in cell death and increase in the nitric oxide (NO) production in the ADCDeltabEnd.3 cells. The increased expression of MMP-2 and MMP-9, and decreased expression of endothelial nitric oxide synthase (eNOS) by ischemic injury was attenuated in ADCDeltabEnd.3 cells. Moreover, the expression of activating transcription factor 3 (ATF3) was increased significantly in ADCDeltabEnd.3 cells. In addition, the suppression of the MMP-2 and MMP-9 expression in ADCDeltabEnd.3 cells was prevented with ATF3 small interfering RNA (siRNA) treatment. These results suggest that the endogenous agmatine in ADCDeltabEnd.3 cells inhibits the MMPs expression mediated via the regulation of eNOS, NO and ATF3.

Angiostatic effects of K252a, a Trk inhibitor, in murine brain capillary endothelial cells

Nerve growth factor (NGF) supports the survival and differentiation of sympathetic and sensory neurons and is also mitogenic for a variety of tumors. K252a, an antagonist of NGF receptor TrkA, was previously used as a pharmacological tool to study NGF actions and as a lead compound for developing anti-tumor drugs. Since recently, NGF was characterized as an angiogenic factor, we sought to investigate the angiostatic properties of K252a on endothelial cells (ECs). For this purpose, we used a murine brain microcapillary ECs model in which we found autocrine release of NGF in the culture medium and activation of TrkA receptor-induced downstream signaling molecules Erk1/2, Akt, and PLCgamma. In this model, we demonstrated the angiostatic property of K252a based on its ability to affect several important angiogenic steps. K252a, but not its cell membrane impermeable analogue K252b at 100 nM: (i) inhibited the proliferation of the ECs by 45 +/- 9%; (ii) reduced by 70 +/- 4% the migration of the ECs measured in a wound-closure model; (iii) reduced by 29 +/- 9% the formation of tube-like structures of the ECs cultured on Matrigel; (iv) stimulated by 100 +/- 25% the collagen deposition by the ECs, a process responsible for the increased endothelial barrier functions expressed by 22 +/- 5% reduction of paracellular permeability and by 17 +/- 3% elevation of transendothelial electrical resistance. These data suggest that NGF/TrkA may represent a target for the development of novel, K252a-derived multikinase inhibitors drugs with anti-tumor and angiostatic dual activities.

Fibroblast growth factor-2 induces the activation of Src through Fes, which regulates focal adhesion disassembly

Cell migration is regulated by focal adhesion (FA) turnover. Fibroblast growth factor-2 (FGF-2) induces FA disassembly in the murine brain capillary endothelial cell line IBE, leading to FGF-2-directed chemotaxis. We previously showed that activation of Src and Fes by FGF-2 was involved in chemotaxis of IBE cells. In this study, we examined the interplay between Src and Fes. FGF-2 treatment decreased the number of FA in IBE cells, but not in cells expressing dominant-negative Fes (denoted KE5-15 cells). FGF-2 induced the activation of Src and subsequent binding to and phosphorylation of Cas in IBE cells, but not in KE5-15 cells. Focal adhesion kinase (FAK) activation and tyrosine phosphorylation by Src were also delayed in KE5-15 cells compared to parental cells. FGF-2 induced activation of Src within FA in IBE cells, but not in KE5-15 cells. Downregulation of Fes or FAK using small interfering RNA diminished Src activation by FGF-2 within FA. These findings suggest that activation of Fes by FGF-2 enhances FAK-dependent activation of Src within FA, promoting FGF-2-induced disassembly of focal adhesions.

Influence of Hydrocortisone on the Mechanical Properties of the Cerebral Endothelium In Vitro

Cerebral endothelial cells accomplish the barrier functions between blood and brain interstitium. Structural features are the tight junctions between adjacent endothelial cells and the formation of marginal folds at the cell-cell contacts. The glucocorticoid hydrocortisone (HC) has been reported to enforce the blood-brain-barrier in vitro measurable by an increase of the transendothelial electrical resistance. This study shows the impact of HC on the mechanical and morphological properties of confluent cell layers of brain microvascular endothelial cells. HC induces an increase in height of these marginal folds and a reduction of the intercellular contact surface. These morphological changes are accompanied by changes in cell elasticity. Staining of fibrous actin indicates that HC induces a reorganization of the actin cortex. The quantitative determination of the local elastic properties of cells reveals for the first time an HC-induced increase of the representative Young’s modulus according to cytoskeletal rearrangements. For this study, cells of two different species, porcine brain capillary endothelial cells and murine brain capillary endothelial cells, were used yielding similar results, which clearly demonstrates that the HC effect on the cell elasticity is species independent.

