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: Laminar shear stress (LSS) boosts medium/large extracellular vesicle (m/lEV) release from HUVECs through activation of Piezo1. (A) In contrast to the static control (SC), LSS (15 dyn/cm 2 ) stimulated substantial EV release from HUVECs as visualized by a confocal microscope ( n = 10 cells from five independent experiments). PKH26 was used to label the membrane structures of EVs and cells. (B) Cumulative EV‐releasing number per cell along with time in response to a single dose of LSS ( n = 100 cells from 10 independent experiments). (C) Cumulative EV‐releasing number per cell along with time in response to increasing doses of LSS ( n = 18 cells from three independent experiments). (D) Quantification of percentage of cells releasing EVs and EV‐releasing number per cell. Left panel: the percentage of cells releasing EVs in total cells ( n = 6). Right panel: the EV‐releasing number per cell ( n = 200 cells from six independent experiments). (E) Single Slice‐SIM imaging revealed that LSS enhanced EV release through membrane budding ( n = 50 cells from three independent experiments). (F) Transmissible electron microscope imaging demonstrated that LSS triggered the release of EVs shed from the membrane of HUVECs ( n = 20 cells from three independent experiments). (G) Scanning electron microscope imaging revealed that EVs were distributed on the cell surface, and some of them were budding from the cell surface with membrane‐continuous necks ( n = 20 cells from three independent experiments). (H) LSS‐induced EVs were Annexin A1‐positive (microvesicle biomarker) ( n = 3). (I) The protocol for isolation of m/lEVs from HUVECs. (J) The morphology of the isolated m/lEVs was imaged using transmission electron microscopy ( n = 4). (K) LSS did not alter the size distribution of m/lEVs as detected by NTA. (L) Quantification of particle number of m/lEVs by NTA ( n = 6). (M) Western blot analysis of LSS‐released m/lEVs with specific biomarkers (Annexin A1, CD9, MMP2, TSG101, ALIX, Calnexin, APOB and cleaved caspase 3). WCL: Whole cell lysate ( n = 4). (N) Yoda1 (Piezo1 agonist, 2 µM) promoted m/lEV release as detected by Western blot ( n = 4). (O) Yoda1 enhanced m/lEV release as visualized by confocal microscope ( n = 200 cells from three independent experiments). (P) GsMTx4 (Piezo1 inhibitor, 5 µM) suppressed LSS‐induced m/lEV release as visualized by confocal microscope ( n = 200 cells from three independent experiments). (Q) LSS elevated Piezo1 phosphorylation ( n = 4). (R) Piezo1 reduction reduced LSS‐induced m/lEV release as visualized by confocal microscope ( n = 200 cells from three independent experiments). In imaging experiments, HUVECs were subjected to either 15 dyn/cm 2 LSS for 300 s or Yoda1 for 10 min, unless otherwise indicated. For Western blot analysis, HUVECs were treated with 15 dyn/cm 2 LSS or Yoda1 for 24 h to ensure adequate EV collection. All inhibitors were added 5 min prior to LSS stimulation. Data are presented as mean ± SEM. ** p < 0.01.

Article Snippet: The following commercially available ELISA kits were used to measure protein levels in plasma of human: Human CD9 (Cusabio, China) and Human Piezo1 Elisa kit (Finetest, China).

Techniques: Shear, Activation Assay, Control, Confocal, Microscopy, Membrane, Imaging, Biomarker Discovery, Isolation, Morphology, Transmission Assay, Electron Microscopy, Western Blot, Analysis, Phospho-proteomics

