IN VITRO MODEL FOR MODULATING THE BLOOD BRAIN BARRIER AND METHODS

OF SCREENING

BACKGROUND OF THE INVENTION

Field of the Invention

[0001] The present invention relates to the blood brain barrier, more particularly to an in vitro blood brain barrier model .

Description of the Related Art

[0002] Endothelial cells (ECs) of brain capillaries form an active permeability and transport system known as the blood-brain barrier (BBB) , which is instrumental in the control of the brain fluid milieu (Engelhardt, 2003) . Complex tight junctions (TJs) between brain ECs constituted by proteins of the claudin (Cldn) family and occludin (OcIn) , seal off the paracellular pathway for blood-borne substances (Furuse and Tsukita, 2006). Whereas Cldn5 is also found in non-barrier endothelium, Cldn3 is predominantly present in brain ECs, where it plays a specific role in the establishment and maintenance of the BBB TJ morphology (Nitta et al., 2003; Wolburg et al . , 2002).

[0003] Concomitantly with the low paracellular permeability, the BBB ECs express specific transporters for glucose, amino acids and, to prevent damage to the brain, members of the multidrug resistance transporter family (also known as ABC transporters; Engelhardt, 2003). ECs rapidly lose their barrier and selective transport properties under pathological conditions in vivo (ischemia, tumor) and upon cultivation in vitro, indicating that the healthy brain context provides induction and maintenance signals for the BBB. Limited knowledge of the nature of these signals and of the molecular regulation of the BBB hampers the development of BBB model systems and patient therapy (Engelhardt, 2006; Liebner et al . , 2000).

[0004] One of the major pathways regulating brain development is the Wnt/wingless pathway, acting via β-catenin stabilization. This favors translocation of β-catenin to the nucleus, binding to transcription factors of the lymphoid enhancer factor/T-cell factor (Lef/TCF) family and modulating gene transcription (Moon, 2005) . The canonical β-catenin driven Wnt signaling pathway and its many components are described in detail in the Figure 1 and in the text found in Barker and Clevers (2006), which is incorporated herein by reference. Table 1 below provides a listing of Wnt protein ligands and Wnt pathway components and their corresponding GENBANK accession numbers, the sequences of which are incorporated herein by reference.

Table 1 . Components of the Wnt Signaling Pathway

* Multiple GENBANK Accession numbers correspond to alternative transcripts.

[0005] Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.

SUMN[ARY OF THE INVENTION

[0006] The present invention provides an in vitro blood brain barrier (BBB) model in which cultured endothelial cells have their cell junctions tightened by means of activation of their Wnt/β-catenin signaling.

[0007] The present invention also provides in vitro methods for screening drug molecules for the ability to pass through the BBB or for screening compounds to determine whether they are effective in restoring or tightening the endothelial cell junctions of cultured endothelial cells, i.e., to form an in vitro BBB model.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 shows a scheme of MBE cultivation and treatments. WT MBE were not treated with TAT-Cre.

[0009] Figures 2A-2B show that canonical Wnt signaling is active in ECs during brain angiogenesis and becomes progressively down regulated during vessel maturation. (Figs. 2Aa and 2Ab) LacZ whole mount stained E9.5 BAT-gal embryos, sectioned and stained for isolectin B4. Black arrows point to nuclear LacZ reporter gene staining, indicating Wnt pathway activation. (Figs. 2Ba-f) Whole mount hindbrain staining for LacZ (reflection) , and IB4 of BAT-gal embryos (E13.5, E17.5) analyzed by confocal microscopy. LacZ was detected as reflection of the 488nm laser, while isolectin was labeled by Alexa488® secondary antibody. White arrowheads indicate LacZ-positive nuclei (see merged image for localization of LacZ-positive nuclei in IB4-positive vessels) . (Figs. 2Bg-I) Staining of brain cryosections from postnatal BAT- gal pups (Pl, P5) for LacZ (antibody staining) and IB4. White arrowheads indicate LacZ-positive nuclei. Positive nuclei outside the vascular system indicate active Wnt Signaling in the brain parenchyma. (Fig. 2C) Quantification of lacZ positive nuclei per lOOμm vessel length. The number of positive nuclei dropped significantly between E15.5 and E17.5 (5 fields per hind brain, 3 brains, ***P=0.0003) . All error bars represent s.e.m.

[0010] Figures 3A-3D show tamoxifen induced Cre expression in Ctnnbl ^lox/lox /PdgfCreERT2 mice faithfully deleted β-catenin in brain vessels of early postnatal mice. Postnatal (Pl) Ctnnbl ^lox/lox /PdgfCreERT2 mice were treated for 3d with 4OHT, sacrificed (P4), the brain dissected, sectioned and stained for β-catenin in a double staining with isolectin B4. (Figs. 3A and 3C) In Cre- mice no effect on the endothelia expression of β- catenin could be observed. β-Catenin could be detected at the junctions of all brain vessels at P4. (Figs. 3B and 3D) Conditional inactivation of Ctnnbl deleted β-catenin in almost all brain vessels at P4. Bar represents lOOμm and 30μm, respectively.

[0011] Figures 4A-4C show conditional inactivation of β- catenin in ECs of postnatal mice led to down regulation of Cldn3 and Cldn5 and increased permeability in brain capillaries. After treatment for 3d with 4OHT Ctnnbl ^lox/lox /PdgfCreERT2 mice were sacrificed (P4, P7, P14), the brain dissected, sectioned and stained for Cldn3, Cldn5 or Plvap, in a double staining with IB4. (Figs. 4Aa-c) In Cre negative mice no effect on the endothelial expression of Cldn5 could be observed. Cldn5 could be detected in almost all brain vessels at Pl, P7 and P14. Left panels show the merge of IB4 and Cldn5, right panels show claudin-5 alone (Figs. Ad-f) . When Cre was active and endothelial β-catenin was deleted, Cldn5 was down regulated in the majority (<80%) of brain capillaries at P4 and P7. At P14 ~50% of brain capillaries were still largely devoid of Cldn5 staining. Left panels show merge of IB4 and Cldn5, right panels show Cldn5 alone; arrowheads point to Cldn5 negative vessels. (Figs. Ba-b) . At P4 brain micro vessels were only weakly positive for Cldn3. See boxed insert for higher magnification. The staining frequency increased at P7 and at P14 most vessels were positive for Cldn3. (Figs. Bc-d) Deletion of β- catenin in Cre positive mice resulted in a marked down regulation of Cldn3 in brain capillaries at P4, P7 and P14. Left panels show the merge of IB4 and Cldn3, right panels show Cldn3 alone. (Figs. Bc-f) The vascular permeability marker Plvap could not be detected in most brain capillaries at P4 and was completely absent at P7 and P14. Left panels show the merge of IB4 and Plvap, right panels show Plvap alone. (Figs. Bg-h) Upon inactivation of β-catenin in Cre positive pups, a strong up regulation of Plvap in most of the vessels (>80%) at P4 and P7 could be observed. At P14 rougly 50% of the vessels showed an up regulation of Plvap. Left panels show the merge of IB4 and Plvap, right panels show Plvap alone; bar represents lOOμm. (Figs. 4Ca and 4Cd) To test the permeability of β-catenin deficient brain capillaries, Evans blue was injected 18h prior tissue harvesting i.p. in Cre negative and positve pups. In Cre- pups at Pl, P7 and P14 only luminal localization of Evans blue could be observed. In Cre+ pups extensive leakage of the dye was apparent in the brain parenchyma at P4 and around micro vessels at P7, respectively.

[0012] Figures 5Aa-I show conditional β-catenin LOF or GOF in ECs in vivo modulates Cldn3 and Plvap expression and permeability in brain capillaries. Brain sections of 4OHT treated Ctnnbl ^lox/lox /Pdgfb-iCreERT2 mice from P14, stained for Cldn3 or Plvap, together with IB4. (Figs. 5Aa, 5Ag) In Cre- mice Cldn3 was detected in the majority of vessels, Plvap was largely absent from all vessels. (Figs. 5Ad, Aj) β-Catenin LOF resulted in Cldn3 down and Plvap up regulation in ~50% of brain capillaries. Left panels show merge of IB4 and Cldn3 or Plvap, right panels show Cldn3 or Plvap alone. Arrowheads point to Cldn3 negative and Plvap positive vessels, respectively. β-Catenin GOF in ECs by 4OHT treatment of Ctnnbl ^{lox(ex3) /lox(ex3)} /Pdgfb-iCreERT2 mice. Brain vessels of E18.5 embryos and P4 pups were analyzed for Cldn3 or Plvap expression, together with IB4. (Figs. Ab, Ac, Ah, Ai) In Cre- mice Cldn3 could only weakly be detected in a subset of brain vessels at E18.5 and at a slightly higher level at P4, whereas Plvap was strongly expressed at E18.5 and to lower level also at P4. (Figs. Ae, Af, Ak, Al) In Cre+ mice Cldn3 was increased at E18.5 and P4. In contrast Plvap was notably down regulated. Left panels show the merge of IB4 and Cldn3 or Plvap, right panels show Cldn3 or Plvap alone. (Fig. 5B) To functionally test barrier properties in β-catenin LOF mice, we injected Evans blue i.p.lδh prior to tissue harvesting. In Cre+ mice at P14 extravasated Evans blue was apparent around many vessels, but not in controls. (Fig. 5N) Significant Plvap regulation at P7 in LOF

(n=3; **P=0.0001; error bar represent s.e.m.) and GOF (n=3; *P=0.0382; error bar represent s.e.m.) could be confirmed by qRT- PCR on the mRNA level. The slightly higher expression of Cldn3 was statistically not significant (ns) . Control was set as 100%

(dashed line) . Bars represent 50μm, see boxed insert for higher magnification .

