Targeting vascular NADPH oxidase 1 blocks tumor angiogenesis through a PPARα mediated mechanism.

Reactive oxygen species, ROS, are regulators of endothelial cell migration, proliferation and survival, events critically involved in angiogenesis. Different isoforms of ROS-generating NOX enzymes are expressed in the vasculature and provide distinct signaling cues through differential localization and activation. We show that mice deficient in NOX1, but not NOX2 or NOX4, have impaired angiogenesis. NOX1 expression and activity is increased in primary mouse and human endothelial cells upon angiogenic stimulation. NOX1 silencing decreases endothelial cell migration and tube-like structure formation, through the inhibition of PPARα, a regulator of NF-κB. Administration of a novel NOX-specific inhibitor reduced angiogenesis and tumor growth in vivo in a PPARα dependent manner. In conclusion, vascular NOX1 is a critical mediator of angiogenesis and an attractive target for anti-angiogenic therapies.

Temozolomide combined with bevacizumab in metastatic melanoma. A multicenter phase II trial (SAKK 50/07)

Background: Single agent DTIC is the standard therapy for metastatic melanoma (MM) with response rates of 5−20%. Temozolomide (Tem) as an oral drug has shown equal efficacy in phase III trials. Preclinical models have shown an inhibitory effect for bevacizumab (Bev) on the proliferation of melanoma cells as well as on sprouting endothelial cells. Therefore, a therapeutic approach that combines angiogenesis inhibitors with cytotoxic agents may provide clinical benefit in MM. Methods: Design: Multicenter phase II trial. Primary endpoint: Clinical benefit (CR, PR and SD) at 12 weeks; secondary endpoints: best overall response by RECIST, response duration, progression free survival, adverse events, survival after 6 months and overall survival. Sample size was calculated according to Simon's two stage optimal design (5% significance level and 80% power) with an overall sample size of 62 patients (pts) to test H0: 20% versus H1: 35% rate of clinical benefit. Response assessment was done every 6 weeks (3 cycles). Eligibility: Stage IV MM, ECOG PS 0−2, no prior treatment for metastatic disease. Treatment regimen: One cycle consisted of Tem at 150 mg/m2 days 1−7 po and Bev at 10 mg/kg day 1 over 30 min iv and was repeated every 2 weeks until progression or unacceptable toxicity. Results: Between January 2008 and April 2009, 62 pts (40 male/22 female) at a median age of 61 years (range 30−86) with stage IV (M1a:4, M1b:12, M1c:46) melanoma were enrolled in 9 centers. The first 50 pts, who received 415 cycles are included in this interim report. The overall response rate was 26% (CR: 1 pt, PR: 12 pts; PR not confirmed yet in 3 pts), and 44% (22 pts) had stable disease over 1.5−7.5 months (median: 3). Only 30% (15 pts) had disease progression at the first evaluation at week 6. The hematological grade 3/4 toxicities according to NCI CTAE 3.0 were thrombocytopenia 10% (5 pts), neutropenia 8% (4 pts), lymphopenia and leucocytopenia each 2% (1 pt). Cumulative non-hematological toxicities grade 3/4 were nausea and fatigue each 6% (3 pts), hypertension, vomiting and hemorrhage, each 4% (2 pts), thrombosis/embolism, infection, constipation, anorexia, elevation of alkaline phosphatase, bilirubin, GGT, ALT and AST each 2% (1 pt). Conclusion: In metastatic melanoma the combination of Tem/Bev is a safe regimen with a promising efficacy and few grade 3/4 toxicities. Updated results of all 62 pts will be presented.

Cancer Cell-Associated MT1-MMP Promotes Blood Vessel Invasion and Distant Metastasis in Triple-Negative Mammary Tumors.

