Antiinflammatory actions of inorganic nitrate stabilize the atherosclerotic plaque

Abstract

Reduced bioavailable nitric oxide (NO) plays a key role in the enhanced leukocyte recruitment reflective of systemic inflammation thought to precede and underlie atherosclerotic plaque formation and instability. Recent evidence demonstrates that inorganic nitrate (NO3-) through sequential chemical reduction in vivo provides a source of NO that exerts beneficial effects upon the cardiovascular system, including reductions in inflammatory responses. We tested whether the antiinflammatory effects of inorganic nitrate might prove useful in ameliorating atherosclerotic disease in Apolipoprotein (Apo)E knockout (KO) mice. We show that dietary nitrate treatment, although having no effect upon total plaque area, caused a reduction in macrophage accumulation and an elevation in smooth muscle accumulation within atherosclerotic plaques of ApoE KO mice, suggesting plaque stabilization. We also show that in nitrate-fed mice there is reduced systemic leukocyte rolling and adherence, circulating neutrophil numbers, neutrophil CD11b expression, and myeloperoxidase activity compared with wild-type littermates. Moreover, we show in both the ApoE KO mice and using an acute model of inflammation that this effect upon neutrophils results in consequent reductions in inflammatory monocyte expression that is associated with elevations of the antiinflammatory cytokine interleukin (IL)-10. In summary, we demonstrate that inorganic nitrate suppresses acute and chronic inflammation by targeting neutrophil recruitment and that this effect, at least in part, results in consequent reductions in the inflammatory status of atheromatous plaque, and suggest that this effect may have clinical utility in the prophylaxis of inflammatory atherosclerotic disease.

Keywords: atherosclerosis; diet; inflammation; nitrate; nitric oxide.

Conflict of interest statement

A.A. is a Director of HeartBeet Ltd.

Figures

Fig. S1.

Effect of dietary nitrite and nitrate on plasma NOx in WT mice. Dietary nitrate caused a dose-dependent elevation of circulating nitrate and nitrite levels, whereas dietary nitrite caused an elevation in nitrite but not nitrate levels. Data are shown as mean ± SEM of n = 8–12. Statistical significance was determined using a one-way ANOVA followed by Dunnett’s multiple posttest represented by ***P < 0.001 compared with control.

Fig. 1.

Dietary nitrate alters plaque composition in ApoE KO mice. Dietary nitrate elevates plasma nitrite and nitrate in NCD and HFD mice (A). A representative image of Oil red O staining in the aortic tree (B) and quantification showing total plaque area (C) and plaque area of the aortic arch (D), both of which were unchanged in response to dietary nitrate. Histological analyses demonstrated a reduction in macrophage accumulation (E), an increase in smooth muscle accumulation (F), and no change in collagen accumulation (G). Representative images of Mac-2, αSMA, and PSR (Picrosirius red) staining from HFD-fed mice (H). (Scale bars, 200 μm.) Data are shown as mean ± SEM of n = 16 for plasma NOx; n = 14–18 for plaque area; n = 9–15 for plaque composition. Statistical significance was determined using unpaired t test represented by *P < 0.05, **P < 0.01, ***P < 0.001, or ****P < 0.001 compared with control. (Mac-2, 1 data point in chow KNO3 and for αSMA, 1 data point in Western KNO3 excluded using Rout’s outlier test).

Fig. 2.

Nitrite reduces leukocyte recruitment in ApoE KO mice. Dietary nitrate suppresses leukocyte rolling and adhesion in NCD-fed mice with no effect in HFD-fed mice (A). Nitrite had no effect on WT leukocyte rolling or adhesion (n = 7) (B), however caused a concentration-dependent reduction in ApoE KO leukocyte rolling and adhesion (n = 5) (C) with no effect on venule blood flow (n = 4/5) (D). All data are expressed as mean ± SEM of (n) mice. Statistical significance was determined using unpaired t test for comparison between KNO3 or KCl treated mice or one-way ANOVA followed by Dunnett’s posttest for comparison of nitrite-induced effects versus baseline and posttest significance represented by *P < 0.05 or **P < 0.01 compared with control.

Fig. 3.

