Nicotinamide Riboside Enhances Endothelial Precursor Cell Function to Promote Refractory Wound Healing Through Mediating the Sirt1/AMPK Pathway

Abstract

Lack of vascularization is directly associated with refractory wound healing in diabetes mellitus (DM). Enrichment of endothelial precursor cells (EPCs) is a promising but challenging approach for the treatment of diabetic wounds. Herein, we investigate the action of nicotinamide riboside (NR) on EPC function for improved healing of diabetic wounds. Db/db mice that were treated with NR-supplemented food (400 mg/kg/d) for 12 weeks exhibited higher wound healing rates and angiogenesis than untreated db/db mice. In agreement with this phenotype, NR supplementation significantly increased the number of blood EPCs and bone marrow (BM)-derived EPCs of db/db mice, as well as the tube formation and adhesion functions of BM-EPCs. Furthermore, NR-supplemented BM-EPCs showed higher expression of sirtuin 1 (Sirt1), phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK), and lower expression of acetylated peroxisome proliferator-activated receptor γ coactivator (PGC-1α) than BM-EPCs isolated from untreated db/db mice. Knockdown of Sirt1 in BM-EPCs significantly abolished the tube formation and adhesion function of NR as well as the expression of p-AMPK and deacetylated PGC-1a. Inhibition of AMPK abolished the NR-regulated EPC function but had no effect on Sirt1 expression, demonstrating that NR enhances EPC function through the Sirt1-AMPK pathway. Overall, this study demonstrates that the oral uptake of NR enhances the EPC function to promote diabetic wound healing, indicating that NR supplementation might be a promising strategy to prevent the progression of diabetic complications.

Keywords: adenosine monophosphate–activated protein kinase; diabetes mellitus; endothelial precursor cells; nicotinamide riboside; sirtuin 1; wound healing.

FIGURE 1

Effects of NR supplement on some diabetes mellitus–related symptoms. (A) Experimental schedule. Db/db mice (4 weeks old) were fed with common food or NR-supplied food (400 mg/kg/d) for consecutive 12 weeks, and the blood glucose was monitored every week. After that, each group mice were further divided into 2 cohorts: excisional wound experiment and BM-EPC isolation experiment. Blood glucose (B), serum insulin (C), body weight (D), subcutaneous fat (E), and serum adiponectin (F) of the mice treated with common food or NR-supplied food for 12 weeks. Values are mean ± SEM, (n = 6). *P < 0.05 vs. control; ^# P < 0.05 vs. db/db. BM-EPC, bone marrow–derived endothelial precursor cell.

FIGURE 2

Effects of NR supplement on wound closure and wound angiogenesis in db/db mice. (A) Full-thickness skin wounds were made in common food and NR-supplied food fed db/db mice. Quantitative analysis of wound closure at indicated time intervals (B). Immunohistochemical analyses of CD31 in day-14 wounds of mice and quantitative study (C). Red arrows point out CD31-positive capillaries (100×, scale bar, 100 μm; 200×, scale bar, 50 μm). Values are mean ± SEM (n = 5). *P < 0.05 vs. control; ^# P < 0.05 vs. db/db.

FIGURE 3

Effects of NR supplement on EPC number and BM-EPC function in db/db mice. (A) NAD concentrations in cultured BM-EPCs (day 5 and day 7 in vitro) isolated from mice. Blood EPC (B) and BM EPC (C) frequency in normal control and db/db mice. Tube formation (D,E) and cell adhesion (F,G) of BM EPC isolated from normal control and db/db mice. D: 50×; scale bar, 100μm; F: 100×; scale bar, 100 μm. Values are mean ± SEM (A, B, C: n = 5; D, E, F, G: n = 6). *P < 0.05 vs. Control; ^# P < 0.05 vs. db/db. BM, bone marrow; BM-EPC, bone marrow–derived endothelial precursor cell.

FIGURE 4

Effect of NR supplement on the expression of Sirt1/AMPK in BM-EPCs. (A) Western blot of Sirt1/AMPK in BM-EPCs isolated from normal control and diabetic mice. (B) Quantitative analysis of Sirt1 and p-AMPK expression taken as in (A). (C) The level of acetylated PGC-1α was measured. Values are mean ± SEM, (n = 3). *P < 0.05 vs. Control; ^# P < 0.05 vs. db/db.

FIGURE 5

Inhibition of Sirt1 with siRNA-Sirt1 abolished the protective effects of NR repletion on BM-EPC function in db/db mice. (A) Experimental schedule. Db/db mice received the NR supplied food for 12 weeks, and then, BM-EPCs were isolated and transfected with siRNA targeting Sirt1. (B,C) Transfection of siRNA–Sirt1 obviously inhibited Sirt1 expression in BM-EPCs from db/db mice. *P < 0.05 vs. siRNA-Sirt1. Tube formation (D) and adhesion abilities of BM-EPCs (E) were measured. Quantitative analysis of tube number (F) and adhesion number (G). (H) Quantitative analysis of Sirt1, and p-AMPK expression was taken as in (I). (I) Western blot of Sirt1/AMPK in BM-EPCs treated with NR in the presence or absence of siRNA–Sirt1. (J) Representative immunoblot images of acetylated PGC-1α. D: 50×; scale bar, 100 μm; E: 100×; scale bar, 100 μm. Values are mean ± SEM, (B, C, H, I, J: n = 3; D–G: n = 5). *P < 0.05. BM-EPC, bone marrow–derived endothelial precursor cell; NS, no significance.

FIGURE 6

Inhibition of AMPK with compound C abolished the protective effects of NR supplement on BM-EPC function in db/db mice. (A) Illustration of experimental protocol. Db/db mice treated with NR for 12 weeks, and then, BM-EPCs were isolated and stimulated with compound C (10 μM). Tube formation (B) and adhesion (C) assay in BM-EPCs treated with NR in the presence or absence of compound C. Quantitated analysis of tube number (D) and adhesion number (E). (F–G) Compound C has no significant effect on Sirt1 expression in BM-EPCs of db/db mice treated with NR. B: 50×; scale bar, 100 μm; C: 100×; scale bar, 100 μm. Values are mean ± SEM (n = 3). *P < 0.05. BM-EPC, bone marrow–derived endothelial precursor cell; NS, no significance.