Blood-brain barrier (BBB) characteristics are induced and maintained by cross-talk between brain microvessel endothelial cells and neighbouring elements of the neurovascular unit. While pericytes are the cells situated closest to brain endothelial cells morphologically and share a common basement membrane, they have not been used in co-culture BBB models for testing drug permeability. We have developed and characterized a new syngeneic BBB model using primary cultures of the three main cell types of cerebral microvessels. The co-culture of endothelial cells, pericytes and astrocytes mimick the anatomical situation in vivo. In the presence of both pericytes and astrocytes rat brain endothelial cells expressed enhanced levels of tight junction (TJ) proteins occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. Further morphological evidence of the presence of interendothelial TJs was provided by electron microscopy. The transendothelial electrical resistance (TEER) of brain endothelial monolayers in triple co-culture, indicating the tightness of TJs reached 400Omegacm(2) on average, while the endothelial permeability coefficients (P(e)) for fluorescein was in the range of 3x10(-6)cm/s. Brain endothelial cells in the new model expressed glucose transporter-1, efflux transporters P-glycoprotein and multidrug resistance protein-1, and showed a polarized transport of rhodamine 123, a ligand for P-glycoprotein. To further characterize the model, drug permeability assays were performed using a set of 19 compounds with known in vivo BBB permeability. Good correlation (R(2)=0.89) was found between in vitroP(e) values obtained from measurements on the BBB model and in vivo BBB permeability data. The new BBB model, which is the first model to incorporate pericytes in a triple co-culture setting, can be a useful tool for research on BBB physiology and pathology and to test candidate compounds for centrally acting drugs.

No abstract

Arodent cardiac side population cell fraction formed clonal spheroids in serum-free medium, which expressed nestin, Musashi-1, and multi-drug resistance transporter gene 1, markers of undifferentiated neural precursor cells. These markers were lost following differentiation, and were replaced by the expression of neuron-, glial-, smooth muscle cell–, or cardiomyocyte-specific proteins. Cardiosphere-derived cells transplanted into chick embryos migrated to the truncus arteriosus and cardiac outflow tract and contributed to dorsal root ganglia, spinal nerves, and aortic smooth muscle cells. Lineage studies using double transgenic mice encoding protein 0–Cre/Floxed-EGFP revealed undifferentiated and differentiated neural crest-derived cells in the fetal myocardium. Undifferentiated cells expressed GATA-binding protein 4 and nestin, but not actinin, whereas the differentiated cells were identified as cardiomyocytes. These results suggest that cardiac neural crest-derived cells migrate into the heart, remain there as dormant multipotent stem cells—and under the right conditions—differentiate into cardiomyocytes and typical neural crest-derived cells, including neurons, glia, and smooth muscle.

Abstract-We recently reported that angiotensin II (Ang II) induced IL-6 mRNA expression in cardiac fibroblasts, which played an important role in Ang II-induced cardiac hypertrophy in paracrine fashion. The present study investigated the regulatory mechanism of Ang II-induced IL-6 gene expression, focusing especially on reactive oxygen species (ROS)-mediated signaling in cardiac fibroblasts. Ang II increased intracellular ROS in cardiac fibroblasts, and the increase was completely inhibited by the AT-1 blocker candesartan and the NADH/NADPH oxidase inhibitor diphenyleneiodonium (DPI). We first confirmed that antioxidant N-acetylcysteine, superoxide scavenger Tiron, and DPI suppressed Ang II-induced IL-6 expression. Because we observed that exogenous H 2 O 2 also increased IL-6 mRNA, the signaling pathways downstream of Ang II and exogenous H 2 O 2 were compared. Ang II, as well as exogenous H 2 O 2 , activated ERK, p38 MAPK, and JNK, which were significantly inhibited by N-acetylcysteine and DPI. In contrast with exogenous H 2 O 2 , however, Ang II did not influence phosphorylation and degradation of IB-␣/␤ or nuclear translocation of p65, nor did it increase NF-B promoter activity. PD98059 and SB203580 inhibited Ang II-induced IL-6 expression. Truncation and mutational analysis of the IL-6 gene promoter showed that CRE was an important cis-element in Ang II-induced IL-6 gene expression. NF-B-binding site was important for the basal expression of IL-6, but was not activated by Ang II. Ang II phosphorylated CREB through the ERK and p38 MAPK pathway in a ROS-sensitive manner. Collectively, these data indicated that Ang II stimulated ROS production via the AT1 receptor and NADH/NADPH oxidase, and that these ROS mediated activation of MAPKs, which culminated in IL-6 gene expression through a CRE-dependent, but not NF-B-dependent, pathway in cardiac fibroblasts. Key Words: angiotensin II Ⅲ interleukin-6 Ⅲ reactive oxygen species Ⅲ mitogen-activated protein kinase Ⅲ cardiac fibroblast C linical, experimental, and genetic data have demonstrated that the renin angiotensin system is linked to the pathogenesis of a variety of cardiac diseases. Expression of angiotensin II (Ang II), the key effector molecule of the renin angiotensin system, is increased under various pathophysiological conditions and stimulates cardiomyocyte hypertrophy and interstitial fibrosis coinciding with accumulation of extracellular matrix. Recent reports had shown that Ang II stimulates membrane-bound NAD(P)H oxidase, which generates reactive oxygen species (ROS) in a variety of nonphagocytic cells. 1 ROS may act as a second messenger that regulates various intracellular signal transduction cascades and the activity of various transcription factors. NF-B and AP-1 are the best characterized transcription factors to be influenced by the cellular oxidation-reduction (redox) state. 2,3 The primary target of activation of NF-B by ROS appears to be the phosphorylation and subsequent degradation of IB. 4 Another well-characterized redox sensitive signali...