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. 2020 Sep 15;12(9):5362-5380.
eCollection 2020.

Renal denervation mitigates atherosclerosis in ApoE-/- mice via the suppression of inflammation

Affiliations

Renal denervation mitigates atherosclerosis in ApoE-/- mice via the suppression of inflammation

Han Chen et al. Am J Transl Res. .

Abstract

Atherosclerosis is a chronic pathological process characterized by the accumulation of inflammation. Overactivation of the sympathetic nervous system accelerates the progression of atherosclerosis. Renal denervation (RDN) reduces the activity of the sympathetic nerve system (SNS) by disrupting sympathetic nerves surrounding renal arteries. We sought to determine whether RDN could mitigate atherosclerosis through the suppression of inflammation. First, we investigated the correlation between plasma norepinephrine concentrations and circulatory inflammation in the progression of atherosclerosis. Then, forty ApoE-/- mice underwent renal denervation or a sham operation after 6 weeks or 12 weeks of feeding with a high-fat diet. The effects of RDN on atherosclerosis in mice were explored. In the development of atherosclerosis, positive correlations were found between SNS activation and the accumulation of circulatory myeloid cells and inflammatory cytokines. In the second part of the study, inhibition of the increase in plaque size was found in both RDN groups compared with that in the sham operation (SO) groups (P<0.05), and RDN also ameliorated inflammation in plaques. Furthermore, RDN attenuated the accumulation of circulating neutrophils and monocytes (P<0.05), which is associated with a significant reduction in levels of several circulating inflammatory cytokines related to hemopoiesis (P<0.05). Flow cytometry analysis revealed comparable levels of neutrophils and monocytes in the bone marrow between all four groups. However, RDN decreased the production and proportions of neutrophils and monocytes in the spleen and reduced splenic sympathetic activity (P<0.05). In summary, our study reveals a novel link between SNS activity and inflammation in atherosclerosis and identifies RDN as a potential anti-inflammatory therapeutic strategy for the treatment of atherosclerosis by restricting the production of splenic immune cells.

Keywords: Renal denervation; atherosclerosis; inflammation; sympathetic nerve system.

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Conflict of interest statement

None.