[0013] Figures 6A-6F show that conditional inactivation of β- catenin did not notably alter Zonula occludens 1/ZO-l and VE-cad localization in brain vessels of early postnatal mice. Postnatal

(Pl) Ctnnbl ^lox/lox /PdgfCreERT2 mice were treated for 3d with 4OHT, sacrificed (P4), the brain dissected, sectioned and stained for VE-cad in a triple staining with ZO-I and isolectin B4. (Figs. 6A, 6C and 6E) In Cre- mice both VE-cad and ZO-I nicely co- localize to interendothelial junctions in brain microvessels .

(Figs. 6B, 6D and 6F) Conditional inactivation of Ctnnbl had no detectable effect on ZO-I and did not alter VE-cad in the majority of brain microvessels. Occasionally VE-cad was observed in vesicular structures, suggesting increased vesicle based permeability of affected vessels at P4. Bar represents lOOμm and 30μm, respectively.

[0014] Figures 7A-7H show that conditional inactivation of β- catenin led to increased junctional localization of plakoglobin in brain vessels of early postnatal mice. Postnatal (Pl) Ctnnbl ^lox/lox /PdgfCreERT2 mice were treated for 3d with 4OHT, sacrificed (P4), the brain dissected, sectioned and stained for Plako in a double staining isolectin B4. (Figs. 7A and 7B) In Cre- mice Plako was detectable at interendothelial junctions in brain microvessels . (Figs. 7E and 7F) Conditional inactivation of Ctnnbl led to increased staining of Plako in the majority of brain microvessels. (Figs. 7C, 7D, 7G, 7H) Late embryonic (E16.5- E18.5) conditional activation of β-catenin in Ctnnbl ^{lox(ex3)/lox(ex3)} /PdgfCreERT2 mice had no visible effect on Plako localization compared to controls. Arrowheads point to representative vessels. Bar represents 50μm and 20μm, respectively.

[0015] Figures 8A-8C show conditional activation of β-catenin transcriptional activity induced Cldn3 (claudin-3) only in brain vessels of early postnatal mice. To test whether β-catenin dependent signaling is also sufficient to induce endothelial barrier properties in vivo, Ctnnbl ^{lox(ex3) /lox(ex3)} mice (Harada et al., 1999) was analyzed. Postnatal (Pl, P4, PIl) Ctnnbl ^{lox(ex3)/lox(ex3)} / PdgfCreERT2 mice were treated for 3d with 4OHT, sacrificed (P4, P7, P14), the brain dissected, sectioned and stained for Cldn3 or Cldn5 in a double staining with IB4. (Figs. 8Aa-c) In Cre- mice no effect on the endothelial expression of Cldn5 could be observed. Cldn5 could be detected in almost all brain vessels at Pl, P7 and P14 (Figs. 8Ad-f) . Conditional activation of Ctnnbl transcriptional activity did not show any further increase in brain vessels at P4, P7 and P14. Left panels show the merge of IB4 and Cldn5, right panels show Cldn5 alone. Bar represents lOOμm. (Figs. 8Ba and 8Bb) Early embryonic (Ell .5-E13.5) activation of β-catenin in Ctnnbl ^{lox(ex3)/lox(ex3)} /PdgfCreERT2 mice increased Cldn3 staining in brain capillaries. Cre- capillaries were devoid of Cldn3 at E13.5. Left panels show merge of IB4 and Cldn3, right panels show Cldn3. (Figs. 8Ca) Concomitantly, activation of β-catenin led to a noticeable decrease of Plvap. Left panels show the merge of IB4 and Cldn3 or Plvap, right panels show Cldn3 or Plvap alone. Boxed insert shown higher magnification of vessels. Bar represents 50μm and 20μm, respectively.

[0016] Figures 9A-9N show Wnt3a conditioned medium (CM) but not WntδaCM increased Cldn3 expression and TJ formation, but decreased Plvap in MBEs. MBEs from WT mice were treated with control, Wnt3a CM or Wnt5a CM in vitro and processed for immunostaining, freeze fracture EM, qRT-PCR and Western blot. (Figs. 9A, 9E and 91) IF staining for Cldn3 in control CM, Wnt3a CM and Wnt5a CM treated MBEs revealed increased junctional staining in WNT3a CM. Bar represents 20μm. (Figs. 9B, 9F and 9J) IF staining for Cldn5 did not show major differences between untreated or Wnt-treated cells. Bar represents 50μm. (Figs. 6D, 6H and 6L) Freeze fracture EM revealed more frequent and elaborated TJ-strands on the P-face in Wnt3aCM treated MBEs. Arrowheads point to TJ ridges on the P-face. Bar represents 200nm. (Fig. 9M) mRNA levels for Cldnl, Cldn3, Cldn5, Cldnl2, OcIn, Plvap and Axin2 measured by qRT-PCR for control, Wnt3a CM and Wnt5a CM treated MBEs. Cldn3 (n=3; P=O.0319), Plvap (n=3; P=O.0098). Axin2 (n=3, **P=0.005); mRNA level of all other tested genes were not significantly altered. Control was set as 100% (dashed line) . (Fig. 9N) The Western blot analysis confirmed the up regulation of Cldn3 upon Wnt3aCM on the protein level. Cldn5 protein levels were not altered by any CM. α-Tubulin/ Tubala served as loading control. (Figs. 9C, 9G and 9K) Immunostaining for active β-catenin showed nuclear localization, in few control/and the majority of Wnt3a CM treated cells, but not in Wnt5a CM treated cells. Arrowheads point to positive nuclei, bar represents 50μm. (Fig. 9N) Western blot for active and total β-catenin, confirming the immunostaining results. Total β-catenin levels do not significantly differ between treatments. No band could be detected in Wnt5a CM treated cells. All error bars represent s.e.m. [0017] Figures 10A-10H show that adherens junction proteins and peripheral TJ proteins were not altered upon Wnt3aCM treatment. Primary MBEs of passage 1 from WT mice were treated with control CM, Wnt3a CM in vitro and processed for immunostaining of VE-cad, total β-catenin/Ctnnbl, junction plakoglobin/Plako and ZO-I. Immunostaining of these adherens and tight junction markers did not show any noticeable alteration between control and Wnt3aCM treatment. Bars represent 17μm and 50μm, respectively.

[0018] Figures 11A-11L show that β-catenin LOF and GOF in vitro confirms its transcriptional requirement for Cldn3 expression in MBEs. MBEs from Ctnnbl ^lox/lox and from Ctnnbl ^{lox(ex3)/lox(ex3)} mice were treated with TAT-Cre in vitro to achieve β-catenin LOF and GOF, respectively. (Figs. 11A-11C, HF- HH) Ctnnbllox/lox MBEs were treated with or without TAT-Cre and stimulated with control CM or Wnt3a CM. Cldn3 revealed stronger junctional staining in Cre-/Wnt3a CM cells compared to Cre+/control CM. TAT-Cre treatment the abrogated this effect of Wnt3a CM Ctnnbl revealed junctional localization and no difference between Cre-/control CM and Cre-/Wnt3a CM, but complete absence of β-catenin in Cre+/Wnt3a CM, confirming the effective deletion of the gene. (Figs. 11D-11E, 11I-11J) Ctnnbl ^{lox(ex3)/lox(ex3)} MBEs were untreated or treated with TAT-Cre and immunostained for Cldn3 and Ctnnbl. Junctional localization and staining intensity of Cldn3 was markedly increase in Cre+ MBEs. Staining of total Ctnnbl revealed an increase of cytoplasmic and nuclear Ctnnbl in TAT-Cre treated cells, indicating transcriptional activity of Ctnnbl. (Fig. HK) mRNA levels by qRT-PCR for Cldn3 in Cre-/Wnt3aCM treated cells from Ctnnbl ^lox/lox (n=3; **P=0.0048) and in Cre+/controlCM cells from Ctnnbl ^{lox(ex3)/lox(ex3)} (n=3; *P=0.0341) mice. mRNA levels of all other tested genes were not significantly altered. Axin2 induction confirmed canonical Wnt signaling in Cre-/Wnt3aCM treated cells from Ctnnbl ^lox/lox (n=3; **P=0.0006) and in Cre+/controlCM cells from _C tnnbl ^{lox(ex3) /lox(ex3)} (n=3; **P=0.0029) mice. Controls were set as 100% (dashed lines); all error bars represent s.e.m. (Fig. HL) Western blot analysis confirmed the up regulation of Cldn3 by Cre-/Wnt3aCM in Ctnnbl ^lox/lox cells. TAT-Cre abrogated the effect of Wnt3aCM. TAT-Cre treatment in Ctnnbl ^{lox(ex3)/lox(ex3)} cells led to the up regulation of Cldn3; transcription and translation of an N-terminal truncated form of β-catenin is detected by the anti-Ctnnbl antibody and represented by the lower band.

[0019] Figures 12A-12F show that activated Ctnnbl can induce Cldn3 and TJ formation in a non-brain derived endothelioma cell line. An ES cell derived endothelioma cell line (100VE) was infected with a control retroviral vector (pBABEpuro, control) or with the same vector containing an N-terminal truncated form of Ctnnbl (ΔNCtnnbl) and processed for immunostaining, freeze fracture electron microscopy, Western blot or qRT-PCR. (Figs. 12A and 12C) Endothelioma cells expressing ΔNCtnnbl showed increased junctional staining for Cldn3 compared to the control. (Figs. 12B and 12D) Freeze fracture electron microscopy frequently revealed TJs in ΔNCtnnbl endotheliomas, whereas in the control TJs were only rarely found. TJs also appeared more elaborate in ΔNCtnnbl cells, showing rows of particles on the P-face, which were largely missing in the control. (Fig. 12E) mRNA levels for Cldn3, Plvap and Axin2 measured by qRT-PCR for control and ΔNCtnnbl endothelioma cells. In ΔNCtnnbl endothelioma cells Cldn3 (n=3; **P=0.0005) and Axin2 (n=3; **P=0.005) were significantly up regulated, whereas Plvap (n=3; *P=0.043) was significantly down regulated. Control was set as 100% (dashed line) ; all error bars represent s.e.m. (Fig. 12F) Western blot analysis confirmed the up regulation of Cldn3 in ΔNCtnnbl-infected endotheliomas, α- Tubulin/Tubala served as loading control.

