SOD2 ameliorates pulmonary hypertension in a murine model of sleep apnea via suppressing expression of NLRP3 in CD11b+ cells

Abstract

Background: High prevalence of obstructive sleep apnea (OSA) in the pulmonary hypertension (PH) population suggests that chronic intermittent hypoxia (CIH) is an important pathogenic factor of PH. However, the exact mechanism of CIH induced PH is not clear. One of the molecules that plays a key role in regulating pulmonary artery function under hypoxic conditions is superoxide dismutase 2 (SOD₂).

Methods: Our study utilized heterozygous SOD₂^-/+ mice firstly in CIH model to explore the exact role of SOD₂ in CIH causing PH. Expression of SOD2 was analyzed in CIH model. Echocardiography and pulmonary hypertension were measured in wild type (WT) and SOD2^-/+ mice under normal air or CIH condition. Hematoxylin-Eosin (H&E) staining and masson staining were carried out to evaluate pulmonary vascular muscularization and remodeling. Micro-PET scanning of in vivo ^99mTc-labelled- MAG3-anti-CD11b was applied to assess CD11b in quantification and localization. Level of nod-like receptor pyrin domain containing 3 (NLRP3) was analyzed by real time PCR and immunohistochemistry (IHC).

Results: Results showed that SOD₂ was down-regulated in OSA/CIH model. Deficiency of SOD2 aggravated CIH induced pulmonary hypertension and pulmonary vascular hypertrophy. CD11b⁺ cells, especially monocytic myeloid cell line-Ly6C⁺Ly6G^- cells, were increased in the lung, bone marrow and the blood under CIH condition, and down-regulated SOD2 activated NLRP3 in CD11b⁺ cells. SOD₂-deficient-CD11b⁺ myeloid cells promoted the apoptosis resistance and over-proliferation of human pulmonary artery smooth muscle cells (PASMCs) via up-regulating NLRP3.

Conclusion: CIH induced down-regulating of SOD2 increased pulmonary hypertension and vascular muscularization. It could be one of the mechanism of CIH leading to PH.

Keywords: CD11b; Chronic intermittent hypoxia; NLRP3; Pulmonary hypertension; SOD2.

Fig. 1

Expression of SOD₂ in the plasma. a. Concentration of SOD₂ in the plasma of both healthy donators (control) and OSA patients (mild, moderate, severe OSA). b. Relative mRNA expression of SOD₂ in the control and chronic intermittent hypoxia (CIH) mice. c. Representative graph of western blot expression of SOD2 compared to β-actin. d. Ratio of SOD₂/β-actin in the group of wild type (WT) mice and SOD2 knockdown mice under control or CIH condition. CON: Wild type mice in the normal air condition. CIH: Chronic intermittent hypoxia. WT: wild type mice. SOD₂^−/+: knock-down of SOD₂ gene heterozygous mice. N = 5 in experiment of b and c. Comparisons between two groups were performed using unpaired T-test after normality distribution test. Comparisons between multiple groups were performed using ANOVA with the Bonferroni test.*p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

PH pressure detection and Echocardiography in WT and SOD2^−/+ mice. a. Right ventricular systolic pressure (RVSP) measured by closed-chest puncture of the right ventricle (RV). b. Ratio of right ventricle to left ventricle plus septum in the four groups. c. Statistical analysis of right ventricle cross-sectional area (RVCSA). d. Representative enchocardiography of RV. e. Statistical analysis of thickness of RV. f. The representative view of PA in color Doppler mode, used to assess flow through the PA, was presented. g. The blood flow velocity of PA was measured by echocardiography. h. The representative enchocardiography of tricuspid annular plane systolic excursion (TAPSE) in the four groups. i. Data of TAPSE. CON: Wild type mice in the normal air condition. CIH: Chronic intermittent hypoxia. WT: wild type mice. SOD₂^−/+: knock-down of SOD₂ gene heterozygous mice. N = 5. Comparisons between multiple groups were performed using ANOVA with the Bonferroni test. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Deficiency of SOD₂ affects the pulmonary vascular remodeling. a. HE staining of pulmonary arterioles in WT and SOD₂^−/+ mice. b. Masson staining of collagen in WT and SOD₂^−/+ mice under CIH condition. c. Co-Immunofluorescence staining of the molecular of α-SMA and VWF. α-SMA was stained as red and VWF was green. Cellular nucleus was stained by DAPI (blue). d. The same lung lobe from WT and SOD₂^−/+ mice were analyzed of pulmonary vessel muscularization. Percentage of area of α-SMA⁺/(α-SMA⁺+VWF⁺) indicated the muscularization degree. Zero-20% represented for no muscularization (N), 20–40% for partly muscularization (P), and 40–100% for fully muscularization (M). e. Heart weight to tibia length ration showed the degree of myocardial hypertrophy in WT and SOD₂^−/+ mice. CON: Wild type mice in the normal air condition. CIH: Chronic intermittent hypoxia. WT: wild type mice. SOD₂^−/+: knock-down of SOD₂ gene heterozygous mice. N = 5. Comparisons between multiple groups were performed using ANOVA with the Bonferroni test. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

