Effects of endothelial progenitor cell-derived microvesicles on hypoxia/reoxygenation-induced endothelial dysfunction and apoptosis - PubMed (original) (raw)

Effects of endothelial progenitor cell-derived microvesicles on hypoxia/reoxygenation-induced endothelial dysfunction and apoptosis

Jinju Wang et al. Oxid Med Cell Longev. 2013.

Abstract

Oxidative stress-induced endothelial dysfunction plays a key role in ischemia/reperfusion injury. Recent evidence indicates that endothelial progenitor cell-derived microvesicles (EPC-MVs) can promote angiogenesis of endothelial cells (ECs). Here, we investigated the potential effects of EPC-MVs on hypoxia/reoxygenation (H/R) injury in human brain microvascular ECs (hb-ECs). MVs were prepared from EPCs cultured in a serum deprivation (SD) medium (starving stress, sEPC-MVs) or SD medium containing tumor necrosis factor- α (TNFα) (apoptotic stress, aEPC-MVs). H/R injury model of hb-ECs was produced by 6 hr hypoxia (1% O2) and 24 hr reoxygenation. The H/R hb-ECs were co-cultured with EPC-MVs. Results showed that (1) H/R hb-ECs were dysfunctional and coupled with increased apoptosis and ROS overproduction; (2) under two different conditions, EPCs displayed remarkable difference in caspase 3 and miR126 expression, which were carried by the corresponsive EPC-MVs; (3) functionally, sEPC-MVs had beneficial effects on H/R hb-ECs, whereas aEPC-MVs had detrimental effects; (4) the diverse effects of sEPC-MVs and aEPC-MVs were associated with the changes in miR126 and eNOS expression and were abolished by PI3K inhibitor. In conclusion, sEPCs-MVs and aEPC-MVs are functionally different on hb-EC apoptosis and dysfunction via their carried RNAs associated with ROS production and PI3K/eNOS/NO pathway.

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Figures

Figure 1

Figure 1

Effects of serum deprivation (SD) alone and SD plus TNF_α_ on EPC apoptosis, caspase 3, and miR126 expression. (a) Representative images showing EPC characterization of Bs-Lectin and Di-LDL double staining. Scale bar: 100 _μ_m. (b) Apoptosis (Annexin V+PI−) of stimulated EPCs. (c) Caspase 3 expression in stimulated EPCs. (d) MiR126 expression in stimulated EPCs. *P < 0.05, versus control; # P < 0.05, versus SD; N = 4/group.

Figure 2

Figure 2

EPC-MV characterization, modification, caspase 3 and miR126 expression. (a) Flow cytometric plots showing Annexin V, CD34 and VEGFR2 expressions (isotype controls: left curves; antibodies: right curves) in EPC-MVs. (b) TEM image showing similar spherical morphology of sEPC-MVs and aEPC-MVs. Scale bar: 500 nm. (c) Summarized data showing effective digestion of EPC-MVs total RNAs by RNase treatment. (d) Caspase 3 and miR126 expression in control MVs (generated from basal condition), sEPC-MVs, and aEPC-MVs. *P < 0.05, versus control; # P < 0.05, versus sEPC-MVs; & P < 0.05, versus aEPC-MVs; N = 4/group. TEM and transmission electron microscopy.

Figure 3

Figure 3

Effects of H/R on hb-EC viability and apoptosis, ROS and NO production, and tube formation. (a) Apoptosis (Annexin V+PI−) and cell viability. (b) ROS and NO production. (c) Tube formation ability. Scale bar: 200 _μ_m. *P < 0.05, versus control; N = 4/group.

Figure 4

Figure 4

Effects of EPC-MVs on miR126 expression, ROS production, and apoptosis in H/R hb-ECs. (a) Representative images showing the merging of PKH26 labeled EPC-MVs with hb-ECs (red: PKH26; blue: DAPI). Scale bar: 100 _μ_m. (b) miR126 expression in H/R hb-ECs cocultured with aEPC-MVs or sEPC-MVs. (c) ROS production of H/R hb-ECs cocultured with aEPC-MVs or sEPC-MVs. (d) Apoptosis of H/R hb-ECs cocultured with aEPC-MVs or sEPC-MVs. *P < 0.05, versus vehicle; + P < 0.05, versus sEPC-MVs or aEPC-MVs; # P < 0.05, versus sEPC-MVs or RNase-sEPC-MVs; N = 4/group.

Figure 5

Figure 5

Effects of EPC-MVs on eNOS expression, NO production, and tube formation in H/R hb-ECs. (a) eNOS production of H/R hb-ECs cocultured with aEPC-MVs or sEPC-MVs. (b) NO production of H/R hb-ECs cocultured with aEPC-MVs or sEPC-MVs. (c) Tube formation ability of H/R hb-ECs cocultured with aEPC-MVs or sEPC-MVs. *P < 0.05, versus vehicle; + P < 0.05, versus sEPC-MVs or aEPC-MVs; # P < 0.05, versus sEPC-MVs or RNase-sEPC-MVs; N = 4/group.

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