Mechanism of the switch from NO to H2O2 in endothelium-dependent vasodilation in diabetes

Abstract

Coronary microvascular dysfunction is prevalent among people with diabetes and is correlated with cardiac mortality. Compromised endothelial-dependent dilation (EDD) is an early event in the progression of diabetes, but its mechanisms remain incompletely understood. Nitric oxide (NO) is the major endothelium-dependent vasodilatory metabolite in the healthy coronary circulation, but this switches to hydrogen peroxide (H₂O₂) in coronary artery disease (CAD) patients. Because diabetes is a significant risk factor for CAD, we hypothesized that a similar NO-to-H₂O₂ switch would occur in diabetes. Vasodilation was measured ex vivo in isolated coronary arteries from wild type (WT) and microRNA-21 (miR-21) null mice on a chow or high-fat/high-sugar diet, and B6.BKS(D)-Lepr^db/J (db/db) mice using myography. Myocardial blood flow (MBF), blood pressure, and heart rate were measured in vivo using contrast echocardiography and a solid-state pressure sensor catheter. RNA from coronary arteries, endothelial cells, and cardiac tissues was analyzed via quantitative real-time PCR for gene expression, and cardiac protein expression was assessed via western blot analyses. Superoxide was detected via electron paramagnetic resonance. (1) Ex vivo coronary EDD and in vivo MBF were impaired in diabetic mice. (2) Nω-Nitro-L-arginine methyl ester, an NO synthase inhibitor (L-NAME), inhibited ex vivo coronary EDD and in vivo MBF in WT. In contrast, polyethylene glycol-catalase, an H₂O₂ scavenger (Peg-Cat), inhibited diabetic mouse EDD ex vivo and MBF in vivo. (3) miR-21 was upregulated in diabetic mouse endothelial cells, and the deficiency of miR-21 prevented the NO-to-H₂O₂ switch and ameliorated diabetic mouse vasodilation impairments. (4) Diabetic mice displayed increased serum NO and H₂O₂, upregulated mRNA expression of Sod1, Sod2, iNos, and Cav1, and downregulated Pgc-1α in coronary arteries, but the deficiency of miR-21 reversed these changes. (5) miR-21-deficient mice exhibited increased cardiac PGC-1α, PPARα and eNOS protein and reduced endothelial superoxide. (6) Inhibition of PGC-1α changed the mRNA expression of genes regulated by miR-21, and overexpression of PGC-1α decreased the expression of miR-21 in high (25.5 mM) glucose treated coronary endothelial cells. Diabetic mice exhibit a NO-to-H₂O₂ switch in the mediator of coronary EDD, which contributes to microvascular dysfunction and is mediated by miR-21. This study represents the first mouse model recapitulating the NO-to-H₂O₂ switch seen in CAD patients in diabetes.

Keywords: Coronary circulation; Coronary dilation; Diabetes; Endothelial dysfunction; Microvascular dysfunction.

Fig. 1

Aortic vasodilation in wild-type and diabetic mice by myography. a. Acetylcholine-induced endothelial dependent dilation (Ach-EDD) in aortic arteries from 9-month-old (9 mo) wild-type (WT) mice, db/db mice, and diet-induced diabetic mice (WT+HFHS) in the presence or absence of L-NAME (NO synthase inhibitor). b. Sodium nitroprusside (SNP)-induced (endothelium-independent) aortic vasodilation for each group. Mo, month old. HFHS, high fat/high sugar. Two-way ANOVA was used for statistical analysis (n=6 mice/group, **P < 0.01 vs. WT 9 mo, ^##P < 0.01 vs. db/db 9 mo, ^$$P < 0.01 vs. WT+HFHS, ^!!P < 0.01 vs. WT 9 mo, and ^&P < 0.05 vs. WT 9 mo)

Fig. 2

Coronary vasodilation in wild-type mice at different ages by myography. a-c. Acetylcholine-induced endothelial dependent dilation (Ach-EDD) in coronary arteries from 3-month-old (3 mo) (a), 9-month-old (9 mo) (b) and 32-month-old (32 mo) (c) wild-type (WT) mice in the presence or absence of L-NAME, Peg-Cat (H₂O₂ scavenger) or indomethacin (inhibitor of prostaglandin synthesis). d. Sodium nitroprusside (SNP)-induced coronary vasodilation for each group. e. The maximum Ach-EDD induced by vasodilators (prostaglandins (PGs), NO, and H₂O₂) in WT mice at different ages. f. The relative contribution of vasodilators (PGs, NO or H₂O₂) to Ach-EDD was calculated in WT mice at different ages. Mo, month old. Two-way ANOVA was used for all statistical analysis (n=6 mice/group, *P < 0.05, **P < 0.01 vs. WT 3 mo [a], vs. WT 9 mo [b], and vs. WT 32 mo [c])

