http://repub.eur.nl/res/aut/23278/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/39275/ 2013-01-23T00:00:00Z The blood- brain barrier (BBB) is crucial in the maintenance of a controlled environment within the brain to safeguard optimal neuronal function. The endothelial cells (ECs) of theBBBpossess specific properties that restrict the entry of cells and metabolites into the CNS. The specialized BBB endothelial phenotype is induced during neurovascular development by surrounding cells of the CNS. However, the molecular differentiation of the BBB endothelium remains poorly understood. Retinoic acid (RA) plays a crucial role in the brain during embryogenesis. Because radial glial cells supply the brain with RA during the developmental cascade and associate closely with the developing vasculature, we hypothesize that RA is important for the induction of BBB properties in brain ECs. Analysis of human postmortem fetal brain tissue shows that the enzyme mainly responsible for RA synthesis, retinaldehyde dehydrogenase, is expressed by radial glial cells. In addition, the most important receptor for RA-driven signaling in the CNS, RA-receptor β (RARβ), is markedly expressed by the developing brain vasculature. Our findings have been further corroborated by in vitro experiments showing RA- and RARβ-dependent induction of different aspects of the brain EC barrier. Finally, pharmacologic inhibition of RAR activation during the differentiation of the murine BBB resulted in the leakage of a fluorescent tracer as well as serum proteins into the developing brain and reduced the expression levels of important BBB determinants. Together, our results point to an important role for RA in the induction of the BBB during human and mouse development. http://repub.eur.nl/res/pub/38840/ 2012-11-01T00:00:00Z Impaired endothelial barrier function results in a persistent increase in endothelial permeability and vascular leakage. Repair of a dysfunctional endothelial barrier requires controlled restoration of adherens junctions, comprising vascular endothelial (VE)-cadherin and associated β-, γ-, α-, and p120-catenins. Little is known about the mechanisms by which recovery of VE-cadherin-mediated cell-cell junctions is regulated. Using the inflammatory mediator thrombin, we demonstrate an important role for the Src homology 2-domain containing tyrosine phosphatase (SHP2) in mediating recovery of the VE-cadherin-controlled endothelial barrier. Using SHP2 substrate-trapping mutants and an in vitro phosphatase activity assay, we validate β-catenin as a bona fide SHP2 substrate. SHP2 silencing and SHP2 inhibition both result in delayed recovery of endothelial barrier function after thrombin stimulation. Moreover, on thrombin challenge, we find prolonged elevation in tyrosine phosphorylation levels of VE-cadherin-associated β-catenin in SHP2-depleted cells. No disassembly of the VE-cadherin complex is observed throughout the thrombin response. Using fluorescence recovery after photobleaching, we show that loss of SHP2 reduces the mobility of VE-cadherin at recovered cell-cell junctions. In conclusion, our data show that the SHP2 phosphatase plays an important role in the recovery of disrupted endothelial cell-cell junctions by dephosphorylating VE-cadherin-associated β-catenin and promoting the mobility of VE-cadherin at the plasma membrane. http://repub.eur.nl/res/pub/32916/ 2012-06-29T00:00:00Z Intestinal epithelial cells (IECs) form a physical barrier between the internal milieu and the intestinal microflora via the expression of tight junctions. TLR-mediated recognition of intestinal microflora by IECs is important for tight junction preservation, production of chemokines, and cell survival. Disturbance of the IEC barrier function results in bacterial invasion and contributes to the development of inflammatory bowel disease. We observed that muramyl dipeptide (MDP), a breakdown product of bacterial peptidoglycan, strongly enhances subsequent Toll-like receptor (TLR) responses in murine colonic epithelium cell lines. Prior exposure to MDP significantly increased the production of chemokines and cytokines and improved the barrier function induced by different TLR2 and TLR4 ligands. shRNA knock-down studies showed that MDP recognition by Nod2 mediated the enhancement of TLR responses. Our studies indicate that Nod2 stimulation by MDP significantly enhances TLR-mediated IEC barrier function and chemokine production. Failure of this protective mechanism may contribute to the increased risk of Crohn's disease in individuals with a loss-of-function mutation in NOD2. http://repub.eur.nl/res/pub/25972/ 2011-07-01T00:00:00Z Abstract Homeostasis of the brain is dependent on the blood–brain barrier (BBB). This barrier tightly regulates the exchange of essential nutrients and limits the free flow of immune cells into the CNS. Perturbations of BBB function and the loss of its immune quiescence are hallmarks of a variety of brain diseases, including multiple sclerosis (MS), vascular dementia, and stroke. In particular, diapedesis of monocytes and subsequent trafficking of monocyte-derived macrophages into the brain are key mediators of demyelination and axonal damage in MS. Endothelin-1 (ET-1) is considered as a potent proinflammatory peptide and has been implicated in the development of cardiovascular diseases. Here, we studied the role of different components of the endothelin system, i.e., ET-1, its type B receptor (ETB) and endothelin-converting enzyme-1 (ECE-1) in monocyte diapedesis of a human brain endothelial cell barrier. Our pharmacological inhibitory and specific gene knockdown studies point to a regulatory function of these proteins in transendothelial passage of monocytes. Results from this study suggest that the endothelin system is a putative target within the brain for anti-inflammatory treatment in neurological diseases. http://repub.eur.nl/res/pub/19548/ 2010-01-15T00:00:00Z RAS oncogenes are among the most frequently mutated genes in human cancer, but effective strategies for therapeutic inhibition of the RAS pathway have been elusive. Sprouty1 (SPRY1) is an upstream antagonist of RAS that is activated by extracellular signal-related kinase (ERK), providing a negative feedback loop for RAS signaling, and other evidence suggests that SPRY1 may have a tumor suppressor function. Studies of RAS status in the human childhood tumor rhabdomyosarcoma (RMS) indicated mutations in approximately half of the tumors of the embryonal rhabdomyosarcoma subtype (ERMS) but not the alveolar subtype (ARMS). ERMS tumors also showed overexpression of SPRY1, which was indeed upregulated by mutant RAS. However, we found that, in the presence of mutant RAS, the function of SPRY1 was changed from an antagonist to an agonist of RAS signaling. Thus, SPRY1 supported formation of activated ERK and mitogen-activated protein/ERK kinase and was essential for ERMS cell proliferation and survival. Conversely, silencing of SPRY1 in ERMS cells (but not ARMS cells) abolished their tumorigenicity in mice. Moreover, silencing of SPRY1 caused regression of established ERMS tumors (but not ARMS tumors) formed in xenograft settings. Our findings argue that SPRY1 inhibition can offer a therapeutic strategy to treat childhood RMS and possibly other tumors carrying oncogenic RAS mutations.