Salidroside exerts angiogenic and cytoprotective effects on human bone marrow-derived endothelial progenitor cells via Akt/mTOR/p70S6K and MAPK signalling pathways

Abstract

Background and purpose: With the increase of age, increased susceptibility to apoptosis and senescence may contribute to proliferative and functional impairment of endothelial progenitor cells (EPCs). The aim of this study was to investigate whether salidroside (SAL) can induce angiogenic differentiation and inhibit oxidative stress-induced apoptosis in bone marrow-derived EPCs (BM-EPCs), and if so, through what mechanism.

Experimental approach: BM-EPCs were isolated and treated with different concentrations of SAL for up to 4 days. Cell proliferation, migration and tube formation ability were detected by DNA content quantification, transwell assay and Matrigel-based angiogenesis assay. Gene and protein expression were assessed by qRT-PCR and Western blot respectively.

Key results: Treatment with SAL promoted cellular proliferation and angiogenic differentiation of BM-EPCs, and increased VEGF and NO secretion, which in turn mediated the enhanced angiogenic differentiation of BM-EPCs. Furthermore, SAL significantly attenuated hydrogen peroxide (H₂O₂)-induced cell apoptosis, reduced the intracellular level of reactive oxygen species and restored the mitochondrial membrane potential of BM-EPCs. Moreover, SAL stimulated the phosphorylation of Akt, mammalian target of rapamycin and p70 S6 kinase, as well as ERK1/2, which is associated with cell migration and capillary tube formation. Additionally, SAL reversed the phosphorylation of JNK and p38 MAPK induced by H₂O₂ and suppressed the changes in the Bax/Bcl-xL ratio observed after stimulation with H₂O₂.

Conclusions and implications: These findings identify novel mechanisms that regulate EPC function and suggest that SAL has therapeutic potential as a new agent to enhance vasculogenesis as well as protect against oxidative endothelial injury.

Keywords: Akt/mTOR/p70S6K; MAPK; angiogenesis; apoptosis; endothelial progenitor cell; reactive oxygen species; salidroside.

Figure 1

Effect of SAL on cellular proliferation of BM-EPCs. (A) Cells were treated with 0 (control), 10, 20, 40 and 80 μM SAL for 24, 48 and 96 h followed by DNA quantification to determine cellular proliferation, n = 5. (B) PCNA expression was measured by Western blot. The immunoblots shown are representative of at least three independent experiments with comparable results. (C) Densitometric analysis of band intensities of PCNA normalized to GAPDH is shown. Data are shown as mean ± SD, *P < 0.05, **P < 0.01 versus control group.

Figure 2

SAL enhances cell migration capacity in a dose-dependent manner. (A and B) Transwell chemotaxis assay was performed on BM-EPCs with PBS (control), 50 ng·mL⁻¹ Bfgf, and 20, 40 and 80 μM SAL in the lower chamber. To quantify the migrated EPCs on the membrane, the upper side of the membrane was washed and wiped with a cotton swab. Transwell membranes were stained with Alexa Fluor 488 phalloidin and the migrated cells were examined using an inverted fluorescence microscopy. The number of migrated cells was quantified by performing cell counts of 10 random fields. Data are presented as mean ± SD, n = 5, **P < 0.01 versus control group. (C and D) BM-EPCs were treated in the absence or presence of indicated concentrations of SAL and bFGF for 48 h, then seeded into the upper chamber of the transwell with culture medium containing 5% FBS in the lower chamber. Data are presented as mean ± SD, n = 5, *P < 0.05, **P < 0.01 versus control group.

Figure 3

SAL promotes cell–matrix adhesion but inhibits cell–cell adhesion of BM-EPCs. (A) BM-EPCs were treated with 0 (control), 20, 40 and 80 μM SAL for 2 days. Cell adhesion to ECM was performed on fibronectin-treated plates for 30 min followed by staining of adherent cells with Hoechst 33258 dye (nuclei, blue). (B) Quantification of attached cells was determined by DNA content using Quant-iT PicoGreen dsDNA Assay Kit. Data are presented as mean ± SD, n = 5, *P < 0.05, **P < 0.01 versus control group. (C and D) BM-EPCs were grown overnight to a confluent monolayer in Endothelial Cell Growth Medium 2 and labelled with Hoechst 33258 dye. Another set of BM-EPCs was treated with 40 or 80 μM SAL for 48 h, fluorescence-labelled with calcein-AM for 1 h, and plated onto the established cell monolayer. Quantification of cell–cell adhesion was performed by counting the number of cells per microscopic field of view that remained attached after 20 min incubation. Data are presented as mean ± SD, n = 5, *P < 0.05, **P < 0.01 versus control group.

Figure 4

Effect of SAL on capillary tube formation in BM-EPCs. (A) After SAL treatment cells were grown on Matrigel™ for 18 h under normal growth conditions, capillary tube formation was observed by inverted light microscopy. (B) Five independent fields were assessed for each well and the average numbers of tubes per 40× magnified field were determined. Data are expressed as mean ± SD, n = 5, *P < 0.05, **P < 0.01 versus control group.

