The effect of fluid shear stress in hydrogen sulphide production and cystathionine γ-lyase expression in human early endothelial progenitor cells

Abstract

Background: Physiological fluid shear stress has been shown to have a beneficial impact on vascular homeostasis. Endothelial progenitor cells (EPCs) make a significant contribution to maintaining endothelial integrity. Therefore, we hypothesised that shear stress-induced endothelium protection plays a role in hydrogen sulphide (H₂S) production and up-regulation of cystathionine γ-lyase (CSE) expression in EPCs.

Methods: Human EPC-derived CSE activity was detected by colorimetric assay, and H₂S production was evaluated by membrane adsorption method. Cell proliferation, migration, and adhesion were assessed by MTT, Transwell, and endothelial cell-mediated adhesion assays, respectively. Real-time polymerase chain reaction (RT-PCR) was carried out to analyse gene expression. Protein expression was analysed by western blot.

Results: Human EPCs were treated with shear stress levels of 5-25 dyn/cm² for up to 3 h, and 25 dyn/cm² for up to 24 h. H₂S production and CSE mRNA expression in the EPCs were increased by shear stress in a dose-dependent manner in vitro. Likewise, time-dependent shear stress also significantly enhanced CSE protein expression. Compared to static condition, shear stress improved EPCs proliferation, migration and adhesion capacity. Knockdown of CSE expression by small interfering RNA substantially eliminated the shear stress-induced above functions of human EPCs in vitro.

Conclusions: This study gives new insight into the regulatory effect of physiological shear stress on the CSE/H₂S system in human EPCs. Our findings may contribute to the development of vascular protective research, although the relevant evidence is admittedly indirect.

Keywords: Shear stress; cystathionine γ-lyase (CSE); endothelial progenitor cells (EPCs); hydrogen sulphide (H2S).

Figure 1

Characterization of cultured human early EPCs. (A) Representative photographs of EPCs on day 7 labelled with FITC-lectin (green), Dil-acLDL (red), and DAPI (blue), and a merged image (×200 magnification). (B) Flow cytometry analysis of the endothelial markers CD31, vWF, KDR, and the monocytic lineage marker CD14 in EPCs (IgG isotype was the control, n=5 per group). Numbers are the mean ± SEM percentage of positive cells for all experiments determined by comparison with the corresponding labelled negative control. EPC, endothelial progenitor cell; FITC, fluorescein isothiocyanate; DAPI, 4’,6-diamidino-2-phenylindole; KDR, kinase-insert domain receptor.

Figure 2

H₂S production by human EPCs in response to different levels of shear stress. *, P<0.05 vs. stationary group. H₂S, hydrogen sulphide; EPC, endothelial progenitor cell.

Figure 3

CSE activity (A), mRNA (B) and protein (C) expression in human EPCs in response to shear stress. *, P<0.05 vs. stationary group. CSE, cystathionine γ-lyase; EPC, endothelial progenitor cell.

Figure 4

Effect of CSE expression on shear stress (SS)-induced functions of human EPCs. (A) Protein expression in EPCs after CSE siRNA or scrambled siRNA transduction (*, P<0.05 vs. EPCs or Scrambled siRNA EPCs, n=5). (B) Quantification analysis of proliferation of human EPCs by SS. (*, P<0.05 vs. EPCs, ^#, P<0.05 vs. SS + EPCs or SS + EPCs + Scrambled siRNA, n=5). (C) Representative photographs and quantification analysis of migration of human EPCs by SS (*, P<0.05 vs. EPCs, ^#, P<0.05 vs. SS + EPCs or SS + EPCs + Scrambled siRNA, n=5). (D) Representative photographs and quantification analysis of adhesion of human EPCs by SS (*, P<0.05 vs. EPCs, ^#, P<0.05 vs. SS + EPCs or SS + EPCs + Scrambled siRNA, n=5). CSE, cystathionine γ-lyase; EPC, endothelial progenitor cell. Scar bars =100 µm.