[0020] Figures 13A-13B show that Cldn3 up regulation requires transcriptionally active rather than junctional localized β- catenin in MBEs in vitro. (Figs. 13Aa-c, 13Ba) MBEs were either treated with with Wnt3a CM or control CM and infected with dnTCF4 adenovirus or a control vector. Wnt3aCM significantly up regulated Cldn3 (n=3; **P<0.0095) and down regulated Plvap (n=3; *P<0.035) on mRNA level, detected by qRT-PCR. Junctional localization of Cldn3 was increased in IF stainings. Infection with dnTCF4 abrogated the effect of Wnt3aCM on Cldn3 and Plvap

(n=3; **P<0.002) was notably increased. Axin2 (n=3; **P<0.004).

(Figs. 13Ad and 13Bb) To dominantly activate β-catenin signaling, MBEs were infected with LefΔN-βCTA lentivirus or a control vector. LefΔN-βCTA significantly up regulated Cldn3 (n=3; **P<0.002) and down regulated Plvap (n=3; **P<0.008) on the mRNA level. Junctional localization of Cldn3 increased in IF stainings. Axin2 (n=3; **P<0.0001).

[0021] Figure 14 is a graph showing that Wnt3aCM up regulated BBB related transporters in MBEs in vitro. Primary MBEs of passage 1 from Ctnnbl ^lox/lox mice were treated with control CM and Wnt3a CM in vitro, total RNA was harvested and processed for qRT- PCR for multidrug resistance transporters Abcblb (also known a MDRl or p-glycoprotein) , Abcg2 and the glucose transporter Slc2al

(also known as Glut-1) . Abcblb did not show a regulation by Wnt3aCM whereas Abcg2 and Slc2al were considerably up regulated, indicating that besides TJs, canonical Wnt pathway activation led to the expression of barrier-related transport systems.

[0022] Figures 15A-15F show that conditional activation of endothelial β-catenin signaling induces barrier properties in ischemia-induced angiogenic retina vessels in vivo. The barrier forming ability of β-catenin stabilization on the vasculature was analyzed in Ctnnbl ^{lox(ex3) /lox(ex3)} /PdgfCreERT2 mice subjected to oxygen-induced retinopathy (0IR) , as a postnatal model for ischemia induced pathological neo-angiogenesis in the CNS (Smith et al., 1994). In 0IR, high levels of the VEGF-A induces neovascularization associated with increased EC proliferation, migration and hyper-permeability (Campochiaro and Hackett, 2003) . As a consequence, the permeability marker Plvap is strongly up regulated both in pre-existing and newly formed vessels after 3d of normoxia, indicating an increase in vascular permeability (Figs 15A-15C) . Activation of endothelial β-catenin reproducibly diminished Plvap expression even two days after the onset of ischemia driven hyper-permeability (Figs. 15D-15F) . In Figs. 15C and 15D, left panels show the merge of Collagen IV (CoIIV) and Plvap, right panels show Plvap alone. These results indicate that activation of Wnt signaling may reduce the detrimental effects of ischemia induced VEGF-A expression on BBB stability. Bars represent 500μm and 250μm, respectively.

[0023] Figures 16A and 16B show graphs of expression analysis of Fgfbpl (Fig. 16A) and Slc22al4 (Fig. 16B) in microvascular brain endothelial cells (MBEs) expressing CCM2 or not in the presence or absence of Wnt3A CM. To further validate the role of β-catenin signaling in inducing BBB properties, MBE derived from mice where the CCM2 gene contained Lox sequences were tested. These cells have been treated in culture with Tat-Cre to induce recombination and therefore a null mutation of the gene. CCM2 null mutation characterizes an hereditary human pathology where BBB is severely affected. CCM2 flox/flox MBEs were isolated, plated and cultured for 7 days until confluence. After splitting (1:2), they were treated with TAT-Cre to achieve CCM2 KO (described in the Materials and Methods of Example 2) . WT or CCM2 KO MBEs were maintained in culture for an additional 7 days in the presence of control conditioned medium (CM) or Wnt3a CM. Cells were lysed for RNA and Affymetrix gene array analysis. Data show that in absence of CCM2 the effect of β-catenin in induction of BBB markers is lost. This supports the concept that Wnt induction of BBB may be affected in pathological conditions.

[0024] Figures 17A-17F show that Wnt3A induces barrier properties in microvascular brain endothelial cells (MBEs) . Fig. 17A shows the timeline where MBEs were cultured for 7 days in the presence or absence of Wnt3a and then the RNA was extracted (indicated as 7 in Figs. 17D-17F) . Fig. 17B shows the timeline where cells were cultured for 7 days with control medium and after the split, Wnt3a was added (indicated as 7 + 7 in Figs. 17D-17F) . In Fig. 17C, the cells were always cultured (14 days), in the presence or absence of Wnt3a (indicated as 14 in Figs. 17D-17F) . mRNA levels for Cldn3 (Fig. 17D), Axin2 (Fig. 17E) and Plvap were measured by quantitative RT-PCR. Data are mean +/- SD of four replicates of a typical experiment out of 3 performed.

[0025] Figures 18A-18D show that the immortalized embryonic endothelial cell line (H5V) is induced to express BBB properties upon treatment with BIO, a GSK3β inhibitor. Fig. 18A shows a timeline where H5V cells were seeded in 6 wells plate and cultured for 7 days. At different time-points (as indicated by the arrows) , cells were treated with 5μM BIO or vehicle (DMSO) and then RNA extracted. Figs. 18B, 18C and 18D show qRT-PCR for Cldn3, Axin2 and Plvap, respectively. Data are means +/- SD of four replicates from a typical experiment out of three performed.

[0026] Figures 19A-19C show the screening with MBEs of GSK3β inhibitors on Axin2 and Cldn3 expression. MBEs were cultured as described in the Materials and Methods section and Figure 16 and after the split 1:2, treated as shown in Fig. 19A with different GSK3-β inhibitors (BIO, SB216763 and LiCl) compared with Wnt3aCM. BIO, SB216763 and LiCl were used at 5μM, lOμM and 1OmM, respectively. mRNA levels for Axin2 (Fig. 19B) and Cld3 (Fig. 19C) were measured by qRT-PCR. The vehicle is DMSO. Data are mean +/- SD of three experiments performed in triplicates.

[0027] Figures 20A-20C show that Wnt3a CM but not Wnt5a CM induces expression of Cyplbl in microvascular brain endothelial cells (MBEs) . In Fig. 2OA, embryonic endothelioma cell lines wild type (WT) or deficient for β-catenin (β-Cat ^K0 ) were tested for the expression of cytochrome P450 family 1, subfamily b, polypeptide 1 (Cyplbl) by qRT-PCR. Fig. 2OB shows a timeline where MBEs were cultured for 7 days with control CM and after the split, Wnt3a CM or Wnt5a CM, which activate or do not activate β- catenin transcription, respectively, were added. After 14 days, RNA was extracted and Axin2 and Cyplbl expression were evaluated by quantitative RT-PCR (qRT-PCR) . Data are means+/- SD of four experiments .

DETAILED DESCRIPTION OF THE INVENTION

[0028] The loss of BBB selective permeability to blood constituents has major pathological consequences for central nervous system (CNS) diseases and conditions such as stroke and tumor derived/associated brain edema. The present invention is based on the present inventors' discovery that canonical, β- catenin driven, Wnt signaling upregulates the endothelial BBB- specific tight junction protein claudin-3 (Cldn3) and barrier- related transporters, whereas permeability-related genes, such as Plvap, are downregulated. Loss of endothelial β-catenin dependent Wnt-signaling in vitro and in vivo leads to rapid loss of specific tight junction components and loss of BBB function.

[0029] One embodiment of the present invention is a method for tightening vascular endothelium that is similar in properties to the BBB, but is independent of the BBB being compromised. This method tightens vascular endothelium such as is found in the retina to treat or inhibit retinopathy associated with endothelial cell proliferation, migration and hyperpermeability (e.g., ischemia-induced retinopathy). Accordingly, patients suffering from this form of retinopathy can be administered an activator of Wnt/β-catenin signaling to tighten the vascular endothelium in the retina, e.g., to reduce the endothelial hyperpermeability associated with such a disorder or condition.

[0030] Preferably, the activator of β-catenin dependent Wnt signaling is administered transiently in the method for tightening the vascular endothelium. This preferred transient administration is administration over a period lasting in the range of one to fourteen days, more preferably one to seven days, most preferably one to five days.

[0031] The activator of Wnt signaling is a Wnt/β-catenin signal promoting agent that stabilizes β-catenin (i.e., inhibits its degradation) . This Wnt/β-catenin signal promoting agent can be a Wnt protein, or an agonist thereof, that is preferably more specific for Wnt/β-catenin signaling in the brain (or that affects the tight junctions of the BBB) than for Wnt/β-catenin signaling elsewhere in the body. The Wnt protein is preferably Wntl (SEQ ID NO : 1 ) , Wnt3 (SEQ ID NO : 2 ) , Wnt3a (SEQ ID NO:3), and Wnt7a (SEQ ID NO:5), more preferably Wnt3a (SEQ ID NO:3) and Wntl (SEQ ID NO:7), most preferably Wnt3a. Non-limiting examples of Wnt agonists include LRP-related polypeptides and fragments and modified fragments of LRP-related polypeptides (see US20050261189, herein incorporated by reference for such polypeptides and fragments thereof) .

