Myeloid ACE2 protects against septic hypotension and vascular dysfunction through Ang-(1–7)-Mas-mediated macrophage polarization

Figures

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  Fig. 1. Imbalance of aortic renin-angiotensin system after CLP-induce sepsis (A), Schematic of the methods for treatment of wild-type (WT) mice with cecal ligation and puncture (CLP) surgery. (B), Survival rate after CLP treatment (n = 24). (C), Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured via the noninvasive tail-cuff method in each group (n = 10). (D), Immunoblotting analysis of ACE, AT1R, ACE2, MasR protein in the aorta of each group, GAPDH was used as an internal control (n = 4). (E), Analysis of ACE, Ang II, and Ang (1–7) levels as well as ACE2 activity in each group (n = 5–6). The data are presented as the mean ± SEM, n represents the samples of each group. Data in (B) were analyzed with Kaplan-Meier; data in C and D were analyzed with 1-way ANOVA by Bonferroni multiple comparison test; data in (E) were analyzed with Mann-Whitney test. NS: No significance.

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  Fig. 2. Overexpression of ACE2 ameliorates sepsis-induced hypotension and vascular dysfunction (A), WT and ACE2 knock-in (ACE2-KI) mice were subjected to CLP surgery. Measurement of aortic ACE2 activity and Ang (1–7) and Ang II levels in each group at 24 h (n = 6). (B), Survival rate of each group after CLP surgery (n = 15). (C), Detection of SBP and DBP in each group by the noninvasive tail-cuff method (n = 5). (D), Concentration-dependent vasodilation of mesenteric artery rings, including endothelium-dependent (Ach 10−9-10−4 mol/L) and non-endothelium-dependent (SNP 10−9-10−5 mol/L) (n = 6). The data are presented as the mean ± SEM, n represents the samples of each group. Data in (A) were analyzed by Mann-Whitney test; data in (B) were analyzed by Kaplan-Meier; data in (C) were analyzed with 2-way ANOVA followed by Tukey multiple comparison test; data in (D) were analyzed by repeated-measures ANOVA.

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  Fig. 3. Deficiency of ACE2 aggravates sepsis-induced hypotension and vascular dysfunction (A), WT and ACE2 knockout (ACE2-KO) mice were subjected to CLP surgery for 24 h. Analysis of aortic ACE2 activity and Ang (1–7) and Ang II levels in each group (n = 6). (B), Survival rate of each group (n = 15). (C), Measurement of SBP and DBP in each group by the noninvasive tail-cuff method (n = 5). (D), Concentration-dependent vasodilation of mesenteric artery rings, including endothelium-dependent (Ach 10−9-10−4 mol/L) and non-endothelium-dependent (SNP 10−9-10−5 mol/L) (n = 6). The data are presented as the mean ± SEM, n represents the samples of each group. Data in (A) were analyzed by Mann-Whitney test; data in (B) were analyzed by Kaplan-Meier; data in (C) were analyzed with 2-way ANOVA followed by Tukey multiple comparison test; data in (D) were analyzed by repeated-measures ANOVA.

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  Fig. 4. Bone marrow-derived cells expressing ACE2 improves CLP-caused hypotension and vascular dysfunction (A), Immunostaining of aorta sections with anti-F4/80 and anti-ACE2 antibodies to examine the localization of ACE2 in macrophages (n = 4). Scale bar, 50 μm. (B), Immunoblotting analysis of ACE2 protein in the bone marrow cells of ACE2-KI mice. β-Actin was used as an internal control (n = 4). (C), An experimental protocol showing WT, ACE2-KI and ACE2-KO bone marrow transplantation to irradiated WT mice. (D), The aortic Ang II level in BMT mice after CLP (left, n = 6) and the aortic Ang (1–7) level in BMT mice after CLP (n = 6) (E), Systolic blood pressure and diastolic blood pressure was measured via the noninvasive tail-cuff method in BMT mice after CLP (n = 8). (F), Concentration-dependent vasodilation of aorta ring, including endothelium-dependent (Ach 10−9-10−4 mol/L) and non-endothelium-dependent (SNP 10−9-10−5 mol/L) (n = 6). The data are presented as the mean ± SEM, n represents the samples of each group. Data in (B) were analyzed by t-test; Data in (D and E) were analyzed by Mann-Whitney test; data in (F) were analyzed by repeated-measures ANOVA.