Figures

Figure 1
Figure 1
SNS sympathetic activity is associated with an increased level of inflammation in atherosclerosis. A. A diagram of the experimental design. B. Representative images and quantification analysis of aortic oil red O staining after 6 and 12 weeks of a high-fat diet (HFD). With the extension of the HFD feeding time, atherosclerotic plaques in the aorta developed gradually. C and D. Circulatory levels of MCP-1 and inflammatory cells, as assessed by flow cytometry after 6 weeks or 12 weeks of a HFD, were increased with prolonged feeding of the HFD. E. A positive correlation was found between the norepinephrine (NE) concentration and the plaque size and circulating inflammatory cells. Data were analyzed by an unpaired t test or 1-way ANOVA and expressed as the mean ± SEM, where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the 6 W groups. n=4-8 per group.
Figure 2
Figure 2
RDN reduces sympathetic activity. A. Representative photomicrographs of hematoxylin and eosin (HE) and tyrosine hydroxylase (TH) staining of intrarenal arteries of ApoE-/- mice from the early and late groups. The intact neural structure in the adventitia was destroyed, and TH intensity, a marker of the activity of the sympathetic nerve system (SNS) in both renal denervation (RDN) groups, was significantly reduced compared with that in the corresponding sham groups. The TH staining intensity was scored on a scale of 0 to 3, where 0 indicated no reaction, 1 indicated a patchy/very weak reaction, 2 indicated a weak to moderate reaction, and 3 indicated a strong reaction. B and C. NE concentrations in the kidneys and peripheral blood of ApoE-/- mice were increased with the extension of HFD feeding, which was alleviated by RDN. D. The plasma renin activity and angiotensin II (Ang II) concentration were comparable in all 4 groups. Aldosterone levels decreased significantly after RDN. Data were analyzed by an unpaired t test or 1-way ANOVA and expressed as the mean ± SEM, where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the corresponding SO group. n=5-10 per group.
Figure 3
Figure 3
RDN attenuates the progression and inflammation of plaques. A. Representative images and quantification of aortic oil red O-staining in ApoE-/- mice from the early and late groups. Six weeks after the operation, atherosclerotic plaques in the aortas of ApoE-/- mice were quantified by oil red O staining. The positive area was decreased in the aortas from both RDN groups. B and C. Representative photomicrographs and quantification of HE, Masson’s trichrome, a-SMA, oil red O and CD68 staining of aortic root cross-sectional lesions of ApoE-/- mice from the early and late groups. RDN delayed the progression and inflammation of atherosclerotic plaques in both the early and late groups as measured by HE, Masson’s trichrome, oil red O and CD68 staining, whereas the a-SMA-positive area remained unchanged by RDN. D. The effect of RDN on the mRNA expression of Ccl-2, Il-6, Tnfα, Ifnγ, Caspase1, Nlrp3, Il-1β and Il-18 was significantly decreased by RDN in ApoE-/- mice from the early and late groups. Data were analyzed by an unpaired t test or 1-way ANOVA and expressed as the mean ± SEM, where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the corresponding SO group. n=5-10 per group.
Figure 4
Figure 4
RDN relieves inflammation in the circulation. A. As assessed by flow cytometry, the levels of circulating neutrophils, monocytes and inflammatory monocytes in ApoE-/- mice from both RDN groups were lower than those in the corresponding SHAM groups, and there were decreased proportions of Ly6C high/low monocytes and M1/M2 macrophages. B. A positive correlation was found between the plasma NE concentration and levels of circulatory neutrophils, monocytes and proinflammatory monocytes in all four groups. C. The increase in the concentration of circulatory MCP-1 in ApoE-/- mice was mitigated by RDN in both the early and late groups. D. Heatmap of 20 inflammatory cytokines within the early and late groups showing an overall decreased level of inflammation in both RDN groups. E. Concentration of inflammatory cytokines with significance measured by cytokine microarrays. F. GO analysis of biological processes identified the regulation of hemopoiesis and cytokine production as major changes within the early and late groups. Data were analyzed by an unpaired t test or 1-way ANOVA and expressed as the mean ± SEM, where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the corresponding SO group. n=5-10 per group.
Figure 5
Figure 5
RDN suppresses the production of inflammatory cells in the spleen. A and B. As assessed by flow cytometry, RDN had no effect on the proportions of neutrophils and monocytes in bone marrow but reduced their proportions in spleen. C. The mRNA expression of genes related to the cell cycle (Ccnb1, Ccnd1, Cdk2, Cdk4 and Cdk6) as well as Scf increased over time and were increased in the sham groups. D. Quantification of proliferating cell nuclear antigen (PCNA)-positive cells in spleen showed the relatively decreased proliferation rate in the RDN groups. E. The increase in the splenic NE concentration in ApoE-/- mice was reduced by RDN. F. Representative photomicrographs of TH staining in the spleen of ApoE-/- mice from the early and late groups. As a marker of SNS activity, the splenic TH intensity in both renal denervation (RDN) groups was significantly reduced compared with that in the corresponding sham groups. Data were analyzed by an unpaired t test or 1-way ANOVA and expressed as the mean ± SEM, where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the corresponding SO group. n=5-10 per group.
Figure 6
Figure 6
Schematic diagram illustrating how RDN plays an anti-inflammatory role in atherosclerosis. The inflammatory cytokines released in atherosclerosis pass through the blood-brain barrier, stimulating the sympathetic nerve center. Excitatory sympathetic nerve signals are then transmitted to the kidney and spleen. RDN reduces the excitability of the SNS. Decreased sympathetic signaling in the spleen attenuates the production of monocytes and neutrophils. As a result, there is a decrease in the number of inflammatory cells released into the blood and infiltrating into the plaque.

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