Journal: Journal of Extracellular Vesicles

Article Title: Shear Stress‐Stimulated Trafficking of Endothelial Piezo1 to Splenic Capsule Boosts Spleen Contraction and Exercise Endurance

doi: 10.1002/jev2.70375

Figure Lengend Snippet: Exercise induces Piezo1 translocation from vascular endothelial cells to the splenic capsule. (A) Schematic of genetically engineered mouse model. (B) Acute exercise‐induced upregulation of Piezo1, Annexin A1, CD9 and MMP2 in circulating m/lEVs was blocked in Piezo1 EC −/− mice ( n = 6). (C) Upper panel: The protocol for collecting m/lEVs at baseline, immediately post‐exercise and at 10 and 100 min of recovery. Lower panel: Exercise induced a significant increase in circulating m/lEV levels, which declined post‐exercise ( n = 6). (D) In vivo imaging identified the spleen as a primary target of exercise‐derived m/lEVs ( n = 6). DiR, a lipophilic dye for labelling m/lEVs to monitor their in vivo distribution. An equivalent concentration of free DiR dye in PBS was prepared as control. (E) Acute exercise decreased Piezo1 content in the aorta and increased Piezo1 content in spleen ( n = 6). (F) Acute exercise‐derived and PKH26‐labeled m/lEVs were enriched in the splenic capsule but not splenic parenchyma ( n = 6). PKH26, a lipophilic membrane dye for labelling m/lEVs isolated from the plasma of exercised mice. Splenic capsule is outlined by white dashed lines. (G) Piezo1 were enriched in the splenic capsule 30 min post‐exercise ( n = 6). α‐SMA, a marker for smooth muscle cells, labelling the splenic capsule. (H) qPCR analysis showed no significant change of Piezo1 mRNA expression in the splenic capsule following acute exercise ( n = 6). (I) In LacZ Piezo1 reporter mice (Piezo1 LacZ/+ ), β‐galactosidase staining revealed undetectable endogenous Piezo1 expression in the spleen in mice with and without acute exercise, whereas it was increased in the aorta following exercise. Splenic capsule is outlined by dashed lines. ( n = 6). (J) Piezo1 was located across various subcellular fractions of the splenic capsule and enriched in the plasma membrane fraction following exercise ( n = 8). Lamin B, ATPase Na + /K + transporting subunit alpha 1 (ATP1A1), and actin were used as markers for the nucleus, plasma membrane and cytoplasm, respectively. (K) Vascular ECs were isolated from Piezo1 f/f and Piezo1 EC −/− mice and treated with Yoda1 for 24 h before m/lEV collection. HUVECs were transfected with Piezo1‐GFP plasmid for 60 h before m/lEV collection. m/lEVs were isolated from vascular ECs or HUVECs and incubated with scSMCs for 30 min. (L) Piezo1 protein levels were increased in recipient scSMCs following incubation with m/lEVs from Yoda1‐stimulated wildtype ECs but not Piezo1 knockout ECs ( n = 4). (M) m/lEV‐carried Piezo1 localized to the plasma membrane and the intracellular space of scSMCs ( n = 4). Data are presented as mean ± SEM. ** p < 0.01.

Article Snippet: The purity of SMCs was confirmed through immunofluorescence staining for α‐smooth muscle actin (α‐SMA, 1:200, 48938S, CST, USA) (>70%).

Techniques: Translocation Assay, In Vivo Imaging, Derivative Assay, In Vivo, Concentration Assay, Control, Labeling, Membrane, Isolation, Clinical Proteomics, Marker, Analysis, Expressing, Staining, Transfection, Plasmid Preparation, Incubation, Knock-Out