Surface Phosphatidylserine Is Responsible for the Internalization on Microvesicles Derived from Hypoxia-Induced Human Bone Marrow Mesenchymal Stem Cells into Human Endothelial Cells.

Previous data have proven that microvesicles derived from hypoxia-induced mesenchymal stem cells (MSC-MVs) can be internalized into endothelial cells, enhancing their proliferation and vessel structure formation and promoting in vivo angiogenesis. However, there is a paucity of information about how the MSC-MVs are up-taken by endothelial cells.MVs were prepared from the supernatants of human bone marrow MSCs that had been exposed to a hypoxic and/or serum-deprivation condition. The incorporation of hypoxia-induced MSC-MVs into human umbilical cord endothelial cells (HUVECs) was observed by flow cytometry and confocal microscopy in the presence or absence of recombinant human Annexin-V (Anx-V) and antibodies against human CD29 and CD44. Further, small interfering RNA (siRNA) targeted at Anx-V and PSR was delivered into HUVECs, or HUVECs were treated with a monoclonal antibody against phosphatidylserine receptor (PSR) and the cellular internalization of MVs was re-assessed.The addition of exogenous Anx-V could inhibit the uptake of MVs isolated from hypoxia-induced stem cells by HUVECs in a dose- and time-dependent manner, while the anti-CD29 and CD44 antibodies had no effect on the internalization process. The suppression was neither observed in Anx-V siRNA-transfected HUVECs, however, addition of anti-PSR antibody and PSR siRNA-transfected HUVECs greatly blocked the incorporation of MVs isolated from hypoxia-induced stem cells into HUVECs.PS on the MVs isolated from hypoxia-induced stem cells is the critical molecule in the uptake by HUVECs

Thrombin promotes fibronectin secretion by bone marrow mesenchymal stem cells via the protease-activated receptor mediated signalling pathways

National Natural Scientific Foundation of China [30971068, 30871018]; National 863 Plans Projects of China [2011AA020101]Introduction: Fibronectin (FN) is commonly used in the development of serum-free media for the expansion of mesenchymal stem cells (MSCs). This study was aimed to observe if thrombin could stimulate FN secretion by human bone marrow MSCs and investigate the potential underlying mechanisms. Methods: PCR was performed to detect the expression of the protease-activated receptors (PARs) in MSCs. After thrombin treatment, the expression level and secretion of FN were observed by RT-PCR, immunofluorescence staining and ELISA, respectively, and the activation of ERK1/2 and NF kappa B pathways was revealed by Western blotting, with or without pre-treatment of small-molecule blockers specific for PAR-1 and -2. The phenotypic and functional activities of thrombin-treated MSCs were also observed. Results: PCR analysis showed that human bone marrow MSCs expressed two subtypes of PARs, PAR-1 and PAR-2. Thrombin treatment enhanced MSCs to express FN at mRNA and protein levels and promoted FN secretion by MSCs, accompanied by potent adherence to the culture plastic. Thrombin induced prompt phosphorylation of ERK 1/2 and NF kappa B p65 and the stimulatory effects of thrombin on FN secretion were blunted by specific inhibitors of these signaling molecules. Blockage to PAR-1 and PAR-2 partially abrogated thrombin-elicited FN secretion by MSCs and ERK 1/2 phosphorylation, whereas that of NF kappa B p65 was unaffected. Moreover, thrombin-treated MSCs maintained the phenotypic features, in vitro osteogenesis and adipogenesis capacities, and inhibitory activity on Phytohemagglutinin-induced allogeneic lymphocyte proliferation. Conclusions: Thrombin could promote FN secretion by MSCs via PAR-mediated ERK 1/2 activation, while NF kappa B might be also involved in an undefined manner

Down-regulation of PSR in HUVECs inhibits their up-taking of MSC-MVs.

<p>(A) PSR expression on HUVECs, PSR siRNA-transfected HUVECs (siRNA) and control RNA-treated HUVECs (siRNA-CTR) was evaluated by flow cytometry. The hollow diagrams represent the fluorescence intensity of cells reacted with the FITC-conjugated antibody. (B) The uptake of CFSE-labeled MSC-MVs was observed with flow cytometry. X-axis: the forward scatter corner signals. Y-axis: CFSE intensity. (C)The t-test showed that the PSR expression was significantly decreased by si-RNA (vs.CTR * P<0.05; vs.siRNA-CTR # P<0.05). (D) PSR siRNA significantly inhibited the internalization of MVs (vs.CTR * P<0.05; vs.siRNA-CTR # P<0.05). These results are representative of the data from three separate experiments.</p

Incorporation of MSC-MVs into HUVECs observed with confocal laser microscopy.

<p>(A) DiI-labeled MSC-MVs were added into the HUVEC culture. The cells were fixed and DAPI-stained, followed by confocal observation. Red: DiI-MVs; Blue: DAPI; Ordinary light: HUVEC; and Merge: the merged images of the three above. Bar: 10μm. (B) The mean fluorescence intensity of DiI in per field. The results are representative of three individual experiments.</p

MSC-MVs were engulfed by HUVECs dose- and time-dependent.

<p>(A) CFSE-labeled MSC-MVs were added to the culture medium of HUVECs at a dose of 10μg/ml and the cells were collected at different time-points. (B) Flow cytometry graphs analysis the mean fluorescent values of tripical experiments, error bars are +/- S.D.,*P<0.001; (C) HUVEC culture was maintained for 12 hours in the presence of graded concentrations of MSC-MVs. X-axis: CFSE fluorescence intensity; Y-axis: forward scatter corner signals showing the size of the gated events. (D) MVs internalization is dose-dependent (*P<0.001, n = 3).</p

Identification of MSC-MVs with electron microscopy (A) and flow cytometry (B).

<p>(A) The bar represents 100nm. (B) MSC-MVs were conjugated with aldehyde/sulfate latex beads and reacted with fluorescein-labeled antibodies or Anx-V. The events were collected with a flow cytometer and the single beads (red) and the doublets of beads (green) were gated for further analysis. The percentages of the positivity in contrast to an isotype antibody are indicated. X-axis: forward scatter corner signals showing the size of the gated events. Beads: Beads were collected for the determination of the gates. Beads+MVs: MVs conjugated with beads were collected for further determination of the gates for analysis. CTR: MVs reacted with a PE-labeled isotype antibody. The results are representative of three individual experiments.</p

Blockage of PSR suppresses the engulfment of MSC-MVs by HUVECs.

<p>(A) PSR expression on the HUVECs was detected with confocal microscopy analysis. PSR: green; DAPI: the nuclear (blue); Bar: 20μm. (B) HUVECs were pre-treated with anti-PSR antibody and cultured in the presence of DiI-labeled MSC-MVs. Twelve hours later, the cells were fixed and observed under a confocal microscope. CTR: HUVEC with DiI-MV; PSR-Antibody: HUVEC+PSR-Antibody with DiI-MV; Bar: 20μm. (C) Blockage of PSR with a specific antibody greatly decreased the internalization of MVs into HUVECs (<i>vs</i>.CTR ^***P<0.0001, n = 2).</p

Western blot analysis on the expression of the exosomal markers CD63, CD81, and CD9 in MSCs and exosome-like vesicles.

<p>Western blot analysis on the expression of the exosomal markers CD63, CD81, and CD9 in MSCs and exosome-like vesicles.</p