Angiopoietin 1 Is Mitogenic for Cultured Endothelial Cells

The angiopoietin (Ang)/Tie2 system is implicated in blood vessel formation and maturation. However, the mitogenic effects of angiopoietins remain to be elucidated. Here, we show that Ang1 is mitogenic for cultured endothelial cells. Ang1 dose-dependently induced the proliferation and increased the labeling index of a murine brain capillary endothelial cell line, IBE cells. Ang1 also increased the labeling index of human umbilical vein endothelial cells (HUVEC). Ang1 up-regulated the expression of cyclin D1 in both of these cells. Ang1 activated mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) in IBE cells and HUVECs. Activated PI3K was associated with c-Fes protein tyrosine kinase in these cells, but not with Tie2. p70 S6 kinase (p70 S6K) was activated by Ang1-treatment, although this activation was blocked by a PI3K inhibitor, LY294002. Simultaneous treatment of cells with PD98059 (MAPK/extracellular regulated kinase kinase inhibitor) and rapamycin (mTOR inhibitor) completely blocked Ang1-induced mitogenic activity for IBE cells and HUVECs. Although Ang2 at high concentration weakly activated Tie2 and p70 S6K, it failed to activate Ras and MAPK, or to induce cell proliferation. Taken together, these findings indicate that Ang1 exerts mitogenic activity on endothelial cells, which requires activation of both MAPK and p70 S6K.

Murine brain capillary endothelial cells exhibit improved barrier properties under the influence of hydrocortisone

Hydrocortisone is known to induce barrier properties in porcine primary cultures of microvascular endothelial cells. Here we present similar effects of hydrocortisone on a serum-free in vitro model based on primary cultured mouse brain endothelial cells. These cells in culture express typical blood–brain barrier properties and the transendothelial electrical resistance is enhanced after the addition of hydrocortisone to the medium in physiological concentrations. The improvement of the barrier is accompanied by changes at the cell borders indicated by immunofluorescence staining of tight junction proteins. Transmission electron microscopy imaging indicates morphological changes at the cell–cell contact zones which correlates to the observed changes in the transendothelial electrical resistance after HC supplementation. Phalloidin staining of F-actin shows a rearrangement to “fiber-like” structures in the longitudinal direction of the cell. These findings together with additional electrical impedance analysis of the monolayer suggest that several changes including cell–cell contact alteration, cell–substrate attachment and cytoskeletal rearrangements cause enhanced barrier properties in this murine endothelial culture. The present data are consistent with earlier findings in a porcine serum-free in vitro model. Thus, evidence is given that the barrier enforcement induced by glucocorticoids is not a species-specific effect and that the barrier improvement is correlated with a change of the cell morphology rather than changes in tight junction protein expression.

Pigment epithelium-derived factor inhibits fibroblast-growth-factor-2-induced capillary morphogenesis of endothelial cells through Fyn

Pigment epithelium-derived factor (PEDF) exerts anti-angiogenic actions. However, the signal-transduction pathways regulated by PEDF remain to be elucidated. We show here that PEDF inhibited fibroblast growth factor 2 (FGF-2) induced capillary morphogenesis of a murine brain capillary endothelial cell line (IBE cells) and of human umbilical-vein endothelial cells (HUVECs) cultured on growth-factor-reduced Matrigel. We previously showed that FGF-2-mediated capillary morphogenesis was blocked by the Src-kinase inhibitor PP2 and that expression of dominant negative Fyn in IBE cells inhibited capillary morphogenesis. We examined the effect of PEDF on kinase activity of Fyn and found that PEDF downregulated FGF-2-promoted Fyn activity by tyrosine phosphorylation at the C-terminus in a Fes-dependent manner. In a stable IBE cell line expressing kinase-inactive Fes (KE5-15 Fes cells), PEDF failed to inhibit FGF-2-induced capillary morphogenesis or Fyn activity. PEDF induced the colocalization of Fyn and Fes in IBE cells expressing wild-type Fes, but not in KE5-15 Fes cells. In addition, wild-type Fes increased the tyrosine phosphorylation of Fyn in vitro, suggesting that Fes might directly phosphorylate Fyn. Expression of constitutively active Fyn (Y531F) in IBE cells exhibited capillary morphogenesis in the absence of FGF-2 and was resistant for PEDF treatment. Our results suggest that PEDF downregulates Fyn through Fes, resulting in inhibition of FGF-2-induced capillary morphogenesis of endothelial cells.