[0032] The Wnt/β-catenin signal promoting agent can also be an inhibitor of glycogen synthase kinase 3β (GSK3β) , an enzyme that is involved in β-catenin inactivation and degradation, or an inhibitor of β-catenin function, including but not limited to, APC, Axin, Chibby, ICAT, Groucho, CtBP and GSK3 binding protein (GBP) . Non-limiting examples of GSK3β inhibitors, include lithium (e.g., LiCl), hymenialdisine, indirubines, pallone, kenpaullone, alsterpallone, azakenpallone, aloisine A, aloisine B, pyrazolopyridine 9, pyrazolapyridine 18, TDZD, GF 109203X, RO 31-8220, etc., are presented in Martinez et al . (2002) and Meijer et al . (2004), both of which are herein incorporated by reference for GSK3β inhibitors. GSK3β inhibitors are certainly well known in the art and are also presented in US Patents 6,057,117 and 6,608,063 and published US applications 2004/0092535 and 20040209878, which are incorporated herein by reference. ATP- competitive, selective GSK-3 inhibitors CHIR-911 and CHIR-837 (also referred to as CT-99021 and CT-98023, respectively) are also known in the art (Ring et al . , 2003; Cline et al . (2002).

[0033] Another embodiment of the present invention provides an in vitro BBB model which comprises cultured endothelial cells, such as brain microvascular endothelial cells (MBEs) , whose cell junctions have been tightened by means of activation of the Wnt/β-catenin signaling in the cultured MBEs, and a method for creating such an in vitro BBB model by growing endothelial cells in culture and causing Wnt/β-catenin signaling to become activated in the cultured endothelial cells. While brain MBEs are known to rapidly lose BBB characteristics in culture, it has been discovered by the present inventors that the cell junctions between cultured MBEs are tightened to form the tight junctions indicative of the BBB by activating Wnt/β-catenin signaling in the cultured MBEs with an activator of Wnt/β-catenin signaling discussed above. This in vitro BBB model satisfies a major need in the art for an in vitro assay using cultured cells to screen for drugs and therapeutic compounds that are able to cross a normal selectively permeable BBB.

[0034] A preferred embodiment of a protocol for isolating and culturing brain microvascular/microvessel endothelial cells is presented hereinbelow in Example 2. One preferred source of endothelial cells is from the mouse brain, but as would be well appreciated by those of skill in the art, this source of MBEs for the in vitro BBB model according to the present invention can be from other commonly used experimental animals from which brain microvascular endothelial cells may be readily obtained. Preferred cultured endothelial cells for use in the in vitro BBB model however also include immortalized endothelial cells, such as prepared by immortalizing endothelial cells (whether obtained from the brain or elsewhere, e.g., heart) with polyoma middle-T antigen as described in Example 2 hereinbelow. An immortalized endothelial cell line has the advantage that large scale screening methods/assays, such as those further embodiments of the present invention, can be standardized (as opposed to using MBEs, which can only be isolated and cultured in small numbers) . Furthermore, the screening data are much more reproducible and consistent over time (i.e., between screening done at different times), whereas with MBEs, one may need to isolate cells from different donors and at different times, which would increase undesirable variability in the data.

[0035] The solid support or surface on which cultured endothelial cells, whether MBEs or immortalized endothelial cells, are grown to confluence to form the in vitro BBB model is a support or surface from which one can determine the presence or absence of tight junctions between endothelial cells, such as by direct or indirect visualization or by determining the permeability of the in vitro BBB to molecules that would normally pass or not pass through the tight junctions of the BBB. Preferred solid supports or surfaces include petri plates, wells of microtiter plates, slides, and filters (e.g., nitrocellulose, nylon membranes, etc, suitable for confluent growth of endothelial cells) . [0036] The induction and maintenance of BBB characteristics in the cultured endothelial cells by an activator of Wnt signaling, i.e., a GSK3β inhibitor, thereby forming the in vitro BBB model, can also be determined by the up-regulation or downregulation of the genes listed in Table 2 hereinbelow, which serve as tools for monitoring the presence of BBB characteristics.

[0037] The in vitro BBB model according to the present invention can be used in a method for screening drug molecules for passage through the BBB when endothelial cells (MBEs or immortalized cells) are cultured on a filter, i.e., a filter membrane (e.g., nitrocellulose filter membrane). Drug molecules to be tested for passage through the BBB are added to the cultured cells after the Wnt/β-catenin signaling is activated in these cultured cells (i.e., by addition of an activator of Wnt/β- catenin signaling) to tighten the cell junctions and thus simulate the BBB. By observing whether or not the tested drug molecule passes through the tight cell junctions of the Wnt/β- catenin signaling activated cells and through the filter on which the cells are cultured, it can be determined if the drug molecule is one which is able to cross the selectively permeable BBB in vivo. This in vitro screening method would be very useful in developing drugs that can cross the BBB and be active in the brain .

[0038] Yet another embodiment of the present invention is a method for screening a compound to determine whether the compound would be effective in restoring or tightening the BBB in patients having a compromised BBB. This method involves culturing endothelial cells (e.g., MBEs, immortalized endothelial cells, etc.) in vitro and screening a test compound to determine if the endothelial cell junctions are tight in the in vitro cell culture of endothelial cells. Determination of tight endothelial cell junctions can be made by any number of methods, including passage/non-passage of a molecule that is known to be incapable of passage across a normal selectively permeable BBB, and stronger junctional staining of Cldn3 relative to a negative control (e.g., absence of test compound) and/or to a positive control (e.g., Wnt3a) .

[0039] While it is possible to administer an activator or inhibitor of Wnt/β-catenin signaling alone, it is preferable to administer it as part of a pharmaceutical preparation/ formulation/composition, where it is mixed with suitable carriers or excipients.

[0040] As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0041] Herein the term "active ingredient" refers to the activator of Wnt/β-catenin signaling accountable for the biological effect sought.

[0042] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to a patient and does not abrogate the biological activity and properties of the administered compound.

[0043] Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. [0044] Techniques for formulation and administration of drugs as active ingredients may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0045] Suitable routes of administration may, for example, include oral, rectal, transmucosal, transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

[0046] Alternatively, one may administer a preparation in a local rather than systemic manner, for example, via injection of the preparation near the BBB of a patient.

[0047] Pharmaceutical compositions for use in the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes and thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0048] For injection, the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0049] For oral administration, the composition can be formulated readily by combining the active ingredient with pharmaceutically acceptable carriers well known in the art. Such carriers enable the therapeutic agent to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP) . If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0050] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active ingredient doses.

[0051] Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

[0052] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner .

[0053] For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0054] The preparations may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0055] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate in oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0056] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

[0057] Pharmaceutical compositions suitable for use in the context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to tighten the vascular endothelium in the patient being treated.

[0058] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0059] For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired effect. Such information can be used to more accurately determine useful doses in humans.

[0060] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or in experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l (1975)) .

[0061] Dosage intervals can also be determined using the MEC value. Preparations should be administered using a regimen, which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably 50-90%.

[0062] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several hours to several days to several weeks or until cure is effected or diminution of the disease state is achieved.

[0063] The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0064] Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration and are not intended to be limiting of the present invention.

EXAMPLE 1

[0065] The blood-brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for brain fluid homeostasis and neuronal function, but little is known about the molecular basis of its development and maintenance.

[0066] Here, the present inventors report that Wnt signaling in brain ECs is active during brain angiogenesis and postnatal vascular maturation. Activation of β-catenin signaling by Wnt3a or by dominant active forms of β-catenin in cultured mouse brain ECs (MBEs) led to a significant increase of Cldn3, to a more in vivo-li ^~ ke appearance of TJ morphology and to a significant decrease in Plvap (also known as meca-32), a marker of leaky vessels (Hallman et al . , 1995). Endothelial specific stabilization of β-catenin in vivo enhanced barrier maturation, while endothelial inactivation of β-catenin caused significant down regulation of Cldn3 and Cldn5 and BBB breakdown. Conditional deletion of β-catenin in ECs in neonatal mice led to a reduction in Cldn3 and Cldn5 expression and vascular leakage. Conversely, during embryonic and early postnatal stages when the BBB is still immature, dominant activation of β-catenin signaling in vivo up-regulated Cldn3 and down-regulated Plvap. Hence, canonical Wnt signaling is necessary to induce barrier properties in brain ECs (BBB characteristics) . In addition, in ischemia- induced retinopathy, β-catenin stabilization prevented vascular hyperpermeability .

[0067] Nonstandard abbreviations used in this Example and throughout the specification are as follows: BBB, blood-brain barrier; TJs, tight junctions; AJs, adherens junctions; E-face, exo-cytoplasmic fracture face; P-face, protoplasmic fracture face; Cldn, claudin; OcIn, occludin; Plvap, plamalemma vesicle associated protein, Lef/TCF, lymphoid enhancer factor/T-cell factor; VE-cad, VE-cadherin; ZO-I, zonula occludens 1; Plako plakoglobin; Ctnnbl, β-catenin; Tubala, tubulin alpha IA; 4OHT, 4-hydroxy-tamoxifen; MBE, mouse brain microvascular EC; CM, conditioned medium; ECGS, EC growth supplement; Gain-of-Function, GOF; Loss-of-Function, LOF.

MATERIALS AND METHODS

[0068] Animals. The following transgenic mouse strains were use: Ctnnbl ^{lox(ex3)/lox(ex3)} (Harada et al . , 1999); Ctnnbl ^lox/lox (Brault et al., 2001); Pdgfb-iCreER (Claxton et al . , 2008); BAT- gal/C57B16 (Maretto et al . , 2003). For MBE preparation, C57B16 wild type (WT) mice were used (Charles River Laboratories; Sulzfeld, Germany) .

