Exercise-Intervened Endothelial Progenitor Cell Exosomes Protect N2a Cells by Improving Mitochondrial Function

Abstract

We have recently demonstrated that exosomal communication between endothelial progenitor cells (EPCs) and brain endothelial cells is compromised in hypertensive conditions, which might contribute to the poor outcomes of stroke subjects with hypertension. The present study investigated whether exercise intervention can regulate EPC-exosome (EPC-EX) functions in hypertensive conditions. Bone marrow EPCs from sedentary and exercised hypertensive transgenic mice were used for generating EPC-EXs, denoted as R-EPC-EXs and R-EPC-EX^ET. The exosomal microRNA profile was analyzed, and EX functions were determined in a co-culture system with N2a cells challenged by angiotensin II (Ang II) plus hypoxia. EX-uptake efficiency, cellular survival ability, reactive oxygen species (ROS) production, mitochondrial membrane potential, and the expressions of cytochrome c and superoxide-generating enzyme (Nox4) were assessed. We found that (1) exercise intervention improves the uptake efficiency of EPC-EXs by N2a cells. (2) exercise intervention restores miR-27a levels in R-EPC-EXs. (3) R-EPC-EX^ET improved the survival ability and reduced ROS overproduction in N2a cells challenged with Ang II and hypoxia. (4) R-EPC-EX^ET improved the mitochondrial membrane potential and decreased cytochrome c and Nox4 levels in Ang II plus hypoxia-injured N2a cells. All these effects were significantly reduced by miR-27a inhibitor. Together, these data have demonstrated that exercise-intervened EPC-EXs improved the mitochondrial function of N2a cells in hypertensive conditions, which might be ascribed to their carried miR-27a.

Keywords: EPC-EXs; exercise; exosomes; hypertension; hypoxia; miR-27a.

Figure 1

Exercise intervention improved the incorporation efficiency of EPC-EXs isolated from hypertensive transgenic mice by N2a cells. (A) representative images showing the incorporation of EX (labeled with PKH26, a red fluorescence) uptake by N2a cells at 8 h, 16 h, and 24 h incubation. Magnification: 200×. (B) summarized data showing the EPC-EX incorporation by N2a cells. Blue: DAPI. Red: PKH26 labeled EXs. * p < 0.05, vs. R-EPC-EX in 16 h; ⁺ p < 0.05, vs. R-EPC-EX in 24 h. Data expressed as mean ± SD, n = 3/group.

Figure 2

Profiling analysis of exosomal miRs and qRT-PCR of miR-27a in EPC-EXs of exercised and sedentary hypertensive mice. (A) representative miR profiling graph showing the changes of miRs in EPC-EXs. MiRs with down- or up-regulated >3-fold are listed. (B) qRT-PCR verified the miR-27a level in EPC-EXs. * p < 0.05, vs. R-EPC-EX. Data are expressed as mean ± SD, n = 3/group.

Figure 3

Exercise-intervened EPC-EXs raised miR-27a level, improved the cell survival ability, and reduced ROS overproduction in N2a cells challenged by Ang II plus hypoxia. (A) miR-27a level in N2a cells co-cultured with or without R-EPC-EXs. (B) summarized data showing the N2a cell viability assessed by MTT assay. (C) representative images of N2a cells of DHE staining (red fluorescence). Scale bar: 100 μm. (D) summarized data of DHE staining assay for ROS production in N2a cells. * p < 0.05, vs. con (no Ang II + hypoxia); ⁺ p < 0.05, vs. Ang II + hypoxia only; ^# p < 0.05, vs. R-EPC-EX; ^ p < 0.05, vs. R-EPC-EX^ET. Data expressed as mean ± SD, n = 3/group.

Figure 4

Exercise-intervened EPC-EXs regulated the MMP in N2a cells challenged by the Ang II plus hypoxia. (A) representative images show the JC-1 staining in N2a cells. Enlarged images are from the dashed box of the merged images. Scale bar: 50 μm. (B) summarized data of JC-1 stain. * p < 0.05, vs. con (no Ang II + hypoxia); ⁺ p < 0.05, vs. Ang II + hypoxia only; ^# p < 0.05, vs. R-EPC-EX; ^ p < 0.05, vs. R-EPC-EX^ET. Data expressed as mean ± SD, n = 3/group.

Figure 5

Exercise-intervened EPC-EXs regulated the expressions of Nox4 and cytochrome c in Ang II plus hypoxia-challenged N2a cells. (A–C) representative bands, and summarized data of cytochrome c and Nox4 levels in N2a cells. * p < 0.05, vs. con (no Ang II + hypoxia); ⁺ p < 0.05, vs. Ang II + hypoxia only; ^# p < 0.05, vs. R-EPC-EX; ^ p < 0.05, vs. R-EPC-EX^ET. Data expressed as mean ± SD, n = 3/group.