Fig. 3

Coronary vasodilation in diabetic mice by myography. a-b. Acetylcholine-induced endothelial dependent dilation (Ach-EDD) in coronary arteries from 3-month-old (3 mo) (a) or 9-month-old (9 mo) (b) db/db mice, and age-matched wild-type (WT) controls in the presence or absence of L-NAME, Peg-Cat, or indomethacin. c. Ach-EDD in coronary arteries from 6-8-month-old WT mice fed HFHS diet (WT+HFHS) and age-matched WT controls in the presence or absence of L-NAME, Peg-Cat, or indomethacin. d. Sodium nitroprusside (SNP)-induced coronary vasodilation for each group. e. The maximum Ach-EDD induced by vasodilators (PGs, NO, and H₂O₂) in db/db mice or WT+HFHS mice. f. The relative contribution of individual vasodilators to Ach-EDD. Mo, month old. HFHS, high fat/high sugar. PGs, prostaglandins. Two-way ANOVA was used for all statistical analysis (n=6 mice/group, *P < 0.05, **P < 0.01 vs. db/db 3 mo [a], vs. db/db 9 mo [b], vs. WT+HFHS [c], or ^#P<0.05, ^##P<0.01 vs. age-matched WT control [a-b] or WT+HFHS [c])

Fig. 4

Induction of miR-21 expression in diabetes. miR-21 expression was determined by qRT-PCR in hearts and aortas from wild-type (WT) and db/db mice (n=5 mice/group) (a), coronary endothelial cells (CEC) from WT and db/db mice (n=3 mice/group) (b), and in healthy or diabetic human CEC treated with high glucose (HG, 25.5 mM) or low glucose (LG, 5.5 mM) (n=3 independent experiments) (c). d-e. miR-21 expression in the endothelium of cardiac tissue from WT or db/db mice was determined by RNAscope in situ hybridization (ISH) (n=4 mice/group). In the representative RNAscope ISH, arrows (white) point to miR-21 (red) expression in endothelial cells (Isolectin-B4, green) (d). The relative expression levels of miR-21 in endothelial cells were quantified by the ratio of miR-21⁺Isolectin-B4⁺ (colocalization of red and green) areas to the number of Isolectin-B4⁺ (green) cells by the Image-Pro Premier image analysis software (n=12 fields, 3 fields/animal). Two-tailed unpaired Student’s t-test was performed for statistical analysis (a, b, and e). Two-way ANOVA was performed for statistical analysis (c). *P < 0.05

Fig. 5

Coronary vasodilation in miR-21 null (miR-21^−/−) mice fed a chow or high fat/high sugar (HFHS) diet by myography. a. Acetylcholine-induced endothelial dependent dilation (Ach-EDD) in coronary arteries from 6–8-month-old chow-fed (a) and HFHS-fed (b) miR-21^−/− mice in the presence or absence of L-NAME, Peg-Cat, or indomethacin. c. Sodium nitroprusside (SNP)-induced coronary vasodilation. d. The maximum Ach-EDD induced by vasodilators (PGs, NO, and H₂O₂) in miR-21^−/− + chow, WT + HFHS, or miR-21^−/− HFHS mice. e. The relative contribution of individual vasodilators to Ach-EDD in miR-21^−/− + chow, WT + HFHS, or miR-21^−/− + HFHS mice. PGs, prostaglandins. Two-way ANOVA was performed for statistical analysis (n=6 mice/group, **P < 0.01 vs. miR-21^−/− + chow [a] and *P< 0.05, **P < 0.01 vs. miR-21^−/− +HFHS [b], or ^#P < 0.05, ^##P < 0.01 vs. WT+HFHS [b])

Fig. 6

Gene expression in mouse coronary arteries and coronary endothelial cells by qRT-PCR. a. Relative mRNA levels in coronary arteries from wild-type (WT) mice, WT mice fed an HFHS diet (WT+HFHS) or db/db mice (n=6 mice/group, **P < 0.01 vs. WT). b. Relative mRNA levels in coronary arteries HFHS diet-fed WT or miR-21^−/− mice (n=6 mice/group, ^#P < 0.05 vs. WT+HFHS). c. Relative mRNA levels in coronary endothelial cells (CEC) from WT and db/db mice (n=3 mice/group, **P < 0.01 vs. WT CEC). Pgc-1α, PPARγ coactivator 1 alpha. Sod1, superoxide dismutase 1. Sod2, superoxide dismutase 2. Cat, catalase. Cav1, caveolin 1. eNos, endothelial nitric oxide synthase. iNos, inducible nitric oxide synthase. One-way ANOVA (a) or unpaired Student’s t-test (b and c) was performed for the statistical analysis.

