Endothelial and mural laminin-α5 contributes to neurovascular integrity maintenance

Background Laminin-alpha 5, a major component of the basal lamina, is predominantly synthesized by endothelial and mural cells (pericytes and vascular smooth muscle cells) in the CNS. Loss of laminin-alpha 5 in either population fails to induce any abnormalities due to functional redundancy. Thus, the functional significance of laminin-alpha 5 in neurovascular integrity remains unknown. Here, we hypothesize that ablation of laminin-alpha 5 in both endothelial and mural cells increases neurovascular permeability. Methods The compound knockout mice were generated by crossing laminin-alpha 5 floxed mice with Tie2-Cre and PDGFR beta-Cre, which target endothelial cells and mural cells, respectively. Neurovascular permeability in these mutants was determined with both exogenous and endogenous tracers. Endothelial paracellular and transcellular permeability was assessed by examining the expression of tight junction proteins and transcytosis-associated proteins. In addition, transmission electron microscopy (TEM) was used to visualize tight junction ultrastructure and endothelial caveolae vesicles. Defects in pericytes and astrocytes were investigated by examining pericyte coverage/contact and astrocyte polarity. Results Elevated neurovascular permeability was observed in the mutants. Subsequent studies found increased Caveolin-1 and decreased major facilitator superfamily domain-containing protein 2a (MFSD2A) expression, but unaltered Claudin-5 or zonula occludens-1 (ZO-1) expression. Consistent with these results, mutant mice exhibited increased endothelial caveolae vesicle number with intact tight junction structure under TEM. Additionally, pericyte coverage and contact were also decreased in the mutant mice, while astrocyte polarity was unaffected. Conclusions These results strongly indicate that endothelial and mural cell-derived laminin-alpha 5 actively maintains neurovascular integrity via the transcellular rather than paracellular mechanism.