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: Inhibition of m/lEV release inhibits the effects of shear stress on promoting endothelial function. (A) Treatment with Yoda1 (Piezo1 agonist, 2 µM) for 24 h increased m/lEV release, whereas both pantethine and Y27632 attenuated this effect ( n = 5). (B, C) Inhibition of m/lEV release by pantethine (50 µM) and Y27632 (2 µM) abolished Yoda1‐induced benefits in HUVECs, as determined by cell migration (B) and cell viability (C) ( n = 5). HUVECs were pre‐treated with pantethine or Y27632 for 5 min followed by Yoda1 treatment for 24 h. (D) Pantethine (150 mg/kg, i.p.) and Y27632 (5 mg/kg, i.p.) were used to test whether inhibiting m/lEV release causes vascular endothelial dysfunction in vivo. Pantethine or Y27632 was injected to mice at 30 min prior to each exercise. The mice were subjected to treadmill exercise at 18 m/min, 60 min/d, 5 days a week, for 1 month. All indices were tested after 1 month of exercise. (E) Schematic diagram of plasma m/lEV isolation and purification. (F) The presence of the EV markers Annexin A1, CD9, MMP2 and TSG101 were determined in SEC fractions 7–9 (500 µL/fraction) using Western blotting. Fractions 7–9 were identified as the m/lEV‐rich fractions ( n = 4). (G) Both pantethine and Y27632 blocked the upregulation of circulating m/lEVs induced by exercise, as determined by NTA ( n = 8). (H) The exercise‐induced increase in exhaustion distance on the treadmill was abolished by pantethine and Y27632 ( n = 8). (I) Pantethine or Y27632 suppressed exercise‐promoted endurance of forelimb grip strength ( n = 6). (J) Pantethine and Y27632 reduced the effect of exercise on decreasing aortic intima‐media thickness ( n = 8). (K) Treatment with pantethine or Y27632 blocked the effect of exercise on reducing blood pressure ( n = 8). (L) Pantethine and Y27632 suppressed the effect of exercise on improving endothelium‐dependent vasodilation in isolated arteries ( n = 6). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01.

Article Snippet: The following commercially available ELISA kits were used to measure protein levels in plasma of human: Human CD9 (Cusabio, China) and Human Piezo1 Elisa kit (Finetest, China).

Techniques: Inhibition, Shear, Migration, In Vivo, Injection, Clinical Proteomics, Isolation, Purification, Western Blot

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 following commercially available ELISA kits were used to measure protein levels in plasma of human: Human CD9 (Cusabio, China) and Human Piezo1 Elisa kit (Finetest, China).

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

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: Elevated Piezo1 levels in m/lEVs are associated with spleen contraction during exercise in healthy adults. (A) Schematic of the experimental protocol for a single bout of treadmill running in healthy adults. (B) Acute exercise reduced the spleen area in both coronal and transverse planes ( n = 20). (C) Piezo1 was visualized by immunoelectron microscope in m/lEVs isolated from blood. (D) The number of circulating m/lEVs was increased after exercise. (E) Exercise upregulated Piezo1 and microvesicle biomarkers (CD9 and Annexin A1) in m/lEVs as detected by western blot ( n = 6). (F) Exercise increased CD9 level in circulating m/lEVs as detected by ELISA kits ( n = 20). (G) Exercise upregulated Piezo1 level in circulating m/lEV as measurement by ELISA kits ( n = 20). (H) Correlation heatmap of spleen contraction (coronal/transverse planes) and candidate biomarkers (CD9, Piezo1, dorsal pedal artery blood flow, etc.). Color intensity denotes correlation strength (red: positive, blue: negative). Values represent Pearson's correlation coefficients ( r ), with asterisks indicating statistical significance ( n = 20). (I) Feature importance ranking in the Ridge model for predicting coronal plane spleen contraction magnitude. Bar length represents contribution weight ( n = 20). (J) Feature importance ranking for transverse plane spleen contraction prediction ( n = 20). Data are presented as mean in the bar graph. Data are presented as the median with interquartile range in the violin plots. * p < 0.05, ** p < 0.01.

Article Snippet: The following commercially available ELISA kits were used to measure protein levels in plasma of human: Human CD9 (Cusabio, China) and Human Piezo1 Elisa kit (Finetest, China).

Techniques: Microscopy, Isolation, Western Blot, Enzyme-linked Immunosorbent Assay