Functional roles for the cancer cell-associated membrane type I matrix metalloproteinase (MT1-MMP) during early steps of the metastatic cascade in primary tumors remain unresolved. In an effort to determine its significance, we determined the in vivo effects of RNAi-mediated downregulation in mammary cancer cells on the migration, blood and lymphatic vessel invasion (LVI), and lymph node and lung metastasis. We also correlated the expression of cancer cell MT1-MMP with blood vessel invasion (BVI) in 102 breast cancer biopsies. MT1-MMP downregulation in cancer cells decreased lung metastasis without affecting primary tumor growth. The inhibition of lung metastasis correlated with reduced cancer cell migration and BVI. Furthermore, cancer cell-expressed MT1-MMP upregulated the expression of MT1-MMP in vascular endothelial cells, but did not affect MT1-MMP expression in lymphatic endothelial cells, LVI, or lymph node metastasis. Of clinical importance, we observed that elevated MT1-MMP expression correlated with BVI in biopsies from triple-negative breast cancers (TNBC), which have a poor prognosis and high incidence of distant metastasis, relative to other breast cancer subtypes. Together, our findings established that MT1-MMP activity in breast tumors is essential for BVI, but not LVI, and that MT1-MMP should be further explored as a predictor and therapeutic target of hematogenous metastasis in TNBC patients. Cancer Res; 71(13); 4527-38. ©2011 AACR.

Glucocorticoids exert direct toxicity on microvasculature: analysis of cell death mechanisms.

Glucocorticoids (GCs) are routinely administered systemically or injected into the eye when treating numerous ocular diseases; however, their toxicity on the retinal microvasculature has not been previously investigated. In this article, the effects of hydrocortisone (Hydro), dexamethasone, dexamethasone-phosphate and triamcinolone acetonide (TA) were evaluated in vitro on human skin microcirculation cells and, bovine endothelial retinal cells, ex-vivo, on flat mounted rat retinas. The degree of GCs induced endothelial cell death varied according to the endothelial cell type and GCs chemical properties. GCs toxicity was higher in skin microvascular endothelial cells and for hydrophobic GC formulations. The mechanism of cell death differed between GCs, Hydro and TA activated the leukocyte elastase inhibitor/L-DNase II pathways but did not activate caspases. The mechanisms of cell death observed in cell cultures were similar to those observed in rat retinal explants. Taken together these results indicate that particular attention should be paid to the potential vascular side effects when administrating GCs clinically and in particular when developing sustained-release intraocular devices.

Cannabidiol protects against hepatic ischemia/reperfusion injury by attenuating inflammatory signaling and response, oxidative/nitrative stress, and cell death.

Ischemia/reperfusion (I/R) is a pivotal mechanism of liver damage after liver transplantation or hepatic surgery. We have investigated the effects of cannabidiol (CBD), the nonpsychotropic constituent of marijuana, in a mouse model of hepatic I/R injury. I/R triggered time-dependent increases/changes in markers of liver injury (serum transaminases), hepatic oxidative/nitrative stress (4-hydroxy-2-nonenal, nitrotyrosine content/staining, and gp91phox and inducible nitric oxide synthase mRNA), mitochondrial dysfunction (decreased complex I activity), inflammation (tumor necrosis factor α (TNF-α), cyclooxygenase 2, macrophage inflammatory protein-1α/2, intercellular adhesion molecule 1 mRNA levels; tissue neutrophil infiltration; nuclear factor κB (NF-κB) activation), stress signaling (p38MAPK and JNK), and cell death (DNA fragmentation, PARP activity, and TUNEL). CBD significantly reduced the extent of liver inflammation, oxidative/nitrative stress, and cell death and also attenuated the bacterial endotoxin-triggered NF-κB activation and TNF-α production in isolated Kupffer cells, likewise the adhesion molecule expression in primary human liver sinusoidal endothelial cells stimulated with TNF-α and attachment of human neutrophils to the activated endothelium. These protective effects were preserved in CB(2) knockout mice and were not prevented by CB(1/2) antagonists in vitro. Thus, CBD may represent a novel, protective strategy against I/R injury by attenuating key inflammatory pathways and oxidative/nitrative tissue injury, independent of classical CB(1/2) receptors.

Constrained peptidomimetics reveal detailed geometric requirements of covalent prolyl oligopeptidase inhibitors.

Prolyl oligopeptidases cleave peptides on the carboxy side of internal proline residues and their inhibition has potential in the treatment of human brain disorders. Using our docking program fitted, we have designed a series of constrained covalent inhibitors, built from a series of bicyclic scaffolds, to study the optimal shape required for these small molecules. These structures bear nitrile functional groups that we predicted to covalently bind to the catalytic serine of the enzyme. Synthesis and biological assays using human brain-derived astrocytic cells and endothelial cells and human fibroblasts revealed that these compounds act as selective inhibitors of prolyl oligopeptidase activity compared to prolyl-dipeptidyl-aminopeptidase activity, are able to penetrate the cells and inhibit intracellular activities in intact living cells. This integrated computational and experimental study shed light on the binding mode of inhibitors in the enzyme active site and will guide the design of future drug-like molecules.