Flow cytometry analysis of percentage of CD11b⁺ cells in WT and SOD₂^−/+ mice. a-d. Representative view of flow cytometry graph of CD11b + cells in the blood of WT and SOD₂^−/+ mice. e. Percentage of CD11b⁺ cells in the peripheral blood mononuclear cells of the four groups analyzed by Flowjo software. f. Percentage of Ly6C⁺Ly6G⁻ cells and Ly6C⁻Ly6G⁺ cells in CD11b⁺ cells in the blood. g. Percentage of CD11b⁺ cells in the pulmonary mononuclear cells of the four groups. h. Percentage of Ly6C⁺Ly6G⁻ cells and Ly6C⁻Ly6G⁺ cells in CD11b⁺ cells in the lung. i. Percentage of CD11b⁺ cells in the bone marrow mononuclear cells of the four groups. j. Percentage of Ly6C⁺Ly6G⁻ cells and Ly6C⁻Ly6G⁺ cells in CD11b⁺ cells in the bone marrow. CON: Wild type mice in the normal air condition. CIH: Chronic intermittent hypoxia. WT: wild type mice. SOD₂^−/+: knock-down of SOD₂ gene heterozygous mice. N = 5. Comparisons between multiple groups were performed using ANOVA with the Bonferroni test. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Micro-PET scanning of in vivo ^99mTc-labelled- MAG3-anti-CD11b in SOD2 deficiency mice. a. Representative radioactive heat map of WT and SOD₂^−/+ mice under condition of normal air and CIH. b. Lung mean-SUV value in the four groups. c. Total reserved SUV in the lung of WT and SOD₂^−/+ mice after CIH intervention. d. Cardiac mean-SUV value in the four groups. e. Total reserved SUV in the heart of WT and SOD₂^−/+ mice after CIH intervention. CON: Wild type mice in the normal air condition. CIH: Chronic intermittent hypoxia. WT: wild type mice. SOD₂^−/+: knock-down of SOD₂ gene heterozygous mice. N = 5. Comparisons between multiple groups were performed using ANOVA with the Bonferroni test. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

Expression of NLRP3 in PH and CIH model. a. mRNA expression of NLRP3 in the lung in WT and SOD₂^−/+ mice under normal air and CIH condition. b. Measurement of histology score of IHC staining of NLRP3 in the lung in the four groups. c and e. IHC staining of NLRP3 in the lung in the four groups. Arrows were pointed to the pulmonary arteriole. The rectangular frame was amplified in the same times in the picture of c and e; d and f. Co-staining of CD11b and NLRP3 by immunofluorescence in the four groups. Blue represented DAPI. Red represented NLRP3. Green represented CD11b. The rectangular frame was amplified in the same times in the picture of d and f. CON: Wild type mice in the normal air condition. CIH: Chronic intermittent hypoxia. WT: wild type mice. SOD₂^−/+: knock-down of SOD₂ gene heterozygous mice. N = 5. Comparisons between multiple groups were performed using ANOVA with the Bonferroni test. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

Co-culture CD11b⁺ myeloid cells and PASMCs. Co-culturing SOD₂-deficient-CD11b⁺ myeloid cells and PASMC cells using transwell plates (CD11b⁺ cells in the upper side; PASMCs in the lower side). a-c. Separation gain rate of CD11b⁺ cells analyzed by FACS. CD11b⁺ cells were separated from C57BL/6 and SOD₂^−/+ mice using magnetic beads. d. PASMCs (the lower side plates) were stained to measure the percentage of tunnel positive cells. e. Brdu positive staining in PASMCs in the four groups. f. mRNA expression of NLRP3 in CD11b⁺ cells. g. mRNA expression of NLRP3 in PASMCs. h. Secretion of IL-1β in the supernatant of the co-culture system. i. Secretion of IL-18 in the supernatant of the co-culture system. CON: Wild type mice in the normal air condition. CIH: Chronic intermittent hypoxia. WT: wild type mice. SOD₂^−/+: knock-down of SOD₂ gene heterozygous mice. N = 5. Comparisons between multiple groups were performed using ANOVA with the Bonferroni test. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 8

Flow chart of the role of SOD2 in chronic intermittent hypoxia induced pulmonary hypertension.