[0069] Preparation of primary mouse brain ECs. Mouse brain microvascular fragments were processed as described previously

(Calabria et al . , 2006; Liebner et al . , 2000). Capillary fragments were seeded in culture medium (DMEM, 20% FCS, lOOμg/ml heparin, 5μg/ml ECGS (homemade from calf brain), 100units/L penicillin/streptomycin, 2mM Glutamin, Ix MEM, ImM Na-Pyruvate) at a density of 1.5xlO ⁴ cells/ml on collagen I coated wells. ECs were selected for puromycin (4μg/ml ) resistance for 2 days, grown to confluency and passaged 1:2 and grown for additional 6d in the presence of control, Wnt3a or Wnt5a conditioned medium

(Fig. 1) . Cells were fixed with methanol for immunofluorescent staining or lysed for RNA and protein, respectively.

[0070] Conditional deletion/activation of β-catenin in vivo and in vitro. ECs were treated with 50μg/ml TAT-Cre for 60 min in DMEM without serum, followed by lOOμM chloroquine for 30min

(Wadia et al . , 2004). The cells were washed with DMEM and cultured with complete medium with or without WntCM (Fig. 1) . [0071] For β-catenin deletion in vivo the Pdgfb-iCreERT2

(Claxton et al . , 2008) line was crossed to mice harboring floxed alleles of β-catenin (Ctnnbl ^lox/lox ) (Brault et al . , 2001) in order to obtain homozygous floxed Ctnnbl ^lox/lox pups of which 50% also carried one Pdgfb-iCreERT2 allele. 4 hydroxy Tamoxifen (4OHT)

(No. H7904, Sigma-Aldrich, St. Louis, MO) was dissolved in absolute ethanol at 20mg/ml, diluted to 4mg/ml in sterile peanut oil and injected i.p. at 20μl/gram into pups 3 days before tissues harvesting. When harvesting embryonic tissues at E13.5, the mothers were injected with lmg of 4OHT at Ell.5 and E12.5. [0072] For β-catenin transcriptional activation (gain of function; GOF) in vivo, Pdgfb-iCreERT2 mice were crossed to Ctnnbl ^{lox(ex3)/lox(ex3)} mice. The Pdgfb-iCreERT2 transgene includes an internal ribosomal entry site (IRES) driving expression of farnesylated EGFP, thus allowing detection of transgenic pdgfb promoter activity and Cre expression. Crosses with the ROSA26R::YFP Cre-reporter mouse line (Srinivas et al . , 2001) were used to optimize 4OHT administration and to assess Cre- recombination frequency (not shown) .

[0073] Cell culture and viral infection of endothelial cells. Culture conditions of wild type endothelioma cell lines was described previously (Balconi et al . , 2000; Liebner et al . , 2004) . Briefly, cells were routinely cultured in MCDB131 with 15% FCS (Hyclone) , endothelial cell growth supplement (5μg/ml; homemade from calf brain), and heparin (100 μg/ml; Sigma-Aldrich) maintenance medium on gelatin-coated tissue culture vessels. For the expression of dominant active β-catenin, cells were infected with murine N-terminal truncated β-catenin (from C. Brancolini, University of Udine, Udine Italy) in pINCO-GFP retroviral vector (ΔNβcat) , or with pINCO-GFP retroviral vector alone, as a control (GFPvc) .

[0074] Lentiviral preparations. The lentiviral construct LEFΔN-βCTA (from K. Vleminckx, VIB - University of Gent, Gent, Belgium) lacking the armadillo repeats with constitutive Tcf-β- catenin transcriptional activity has been used (Vleminckx et al . , 1999) . The day after splitting the cells, they were infected with lenti-GFP or lenti-LEFΔN-βCTA and then after one weeks cells were fixed or extracted. Lentiviral and packaging plasmids were donated by L. Naldini (HSR-TIGET, San Raffaele Telethon Institute for Gene Therapy, Milan, Italy) . Lentiviral vectors were produced as described in Dull et al . (1998) . Two consecutive cycles of infection (5 h and overnight) were performed with a multiplicity of infection of 300 in 1 ml of complete culture medium.

[0075] The adenoviral construct dnTCF4 has been provided by S.J. George (Bristol Heart Institute, Bristol, UK) (Quasnichka et al . , 2006) . The day after splitting one set of cells was treated with Wnt3aCM and when they reached confluency they were infected with Ad-GFP or Ad-dnTcf4. 72h after the infection cells were fixed or extracted for qRT-PCR.

[0076] Evan's Blue permeability. A 1% sterile solution of Evan's Blue (Sigma) in PBSa was injected i.p. 18-24hours before the animals were harvested. To remove the Evan's Blue from the vasculature, the mice were perfused with PBSa while under terminal anaesthesia. Frozen sections of brain (12μm) and skin (15μm) were dried onto + slides for approx lOmin at 37 ⁰ C. They were then fixed in +4 ⁰ C acetone for 2min, air dried, dipped in xylene and mounted in DPX (Hamer et al . , 2002) .

[0077] Antibodies. Mouse mAbs were as follows: anti-α-tubulin and anti-claudin-5 (Invitrogen; invitrogen.com), anti-β-catenin (BD Transduction; bdbiosciences.com), anti-active-β-catenin (8E7) (Upstate; millipore.com); Rat anti-PECAM/CD31 clone MEC13.3 mAb has been described previously (Vecchi et al . , 1994). Rat anti-VE- cadherin clone BV13 mAb has been described previously (Lampugnani et al . , 2002). Rat monoclonal anti meca-32/Plvap mAb was kindly provided by Dr. Rupert Hallmann (Hallmann et al . , 1995) .

[0078] Polyclonal Abs were as follows: goat anti-β- galactosidase (Abeam; abcam.com); rabbit anti-claudin-3, rabbit anti-ZO-1 (Invitrogen); Rabbit anti-collagen IV (Abd Serotec; ab- direct.com). Biotinylated isolectin B4, (Sigma). ALEXAFLUOR™ (AF) direct conjugated isolectin AF 568 and AF 488 (Invitrogen).

[0079] Secondary Abs were as follows: Appropriated Abs and streptavidin conjugates ALEXAFLUOR™ 350, 488, 555, 568 and 633 were from Invitrogen (the anti-mouse Abs were highly cross- absorbed against non specific mouse proteins) . For nuclear stain on brain sections Hoechst 333258 was diluted to lug/ml in PBT and incubated at RT for 5 minutes. Alternatively TOTO-3 (1:1,000; Invitrogen) was used.

[0080] Immunohistochemistry. β-Galactosidase staining of BAT- GaI embryos was carried out as previously described (Liebner et al . , 2004). LacZ was either detected as reflection of the 488nm laser or by antibody staining. Eyes were fixed in PFA for 5 minutes on ice and retinas were subsequently dissected in cold PBSa. Retinas were then fixed in +4 ⁰ C methanol and stored at - 2O ⁰ C. Before staining, the retinas were placed in PBSa to re- hydrate. Retinas were incubated in blocking buffer (1% BSA + 0.5% Triton IOOX in PBSa) for one hour. Hindbrains were dissected out from PFA fixed embryos and were washed in PBS and subjected to isolectin and immunofluorescent staining with anti collagen IV, claudin-5, Meca-32/Plvap, VE-cad, ZO-I and β-catenin was carried out as previously described (Gerhardt et al . , 2003). Appropriate ALEXAFLUOR™ conjugated secondary antibodies were used for detection .

[0081] Flat mounted hindbrains and retinas were analyzed by fluorescence microscopy using a Nikon 8Oi microscope equipped with a digital camera (Nikon DS-5Mc) . Measurements were carried out by using Nikon NIS Elemnts 3.0 software. Alternatively, images were captured by confocal laser scanning microscopy using a Nikon Clsi or a Leica LCS NT. Images were processed using ImageJ and Adobe Photoshop CS ^® .

[0082] RNA preparation and quantitative qRT-PCR. Total RNA from cultured cells was prepared with Qiagen RNeasy Mini Kit (Qiagen; qiagen.com) according to manufacturers protocol. RNA was reverse-transcribed with the Transcriptor First Strand cDNA Sythesis Kit (Roche; roche-applied-science.com) using lμg of total RNA per 20μl reaction. For real-time PCR reactions ABSOLUTE™ qRT-PCR SYBR ^® Green Fluorescein Mix (Thermo; thermo.com) was used as described in the protocol on a DNA Engine Opticon 2 (BioRad; bio-rad.com).