Gene transfer of a soluble IL-1 type 2 receptor-Ig fusion protein improves cardiac allograft survival in rats.

OBJECTIVE: Interleukin-1 (IL-1) mediates ischemia-reperfusion injury and graft inflammation after heart transplantation. IL-1 affects target cells through two distinct types of transmembrane receptors, type-1 receptor (IL-1R1), which transduces the signal, and the non-signaling type-2 receptor (IL-1R2), which acts as a ligand sink that subtracts IL-1beta from IL-1R1. We analyzed the efficacy of adenovirus (Ad)-mediated gene transfer of a soluble IL-1R2-Ig fusion protein in delaying cardiac allograft rejection and the mechanisms underlying the protective effect. METHODS: IL-1 inhibition by IL-1R2-Ig was tested using an in vitro functional assay whereby endothelial cells preincubated with AdIL-1R2-Ig or control virus were stimulated with recombinant IL-1beta or tumor necrosis factor-alpha (TNF-alpha), and urokinase-type plasminogen activator (u-PA) induction was measured by zymography. AdIL-1R2-Ig was delivered to F344 rat donor hearts ex vivo, which were placed in the abdominal position in LEW hosts. Intragraft inflammatory cell infiltrates and proinflammatory cytokine expression were analyzed by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. RESULTS: IL-1R2-Ig specifically inhibited IL-1beta-induced u-PA responses in vitro. IL-1R2-Ig gene transfer reduced intragraft monocytes/macrophages and CD4(+) cell infiltrates (p<0.05), TNF-alpha and transforming growth factor-beta (TGF-beta) expression (p<0.05), and prolonged graft survival (15.6+/-5.7 vs 10.3+/-2.5 days with control vector and 10.1+/-2.1 days with buffer alone; p<0.01). AdIL-1R2-Ig combined with a subtherapeutic regimen of cyclosporin A (CsA) was superior to CsA alone (19.4+/-3.0 vs 15.9+/-1.8 days; p<0.05). CONCLUSIONS: Soluble IL-1 type-2 receptor gene transfer attenuates cardiac allograft rejection in a rat model. IL-1 inhibition may be useful as an adjuvant therapy in heart transplantation.

