A pericyte-derived angiopoietin-1 multimeric complex induces occludin gene expression in brain capillary endothelial cells through Tie-2 activation in vitro
A pericyte-derived angiopoietin-1 multimeric complex induces occludin gene expression in brain capillary endothelial cells through Tie-2 activation in vitro
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DOI：
10.1111/j.1471-4159.2004.02343.x
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发表时间：
2004-04-01
影响因子：
4.7
通讯作者：
Terasaki, T
中科院分区：
文献类型：
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作者：
Hori, S;Ohtsuki, S;Terasaki, T
尽管血脑屏障(BBB)上的紧密连接(TJ)对于阻止化合物非特异性进入中枢神经系统非常重要，但调节TJ维持的分子机制仍不清楚。因此，本研究的目的是利用条件永生化的成年大鼠脑毛细血管内皮细胞(TR-BBB13)、II型星形胶质细胞(TR-AST4)和脑周细胞(TR-PCT1)细胞系，鉴定来自星形胶质细胞和周细胞的调节occludin表达的分子，这些细胞包裹着脑微血管。TransFilter与tr-AST4细胞共培养，并暴露于tr-ast4细胞条件培养液(AST-CM)或tr-PCT1细胞条件培养液(PCT-CM)中，可增加tr-BBB13细胞的occludin mRNA。血管生成素-1中和抗体可显著抑制PCT-CM诱导的封闭素上调，而AST-CM对其上调无明显影响。免疫沉淀和免疫印迹分析证实，多聚体Angiopoietin-1是由Trp-PCT1细胞分泌的，并通过Tie-2的酪氨酸磷酸化在TrBB13细胞中诱导阻滞素mRNA的表达。一项分级的AST-CM研究表明，分子量在30-100 kDa范围内的因素导致了闭塞素的诱导。相反，转化生长因子β1降低了阻滞素的mRNA表达，在低氧处理后，其在TR-AST4细胞中的表达上调。总之，体外血脑屏障模型研究表明，周细胞来源的多聚体血管生成素-1/Tie-2途径诱导了occludin的表达。
Although tight-junctions (TJs) at the blood-brain barrier (BBB) are important to prevent non-specific entry of compounds into the CNS, molecular mechanisms regulating TJ maintenance remain still unclear. The purpose of this study was therefore to identify molecules, which regulate occludin expression, derived from astrocytes and pericytes that ensheathe brain microvessels by using conditionally immortalized adult rat brain capillary endothelial (TR-BBB13), type II astrocyte (TR-AST4) and brain pericyte (TR-PCT1) cell lines. Transfilter co-culture with TR-AST4 cells, and exposure to conditioned medium of TR-AST4 cells (AST-CM) or TR-PCT1 cells (PCT-CM) increased occludin mRNA in TR-BBB13 cells. PCT-CM-induced occludin up-regulation was significantly inhibited by an angiopoietin-1-neutralizing antibody, whereas the up-regulation by AST-CM was not. Immunoprecipitation and western blot analyses confirmed that multimeric angiopoietin-1 is secreted from TR-PCT1 cells, and induces occludin mRNA, acting through tyrosine phosphorylation of Tie-2 in TR-BBB13 cells. A fractionated AST-CM study revealed that factors in the molecular weight range of 30-100 kDa led to occludin induction. Conversely, occludin mRNA was reduced by transforming growth factor beta1, the mRNA of which was up-regulated in TR-AST4 cells following hypoxic treatment. In conclusion, in vitro BBB model studies revealed that the pericyte-derived multimeric angiopoietin-1/Tie-2 pathway induces occludin expression.