Journal: Stem cell research & therapy

Article Title: New mechanism for mesenchymal stem cell microvesicle to restore lung permeability: intracellular S1P signaling pathway independent of S1P receptor-1.

doi: 10.1186/s13287-022-03177-4

Figure Lengend Snippet: Fig. 4 MSC MV restored the rearrangement of the cytoskeleton protein F-action, the loss of tight junction protein ZO-1, and adherens junction protein VE-cadherin and β-catenin of LPS injured HLMVEC independent of S1P receptor-1. HLMVEC were seeded on cell slides and stained with A phalloidin (green) for F-actin, B Goat anti-rabbit IgG H&L (Alexa flour®488) (green) for ZO-1, and C Goat anti-mouse IgG H&L (Alexa flour®488) (green) for VE-cadherin. F-actin staining in HLMVEC of the control group showed a typical peripheral distribution, and VE-cadherin and ZO-1 staining were strong at the junctions between cells. After being exposed to LPS for 24 h, F-actin of cells reorganized into the center of the cell to form “actin stress fibers,” and the staining of cell adhesion junction protein VE-cadherin and cell tight junction protein ZO-1 was lost, resulting in an increase in cell pore size, which may be the reason for the increased protein permeability. The administration of MSC MV largely prevented the reorganization of cytoskeleton protein F-actin into “actin stress fibers” and restored the staining of VE-cadherin and ZO-1 between HLMVEC cells damaged by LPS. Images are representative for each condition run in triplicates. DAPI (blue) was used to stain the cell nuclei. D Western blot analyses showed that the loss in VE-cadherin total protein levels with LPS injury was partially restored by MSC MV treatment. E Western blot analyses showed that the loss in ZO-1 total protein levels with LPS injury was partially restored by MSC MV treatment. F Western blot analyses showed that the loss in β-catenin total protein levels with LPS injury was also partially restored by MSC MV treatment. The therapeutic effect of MSC MVs on the cytoskeleton structure and cell junction proteins was not abolished by adding S1P receptor-1 antagonist W123. Data are presented as mean ± SD, N = 3, *p < 0.05 versus control using ANOVA with post hoc Tukey HSD test. Abbreviations: MSC, mesenchymal stem cell; MV, microvesicles; LPS, lipopolysaccharide; HLMVEC, human lung microvascular endothelial cells; S1P, sphingosine-1-phosphate; and ANOVA, analysis of variance

Article Snippet: Then, PVDF membranes were blocked in TBST containing 5% skim milk for 1 h at room temperature followed by primary antibodies for VE-cadherin (1:1000 dilution, Cell Signaling), ZO-1 (1:1000, Proteintech), Rac1 (1:1000, Proteintech), β-catenin (1:1000, Arizo), SPHK (1:500, HuaAn Biotechnology, Hangzhou, China), CD63 (1:1000, Abcam), TSG101 (1:1000, Abcam) or β-actin (1:1000, Santa Cruz, USA) as loading control at 4 °C overnight.

Techniques: Staining, Control, Pore Size, Permeability, Western Blot

Journal: Acta physiologica (Oxford, England)

Article Title: Increased R-spondin 3 contributes to aerobic exercise-induced protection against renal vascular endothelial hyperpermeability and acute kidney injury.

doi: 10.1111/apha.14036

Figure Lengend Snippet: FIGURE 5 RSPO3 ameliorates LPS-induced hyperpermeability of renal endothelial cells by suppression of MMPs expression. (A) Mice were intraperitoneally injected with LPS (12 mg/kg) with or without recombinant RSPO3 protein (0.25 mg/kg). The control group was injected with saline. Kidney samples were collected 24 h after LPS injection. The mRNA levels of MMP family members including MMP-3, MMP-7, MMP-9, MMP-13, and MMP-14 were determined by qRT-PCR (n = 7, two-way ANOVA with Bonferroni's post-tests). (B) HMVECs were treated with LPS (1 μg/mL) in the presence or absence of RSPO3 (100 ng/mL) for 24 h. The mRNA levels of MMP-3, MMP-7, MMP-9, MMP-13, and MMP-14 were determined by qRT-PCR (n = 4, one-way ANOVA with Bonferroni's post-tests). (C,D) HMVECs were treated with LPS (1 μg/mL) in the presence or absence of BB2516 (10 μM) for 24 h (n = 4, one-way ANOVA with Bonferroni's post-tests). (C) Protein levels of ZO-1, VE-cadherin, SDC-1, and SDC-4 were examined by Western blots. Corresponding histograms were shown on the right panel of the representative protein bands. (D) Permeability of confluent HMVEC monolayer was determined by 10-kDa FITC-dextran flux. (E,F) HMVECs were treated with LPS (1 μg/mL) in the presence or absence of RSPO3 (100 ng/mL) for 24 h (n = 4, one-way ANOVA with Bonferroni's post-tests). (E) Protein levels of ZO-1, VE-cadherin, SDC-1, and SDC-4 were examined by Western blots. Corresponding histograms were shown on the right panel of the representative protein bands. (F) Permeability of confluent HMVEC monolayer was determined by 10-kDa FITC-dextran flux. Data were expressed as mean ± SEM. *p < 0.05, †p < 0.01, ‡p < 0.001.

Article Snippet: Membranes were incubated with primary antibody against β- actin (1:5000; Sigma Aldrich, MO, USA), ZO- 1 (1:500; Proteintech, Chicago, USA), VE- cadherin (1:500; Proteintech), SDC- 1 (1:500; Proteintech), SDC- 4 (1:1000; Abcam, Cambridge, MA, USA), and RSPO3 (1:500; ABclonal, Wuhan, Chain) at 4°C overnight.

Techniques: Expressing, Injection, Recombinant, Control, Saline, Quantitative RT-PCR, Western Blot, Permeability