CXCL1–CXCR2 axis mediates angiotensin II-induced cardiac hypertrophy and remodelling through regulation of monocyte infiltration

CXCL1–CXCR2 axis mediates angiotensin II-induced cardiac hypertrophy and remodelling through... Abstract Aims Chemokine-mediated monocyte infiltration into the damaged heart represents an initial step in inflammation during cardiac remodelling. Our recent study demonstrates a central role for chemokine receptor CXCR2 in monocyte recruitment and hypertension; however, the role of chemokine CXCL1 and its receptor CXCR2 in angiotensin II (Ang II)-induced cardiac remodelling remain unknown. Methods and results Angiotensin II (1000 ng kg−1 min−1) was administrated to wild-type (WT) mice treated with CXCL1 neutralizing antibody or CXCR2 inhibitor SB265610, knockout (CXCR2 KO) or bone marrow (BM) reconstituted chimeric mice for 14 days. Microarray revealed that CXCL1 was the most highly upregulated chemokine in the WT heart at Day 1 after Ang II infusion. The CXCR2 expression and the CXCR2+ immune cells were time-dependently increased in Ang II-infused hearts. Moreover, administration of CXCL1 neutralizing antibody markedly prevented Ang II-induced hypertension, cardiac dysfunction, hypertrophy, fibrosis, and macrophage accumulation compared with Immunoglobulin G (IgG) control. Furthermore, Ang II-induced cardiac remodelling and inflammatory response were also significantly attenuated in CXCR2 KO mice and in WT mice treated with SB265610 or transplanted with CXCR2-deficienct BM cells. Co-culture experiments in vitro further confirmed that CXCR2 deficiency inhibited macrophage migration and activation, and attenuated Ang II-induced cardiomyocyte hypertrophy and fibroblast differentiation through multiple signalling pathways. Notably, circulating CXCL1 level and CXCR2+ monocytes were higher in patients with heart failure compared with normotensive individuals. Conclusions Angiotensin II-induced infiltration of monocytes in the heart is largely mediated by CXCL1–CXCR2 signalling which initiates and aggravates cardiac remodelling. Inhibition of CXCL1 and/or CXCR2 may represent new therapeutic targets for treating hypertensive heart diseases. Chemokine CXCL1 , Receptor CXCR2 , Monocyte infiltration , Angiotensin II , Inflammation , Cardiac remodelling Translational perspective Our present study reveals that CXCL1–CXCR2 axis is essential for monocyte infiltration into the heart and cardiac remodelling induced by angiotensin II. Blocking of CXCL1 and CXCR2 activation or ablation of CXCR2 effectively prevents adverse cardiac remodelling. Thus, pharmacological targeting of CXCL1–CXCR2 signalling may represent a novel therapeutic strategy for treating hypertensive heart diseases. Introduction Adverse cardiac remodelling is now recognized as a determinant of the clinical course of heart failure (HF), which is still one of the leading causes of death worldwide.1 This process is mainly influenced by haemodynamic load and neuro-hormonal activation.2 Among the neuro-humoral factors, angiotensin (Ang) II, a key component of the renin–angiotensin system (RAS), plays an important role in the pathogenesis of cardiac remodelling in a variety of diseases.3 Interestingly, pharmacological or genetic interventions that inhibit RAS activity have been shown to reduce cardiac inflammation and improve HF.3 There is growing evidence to support the critical role of inflammatory cells especially monocytes/macrophages in the pathophysiology of HF in both animal and human research.4 Therefore, it is important to identify the mechanisms that attract these cells into the heart after hypertensive stress to prevent cardiac dysfunction. Chemokines are a family of inflammatory cytokines that have ability to induce the directional migration and activation of leucocytes. They are classified into four distinct classes, based on a shared cysteine motif: CC (CCL1–28), CXC (CXCL1–17), CX3C (CX3C1), and C (XCL1-2). Chemokines exert their biological functions via cell–surface receptors, which are designated as CCRs, CXCRs, CX3CRs, and XCRs. Increasing evidence suggests that these chemokines play a fundamental role in recruiting neutrophils, monocytes, and