Astrocytes (AC) are essential for brain homeostasis. Much data suggests that AC support and protect the vascular endothelium, but increasing evidence indicates that during injury conditions they may lose their supportive role resulting in endothelial cell activation and BBB disturbance. Understanding the triggers that flip this switch would provide invaluable information for designing new targets to modulate the brain vascular compartment. Hypoxia-inducible factor-1 (HIF-1) has long been assumed to be a culprit for barrier dysfunction as a number of its target genes are potent angiogenic factors. Indeed AC themselves, reservoirs of an array of different growth factors and molecules, are frequently assumed to be the source of such molecules although direct supporting evidence is yet to be published. Being well known reservoirs of HIF-1 dependent angiogenic molecules, we asked if AC HIF-1 dependent paracrine signaling drives brain EC disturbance during hypoxia. First we collected conditioned media from control and siRNA-mediated HIF-1 knockdown primary rat AC that had been exposed to normoxic or hypoxic conditions. The conditioned media was then used to culture normoxic and hypoxic (1% O2) rat brain microvascular EC (RBE4) for 6 and 24 h. Various activation parameters including migration, proliferation and cell cycling were assessed and compared to untreated controls. In addition, tight junction localization and barrier stability per se (via permeability assay) was evaluated. AC conditioned media maintained both normoxic and hypoxic EC in a quiescent state by suppressing EC metabolic activity and proliferation. By FACs we observed reduced cell cycling with an increased number of cells in G0 phase and reduced cell numbers in M phase compared to controls. EC migration was also blocked by AC conditioned media and in correlation hypoxic tight junction organization and barrier functionality was improved. Surprisingly however, AC HIF-1 deletion did not impact EC responses or barrier stability during hypoxia. This study demonstrates that AC HIF-1 dependent paracrine signaling does not contribute to AC modulation of EC barrier function under normoxic or hypoxic conditions. Thus other cell types likely mediate EC permeability in stress scenarios. Our data does however highlight the continuous protective effect of AC on the barrier endothelium. Exploring these protective mechanisms in more detail will provide essential insight into ways to prevent barrier disturbance during injury and disease.

中文翻译：

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星形胶质细胞特异性缺氧诱导因子 1 (HIF-1) 在体外缺氧期间不会破坏内皮屏障

星形胶质细胞 (AC) 对大脑稳态至关重要。许多数据表明 AC 支持和保护血管内皮，但越来越多的证据表明，在损伤条件下，它们可能会失去支持作用，导致内皮细胞激活和 BBB 紊乱。了解触发此开关的触发器将为设计调节脑血管室的新目标提供宝贵的信息。缺氧诱导因子-1 (HIF-1) 长期以来一直被认为是屏障功能障碍的罪魁祸首，因为其许多靶基因是有效的血管生成因子。事实上，AC 本身是一系列不同生长因子和分子的储存库，通常被认为是这些分子的来源，尽管尚未公布直接的支持证据。作为众所周知的 HIF-1 依赖性血管生成分子的储存库，我们询问 AC HIF-1 依赖性旁分泌信号是否在缺氧期间驱动脑 EC 紊乱。首先，我们从暴露于常氧或缺氧条件的对照和 siRNA 介导的 HIF-1 敲低原代大鼠 AC 中收集条件培养基。然后使用条件培养基培养常氧和缺氧 (1% O2) 大鼠脑微血管 EC (RBE4) 6 和 24 小时。评估各种激活参数，包括迁移、增殖和细胞周期，并与未处理的对照进行比较。此外，对紧密连接定位和屏障稳定性本身（通过渗透性测定）进行了评估。AC 条件培养基通过抑制 EC 代谢活动和增殖将常氧和缺氧 EC 维持在静止状态。通过 FAC，我们观察到细胞周期减少，与对照组相比，G0 期细胞数量增加，M 期细胞数量减少。EC 迁移也被 AC 条件培养基阻止，并且相关的缺氧紧密连接组织和屏障功能得到改善。然而，令人惊讶的是，AC HIF-1 缺失在缺氧期间不会影响 EC 反应或屏障稳定性。该研究表明 AC HIF-1 依赖性旁分泌信号在常氧或缺氧条件下对 EC 屏障功能的 AC 调节没有贡献。因此，其他细胞类型可能在压力情况下介导 EC 通透性。然而，我们的数据确实强调了 AC 对屏障内皮的持续保护作用。