Role of the CCL5 and Its Receptor, CCR5, in the Genesis of Aldosterone-Induced Hypertension, Vascular Dysfunction, and End-Organ Damage

Abstract

Background: Aldosterone has been described to initiate cardiovascular diseases by triggering exacerbated sterile vascular inflammation. The functions of CCL5 (C-C motif chemokine ligand 5) and its receptor CCR5 (C-C motif chemokine receptor 5) are well known in infectious diseases, their contributions to aldosterone-induced vascular injury and hypertension remain unknown.

Methods: We analyzed the vascular profile, blood pressure, and renal damage in wild-type (CCR5^+/+) and CCR5 knockout (CCR5^-/-) mice treated with aldosterone (600 µg/kg per day for 14 days) while receiving 1% saline to drink. Vascular function was analyzed in aorta and mesenteric arteries, blood pressure was measured by telemetry and renal injury and inflammation were analyzed via histology and flow cytometry. Endothelial cells were used to study the molecular signaling whereby CCL5 induces endothelial dysfunction.

Results: Aldosterone treatment resulted in exaggerated CCL5 circulating levels and vascular CCR5 expression in CCR5^+/+ mice accompanied by endothelial dysfunction, hypertension, and renal inflammation and damage. CCR5^-/- mice were protected from these aldosterone-induced effects. Mechanistically, we demonstrated that CCL5 increased NOX1 (NADPH oxidase 1) expression, reactive oxygen species formation, NFκB (nuclear factor kappa B) activation, and inflammation and reduced NO production in isolated endothelial cells. These effects were abolished by antagonizing CCR5 with Maraviroc. Finally, aorta incubated with CCL5 displayed severe endothelial dysfunction, which is prevented by blocking NOX1, NFκB, or CCR5.

Conclusions: Our data demonstrate that CCL5/CCR5, through activation of NFκB and NOX1, is critically involved in aldosterone-induced vascular and renal damage and hypertension placing CCL5 and CCR5 as potential therapeutic targets for conditions characterized by aldosterone excess.

Keywords: NADPH oxidases; aldosterone; chemokines; chemokines receptors; oxidative stress.

Figure 1.. Aldosterone increases CCL5 circulating levels, CCR5 vascular expression inducing endothelial dysfunction.

Inflammatory profile in plasma, measured by proteome profiler mouse cytokine array and presented as heat map (A) (pool of 6 mice per group); CCL5 levels, measured by ELISA (B); and chemokine receptors expression in aortae measured by RT-PCR (C) (n=3), from CCR5^+/+ mice treated with vehicle or aldosterone (600 μg/kg/day for 14 days). Chemokine receptors expression, measured by RT-PCR, in endothelial cells (MEC) treated with vehicle or aldosterone (0.1μM) in the presence or not of mineralocorticoid receptor (Eplerenone, 1μM) (D) (n=4). Concentration-effect curves to acetylcholine in aortae from CCR5^+/+ mice incubated with vehicle or CCL5 (1, 10 and 100ng/ml, 24h) (E) (n=4). Concentration-effect curves to acetylcholine in aortae from CCR5^+/+ mice incubated with vehicle or CCL5 (100ng/ml, 24h) in the presence of CCR5 antagonist (Maraviroc, 40μM) (F) (n= 3-7). Values represent means ± SEM. Student t test or ANOVA test. *p<0.05 vs. Vehicle; #p<0.05 vs. CCL5.

Figure 2.. CCR5 deficiency prevents endothelial dysfunction and vascular inflammation induced by aldosterone.

Concentration-effect curves to acetylcholine (A) and sodium nitroprusside (B)in aortae (n=4-7); phosphorylated (p65 subunit) and total NFκB expression, analyzed by western blot (C) (n=4) and inflammatory markers, measured by RT-PCR (D) (n=4), in aortae from CCR5^+/+ and CCR5^−/− mice treated with vehicle or aldosterone (600 μg/kg/day + saline for 14 days). Phosphorylated (p65 subunit) and total NFκB expression analyzed by western blot in aortae from CCR5^+/+ mice incubated with vehicle or CCL5 (100ng/ml, 24h) (E) (n=4-5) and concentration-effect curves to acetylcholine (F) in aortae from CCR5^+/+ mice incubated with vehicle or CCL5 (100ng/ml, 24h) in the presence of an IκK/IKB inhibitor (BMS-345541, 5μM) (n=4-5). Values represent means ± SEM. Student t test or ANOVA test. *p<0.05 vs. CCR5^+/+ or Vehicle; #p<0.05 vs. CCR5^+/+_Aldo or CCL5.