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  Fig. 5. ACE2 inhibits CLP-induced oxidative stress, NO production and macrophage polarization in vivo (A), WT and ACE2 knockin (ACE2-KI) mice were subjected to CLP surgery for 24 h. Dihydroethidium (DHE) staining of the aortic segments (left, scale bar, 50 μm) and quantification (right, n = 6). (B), qPCR analysis of NOX2 mRNA expression level in the aorta (n = 6). (C), The MDA level in aorta (n = 6). (D), Analysis of serum total nitrite levels (n = 5–8). (E), Immunoblotting analysis of 3-NT protein in the aorta. β-Actin was used as an internal control (n = 4). (F), Immunohistochemical staining of 3-nitrotyrosine (NT) in the aorta (left, scale bar, 50 μm) and quantification of 3-NT intensity (right, n = 5). (G), Immunostaining of aortic segments with anti-Mac-2 antibody (left, scale bar, 50 μm) and quantification of Mac-2-positive macrophages (right, n = 6). (H), qPCR analysis of iNOS mRNA expression level in the PBLs (n = 6). (I), qPCR analysis of the mRNA levels of IL-1β, IL-6, TNF-α and IFN-γ and ARG1 and YM1 in the aorta (n = 6). The data are presented as the mean ± SEM, n represents the samples of each group. Data in (A) through (C) and (F) and (H) and (I) were analyzed by…

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  Fig. 6. ACE2 expression on myeloid cells suppresses vascular oxidative stress, NO production and macrophage polarization (A), WT, ACE2-KI and ACE2-KO BM cell were transplantation to irradiated WT mice and subjected to CLP for 24 h. DHE staining of the aortic segments (left, scale bar, 50 μm) and quantification (right, n = 6). (B), Immunostaining of aortic segments with anti-Mac-2 antibody (left, scale bar, 50 μm) and quantification of Mac-2-positive macrophages (right, n = 6). (C), Analysis of serum total nitrite levels (n = 8). (D), qPCR analysis of NOX2 mRNA expression level in the aorta (n = 6). (E), qPCR analysis of the mRNA levels of IL-1β, IL-6, TNF-α and IFN-γ and ARG1 and YM1 in the aorta (n = 6). The data are presented as the mean ± SEM, n represents the samples of each group. Data in (A) and (B) were analyzed with 2-way ANOVA followed by Tukey multiple comparison test; data in (C) through (E) were analyzed by Mann-Whitney test.

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  Fig. 7. ACE2 attenuates CLP-induced macrophage polarization and endothelial cell injury through Ang (1–7)-MasR-dependent mechanism (A), BMDMs isolated from WT and ACE2-KI mice were stimulated with LPS (1000 ng/ml) for 24 h. DHE staining of ROS production in BMDMs (left), and the quantification of DHE fluorescence intensity in each group (right, n = 3). Scale bar, 50 μm. (B), qPCR analysis of IL-1β, IL-6, TNF-α, ARG1 and YM1 mRNA levels in BMDMs (n = 3). (C), Immunoblotting analysis of phosphorylated (P)-p65, p65, P-STAT1 and STAT1 proteins in BMDMs (left), and the quantification of protein intensity in each group (n = 3). β-Actin was used as an internal control. (D), BMDMs isolated from WT and ACE2-KI mice were pretreated with or without MasR inhibitor A779 (10 μM) for 30 min and then stimulated with LPS (1000 ng/ml) or saline for additional 6 h and placed in transwell inserts above HUVECs for 24 h. (E), qPCR analysis of E-selectin, ICAM-1 and VCAM-1 mRNA expression levels of HUVECs co-cultured with WT or ACE2-KI BMDMs (n = 3). (F), The detection of total nitrite level in media supernatant released by HUVECs (n = 3). (G), qPCR analysis of NOX1 and NOX4 mRNA expression levels of HUVECs…

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  Fig. 8. Analysis of the RAAS components in plasma of patients with sepsis and hypotension Schematic model for ACE2 in sepsis-induced hypotension and vascular dysfunction. Sepsis reduces myeloid ACE2 expression, which blocks Ang II hydrolysis and Ang (1–7) production, resulting in inhibition of MasR and activation of NF-kB and STAT1 signals, this promotes macrophage M1 polarization and the generation of pro-inflammatory cytokines, ROS, and NO, thereby leading to endothelial dysfunction and hypotension in wild-type mice. In contrast, ACE2 overexpression in mice ameliorates sepsis-induced effects. These data highlight that selective activation of ACE2 may represent promising therapeutic option for sepsis and its related complications.