T‐cell factor‐4‐dependent up‐regulation of fibronectin is involved in fibroblast growth factor‐2‐induced tube formation by endothelial cells

Deletion of fibronectin or its receptor, alpha(5) integrin, interferes with the formation of a functional circulation in mice. We hypothesized that alpha(5)beta(1) integrin/fibronectin interaction may be involved in differentiation of endothelial cells during angiogenesis. We examined the effect of blocking antibody against alpha(5)beta(1) integrin in fibroblast growth factor-2 (FGF-2)-induced angiogenesis by Matrigel plug assay. Although the antibody did not inhibit the recruitment of endothelial cells into plugs, it inhibited organization of lumen-containing capillaries. The antibody also inhibited FGF-2-induced tube formation by murine brain capillary endothelial cells (IBE cells) cultured in type I collagen gels. We previously showed that FGF-2 failed to induce tube formation by IBE cells expressing kinase-dead c-Fyn (KDFyn cells). Association with beta-catenin enhances the transcriptional activity of T-cell factor-4 (TCF-4), which up-regulates the expression of fibronectin. FGF-2 induced association of beta-catenin with TCF-4 and up-regulation of fibronectin in IBE cells, but not in KDFyn cells. Expression of mutant TCF-4, which does not associate with beta-catenin, inhibited FGF-2-induced tube formation and expression of fibronectin in IBE cells. FGF-2-induced tyrosine phosphorylation of beta-catenin, and association with TCF-4 was increased in IBE cells, but not in KDFyn cells. Taken together, interaction of alpha(5)beta(1) integrin and fibronectin is involved in FGF-2-induced tube formation by endothelial cells and up-regulation of fibronectin through TCF-4 seemed to be involved in this process.

Role of focal adhesion formation in migration and morphogenesis of endothelial cells

Cell motility and morphogenesis are regulated by a balance between formation and disassembly of stress fibers and focal adhesions. To understand the mechanisms underlying these cellular responses in angiogenesis, we studied the Rho family protein-driven pathways in FGF-2-induced chemotaxis and capillary morphogenesis of murine brain capillary endothelial cell line, IBE cells. Cells seeded onto fibronectin-coated surface migrated toward FGF-2. Expression of dominant negative Rho A (DNRho) or kinase-dead p21-activated kinase 1 (KDPAK1), or treatment with Y27632 inhibited chemotaxis in association with the lack of FGF-2-induced decrease in focal adhesions. On Matrigel, DNRho and Y27632 induced FGF-2-independent capillary morphogenesis despite loss of stress fiber formation. KDPAK1 cells formed stress fibers and showed capillary morphogenesis in response to FGF-2. Increase in focal adhesions was closely associated with capillary morphogenesis. Our results suggest that formation or disassembly of focal adhesions seems to determine the motility or morphogenesis of endothelial cells.

Fibroblast Growth Factor-2-mediated Capillary Morphogenesis of Endothelial Cells Requires Signals via Flt-1/Vascular Endothelial Growth Factor Receptor-1: POSSIBLE INVOLVEMENT OF c-Akt

Capillary morphogenesis is a crucial angiogenic response of endothelial cells. Although fibroblast growth factor-2 (FGF-2) potently induces capillary morphogenesis, the contribution of vascular endothelial growth factor-A (VEGF-A) in this response has not been clarified well. Here we examined the role of VEGF signaling in FGF-2-induced capillary morphogenesis by murine brain capillary endothelial cells (IBE cells) and human umbilical vein endothelial cells. FGF-2-treated IBE cells rapidly extended on Matrigel in association with actin reorganization. Chimeric protein, of which the extracellular domain of VEGF receptor-1 (VEGFR-1) fused to immunoglobulin Fc, inhibited FGF-2-induced cell extension, resulting in decreased capillary morphogenesis. Blocking antibody against VEGFR-1 inhibited FGF-2-induced capillary formation. Also, anti-VEGF-A antibody inhibited FGF-2-induced capillary morphogenesis, which was restored by the addition of placental growth factor-1. Similar results were obtained by the experiments with human umbilical vein endothelial cells. Expression of kinase-inactive c-Akt in IBE cells showed impaired capillary morphogenesis promoted by FGF-2. Conversely, stable cell lines expressing activated c-Akt demonstrated ligand-independent capillaries, which were resistant to the treatment with anti-VEGFR-1 blocking antibody. Upstream of c-Akt, calmodulin-dependent signals seemed to be involved. Taken together, signals via VEGFR-1 were required for FGF-2-induced capillary morphogenesis by endothelial cells, and c-Akt activity seemed to be involved in this process.

