A genomic comparison of in vivo and in vitro brain microvascular endothelial cells.
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The blood-brain barrier (BBB) is composed of uniquely differentiated brain microvascular endothelial cells (BMEC). Often, it is of interest to replicate these attributes in the form of an in vitro model, and such models are widely used in the research community. However, the BMEC used to create in vitro BBB models de-differentiate in culture and lose many specialized characteristics. These changes are poorly understood at a molecular level, and little is known regarding the consequences of removing BMEC from their local in vivo microenvironment. To address these issues, suppression subtractive hybridization (SSH) was used to identify 25 gene transcripts that were differentially expressed between in vivo and in vitro BMEC. Genes affected included those involved in angiogenesis, transport and neurogenesis, and real-time quantitative polymerase chain reaction (qPCR) verified transcripts were primarily and significantly downregulated. Since this quantitative gene panel represented those BMEC characteristics lost upon culture, we used it to assess how culture manipulation, specifically BMEC purification and barrier induction by hydrocortisone, influenced the quality of in vitro models. Puromycin purification of BMEC elicited minimal differences compared with untreated BMEC, as assessed by qPCR. In contrast, qPCR-based gene panel analysis after induction with hydrocortisone indicated a modest shift of 10 of the 23 genes toward a more 'in vivo-like' gene expression profile, which correlated with improved barrier phenotype. Genomic analysis of BMEC de-differentiation in culture has thus yielded a functionally diverse set of genes useful for comparing the in vitro and in vivo BBB. (+info)
Retinoic acid inhibits Th17 polarization and enhances FoxP3 expression through a Stat-3/Stat-5 independent signaling pathway.
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CD4(+) helper T (Th) cells play a crucial role in the delicate balance between host defense and autoimmune disease. Two important populations of helper T cells are the proinflammatory, interleukin-17 (IL-17)-producing (Th17) cells and the anti-inflammatory forkhead box P3-positive (FoxP3(+)) T regulatory (Treg) cells. Here we show that all-trans retinoic acid (ATRA) and other agonists of the retinoic acid receptor alpha (RARalpha) inhibit the formation of Th17 cells and promote FoxP3 expression. Conversely, inhibition of retinoic acid signaling constrains transforming growth factor beta (TGF-beta1) induction of FoxP3. The effect of ATRA is mediated independently of IL-2, signal transducer and activator of transcription 5 (Stat5) and Stat3, representing a novel mechanism for the induction of FoxP3 in CD4 T cells. As previous studies have shown that vitamin A derivatives are protective in animal models of autoimmune disease, the current data suggest a previously unrecognized role for RARalpha in the regulation of CD4(+) T-cell differentiation and provide a mechanism for the anti-inflammatory effects of retinoic acid. (+info)
Aromatic residues in the C-terminal domain 2 are required for Nanog to mediate LIF-independent self-renewal of mouse embryonic stem cells.
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Nanog was identified by its ability to sustain the LIF-independent self-renewal of mouse embryonic stem (ES) cells and has recently been shown to play a role in reprogramming adult fibroblasts into pluripotent stem cells. However, little is known about the structural basis of these remarkable activities of Nanog. We have previously identified an unusually strong transactivator named CD2 at its C terminus. Here we demonstrate that CD2 is required for Nanog to mediate ES cell self-renewal. Furthermore, deletion and point mutation analysis revealed that CD2 relies on at least seven aromatic amino acid residues to generate its potent transactivating activity. A mutant Nanog bearing alanine substitutions for these seven residues fails to confer LIF-independent self-renewal in mouse ES cells. Substitution of CD2 by the viral transactivator VP16 gave rise to Nanog-VP16, which is 10 times more active than wild-type Nanog in ES cells. Surprisingly, the expression of Nanog-VP16 in mouse ES cells induces differentiation and is thus unable to sustain LIF-independent self-renewal for mouse ES cells. Taken together, our results demonstrate that the CD2 domain of Nanog is a unique transactivator that utilizes aromatic residues to confer specific activity absolutely required for ES self-renewal. (+info)
Epithelial-to-mesenchymal transition is a potential pathway leading to podocyte dysfunction and proteinuria.
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