[0083] Primers used in quantitative qRT-PCR. The following primers were used for identification of Cldnl (sense 5'GAT GTG GAT GGC TGT CAT TGG 3' (SEQ ID NO : 7 ) , anti-sense 5' ACA CCT CCC AGA AGG CAG AGG 3' (SEQ ID NO : 8 ) ) , Cldn3 (sense 5' CGT ACA AGA CGA GAC GGC CAA G 3' (SEQ ID NO: 9), anti-sense 5' CAC GTA CAA CCC AGC TCC CAT C 3' (SEQ ID NO:10)), Cldn5 (sense 5' ATG TCG TGC GTG GTG CAG AGT 3' (SEQ ID NO: 11), anti-sense 5' GCG CCG GTC AAG GTA ACA AAG 3' (SEQ ID N0:12)), Cldnl2 (sense 5' CAG ACA GGC TGC TTG GAG AAA C 3' (SEQ ID N0:13), anti-sense 5' AGG CAA TAC CAC ACA GGA AGG A 3' (SEQ ID N0:14)), OcIn (sense 5' GTG AAT GGC AAG CGA TCA TAC C 3' (SEQ ID NO: 15), anti-sense 5' TGC CTG AAG TCA TCC ACA CTC A 3' (SEQ ID N0:16)), Plvap (sense 5' GAC TAC GCG ACG TGA GAT GGA 3' (SEQ ID NO: 17), anti-sense 5' AGG ATG ATA GCG GCG ATG AAG 3' (SEQ ID N0:18)), and Axin2 (5' GCC GAC CTC AAG TGC AAA CTC -3' (SEQ ID NO: 19), anti-sense 5' GGC TGG TGC AAA GAC ATA GCC 3' (SEQ ID NO:20)) and as reference for the house keeping genes RNA polymerase II (sense 5' ATG AGC TGG AAC GGG AAT TTG A 3' (SEQ ID N0:21), anti-sense 5' ACC ACT TTG ATG GGA TGC AGG T 3' (SEQ ID NO:22)) and G6PDH (sense 5' GGA CGA CAT CCG AAA GCA GAG T 3' (SEQ ID NO:23), anti-sense 5' GAA TAG ACG GTT GGC CTG CAT C 3' (SEQ ID NO: 24) .

[0084] The following qRT-PCR conditions were applied: 15min at 95°C; 40 cycles of 15sec at 94°C, 30sec at 60 ⁰ C, and 30sec at 72 ⁰ C. To confirm that the expected product was generated and to distinguish specific from nonspecific products and primer-dimers, a melting curve analysis was performed after amplification by a gradual increase in temperature from 50 ⁰ C to 95°C at a rate of 0.1°C/s. The appearance of a single, narrow peak indicated a specific amplification product, which was additionally checked for its correct size by agarose gel electrophoresis. For intron- less genes, -RT controls were performed to exclude the possibility of genomic background.

[0085] OJi?. Oxygen induced retinopathy was induced in Ctnnbl ^{lox(ex3)/lox(ex3)} x Pdgfb-iCreERT2 mice essentially as described previously (Smith et al . , 1994). However, the 75% Oxygen treatment was performed for only 3 days, between P7 and PlO, after which animals were returned to room air for additional 5 days until tissue harvesting at P15. This modification results in similar pathology and hyperpermeability as after 5 days of 75% oxygen, but increased lethality of the lactating females can be avoided. 4OHT was administered on day 12, i.e. 2 days after return to room air to allow hyperpermeability and neovascularization to develop before stabilization of β-catenin. Oxygen enriched environment was maintained in purpose designed variable oxygen level isolator system (Quantum Air Technology, Rossendale, Lancashire, UK) . Oxygen levels were controlled by ProOx 110 oxygen regulator (Biospherix, Redfield, NY, USA) .

[0086] Western blotting. Western blot analysis was carried out according to standard protocols. Briefly, confluent cells were washed with ice cold PBS and scraped in lysis buffer (5OmM Tris and 15OmM NaCl, pH7.4, containing 1% Triton X-IOO, 1% Nonidet P- 40, 0.5% sodium-deoxycholate, 0.1% sodium-dodecyl-sulfate, ImM phenylmethylsulfonyl fluoride, 15μg/ml leupeptin, 71μg/ml phenanthrolyne, and 20U/ml aprotine (Sigma) . The insoluble material was removed by centrifugation at 12,000rpm for 10 min. Alternatively, cultured cells were lysated by boiling in a modified Laemli sample buffer (2% SDS, 20% glycerol, and 125mM Tris-HCl, pH 6.8). The protein content was measured according to the BCA method (Pierce; piercenet.com). Total cell lysates were separated by SDS-PAGE under reducing conditions, transfered to a nitrocellulose membrane and analyzed in immunoblot with specific antibodies .

[0087] Freeze-fracturing. Freeze-fracturing on glutaraldehyde fixed ECs was carried out as previously described (Liebner et al . , 2000). Shock-frozen cells were transferred to a Balzers freeze-fracturing device BAF 400D (Bal-Tec; bal-tec.li), fractured at about 10 ^~6 mbar and -150 ⁰ C, and shadowed with platinum/carbon (2.5nm, 45°) and carbon (25nm, 90°). The replicas were cleaned in 13% sodium hypochlorite, washed several times in distilled water, mounted on Pioloform-coated copper grids and observed in a Zeiss EMlO electron microscope (Zeiss; smt.zeiss.com). Negatives were digitized and images were arranged in Adobe Photoshop CS ^® for Macintosh.

[0088] Statistical analysis. Results are presented as mean ± s.e.m. Two tailed Student's t-test was used to analyze the difference between two groups. Values were regarded significant at P<0.05.

RESULTS AND DISCUSSION

Active endothelial β-catenin signaling correlates with CNS vascularization, BBB development and maturation.

[0089] In BAT-gal reporter mice for Wnt/β-catenin signaling, strong LacZ-reporter activity was observed in ECs starting at E9.5, when the perineural vascular plexus is fully developed, up to E15.5 (Figs. 2A, 2Ba-c) . Throughout brain angiogenesis, numerous ECs in the immature network were positive for reporter gene expression (Figs. 2Ba-c) . From E15.5 to E17.5 the number of LacZ positive nuclei decreased significantly (Figs. 2Bd-f; quantification in Fig. 2C) to a level which was maintained during early postnatal states in vessels in the cortex (Figs. 2Bg-I; quantification in Fig. 2C). Adult brain vessels rarely displayed LacZ positive nuclei, suggesting that Wnt signaling activity is required for BBB maturation but is low when the BBB is fully mature (data not shown; quantification in Fig. 2C).

Conditional activation or inactivation of endothelial β-catenin in vivo modulates Cldn3 and Plvap expression and BBB permeability .

[0090] In order to understand the role of β-catenin in BBB maturation and maintenance, β-catenin was selectively deleted in ECs (Loss-of-Function, LOF) at three time points during the first two weeks of postnatal development, corresponding to stages of progressive BBB maturation and to the phase of postnatal Wnt signaling reporter activity (Kniesel et al . , 1996). For this purpose, βcat ^lox/lox mice were crossed with the tamoxifen-inducible Pdgfb-iCreERT2 mice (Claxton et al . , 2008; Brault, 2001). Cre expression was monitored by EGFP expression (the Pdgfb-iCreERT2 allele includes an IRES-EGFP sequence) and recombination was tracked by cross-breeding with the ROSA26R::YFP Cre-reporter mouse line (not shown) (Srinivas et al . , 2001) . After induction of Cre-mediated recombination by 4-hydroxy-tamoxifen (4OHT) in βcat ^lox/lox /Pdgfb-iCreERT2 mice, immunolobeling for β-catenin demonstrated complete absence of the protein in nearly all vessels at P4 (Figs. 3A-3D) and P7, and -50% at P14 (not shown). In Cre brains, Cldn3 was expressed at low levels in brain vessels at P4 and P7 (Fig. 4) and consistently expressed in all brain vessels at P14 (Fig. 5Aa). In Cre-positive littermates, Cldn3 was strongly reduced in most brain vessels at P4 and P7, and in approximately half of the vessels at P14 (Fig. 5Ad). Conversely, Pvlap was poorly expressed in all brain vessels of control animals (Fig. 5Ag and Fig. 4) was strongly upregulated when β- catenin was inactivated (Figs. Aj and 4). To functionally test barrier properties, Evans blue was injected i.p. 18h prior to tissue harvesting. At P14, extravasated Evans blue was apparent around many vessels following endothelial β-catenin inactivation, but not in control samples (Fig. 5B) . An effect which was even more pronounced at earlier (P4, P7) postnatal stages (Figs. 40- 4R) . Cldn5 was consistently expressed in Cre-, but was markedly reduced or absent from most Cre+ vessels at P4 and 7, and strongly reduced in -50% of the vessels at P14, suggesting that loss of endothelial β-catenin affected the molecular composition of TJs (Figs. 4Aa-c, 4Ad-f) . VE-cadherin (VE-cad) and ZO-I were unchanged and localized to all inter-endothelial junctions (Fig. 6), while plakoglobin (Plako) was slightly increased at adherens junctions (AJs) of Cre+ samples, confirming previous data showing that plakoglobin can substitute for the loss of β-catenin at AJs in ECs (Cattelino et al . , 2003). (Fig. 7).

[0091] To selectively activate β-catenin transcriptional activity in ECs (Gain-of-Function, GOF) we crossed Ctnnbl ^{lox(ex3) /wt} mice with Pdgfb-iCreERT2 mice (Claxton et al . , 2008; Harada et al., 1999). At E18.5 and at P4, Cre- mice showed only faint Cldn3 staining in brain vessels, whereas Plvap was strongly expressed, confirming the immaturity of the BBB at late embryonic and early postnatal stages (Figs. 5Ab-c, 5C, 5Ah-i) . Cre-mediated β- catenin activation, increased Cldn3 junctional staining at E18.5 and at P4 (Figs. Ae-f) and decreased Plvap (Figs. Ak-I). qRT-PCR confirmed significant down regulation of Plvap mRNA in Cre+ samples (Fig. 5C) . Also Cldn3 mRNA level appeared to be increased although statistical analysis failed to show significant regulation. The low abundance of this gene in total brain RNA samples is a likely cause for the observed variability and isolation of ECs prior to RNA extraction may be required to achieve adequate sensitivity in vivo. Nevertheless, even at earlier embryonic stages (E13.5), activation of β-catenin transcription resulted in detectably stronger junctional Cldn3 staining and reduction of Plvap in brain vessels (Fig. 8) . Cldn5 expression and localization was not changed between Cre+ and control vessels (Fig. 8).

[0092] Together these results illustrate that endothelial β- catenin is required for BBB maturation and function in vivo and that transcriptional activation of β-catenin accelerates BBB maturation .