Role of forkhead transcription factor FOXC2 in lymphatic vascular developement

Le système vasculaire lymphatique est le second réseau de vaisseaux du corps humain. Sa fonction principale est de retourner le fluide interstitiel excédentaire au système cardiovasculaire. Il est également impliqué dans la défense immunitaire de l'organisme, ainsi que dans le transport initial des graisses alimentaires. De multiples pathologies sont associées au dysfonctionnement du développement vasculaire lymphatique, dont les lymphoedèmes. Un des gènes clés dans le contrôle de l'étape de maturation du système lymphatique est le facteur de transcription FOXC2. De précédentes études utilisant des modèles génétiques mutins déficients en Foxc2 ont montré son rôle dans la régulation du processus de spécification des vaisseaux lymphatiques en capillaires versus vaisseaux collecteurs, ainsi que dans la formation des valves lymphatiques. Chez l'homme, les mutations dans le gène FOXC2 causent le syndrome lymphoedème- distichiasis. Dans ce travail, nous avons étudié les mécanismes moléculaires qui régulent l'expression et l'activité de FOXC2 dans les vaisseaux lymphatiques. Nous avons découvert que la fonction de FOXC2 est régulée par phosphorylation de la protéine, qui détermine son activité transcriptionnelle au niveau génomique, jouant ainsi un rôle important dans le développement vasculaire in vivo. Les vaisseaux lymphatiques sont soumis à des forces de stress générées par le flux de la lymphe (FSS). Nous avons donc testé l'hypothèse que ces forces contribuent à la morphogenèse et à l'organisation des vaisseaux lymphatiques. In vitro, les cellules endothéliales lymphatiques répondent aux forces mécaniques, qui induisent l'expression de FOXC2, activent la voie de signalisation Ca2+/calcineurin/NFATcl et régulent l'expression de la protéine de jonction gap connexin37. Nous avons également montré que le stress de flux mécanique, FOXC2, calcineurin/NFATcl et connexin37 coopèrent dans le contrôle de la maturation des vaisseaux lymphatiques in vivo. En dernier lieu, nous avons cherché à identifier les récepteurs de surface cellulaires permettant le transfert du signal de stress mécanique qui induit l'expression de FOXC2. Nous présentons ici des données préliminaires, qui suggèrent le rôle de la voie de signalisation TGFß ainsi que l'implication des jonctions adhérentes dans ce processus. En conclusion, la présente étude met en lumière les mécanismes de l'activité de FOXC2 dans les cellules endothéliales lymphatiques et l'importance du rôle des forces mécaniques de flux dans le contrôle de son l'expression, ainsi que dans le développement et la fonction du système vasculaire lymphatique. - The lymphatic vascular system is a second vascular system of human body. Its main fonction is to transfer excess interstitial fluid back to cardiovascular system. In addition, it is involved in immune defense and responsible for the uptake of dietary fat. A number of pathologies called lymphedemas are associated with lymphatic vascular system dysfunction. Hereditary lymphedemas are caused by mutations in genes controlling lymphatic vascular development. One of the key genes responsible for lymphatic vascular maturation is forkhead transcription factor FOXC2. Previous studies of Foxc2 knockout mice showed that Foxc2 controls the process of lymphatic capillary versus collecting vessel fate specification and formation of lymphatic valves. Importantly, mutations in FOXC2 cause human lymphedema-distichiasis syndrome. In this work we investigated the molecular mechanisms regulating the expression and activity of FOXC2 in lymphatic vasculature. We discovered that FOXC2 function is regulated by phosphorylation. We describe how phosphorylation controls FOXC2 transcriptional activity on a genome-wide level and show that FOXC2 phosphorylation plays an important role in vascular development in vivo. Lymphatic vessels are subjected to fluid shear stress (FSS). Therefore we investigated whether mechanical forces contribute to lymphatic vascular patterning and morphogenesis. We found that FSS induces the expression of FOXC2, activates Ca2+/calcineurin/NFATcl signaling and induces the expression of gap junction protein connexin37 in lymphatic endothelial cells in vitro. Importantly, we were able to show that shear stress, FOXC2, calcineurin/NFATcl and connexin37, control maturation of lymphatic vessels in vivo. Finally, we searched for cell surface receptors that mediate the induction of FOXC2 by shear stress, and we present some preliminary data, suggesting the role of TGF-beta signaling and adherens junctions in this process. In conclusion, the present study sheds light on the mechanisms of FOXC2 activity and suggests an important role of mechanical forces in controlling FOXC2 expression as well as lymphatic system development and function.

Delivery of antisense oligonucleotide to the cornea by iontophoresis.

We wished to evaluate the potential of iontophoresis to promote the delivery of antisense oligonucleotides (ODN) directed at the vascular endothelial growth factor (VEGF)-R2 receptor (KDR/Flk) to the cornea of the rat eye. Fluorescence (CY5)-labeled ODNs in phosphate-buffered saline (PBS) (20 microM) were locally administered to rat eyes, and their fate within the anterior segment was studied. Thirty-four male, 5-week-old Wistar rats were used for all experiments. The rats were divided in four groups. In group I (12 rats, 12 eyes), the ODNs (20 microM) were delivered by iontophoresis (300 microA for 5 minutes) using a specially designed corneal applicator. In group II (12 rats, 12 eyes), the ODNs (20 microM) were delivered using the same applicator, but no electrical current was applied. In group III (6 rats, 6 eyes), a corneal neovascular reaction was induced prior to the application of ODNs (20 microM), and iontophoresis electrical current was delivered as for group I rats. Group IV (4 rats, 4 eyes) received ODN (60 microM) iontophoresis application (300 microA for 5 minutes) and were used for ODN integrity studies. The animals were killed 5 minutes, 90 minutes, and 24 hours after a single ODN application and studied. Topically applied ODNs using the same iontophoresis applicator but without current do not penetrate the cornea and remain confined to the superficial epithelial layer. ODNs delivered with transcorneoscleral iontophoresis penetrate into all corneal layers and are also detected in the iris. In corneas with neovascularization, ODNs were particularly localized within the vascular endothelial cells of the stroma. ODNs extracted from eye tissues 24 hours after iontophoresis remained unaltered. The iontophoresis current did not cause any detectable ocular damage under these conditions. Iontophoresis promotes the delivery of ODNs to the anterior segment of the eye, including all corneal layers. Iontophoresis of ODNs directed at VEGF-R2 may be used for the design of specific antiangiogenic strategy in diseases of the cornea.