Figure 5

SAL enhances the secretion of VEGF and the production of NO in BM-EPCs. Cells were cultured until confluency in 96-well plates and subsequently stimulated with 0 (control), 20, 40 and 80 μM SAL. (A) Cell culture supernatants were collected after 24 and 48 h and VEGF secretion was determined by elisa, n = 5. (B) To assess NO production, cell culture supernatants were collected 2, 5, 8 and 10 days after SAL stimulation and analysed by Griess assay, n = 4. (C) Cell culture supernatants were collected 48 h after SAL stimulation with or without 10 μM L-NAME, NO levels were measured by Griess assay, n = 3. (D and E) BM-EPCs were treated with 10 μM L-NAME and 80 μM SAL for 48 h. Cell migratory ability was evaluated by performing cell counts in 10 random fields. Capillary-like tube formation were determined by assessing average numbers of tubes per 40× magnified field, n = 3. All data are presented as mean ± SD, *P < 0.05, **P < 0.01 versus control group.

Figure 6

SAL protects BM-EPCs from H₂O₂-induced cell damage and ROS production. (A) Cells were pretreated with 0 (control), 40, 80 μM SAL and stressed by 1 mM H₂O₂. Cell survival was monitored by calcein AM/PI double staining and analysed qualitatively by fluorescence microscopy. Representative micrographs from each treatment group are shown. (B) After the treatment with SAL and induction by 1 mM H₂O₂, BM-EPCs were washed with PBS and cellular viability was evaluated by quantification of DNA content, n = 6. (C) Cell death was evaluated by LDH assay, n = 4. (D) Cells were pretreated with 40 and 80 μM SAL for 48 h, labelled with 30 μM DCFH-DA and subsequently stressed with H₂O₂ for 6 h prior to fluorescence microscopy. (E) Production of ROS was quantified by the amount of DCF formed in the BM-EPCs. The fluorescence intensity was measured using a microplate reader, n = 6. (F) Cells were treated with 40 and 80 μM SAL for 48 h, followed by H₂O₂ treatment for 6 h. NADPH oxidase activity was measured colourimetrically using NADP/NADPH assay kit, n = 5. (G and H) Cells were treated with 80 μM SAL for 48 h, and then stimulated with H₂O₂ for 6 h after pretreatment with 10 μM DPI. ROS was quantified by DCF formation as described earlier (n = 4), and cellular viability was evaluated by quantification of DNA content (n = 3). All data are expressed as mean ± SD. *P < 0.05, **P < 0.01 versus H₂O₂ group.

Figure 7

SAL inhibits H₂O₂-induced cell apoptosis. BM-EPCs were pretreated with 0 (control), 40, 80 μM SAL for 48 h and stressed by 1 mM H₂O₂. (A) Then cells were labelled with the fluorescent dye Rhodamine 123, the superoxide indicator DHE and the nucleic acid stain Hoechst 33258, n = 4. (B) BM-EPCs were loaded with 5 μM Rhodamine 123. After 20 min incubation in the dark, cells were washed twice with PBS, and the plates were immediately read using a fluorescent plate reader, n = 5. (C) Cells were probed with 10 μM DHE for 30 min, fluorescence intensity was determined using a fluorescent plate reader, n = 4. (D) Cells were harvested and labelled with a combination of annexin V-FITC and PI. H₂O₂-induced apoptosis was determined by flow cytometry, n = 3. All data are expressed as mean ± SD. *P < 0.05, **P < 0.01 versus H₂O₂ group.

Figure 8

Gene expression and signal pathway analysis of SAL-treated BM-EPCs. (A) BM-EPCs were cultivated in endothelial basal medium with or without 80 μM SAL for 2 and 10 days. Gene expression levels of VEGF, VEGFR2, eNOS, VE-cadherin, vWF and PECAM-1 were assessed via quantitative real-time PCR. Data were normalized to GAPDH expression and fold changes were calculated by the 2^−ΔΔCT method; n = 5, *P < 0.05, **P < 0.01 versus control group. (B) BM-EPCs were treated with 0 (control), 20, 40 and 80 μM SAL for 48 h and then harvested. Total cell lysates were prepared and subjected to SDS-PAGE, followed by Western blot analysis. The immunoblots shown are representative of at least three independent experiments with comparable results. Densitometric analysis was done using Quantity One software and shown in the lower panel. *P < 0.05, **P < 0.01 versus control group. (C and D) BM-EPCs were pretreated with either 20 μM LY294002 or 10 μM U0126 for 90 min, then cultured with 0 (control) or 80 μM SAL for 48 h. Protein expression was analysed by Western blot analysis. Densitometric analyses of band intensities normalized to the total proteins or GAPDH are shown in the right panels, n = 5. **P < 0.01 versus SAL 80 group. (E and F) BM-EPCs were treated under the same conditions described earlier, cell migratory ability and capillary-like tube formation were determined as described above, n = 3. *P < 0.05, **P < 0.01 versus SAL 80 group. All data are presented as mean ± SD.

Figure 9

Gene expression and signal pathway analysis of H₂O₂-treated BM-EPCs. (A) BM-EPCs were treated with 80 μM SAL for 2 days, then incubated with 1 mM H₂O₂ for 6 h and gene expression levels of Nox4, STAT3, VEGF, VEGFR2, eNOS were determined, n = 3. (B) Cells were incubated without or with 80 μM SAL for 48 h before stimulation with 1 mM H₂O₂. Total protein (30 μg) was isolated from the cells, applied to Western blotting and probed with specific antibodies. The immunoblots shown here are representative of at least three independent experiments with similar results. (C and D) Densitometric analyses of band intensities normalized to the total proteins or GAPDH are shown. All data are presented as mean ± SD. *P < 0.05, **P < 0.01 versus H₂O₂ group.