Figure 3.. CCL5 via CCR5 induces endothelial cell activation and immune cell adhesion.

Photomicrography and fluorescence intensity depicting labeled macrophages (calcein-AM probe, green) and endothelial cells (MEC, DAPI, blue). MEC were treated with vehicle, Maraviroc (40μM, 30 minutes prior CCL5 incubation), CCL5 (100ng/ml, 24h) or CCL5 plus Maraviroc. Scale bar = 100μm (A) (representative of 4 independent experiments). Fluorescence intensity of lysate cells after treatments described above (B) (n=4), and inflammatory markers, measured by RT-PCR, in MEC treated with vehicle or CCL5 (100ng/ml) in the presence of Maraviroc (40μM) (C) (n=4). Values represent means ± SEM. ANOVA test.

Figure 4.. Aldosterone induces hypertension and kidney injury via CCR5.

Systolic blood pressure (A) and diastolic blood pressure (B), measured via radiotelemetry, in CCR5^+/+ and CCR5^−/− mice treated with vehicle or aldosterone (600 μg/kg/day and saline for 14 days) (n=4). Immunofluorescence for podocyte marker synaptopodin (Synap; green), endothelial marker endomucin (Endom; red), and nuclei (DAPI; blue), α-smooth muscle actin (α-SMA; green), fluorescein-labeled collagen III (red) and Lotus tetragonolobus lectin (LTA; green), fibrosis was also measured by Trichrome stain. Images were obtained from kidney sections of CCR5^+/+ and CCR5^−/− mice treated with vehicle or aldosterone (n=4). The images shown are representative of 3 independent experiments. Scale bar = 20, 30, or 50μm (C). Kidney injury markers, measured by RT-PCR in CCR5^+/+ and CCR5^−/− mice treated with vehicle or aldosterone (D) (n=4), and proteinuria levels in CCR5^+/+ and CCR5^−/− mice treated with vehicle or aldosterone measured by 10% SDS-PAGE gels followed by staining with Coomassie Brilliant. Albumin was used as a control analyzed (E) (n=4) The images shown are representative of 4 independent experiments. Values represent means ± SEM. Student t test or ANOVA test. *p<0.05 vs. CCR5^+/+.

Figure 5.. Aldosterone, via CCR5, promotes endothelial dysfunction by NOX1-dependent mechanisms.

Representative western blot (A) to NOX1 (i), NOX2 (ii) and NOX4 (iii) expression (n=4); ROS generation, measured Amplex red assay (B); and concentration-effect curves to acetylcholine, in the presence of NOXA1ds (10μM) (C) (n=4), in aortae from CCR5^+/+ and CCR5^−/− mice treated with vehicle or aldosterone (600 μg/kg/day for 14 days). Representative western blot for NOX1 (i); ROS generation (ii) (D) (n=4-5); and concentration-effect curves to acetylcholine in aortae from CCR5+/+ mice, in the presence of NOX1 inhibitor (NOXA1ds, 10μM) and/or vehicle or CCL5 (100ng/ml) (E) (n=4-7). Representative western blot to NOX1 (i) and ROS generation (ii), in endothelial cells (MEC) treated with vehicle or CCL5 (100ng/ml) in the presence of Maraviroc (40μM) and NOXA1ds (10μM) (F) (n=3). Values represent means ± SEM. ANOVA test. *p<0.05 vs. CCR5^+/+ or Vehicle; #p<0.05 vs. CCR5^+/+_Aldo or CCL5.

Figure 6.. CCL5 induces an increase in NOX1 expression and NFKB activity by a positive feedback mechanism in endothelial cells.

Representative western blot to NOX1 expression (A) N=4) and ROS generation, measured by Amplex red (B) (N=3), in endothelial cells (MEC) treated with vehicle or CCL5 (100ng/ml) (N=4), in the presence of an IκK/IKB inhibitor (BMS-345541, 5μM). Representative western blot for phosphorylated and total NFκB expression in MEC treated with vehicle or CCL5, in the presence of NOXA1ds (10μM) (C) (n=4). Values represent means ± SEM. ANOVA test.