Leukocytes, inflammation, and angiogenesis in cancer: fatal attractions.

Leukocytes are cells of defense. Their main function is to protect our body against invading microorganisms. Some leukocytes, in particular, polymorphonuclear and monocytes, accumulate at sites of infection and neutralize pathogens through innate mechanisms. The blood and lymphatic vascular system are essential partners in this defensive reaction: Activated endothelial cells promote leukocyte recruitment at inflammatory sites; new blood vessel formation, a process called angiogenesis, sustains chronic inflammation, and lymphatic vessels transport antigens and antigen-presenting cells to lymph nodes, where they stimulate naive T and B lymphocytes to elicit an antigen-specific immune response. In contrast, leukocytes and lymphocytes are far less efficient in protecting us from cancer, the "enemy from within." Worse, cancer can exploit inflammation to its advantage. The role of angiogenesis, leukocytes, and inflammation in tumor progression was discussed at the second Monte Verità Conference, Tumor Host Interaction and Angiogenesis: Basic Mechanisms and Therapeutic Perspectives, held in Ascona, Switzerland, October 1-5, 2005. (Conference chairs were K. Alitalo, M. Aguet, C. Rüegg, and I. Stamenkovic.) Eight articles reporting about topics presented at the conference are featured in this issue of the Journal of Leukocyte Biology.

The blood-brain barrier: cellular basis.

Perfusion experiments with horseradish peroxidase have established that the morphological substrate of the blood-brain barrier is represented by microvascular endothelial cells. They are characterized by complexly arranged tight junctions and a very low rate of transcytotic vesicular transport. They express transport enzymes, carrier systems and brain endothelial cell-specific molecules of unknown function not expressed by any other endothelial cell population. These blood-brain barrier properties are not intrinsic to these cells but are inducible by the surrounding brain tissue. Type I astrocytes injected into the anterior eye chamber of the rat or onto the chick chorioallantoic membrane are able to induce a host-derived angiogenesis and some blood-brain barrier properties in endothelial cells of non-neural origin. Recently we have shown that this cellular interaction is due to the secretion of a soluble astrocyte derived factor(s). Astrocytes are also implicated in the maintenance, functional regulation and the repair of the blood-brain barrier. Complex interactions between other constituents of the microenvironment surrounding the endothelial cells, such as the basement membrane, pericytes, nerve endings, microglial cells and the extracellular fluid, take place and are required for the proper functioning of the blood-brain barrier, which in addition is regionally different as reflected by endothelial cell heterogeneity.

Interplay between connexin40 and nitric oxide signaling during hypertension.

Connexins (Cxs) and endothelial nitric oxide synthase (eNOS) contribute to the adaptation of endothelial and smooth muscle cells to hemodynamic changes. To decipher the in vivo interplay between these proteins, we studied Cx40-null mice, a model of renin-dependent hypertension which displays an altered endothelium-dependent relaxation of the aorta because of reduced eNOS levels. These mice, which were either untreated or subjected to the 1-kidney, 1-clip (1K1C) procedure, a model of volume-dependent hypertension, were compared with control mice submitted to either the 1K1C or the 2-kidney, 1-clip (2K1C) procedure, a model of renin-dependent hypertension. All operated mice became hypertensive and featured hypertrophy and altered Cx expression of the aorta. The combination of volume- and renin-dependent hypertension in Cx40-/- 1K1C mice raised blood pressure and cardiac weight index. Under these conditions, all aortas showed increased levels of Cx40 in endothelial cells and of both Cx37 and Cx45 in smooth muscle cells. In the wild-type 1K1C mice, the interactions between Cx40 and Cx37 with eNOS were enhanced, resulting in increased NO release. The Cx40-eNOS interaction could not be observed in mice lacking Cx40, which also featured decreased levels of eNOS. In these animals, the volume overload caused by the 1K1C procedure resulted in increased phosphorylation of eNOS and in a higher NO release. The findings provide evidence that Cx40 and Cx37 play an in vivo role in the regulation of eNOS.