Activation of endothelial β-catenin signaling induces barrier characteristics in vitro

[0093] The present inventors asked whether activation of β- catenin/Wnt signaling could reconstitute BBB characteristics in cultured mouse brain microvascular ECs (MBEs) . Since it is essentially impossible to culture MBEs from embryos or pups, an in vitro assay was developed using MBEs from young adult mice, which are known to rapidly lose BBB characteristics in culture

(Liebner et al . , 2000). With these cells, the effect of β- catenin signaling on BBB reconstitution to mimic the in vivo conditions described above was studied. Passaged MBEs were cultured for 6d in either control or conditioned medium (CM) of L-cells, producing Wnt3a, which acts through β-catenin (Willert et al . , 2003), or Wnt5a, which does not stabilize β-catenin in ECs (Chen et al . 2003) (Fig. 1).

[0094] Treatment of MBEs with Wnt3a CM resulted in markedly stronger junctional staining of Cldn3 compared to control CM

(Figs. 9A and 9E). The localization and staining intensity of Cldn5, ZO-I, Plako and VE-cad was unaffected (Figs. 6B, 6F and Fig. 10) . Freeze fracture analysis revealed more TJ strands in Wnt3aCM treated cells, regularly showing areas of high P-face association of TJ particles, recombing TJs of BBB ECs in vivo.

(Figs. 9D and 10H) . As a control, MBEs were activated with Wnt5a CM, which closely correspond to control conditions concerning Cldn3, Cldn5 and active β-catenin staining, as well as TJ- particle distribution on the P-face (Fig. 9I-9L) . qRT-PCR and Western blot confirmed the up regulation of Cldn3 in Wnt3a CM, but not in Wnt5A CM stimulated cells on the mRNA and protein level, respectively (Figs. 9M and 9N). As expected, nuclear immunolabeling and Western blot of activated β-catenin and induction of the canonical Wnt target gene Axin2 (Figs. 9C, 9G, 9K, 9M and 9N) was strongest in Wnt3aCM treated cells, confirming β-catenin stabilization by Wnt3aCM. None of these parameters were induced by WntδaCM.

[0095] Other junctional proteins including Cldn5, VE-cad, Ctnnbl, Plako, ZO-I, OcIn, Cldnl and Cldnl2 were not modified in distribution and expression (Figs. 9 and 10).

[0096] To directly validate the role of β-catenin in Cldn expression, β-catenin were genetically deleted or stabilzed in ECs in vitro by harvesting brain ECs from Ctnnbl ^{1 lox} and Ctnnbl ^{lox(ex3)/lox(ex3)} mice, respectively (Brault et al . , 2001; Harada et al . , 1999). Cells were treated with cell permeable TAT-Cre to achieve loxP site mediated recombination, thus generating β-catenin LOF and GOF in vitro (Wadia et al . , 2004).

[0097] Ctnnbl ^lox/lox cells grew and behaved as wild type cells, showing Cldn3 up regulation upon Wnt3aCM treatment (Figs. HA, HB, HK and HL) , an effect abrogated by the loss of β-catenin (Figs. HC, HH, HK and HL).

[0098] In contrast, β-catenin stabilization led to an increase in Cldn3 junctional staining, mRNA and protein levels (Figs. HD, HE, HI, HJ, HK and HL) . A double band for β-catenin in the Cre+/Ctnnbl ^Δex3/Δex3 cell extract, demonstrates the effective, although not complete recombination of exon3 by Cre recombinase. Thus, the effects of β-catenin stabilization are comparable to those of stimulating the cells with Wnt3aCM .

[0099] When a non-brain-derived endothelioma cell line was infected (Lampugnani et al . , 2002), exhibiting low barrier properties, with N-terminal truncated β-catenin, a significative increase in Cldn3 and barrier properties (freeze fracture analysis and decrease in Pvlap) was observed, suggesting that elevated β-catenin transcriptional activity may induce BBB properties also in non-brain derived ECs. (Fig. 12).

Cldn3 up-regulation requires transcriptionally active rather than junctional localized β-catenin in MBEs in vitro

[00100] To better discriminate the effects of junctional versus transcriptional active β-catenin on Cldn3 regulation, MBEs were infected with an adenoviral vector containing a dominant negative form of TCF4 (dnTCF4) (Wuasnichka et al . , 2006), which inhibits signaling but does not affect the junctional pool of β-catenin. Alternatively, MBEs were infected with a lentiviral vector containing LefΔN-βCTA, which dominantly induces β-catenin signaling without binding to VE-cad (Vleminckx et al . , 1999). Adenoviral infection with dnTCF4 completely abrogated the Wnt3a mediated up regulation of Cldn3 and Axin2 in primary MBEs, whereas Plvap was significantly up regulated (Figs. 13Aa-c and 13B) .

[00101] Lentiviral infection of the dominant active LefΔN-βCTA construct had principally the same, but notably stronger, effect than Wnt3aCM, namely Cldn3 up and Plvap down regulation (Figs. 13Ad and 13Bb) . Together these results show that the transcriptional activity of β-catenin is necessary and sufficient to up regulate Cldn3 and down regulated Plvap, leading to increased barrier properties of brain ECs.

[00102] Cldn-3 is an important downstream effector of β-catenin signaling while Cldn5, although affected in vivo by the loss of β-catenin, appears to be ubiquitous and more stably expressed.

[00103] Although newborn Cldn-5 null mice die immediately after birth for defective BBB (Nitta et al . , 2003), data reported here and in previous work (Wolburg et al . , 2002) strongly suggest that Cldn-3 is a major determinant of BBB, cooperating with Cldn5 in maintaining low permeability. The precise mechanism through which β-catenin regulates the expression of Cldn3 is yet to be defined, but the promoter contains consensus binding domains for the Tcf/LEF-β catenin transcriptional complex (own unpublished work) . Recently, it was demonstrated in the laboratories of the present inventors that β-catenin can stabilize FOXO-I binding to the Cldn5 promoter, contributing to its repression (Taddei et al . , 2008). In the experimental results in this example, β-catenin in vivo was found to be required to maintain high levels of Cldn5 expression in brain microvasculature . Although the exact mechanism of this regulation in brain microvasculature is not known, it is likely that the transcriptional role of β-catenin varies depending on interacting transcription factors expressed at specialized region of the vascular tree. Other BBB-related claudins, such as Cldnl and Cldnl2 were not significantly modified by β-catenin signaling.

[00104] In this example, the present inventors mostly focus on the role of canonical Wnt signaling on vascular junction organization and permeability control however, other barrier- associated may also be controlled by Wnt/β-catenin . Recently, the transporters p-glycoprotein (p-gp) and MDR-I were shown to be up regulated by specific GSK3β inhibitors in rat brain ECs (Lim et al . , 2008). Preliminary results from the laboratory of the present invetnors did not confirm a regulation of p-gp and MDR-I, but indicate that Abcg2 and the glucose transporter Slc2al were notably up regulated in MBEs by Wnt3aCM (Fig. 14) Although further studies need to elucidate the entire set of BBB-related genes directly or indirectly regulated by Wnt/β-catenin, these findings fit well with the concomitant down regulation of Plvap by active β-catenin in vitro and in vivo. Plvap is associated to vessels with high vesicular transport, and negatively correlates with the development of the BBB (Hallmann et al . , 1995). Conversely, BBB microvessels are known to up regulate Plvap under pathological conditions such as Alzheimers disease, brain tumors and stroke (Carson-Walter et al . , 2005; Sparks et al . , 2000).

[00105] To test the relevance of active β-catenin signaling under pathological conditions, the oxygen induced retinopathy (OIR) model (Smith et al . , 1994) was applied to Ctnnbl ^{lox(ex3)/lox(ex3)} /Pdgfb-iCreERT2 mice. Ischemia induced pathological hyperpermeability in the OIR model, indicated by high Plvap expression in retinal vessel, was notably down regulated in Cre+ mice, suggesting that activation of β-catenin signaling represents a novel therapeutic opportunity to control vascular hyperpermeability in CNS ischemia (Fig. 15).

[00106] In conclusion, this is the first and direct evidence for the role of the canonical Wnt pathway as a key regulator of the BBB phenotype in ECs. The BBB is indispensable for brain function and understanding of the molecular mechanisms which determine barrier properties in ECs.

EXAMPLE 2

In vitro blood brain barrier model MATERIALS AND METHODS

[00107] Cell isolation and culture. Primary Mouse Brain Endothelial cells (MBEs) were isolated as previously described (Calabria et al . , 2006; Liebner et al . , 2008). Capillary fragments isolated from mouse brain, were seeded in culture medium (DMEM, 20%FCS, 100 μg/ml heparin, 5 μg/ml endothelial cells growth supplement (ECGS-see Example I)) at a density of 1.5 x 10 ⁴ cells/ml on a collagen I-coated wells. Endothelial cells were selected for puromycin (4 μg/ml) resistance for 2 days, grown to confluency and passaged 1:2, and grown for an additional 6-7 days. Cells were then lysed for RNA preparation.

[00108] Conditional deletion of CCM2 gene in vitro. Primary Mouse Brain Endothelial cells were isolated as described above from CCM2 flox/flox mice obtained from E. Tournier- Lasserve (INSERM, Paris, France; Boulday et al . , 2009) and then treated as described for Ctnnbl ^lox/lox or Ctnnbl ^lox < ^ex3 > ^Aoχ < ^eχ3) _

[00109] Immortalized line of endothelial cells. H5V endothelial cells (ECs) were isolated from the heart microcirculation of E15 embryos, immortalized with polyoma middle-T antigen (Garlanda et al . , 1994) and cultured as previously described (Balconi et al . , 2000) . Briefly, the culture medium was DMEM, 10%FCS, 100 μg/ml heparin, 5 μg/ml ECGS. Immortalized ECs were plated in 35mm Petri dishes at 30,000 cells/cm ² and were allowed to growth until confluence. The cells were then lysed for RNA preparation.