Sonic Hedgehog Induces Capillary Morphogenesis by Endothelial Cells through Phosphoinositide 3-Kinase

Sonic hedgehog (Shh) acts as a morphogen in many cell types. Recent studies have shown that hedgehog signaling is involved in vascular development as well as postnatal angiogenesis. However, the direct action of Shh on cultured endothelial cells has not been clearly shown. To address this issue, we examined the effect of Shh on morphological changes by murine brain capillary endothelial cells (IBE cells) and human umbilical endothelial cells (HUVECs). Shh induced capillary morphogenesis by these cells. The effect was inhibited by cyclopamine or pertussis toxin. Shh-induced capillary morphogenesis was also blocked by LY294002, a phosphoinositide 3-kinase (PI3-kinase) inhibitor. Shh rapidly increased PI3-kinase activity in IBE cells and HUVECs; this activity was inhibited by cyclopamine. Nuclear localization of Gli1 was increased in Shh-treated IBE cells, which was not affected by LY294002. Actinomycin D and cycloheximide inhibited Shh-induced capillary morphogenesis. In IBE cells expressing kinase-inactive c-Fes, Shh failed to stimulate PI3-kinase activity and capillary morphogenesis. Considered collectively, Shh induced capillary morphogenesis of endothelial cells through both rapid activation of c-Fes/PI3-kinase pathways and transcriptionally regulated pathways.

Stromal Cell-derived Factor-1α Induces Tube-like Structure Formation of Endothelial Cells through Phosphoinositide 3-Kinase

Stromal cell-derived factor-1alpha (SDF-1alpha) is a CXC chemokine, which induces tube formation of endothelial cells. Although SDF-1alpha transduces signals via CXC receptor 4 (CXCR4), resulting in activating a panel of downstream signaling molecules, such as phosphoinositide 3-kinase (PI3-kinase), little is known about the SDF-1alpha-mediated signaling pathways leading to tube formation. Here we examined the signal transduction pathway involved in SDF-1alpha-mediated tube formation by primary human umbilical endothelial cells and murine brain capillary endothelial cell line (IBE (immortalized murine brain capillary endothelial) cells). SDF-1alpha stimulated tube formation by IBE cells, which was blocked by LY294002 and pertussis toxin, suggesting that PI3-kinase and G(i) protein were involved in this process. SDF-1 also stimulated tube formation of human umbilical endothelial cells, and the response was LY294002-sensitive. SDF-1alpha activated PI3-kinase in IBE cells. In stable IBE cell lines expressing either the mutant p85 subunit of PI3-kinase (denoted Deltap85-8 cells), which lacks association with the p110 subunit, or kinase-inactive c-Fes (denoted KEFes 5-15 cells), SDF-1alpha failed to activate PI3-kinase and to stimulate tube formation. SDF-1alpha-induced tube formation was inhibited by an antibody against murine vascular endothelial cadherin. The antibody as well as LY294002 attenuated SDF-1alpha-mediated compact cell-cell contact, which proceeded to tube formation. Taken together, SDF-1alpha induces compact cell-cell contact through PI3-kinase, resulting in tube formation of endothelial cells.

Negative regulation of urokinase-type plasminogen activator production through FGF-2-mediated activation of phosphoinositide 3-kinase.

Activation of phosphoinositide 3-kinase (PI3-kinase) is involved in many cellular responses. FGF-2 is one of the potent inducers of urokinase-type plasminogen activator (uPA) production in endothelial cells. However, little is known about the molecular mechanisms underlying FGF-2-mediated uPA production. Here we examined the signal transduction pathways involved in the regulation of uPA production by FGF-2-treatment. FGF-2 potently upregulated uPA production in murine brain capillary endothelial cells (IBE cells), as well as porcine aortic endothelial (PAE) cells and L6 myoblasts ectopically expressing FGFR1. PI3-kinase inhibitors, wortmannin and LY294002, both enhanced FGF-2-dependent uPA production by these cells. Stable expression of activated mutant p110alpha catalytic subunit of PI3-kinase into IBE cells decreased FGF-2-mediated uPA production, suggesting that PI3-kinase exhibited the negative regulatory effect on uPA production. No increase in FGF-2-induced PI3-kinase activity was observed in proteins immunoprecipitated by anti-phosphotyrosine antibody. Although stable expression of deleted mutant p85alpha regulatory subunit, which lacks association with p110 catalytic subunit, in IBE cells showed no dominant negative effect, transient expression of dominant negative Ras inhibited FGF-2-mediated PI3-kinase activation. These results suggest that only activated Ras contributed the FGF-2-mediated PI3-kinase activation. In cells stably expressing mutant p85alpha subunit, FGF-2 efficiently induced uPA production. Taken together, activation of PI3-kinase by FGF-2 is Ras-dependent and results in down-regulation of uPA production.