Fig. 7

Assessment of NO, H₂O₂, and superoxide levels. Serum levels of H₂O₂ (a) as well as nitrite and nitrate (NOx) metabolites (b) were measured in wild-type (WT), WT+HFHS, db/db and miR-21^−/− +HFHS mice (n=6 mice/group, **P < 0.01 vs. WT, ^##P < 0.01 vs. WT+HFHS). c-d. Coronary endothelial cells (CEC) were isolated from WT or miR-21^−/− mice and treated with high lipid (HL, 150 μM palmitate, linoleic acid, oleic acid, and 10 μg/ml cholesterol) or high glucose (HG, 25 mM) for 24 h. Superoxide detection was performed using DMPO (50 mM) spin-trapped and electron paramagnetic resonance (c), and superoxide levels were quantified (d) (n=4-6, **P < 0.01 vs. WT CEC, ^##P < 0.01 vs. WT CEC+HG). HFHS, high fat/high sugar. One-way (a-b) or two-way ANOVA (d) was performed for statistical analysis

Fig. 8.

Myocardial blood flow (MBF) and cardiac work (CW) at baseline, and during norepinephrine-induced metabolic hyperemia. a. Workflow for NE-induced stress test and MBF measurements. Mice were given norepinephrine (NE) at 2.5 μg/kg/min (NE 2.5) or 5.0 μg/kg/min (NE 5.0) to induce hyperemia. b. MBF in wild-type (WT), db/db and WT+HFHS mice in the absence of inhibitors (n=5-6, *P < 0.05 vs. 3-month-old WT mice (3 mon)). c-e. MBF in WT (c), HFHS-fed WT (d) and db/db mice (e) treated with or without L-NAME or Peg-Cat during NE-induced metabolic hyperemia (n=5-7 mice/group, *P < 0.05 vs. WT 3 mo). f-i. Relationship between CW (double product of heart rate, beats/min and mean arterial pressure, mmHg) and MBF in mice shown in (b-e) (n=5-7). Min, minutes. Mo, month old. HFHS, high fat/high sugar. Two-way ANOVA (b-e) was performed, and linear regression (f-i) analysis was performed (*P < 0.05 vs WT 3 mo, ^# P < 0.05 vs WT+HFHS, ^$P < 0.05 vs db/db 3 mo)

Fig. 9.

Gene expression in mouse coronary endothelial cells (CEC). a. Pgc-1α mRNA levels in wild-type (WT) and miR-21^−/− CEC in the presence of low glucose (LG; 5.5 mM) or high glucose (HG; 25.5 mM) (n=4-6 mice/group, *P < 0.05). b. Relative miR-21 expression levels in WT CEC transduced with adenovirus expressing GFP (Ad-GFP; control) or PGC-1α (Ad-PGC-1α) under HG for treatment for 72 h. (n=4-5, **P < 0.01). c. Pparα, Sod2, Cat and eNos expression in WT CEC treated with HG, and miR-21^−/− CEC treated with HG in the presence or absence of a PGC-1α inhibitor SR-18292 (SR; 20 μM) (n=5-11, *P< 0.05). Pgc-1α, PPARγ coactivator 1 alpha. Sod2, superoxide dismutase 2. Cat, catalase. eNos, endothelial nitric oxide synthase. Unpaired Student’s t-test (a, b) or one-way ANOVA (c) was used for statistical analysis

Fig. 10.

miR-21 regulates PPARα, eNOS, and PGC-1α expression. a-d. Western blot assays were performed using cardiac tissue lysates. Representative Western blot images are presented (a, b) and protein levels were quantified (c, d) (n=4 mice/group. *P < 0.05. A two-tailed unpaired Student’s t-test was performed for statistical analysis. e. Functional protein association networks via STRING shows the signaling pathway linking PGC-1α and PPARα to the ROS-related genes that were detailed by qPCR in Fig. 6