[00110] Cell treatment. Microvascular brain endothelial cells or H5V cells, cultured as described above, were grown to confluence and exposed to various agents that activate β-catenin signaling for the periods of time stated. Agents used to activate β-catenin signaling include Wnt3a-conditioned medium (CM) and the following GSK 3β inhibitors: 6-bromoindirubin-3' - oxime (BIO) (Chemicon; Meijer et al . , 2003), LiCl (Merck; Taddei et al., 2008) and SB216763 (Sigma; Meijer et al . , 2004). Control cells were treated with an equal volume of DMSO (vehicle) .

[00111] RNA preparation and qRT-PCR were as described in the Materials and Methods section of Example 1.

[00112] Affymetrix gene array analysis. This analysis was performed as described in Taddei et al . (2008) .

RESULTS

[00113] The present inventors developed a list of genes which define BBB identity of cultured mouse brain endothelial cells. These genes are up-regulated by Wnt pathway activation and by treatment of the cells with 5μM 6-bromindirubin-3' -oxime (BIO), a GSK3β inhibitor. This list of genes is presented in Table 2 below. The biological characteristics of these genes are also presented in the footnote section of Table 2. The genes have been identified first by Affymetrix gene array analysis and then validated by RT-PCR, and they correspond to proteins such as transporters of cell nutrients, junction proteins and others which are known to be expressed specifically in BBB endothelial cells. The genes in Table 2 are tools for monitoring the induction and maintenance of BBB characteristics in cultured cells. Therefore, they can be used for quality control of BBB endothelial cells. Validation of this list of genes was made on cultured endothelial cells where a gene responsible for a human hereditary disease called cerebral cavernous malformation (CCM) has been deleted (Labauge et al . , 2007) . An example of two genes from the list in Table 2 which are not increased in expression by Wnt activation in CCM mutant cells (CCM2 KO cells which induces pathological malformation) is shown in Figs. 16A and 16B. The up-regulation of these two genes, Fgfbpl and Slc22al4, due to β- catenin activation is completely abrogated in CCM2 KO MBEs.

Table 2

FOOTNOTES :

SLC= Solute Carrier Transports

Hydrophilic nutrients, such as amino acid, are actively transported through the brain endothelium by a variety of membrane transporters, notably the large family of SLC

ABC= ATP Binding Cassette

Efflux transporters of the ABC family transport a large panel of lipophilic molecules, in particular xenobiotics. The most active ABC-transporters are P-glicoprotein (P-gp) encoded by the multidrug resistance gene (MDRl or ABCBl), multidrug resistance protein proteins (MRPs, or ABCC protein) and the brest cancer resistance protein (BCRP or ABCG2)

Nitric oxide (NO) is a potent mediator in biologic processes such as neurotransmission, inflammatory response, and vascular homeostasis. NOSTRIN binds the enzyme responsible for NO production, endothelial NO synthase (ENOS; MIM 163729), and triggers the translocation of ENOS from the plasma membrane to vesicle-like subcellular structures, thereby attenuating ENOS- dependent NO production.

FGF-BPl= it is involved in the FGF bioactivation. Extracellular chaperone molecule for FGFs. FGF-BPl expression is highly tissue specific; it is express in most brestcancer, colon cancer, SCC. It is an agiogenic switch molecule. Nat. Med 1997, 3(10): 1137. Int J Biochem Cell Biol, 2006;38(9) : 1463. SLCO1C1/OATP1C1= Organic ion transporter. Primary Thyroid Hormone transporter at the BBB. Particularly expressed in RAT and Mouse cerebral microvessels. Organic anion transporting polypeptides (OATPs) are expressed in most mammalian tissues and mediates cellular up-take of a variety of organic compounds such as bile salts, steroid conjugated, oligopeptides and a large list of drugs probably by acting as anion exchangers. Endocrinology 2008; 149 (12) :6251 Pde7b= cAMP specific cyclic nucleotide phosphodiesterase. Member of the phosphodiesterase family with highj affinity and specificity for cAMP. Highly expressed in pancreas, brain, heart, thyroid etc. Pde families contain many splice variants that mostly are unique in tissue-expression patterns, gene regulation, subcellular localization. "Overview of PDEs and their regulation" Or. Res. 2007; 100:309

SLCOla4/OATPla4/OATP2= Organic anion transporter. Partially localize to the lumen. Luminal/abluminal localization of different transporters; see Neuroscience 2008; 155(2) :423. SLC35f2= Transporter protein, unknown function. Viral receptor/feline homologue (PNAS, 2007; 104(26) : 11032) SLCOla5/OATP3/Slc21a7= organic anion transporting polypetides: transports various CNS-acting endogenous compound, es. Thyroid hormons, Prostaglandin E2. Slc22al4= Organic cation transporter. No biological data available.

BEX1/Rex3/Bex4= Brain Expressed X-linked gene. Expressed in several tissues, involved in cell growth and differentiation. In KO mice, a defect in muscle regeneration has been observed. Elevated and prolonged cell proliferetion and also delayed in cell differentiation in KO vs WT after cardiotoxin tratment. Biochem Biophys Res Com 2007; 363(2): 405. Bexl gene is highly expressed in neuroendocrine tumor. BJM 2008; 99: 1330 FLVCR2/CCT/Mfsd7c= Transporter specific for calcium- chelator complex.

Gprl26/DREG=G protein-couple receptor (GPCR). It is expressed at high levels in the heart and somite during embryogenesis and in the adult lung.

Foxql/Hfhl= in adult it is higly expressed in kidney and stomach. In KO mice has been observed lack of gastric secretion : Impairment of the fusion of cytoplasmic tubovesicles to the apical membrane of secretory canaliculi.

Cyplbl= Cytochrome P450 (CYP) family 1, subfamily b, polypeptide 1

Cytochrome P450 (CYP) enzymes are membrane-bound, heme- containing terminal oxidases that can catalyze a large number of chemical reactions and can use an almost unlimited number of biologically occurring and synthetic compounds. Cyplbl is highly expressed in endothelia cells of the blood-brain barrier (Dauchy et al. 2008, J Neurochem 107(6) : 1518-28)

[00114] The present inventors have developed a protocol to maintain BBB characteristics in freshly cultured mouse endothelial cells from brain microcirculation in the absence of co-culture with astrocytes to generate a preferred embodiment of the in vitro BBB model according to the present invention. In normal conditions, cultured brain endothelial cells lose BBB characteristics within 4-5 days. The present inventors however are able to prolong this time up to at least 14 days using the protocol provided below. A typical example of BBB marker expression is presented in Figures 17D-17F. While Axin2 is a marker of Wnt signaling, Claudin3 (Cldn3) is a marker of BBB and Plvap is a marker of loss of BBB. Therefore, the expression of Plvap is decreased when the BBB is established. The results shown in Figs. 17D-17F confirm that Wnt3a induces significant Axin2 and Claudin3 up-regulation and Plvap downregulation .

Protocol for isolation and culture of mouse brain microvessel endothelial cells (MBE)

• Remove brain from mice

• Remove meninges and white matter

• Dissect the cortices with forceps and mince the tissue with surgical blades.

• Digest with collagenase Type II for Ih at 37°C

• Centrifuge 10 min, 1200 rpm

• BSA gradient to obtain a microvessel enriched cell pellet (20 min, 2600 rpm, 4°C)

• Discard myelin and further digest the pellet with collagenase/dispase and Dnase I (15min, 37°C).

• Centrifuge 10 min, 1200 rpm and re-suspend the cells in complete culture medium.

• The microvessel fragments are seeded on collagen I coated dishes and cultured for 2 days with puromycin- containing medium. The puromycin concentration of 4ug/ml removed contaminating cell types without any noticeable MBE growth inhibition.

• Culture MBE for 5-6 days with normal culture medium.

• Split the cells (ratio 1:2) and culture for 6-7 days on collagen I coated dishes, in either control or conditioned medium of L cells, producing Wnt3a which acts through β-catenin. • Replace medium every 2 day.

[00115] An immortalized endothelial cell line (H5V) was developed by the present inventors. By treating this immortalized endothelial cell line with the GSK3-β inhibitor, BIO, the present inventors obtained a cell line which expresses BBB properties and which can be used as a preferred embodiment for large screenings of neurologically active drugs to assess their ability to cross the BBB. In addition, this H5V cell line can also be used to screen compounds which may restore or tighten BBB. The expression of BBB genes Cldn3, Axin 2 and Plvap in this cell line is reported in Figs. 18B-18D. H5V cells exposed to 5μM BIO for 6 days showed significantly increased expression of Cldn3 and Axin 2 compared with the vehicle (DMSO) . Treatment with BIO thus resulted in activation of β-catenin signaling (Axin2 up- regulation) and induction of barrier characteristics in vitro (up-regulation of Cldn3 and down regulation of Plvap) .

[00116] Three different compounds (BIO, LiCl, SB216763) which can induce BBB characteristics by stabilizing β-catenin were compared against Wnt3a to confirm that the in vitro BBB model according to the present invention can be used to identify such compounds. The results from a typical experiment are presented in Figs. 19B and 19C. From this experiment it is not only confirmed that the presently claimed in vitro screening method is capable of identifying compounds that tighten or restore BBB characteristics but also that these reagents can be used to maintain endothelial BBB characteristics in the in vitro BBB model .

[00117] Having now generally described the invention, the same will be more readily understood through reference to the following example which is provided by way of illustration and is not intended to be limiting of the present invention.

[00118] Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

[00119] While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

[00120] All references cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.

[00121] Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.

[00122] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein) , readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

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