Transport of Drugs Across the Blood-Brain Barrier by Nanoparticles

Poly(butyl cyanoacrylate) nanoparticles coated with polysorbate 80 (Tween 80) enable the transport of a number of drugs across the blood-brain barrier (BBB) into the brain following intravenous injection. These drugs include the hexapeptide endorphin dalargin, the dipeptide kytorphin, loperamide, tubocurarine, doxorubicin, and the NMDAreceptor antagonists MRZ 2 / 576 and MRZ 2 / 596. After binding to the polysorbate-coated particles, dalargin as well as loperamide exhibited a dose-dependent antinociceptive effect after i.v. injection as determined by the tail-flick as well as by the hot plate test. This effect was accompanied by a Straub reaction and was totally inhibited by pretreatment with naloxone, indicating that it is a central effect and not peripheral analgesia. After brain perfusion of rats with tubocurarine bound to the polysorbate-coated nanoparticles epileptic spikes were observable in the EEC of the rats but not with the controls. Other very interesting results were obtained with the NMDA-receptor antagonists MRZ 2 / 576 and MRZ 2 / 596. The very short anticonvulsive response of MRZ 2 / 576 was increased from below 30 to 300 min, and the transport across the BBB of the non-penetrating MRZ 2 / 596 was enabled after i.v. injection. Intravenous injection of polysorbate 80-coated nanoparticles loaded with doxorubicin (5 mg / kg) achieved very high brain levels of 6 μg / g brain tissue while all the controls, including uncoated nanoparticles and doxorubicin solutions mixed with polysorbate, did not reach the analytical detection limit of 0.1 μg / g. Moreover, experiments with the extremely aggressive glioblastoma 101 / 8 transplanted intracranially showed a long term survival for 6 months of 40 % of the rats after intravenous injection of the polysorbate 80-coated nanoparticle preparation (3 x 1.5 mg / kg). The surviving animals were sacrificed after this time and showed total remission by histological investigation. Untreated controls died within 10 - 20 days, the animals in the six other control groups between 10 - 50 days. The mechanism of the drug transport across the blood-brain barrier with the nanoparticles requires further elucidation. The most likely mechanism at present appears to be endocytotic uptake by the brain capillary endothelial cells followed either by release of the drugs in these cells and diffusion into the brain or by transcytosis. Endocytotic uptake of the polysorbate-coated nanoparticles but not of uncoated particles has been shown with bovine, murine, rat, and human brain capillary endothelial cells. After injection of the nanoparticles, apolipoprotein E (apo E) or apo B adsorption of the particles seems to occur as already shown in vitro, followed by interaction with the LDL receptor and endocytotic uptake. This scenario is rather likely since both apolipoproteins can interact with the LDL-receptor. They may then be taken up like the naturally occurring lipoprotein particles.

Role of c-Fyn in FGF-2-Mediated Tube-like Structure Formation by Murine Brain Capillary Endothelial Cells

Tube formation of endothelial cells is an important step of angiogenesis. However, little is known about the molecular mechanisms underlying growth factor-mediated tube formation by endothelial cells. FGF-2 stimulates tube formation by a murine brain capillary endothelial cell line, IBE cells, when cultured on collagen gels (differentiation-associated culture condition), whereas cells proliferate and migrate without forming tube on fibronectin-coated surface (proliferation/migration-associated condition). To elucidate FGF-2-mediated signal transduction pathways leading to tube formation by endothelial cells, we focused on the contribution of Src family kinases. Src family kinase inhibitor PP2 attenuated FGF-2-induced tube formation. Stable expression of kinase-inactive c-Src in IBE cells demonstrated no dominant negative effect on FGF-2-induced tube formation. In vitro kinase assay revealed that c-Fyn was activated by FGF-2 only in cells cultured on collagen gels. Three independent cell lines, expressing kinase-inactive c-Fyn, all exhibited attenuation of FGF-2-mediated tube formation. However, FGF-2-mediated proliferation or migration was not clearly perturbed in these cells. These results show the first time that c-Fyn plays a pivotal role in tube formation by endothelial cells.