Dietary nitrate reduces neutrophil recruitment in acute inflammation. Dietary nitrate did not alter baseline peritoneal cell numbers (n = 10) (A) but did reduce TNF-α (n = 10) (B) and zymosan (n = 17) (C) -induced neutrophil recruitment into the peritoneal cavity at 4 and 24 h. This effect was associated with reduced MPO activity in the (D) cell pellet (n = 6) and (E) mesentery (n = 6–10) at both time points. Data are shown as mean ± SEM and analyzed using one-way ANOVA followed by Dunnett’s multiple posttest represented by *P < 0.05, **P < 0.01, or ***P < 0.001 compared with control.

Fig. S2.

Effect of inorganic nitrate upon recruitment of specific leukocyte subsets in to the peritoneal cavity in WT mice. Resident (A–C) and inflammatory monocytes (D–F) in response to PBS (n = 10), TNF-α (n = 10), or zymosan (n = 17). Data are shown as mean ± SEM and analyzed using one-way ANOVA (no significant differences).

Fig. 4.

Dietary nitrate suppresses TNF-α–induced neutrophil and inflammatory monocyte recruitment in ApoE KO mice. Dietary nitrate attenuated TNF-α–induced leukocyte recruitment (4 h) into the peritoneal cavity (A), and specifically a reduction in neutrophil numbers (B) with a representative scatter plot of neutrophil identification shown in C. This was associated with reduced MPO activity (D) and CD11b expression (E) with a representative histogram of neutrophil CD11b expression, blue and green representing control and nitrate-fed, respectively (F). Furthermore, dietary nitrate treatment caused a reduction of inflammatory monocyte recruitment into the peritoneal cavity at 24 h after TNF-α (G) and an elevation of IL-10 4 h after TNF-α in peritoneal lavage fluid (H). Finally, qRT-PCR for IL-10 mRNA of aortic arch from NCD or HFD ApoE KO mice demonstrates an increase of expression in NCD-fed mice (I). Data are shown as mean ± SEM of n = 8–10 mice. Statistical significance was determined using unpaired t test represented by *P < 0.05 or **P < 0.01 compared with control. (IL-10, 1 data point in KCl excluded using Routs outlier test).

Fig. S3.

Inorganic nitrate causes modest attenuation of plasma chemokine levels. Inorganic nitrate reduced plasma CXCL1 in NCD-fed mice with no effect in mice fed HFD (A). Plasma CXCL2 was decreased in NCD- and HFD-fed mice, but significantly only in the latter (B). CCL2 was reduced in both diets (C). There was a trend toward reduction of CCL5 expression in both diets (D). Data are shown as mean ± SEM of n = 19–26. Statistical significance was determined using unpaired t test represented by *P < 0.05 compared with KCl in each treatment group. The unequal groups relate to technical failures during the assay.

Fig. S4.

Inorganic nitrate has no effect on mRNA expression of various chemokines. qRT-PCR of aortic arch from NCD or HFD ApoE KO mice showed no change in mRNA levels of CXCL1 (A), CXCL2 (B), CXCL5 (C), CCL2 (D), CX3CL1 (E) by inorganic nitrate. Data are shown as mean ± SEM of n = 8–11. Statistical significance was determined using unpaired t test; no significant differences were found.

Fig. 5.

Liver and erythrocyte XOR nitrite reductase activity is enhanced in ApoE KO mice. Liver from ApoE KO mice exhibited greater nitrite reductase activity vs. WT at pH 7.4 (A) and pH 6.8 (B). The same pattern was observed in erythrocytes (G and H). Allopurinol had no effect on nitrite reductase activity from liver (C and D) or erythrocytes (I and J) from WT mice, in comparison ApoE KO mice exhibited reduced nitrite reductase activity in liver (E and F) and erythrocytes (K and L). Data are shown as mean ± SEM of n = 5. Statistical significance was determined using two-way ANOVA represented by #P < 0.05, ##P < 0.01, or ###P < 0.001, followed by Sidak’s multiple posttest represented by *P < 0.05, **P < 0.01, or ***P < 0.001, significantly different from WT or control.

Fig. 6.

Liver and erythrocyte XOR protein expression and activity is enhanced in ApoE KO mice. XOR expression in liver (A) and erythrocytes (B) and liver conventional XOR activity (C). Data are shown as mean ± SEM of n = 8. Statistical significance was determined using unpaired t test represented by *P < 0.05 or **P < 0.01 compared with WT.