Journal: Life Science Alliance

Article Title: Brain vascular stability relies on PAK2–cilia–PDGF-BB–HSPGs on basolateral side of endothelium

doi: 10.26508/lsa.202503460

Figure Lengend Snippet: (A) Human microvascular brain ECs cultured in a microfluidic chamber and stained for ciliary marker ARL13b and acetylated α-tubulin in the cross-sectional plane; the white dotted box was enlarged in the middle. (B) Transverse plane of the vessel tube. White arrowheads indicate ARL13b cilia projecting into the basolateral space. Scale bars: 100 μm. (C, C′) Fluorescent image of a 52-hpf transgenic zebrafish Tg( flk :mCherry; b-actin :Arl13b-GFP) expressing mCherry in the vasculature and mouse Arl13b-GFP fusion protein in the cilia. (C′) White boxed area is enlarged in (C′). White arrows in (C′) point to basolateral zebrafish endothelial cilium location. The scale bar in (C, C′) is 50 μm. (D) Quantification of zebrafish endothelial cilia (luminal versus abluminal location) in metencephalic artery (MTA) and middle cerebral vein (MCeV). N = 4 for each vessel group was quantified for the presence of abluminal versus luminal cilia. An unpaired t test showed no difference in the cilium location between the two vessel groups. (E) Immunohistochemistry for abluminal cilia from representative cortical microvessels from ARL13b-EGFP transgenic adult mice ( C57BL/6 background) showing luminal versus abluminal cilium localization stained with anti-GFP. The black arrow points to Arl13b-positive abluminal cilia. Scale bars: 100 μm. (E′) Quantification of Arl13b cilium length on the abluminal versus luminal side of mouse endothelial vessels. (E″) Frequency of cilia on the abluminal versus luminal side of mouse endothelial vessels. A total of 30 endothelial vessel-like structures were quantified. (F) Immunofluorescence staining of a 22-wk-old human brain for ARL13b protein (green) and CD31 vessel staining (red). White arrows point to cilia in the basolateral side of the vessel. Scale bars: 10 μm. (F′) Quantification of endothelial cilial frequency in the ventricular zone (VZ) and subventricular zone (SVZ) in human brain sections. Scale bar in (F) is 10 μm.

Article Snippet: To generate 3D EC vessels, we used the Nortis ParVivo triple-channel microfluidic chips, which we will refer to as the microphysiological systems (MPS).

Techniques: Cell Culture, Staining, Marker, Transgenic Assay, Expressing, Immunohistochemistry, Immunofluorescence