ErbB Ligands in Angiogenesis

The formation of new blood vessels, i.e. angiogenesis, is an important phenomenon during normal development and wound repair, as well as during various pathological processes, such as tumor growth and metastasis. Specific growth factors regulate angiogenesis by modulating the different cellular functions of endothelial cells (EC), and periendothelial cells, such as pericytes (PC) and smooth muscle cells (SMC), which interact with ECs in a paracrine manner. ErbB receptors form a subgroup of transmembrane receptor tyrosine kinases that interact with growth factors of the epidermal growth factor (EGF) family. ErbB receptors regulate behaviour of a variety of normal as well as tumor cell types. Cancer drugs that target epidermal growth factor receptor (EGFR, ErbB1) or ErbB2 receptor have been approved for clinical use. It has been speculated that part of the antitumor activity of ErbB inhibitor compounds result from an antiangiogenic mechanism. The results presented here indicate a role for endothelial-derived EGF-like growth factors heparin binding EGF-like growth factor (HB-EGF) and neuregulin-1 (NRG-1) in the paracrine regulation of angiogenesis. HB-EGF, EGFR and ErbB2 are shown to mediate interaction between ECs and SMCs in vitro, and gefitinib, an inhibitor of EGFR kinase activity, suppresses recruitment of PCs/SMCs in vivo. NRG-1 is shown to regulate EC functions in vitro and angiogenesis in vivo by indirect mechanisms involving vascular endothelial growth factor-A (VEGF-A) and VEGF receptor-2 (VEGFR-2). Furthermore, EGFR activity is demonstrated to regulate recruitment of bone marrow-derived perivascular cells during tumor neovascularization in vivo. These results indicate that ErbB signaling is involved in the cellular processes of new blood vessel formation. This study gives new information about the role of ErbB ligands and receptors in angiogenesis and vasculogenesis and about the mechanisms by which ErbB inhibitor drugs such as gefitinib affect tumor growth.

Targeting the tumor vasculature to enhance T cell activity.

T cells play a critical role in tumor immune surveillance as evidenced by extensive mouse-tumor model studies as well as encouraging patient responses to adoptive T cell therapies and dendritic cell vaccines. It is well established that the interplay of tumor cells with their local cellular environment can trigger events that are immunoinhibitory to T cells. More recently it is emerging that the tumor vasculature itself constitutes an important barrier to T cells. Endothelial cells lining the vessels can suppress T cell activity, target them for destruction, and block them from gaining entry into the tumor in the first place through the deregulation of adhesion molecules. Here we review approaches to break this tumor endothelial barrier and enhance T cell activity.

The embryonic blood-CSF barrier has molecular elements for specific glucose transport and for the general transport of molecules via transcellular routes.

In vertebrates, early brain development takes place at the expanded anterior end of the neural tube, which is filled with embryonic cerebrospinal fluid (E-CSF). We have recently identified a transient blood-CSF barrier that forms between embryonic days E3 and E4 in chick embryos and that is responsible for the transport of proteins and control of E-CSF homeostasis, including osmolarity. Here we examined the presence of glucose transporter GLUT-1 as well the presence of caveolae-structural protein Caveolin1 (CAV-1) in the embryonic blood-CSF barrier which may be involved in the transport of glucose and of proteins, water and ions respectively across the neuroectoderm. In this paper we demonstrate the presence of GLUT-1 and CAV-1 in endothelial cells of blood vessels as well as in adjacent neuroectodermal cells, located in the embryonic blood-CSF barrier. In blood vessels, these proteins were detected as early as E4 in chick embryos and E12.7 in rat embryos, i.e. the point at which the embryonic blood-CSF barrier acquires this function. In the neuroectoderm of the embryonic blood-CSF barrier, GLUT-1 was also detected at E4 and E12.7 respectively, and CAV-1 was detected shortly thereafter in both experimental models. These experiments contribute to delineating the extent to which the blood-CSF embryonic barrier controls E-CSF composition and homeostasis during early stages of brain development in avians and mammals. Our results suggest the regulation of glucose transport to the E-CSF by means of GLUT-1 and also suggest a mechanism by which proteins are transported via transcellular routes across the neuroectoderm, thus reinforcing the crucial role of E-CSF in brain development.