Trimethylamine-N-Oxide Impedes Late Endothelial Progenitor Cell-Mediated Revascularization by Triggering Mitochondrial Apoptosis via Suppression of MnSOD

Abstract

Background and Aims: Trimethylamine-N-oxide (TMAO) is recognized as a novel marker and mediator of atherosclerotic cardiovascular disease (ASCVD). Endothelial progenitor cells (EPCs) are crucial for maintaining vascular homeostasis. Impaired EPC numbers and function correlate with increased adverse cardiovascular events. The aim of this study was to decipher the effect of TMAO on late EPCs (LEPCs) and its underlying molecular mechanism. Methods and Results: In vitro migration and tubulogenic capacities of LEPCs were attenuated by TMAO in a dose-dependent manner, accompanied by inhibition of manganese superoxide dismutase (MnSOD) and mitochondrial damage. TMAO-induced mitochondrial damage provoked proinflammatory responses (increased levels of IL-6, IL-1b, ICAM-1, E-sel, and TNF-α) and autophagic cell death (confirmed by western blot immunofluorescent staining and transmission electron microscopy) in LEPCs. Overexpression of MnSOD through adenovirus transfection reversed TMAO-related LEPCs dysfunction. To study the effect of TMAO on LEPC-mediated vascular repair in vivo, a hind limb ischemia model was established in nude mice, and LEPCs were injected in the ischemic hind limb. Laser Doppler imaging of mouse ischemic hindlimbs at 21 days indicated that TMAO treatment inhibited LEPCs-mediated blood flow recovery, which was restored by MnSOD overexpression. Immunohistology analyses further revealed consistent alterations in capillary density determined by CD31 staining. Conclusions: TMAO induces mitochondrial damage in LEPCs via MnSOD suppression, which leads to cell dysfunction, proinflammatory activation, and autophagic cell death in vitro and impaired LEPCs-mediated revascularization in vivo. Overexpression of MnSOD restores TMAO-induced LEPCs dysfunction and further enhances LEPC-mediated revascularization in the ischemic hind limbs in nude mice.

Figure 1

TMAO impairs LEPC function and induces mitochondrial damage in a dose-dependent manner. (a) Representative flow cytometry analyses of EC markers (CD31, CD309, and CD34) and monocyte marker CD45 in LEPCs. (b) Quantification of CCK-8 cytotoxicity assay. (c) Representative images and quantification of scratch-wound assay to determine migration area over 24 h. Scale bar: 100 μm (n = 3). (d) Representative images and quantification of transwell assay of migration capacity. Scale bar: 100 μm (n = 5). (e) Representative and quantification of flow cytometry analyses of JC-1 staining to determine mitochondrial membrane potential (n = 3) (Mean ± SEM; ⁣^∗p < 0.05, ⁣^∗∗p < 0.01, ⁣^∗∗∗p < 0.001, and ⁣^∗∗∗∗p < 0.0001 vs. NC).

Figure 2

TMAO provokes the proinflammatory response, autophagy, and apoptosis in LEPCs. (a) RT-PCR analyses of proinflammatory cytokines mRNA levels in LEPCs treated with or without TMAO (n = 3). (b) Western Blotting analyses of Bcl-2 and Bax levels in LEPCs treated with or without TMAO. (c) Autophagy flux was detected by confocal microscopy in LEPCs transfected with Ad-mRFP-GFP-LC3 (GFP indicates autophagosome; mRFP indicates autolysosome. Scale bar: 20 μm; n = 3). (d) Autophagic vacuoles and autolysosomes were quantified by transmission electron microscopy; red arrows denote autophagosome, and yellow arrows indicate autolysosomes. Scale bar: 0.2 μm (Mean ± SEM; ⁣^∗p < 0.05, ⁣^∗∗p < 0.01, ⁣^∗∗∗p < 0.001, and ⁣^∗∗∗∗p < 0.0001 vs. NC).

