The contribution of endothelial basement membrane laminins and immune cells to functional integrity of the neurovascular unit

脑中的血管与周围神经元之间的功能性连接通过神经元对局灶性缺血的快速反应来说明，并且通过内皮细胞、星形胶质细胞和神经元以及位于内皮下方并标记CNS实质边界的两种不同的基底膜（BM）（分别称为内皮BM和实质BM）来表现。这些细胞层和非细胞层共同形成血管和神经元之间的结构连续体，现在通常称为神经血管单元（NVU）。虽然NVU的细胞成分是常见的研究主题，但NVU的BM及其对其结构和功能完整性的贡献却知之甚少。我们已经表明，内皮和实质BM的生物化学不同，主要是在其层粘连蛋白亚型的组成，层粘连蛋白α 4和α 5的特点，内皮BM和层粘连蛋白α 2和α 1的特点，实质BM。我们实验室的神经炎症研究还表明，两种BM对白细胞外渗穿过CNS毛细血管后小静脉的影响不同，层粘连蛋白α 5和α 4充当浸润白细胞的迁移线索，而实质BM充当CNS实质的有效屏障。在短暂性大脑中动脉闭塞（MCAO）（一种缺血性中风模型）中使用层粘连蛋白α 4敲除（KO）小鼠（Lama 4-/-）和内皮细胞特异性层粘连蛋白α 5条件性KO小鼠的更近期的研究进一步特别地涉及层粘连蛋白α 5在内皮细胞屏障特性中的作用。此外，MCAO研究揭示了免疫细胞在血管腔内和BM层之间以尚未确定的方式意外积聚，表明血管腔内和/或血管周围区域内的免疫细胞可以与周围CNS实质沟通。我们在这里建议集中于NVU的内皮部分，重点是内皮细胞BM层粘连蛋白，层粘连蛋白α 4和α 5，以及它们如何影响NVU的功能完整性。此外，我们将研究缺血性卒中后免疫细胞与脑血管相互作用的分子机制。这些研究将有助于理解正常NVU功能的重要因素，并可能为开发更有效的治疗方法开辟新的途径，以最大限度地减少缺血性卒中后的脑损伤。

The functional connection between blood vessels in the brain and the surrounding neurons is illustrated by the rapid response of neurons to focal ischemia, and is manifested by endothelial cells, astrocytes and neurons, but also two distinct basement membranes (BMs) that underlie the endothelium and marker the border to the CNS parenchyma (known as the endothelial and parenchymal BMs, respectively). These cellular and acellular layers collectively form a structural continuum between blood vessel and neurons that is now commonly referred to as the neurovascular unit (NVU). While the cellular constituents of the NVU are common topics of research, the BMs of the NVU and their contribution to its structural and functional integrity are poorly understood. We have shown that the endothelial and parenchymal BMs are biochemically distinct, varying principally in their laminin isoforms composition, with laminin alpha4 and alpha5 characterising the endothelial BM and laminin alpha2 and alpha1 characterizing the parenchymal BM. Neuroinflammation studies in our lab have also shown that the two BMs impact differently on leukocyte extravasation across CNS postcapillary venules, with laminin alpha5 and alpha4 acting as migration cues for infiltrating leukocytes and the parenchymal BM acting as the effective barrier to the CNS parenchyma. More recent studies using laminin alpha4 knockout (KO) mice (Lama4-/-), and endothelial cell specific laminin alpha5 conditional KO mice in transient middle cerebral artery occlusion (MCAO), an ischemic stroke model, have further implicated specifically laminin alpha5 in endothelial cell barrier properties. In addition, the MCAO studies revealed an unexpected accumulation of immune cells within vessel lumens and between BMs layers, in an as yet undefined manner, suggesting that immune cells within the vessel lumen and/or perivascular zone can communicate with the surrounding CNS parenchyma. We here propose to focus on the endothelial portion of the NVU, with emphasis on the endothelial cell BM laminins, laminin alpha4 and alpha5, and how they influence the functional integrity of the NVU. In addition, we will investigate the molecular mechanism/s of immune cell interaction with cerebral vessels following ischemic stroke. These studies will contribute to the understanding of factors important for normal NVU function and are likely to open new avenues for the development of more effective therapies to minimize brain damage after ischemic stroke.