Dietary Nitrate Supresses Leukocyte Recruitment and Activation in Apolipoprotein E Knockout Mice

Reduced bioavailable NO, resulting in enhanced leukocyte activation and smooth muscle hypertrophy, plays a pivotal role in atherosclerotic plaque formation. Recent evidence suggests that elevation of circulating inorganic nitrite, accomplished via dietary nitrate (NO 3 − ) intake, may offer a mode of NO delivery that could restore NO‐dependent protection in atherosclerosis. We assessed if this might be the case in the atherosclerotic prone apolipoprotein E (ApoE) KO mouse model. Methods WT and ApoE KO littermate mice were fed either potassium nitrate (KNO 3 , 15 mM in the drinking water) or equimolar KCL for 2 weeks prior to assessment of inflammatory responses following TNFα stimulation (300ng, i.p., 4h) or were fed the salt solutions for 12 weeks whilst on either a chow or high fat ‘western’ diet for assessment of atheroma formation, leukocyte activation/reactivity and chemokine expression. Results ApoE KO mice exhibited lower levels of tissue and plasma nitrate (WT, 37.7 ± 2.3 μM; ApoE KO 20.0 ± 2.8 μM, P<0.0001, n=12/15) and nitrite (WT, 0.56 ± 0.07 μM; ApoE KO 0.45±0.05 μM, n=11) compared to WT; a difference overcome by KNO 3 feeding. Whilst plaque size was not altered by nitrate treatment in ApoE KO mice there was a reduction in macrophage accumulation within the plaque in chow and ‘western’ fed mice, with an ~4‐fold elevation in smooth muscle accumulation in the latter group (see table below). KNO 3 ‐treated chow fed ApoE KO mice had reduced circulating leukocyte numbers (KCL, 2.0 ± 0.2 cells/ml; KNO 3 , 1.5 ± 0.1 cells/ml, P<0.05 n=12/14) and reduced microvascular leukocyte rolling (KCL, 39.0 ± 4.9 cells/min; KNO 3 , 25.7 ± 2.8 cells/min, P<0.05, n=10,) and adhesion (KCL, 6.3 ± 0.9 cells/100μm; KNO 3 , 3.8 ± 0.4 cells/100μm, P<0.05, n=10), with a similar trend observed in 'western’ fed mice. In addition, there was attenuated circulating neutrophil CD11b expression (~28%, P<0.05; n=12) and plasma chemokine levels including CXCL1 and CCL2 (P<0.05) with a trend for reduced CCL5 expression in nitrate fed mice. In ApoE KO, TNFα elicited inflammatory cell recruitment into the peritoneal cavity (6.1 ± 0.2×10 6 cells) that was modestly attenuated by KNO 3 treatment (5.4 ± 0.1×10 6 cells; P<0.01; n=10) and specifically due to a suppression of neutrophil recruitment (~22%) associated with reductions in both myeloperoxidase activity (~35%) and CD11b expression (~36%, P<0.01, n=9) in KNO 3 fed mice vs KCL. Conclusion Our data suggests that dietary nitrate intervention in ApoE KO mice restores plasma nitrite levels and supresses inflammation by targeting leukocyte margination into the circulation and likely recruitment into the lesional site. We speculate that this activity may have clinical utility in the prevention of atherosclerosis. Support or Funding Information This work was funded by the British Heart Foundation. 1 Comparison of plaque area, macrophage and smooth muscle accumulation in chow and ‘western’ fed ApoE KO mice treated with KCL or KNO 3 . Data shown as mean ± s.e.m and analysed using an unpaired Student t‐test.Total area of plaque (n=12/15) Macrophage staining area (n=8/10) Smooth muscle actin staining area (n=10)Chow KCL 2.8±0.6 N.S. 0.011±0.003 P<0.05 ‐KNO 3 2.6±0.3 0.003±0.001 ‐ Western KCL 5.9±0.6 N.S. 0.027±0.004 P<0.01 0.0009±0.0003 P<0.05 KNO 3 7±0.7 0.014±0.003 0.004±0.001