Figure 3

TMAO-induced functional deterioration of LEPCs by inhibiting MnSOD. (a) Western Blotting analyses of MnSOD expression in LEPCs treated with or without TMAO. (n = 3, ⁣^∗p < 0.05 and ⁣^∗∗p < 0.01 vs. NC). (b) Representative images and quantification of scratch-wound assay to determine migration area over 24 h. Scale bar: 100 μm (n = 3, ⁣^∗∗p < 0.01 vs. Ad-NC + PBS, ⁣^∗p < 0.05 vs. Ad-NC + TMAO). (c) Representative images and quantification of transwell assay of migration capacity. Scale bar: 100 μm (n = 4, ⁣^∗∗∗p < 0.001 vs. Ad-NC + PBS, ⁣^∗∗p < 0.01 vs. Ad-NC + TMAO). (d) Representative images and quantification of matrigel tube formation assay over 6 h in LEPCs. Scale bar: 50 μm (n = 3, ⁣^∗∗∗p < 0.001 vs. Ad-NC + PBS, ⁣^∗p < 0.05 vs. Ad-NC + TMAO) (Mean ± SEM).

Figure 4

MnSOD overexpression reverses TMAO-induced proinflammatory response and mitochondrial apoptosis in LEPCs. (a) RT-PCR analyses of proinflammatory cytokines mRNA levels in LEPCs (n = 3, ⁣^∗∗∗p < 0.001 vs. Ad-NC + TMAO). (b) Representative images and quantification of low cytometry analyses of cell apoptosis in LEPCs (n = 3, ⁣^∗∗∗p < 0.001 vs. Ad-NC + PBS, ⁣^∗p < 0.05 vs. Ad-NC + TMAO). (c) Western Blotting analyses of Bcl-2 and Bax level in LEPCs (n = 3, ⁣^∗∗∗p < 0.001 vs. Ad-NC + PBS, ⁣^∗∗p < 0.01 vs. Ad-NC + TMAO; ⁣^∗p < 0.05 vs. Ad-MnSOD+TMAO). (d) Representative and quantification of JC-1staining to determine mitochondrial membrane potential. Scale bar: 50 μm (n = 3, ⁣^∗p < 0.05 vs. Ad-NC + TMAO). (e) Autophagy flux was detected by confocal microscopy in LEPCs transfected with Ad-mRFP-GFP-LC3 (GFP indicates autophagosome; mRFP indicates autolysosome. Scale bar: 0.5 μm. n = 3, ⁣^∗p < 0.05 vs. Ad-NC + TMAO). (f) Autophagic vacuoles and autolysosomes were quantified by transmission electron microscopy (red arrows denote autophagosome; yellow arrows indicate autolysosomes. Scale bar: 0.5 μm. n = 3, ⁣^∗∗∗p < 0.001 vs. Ad-NC + TMAO; ⁣^∗∗p < 0.01 vs. Ad-MnSOD+TMAO) (Mean ± SEM).

Figure 5

MnSOD overexpression restores TMAO-induced impaired reparative capacity of LEPCs in vivo. (a) Reperfusion assessed by laser Doppler imaging at 21 days postligation with blood flow quantified as a ratio in ischemic versus healthy control limb (n = 5, ⁣^∗p < 0.05 and ⁣^∗∗p < 0.01 vs. Ad-NC + PBS, #p < 0.05 vs. Ad-NC + TMAO). (b) Capillary density was measured by immunohistochemical staining (n = 10, ⁣^∗∗p < 0.01 vs. Ad-NC + PBS, ⁣^∗p < 0.05 vs. Ad-MnSOD+TMAO) (Mean ± SEM).

Figure 6

Schematic illustration. (a) Trimethylamine-N-oxide triggers mitochondrial apoptosis of LEPCs via suppression of MnSOD. (b) The experimental flow of revascularization in hind limb ischemic mice.