lymphocytes to the injured heart.5 Interestingly, several chemokines [MIP-1a, MIP-1β, RANTES, CXCL1 and interleukin (IL)-8, and CXCL5] and their receptors (CCR1, CCR2, CCR5, CCR7, CXCR1, CXCR2, and CX3CR) have been found in animal models and patients with hypertrophy and HF, and some of them are closely related to the disease severity.6,7 Among them, CXC chemokine CXCL1 and its receptor CXCR2 play a critical role in promoting the recruitment of neutrophils and monocytes/macrophages into the injured heart and arterial wall, thereby inducing myocardial infarction, ischaemia/reperfusion injury, atherosclerosis, and hypertension.8–10 Recent studies have demonstrated that monocytes have been implicated in the pathogenesis of Ang II-induced hypertension and cardiac remodelling.4,11–13 Therefore, it is important to explore whether the CXCL1–CXCR2 axis induces recruitment of monocytes into the heart and has a critical role in Ang II-induced cardiac remodelling. In this study, we showed that bone marrow (BM)-derived CXCR2+ monocytes are involved in Ang II-induced adverse cardiac remodelling and dysfunction. The levels of CXCL1 and CXCR2 were markedly upregulated in Ang II-induced hypertrophic heart and blood of HF patients. The inhibition of CXCL1 and CXCR2 or deletion of CXCR2 significantly reduced Ang II-induced infiltration of monocytes into the heart thereby improving cardiac remodelling and dysfunction. Therefore, these results provide novel evidence supporting that inhibition of CXCL1–CXCR2 signalling exerts a cardio-protective role and representing a new therapeutic target for hypertensive cardiac remodelling. Methods Animals Wild-type (WT) mice (C57BL/6J, male) and CXCR2 knockout mice [CXCR2 KO, B6.129S2(C)-Cxcr2tm1Mwm/J] at 8–12 weeks of age were used to establish the cardiac remodelling model by subcutaneous infusion of Ang II at a dose of 1000 ng/kg/min as described previously.13 The anti-CXCL1 antibody (100 μg/mice/day) and CXCR2 inhibitor SB265610 (2 mg/kg/day) were administrated intraperitoneally to WT mice beginning 1 day before Ang II infusion and continued during Ang II treatment (Supplementary material online). Flow cytometry The inflammatory cells in the heart tissues were analysed by flow cytometry (Supplementary material online). Histological study Heart samples, fixed in 4% paraformaldehyde and embedded in paraffin, were used for the quantification of cardiac hypertrophy, fibrosis, and inflammatory cells (Supplementary material online). Bone marrow chimeric mice Chimeric mice were used to examine the contribution of BM-derived CXCR2-positive cells to cardiac hypertrophic remodelling (Supplementary material online). Human study populations We explored the patients with HF with reduced ejection fraction (EF%) (n = 32) and age- and sex-matched control subjects (n = 30) in a monocentric clinical cohort between August 2017 and October 2017. Heart failure patients were diagnosed according to the 2016 ESC Guidelines.1 The baseline characteristics of control subjects and patients are indicated in Table 1 (Supplementary material online). Table 1 Baseline characteristics of normotensive control subjects and heart failure patients Parameters Normotensive Heart failure P-value Controls (n = 32) Patients (n = 30) Age (years) 53.84 ± 7.14 71.27 ± 13.57 <0.001 Male, n (%) 19 (59.4) 16 (53.3) 0.634 LVEF (%) 65.00 ± 1.88 44.47 ± 12.68 <0.001 Septum (mm) 9.66 ± 0.94 10.67 ± 1.95 0.009 LVEDD (mm) 45.63 ± 1.91 60.10 ± 11.50 <0.001 LV posterior wall (mm) 9.31 ± 0.78 9.97 ± 1.19 0.310 BNP (pg/mL) 26.76 ± 15.37 1151.83 ± 1669.10 <0.001 Systolic blood pressure (mmHg) 121.19 ± 13.08 135.63 ± 20.94 0.014 Diastolic blood pressure (mmHg) 76.16 ± 8.97 78.40 ± 15.05 0.632 Heart rate (b.p.m.) 67.66 ± 7.32 87.27 ± 19.41 <0.001 Total cholesterol (mmol/L) 5.65 ± 1.53 3.85 ± 0.99 0.214 LDL cholesterol (mmol/L) 3.02 ± 1.04 2.14 ± 0.61 0.133 HDL cholesterol (mmol/L) 1.56 ± 0.34 1.07 ± 0.38 <0.001 Triglycerides (mmol/L) 1.44 ± 0.76 1.31 ± 0.89 0.375 Creatinemia (µmol/L) 62.45 ± 15.00 102.02 ± 52.66 <0.001 Fasting blood glucose (mmol/L) 5.47 ± 1.05 6.43 ± 2.40 0.001 White blood cell count (109/L) 5.85 ± 1.64 7.15 ± 2.11 0.009 Parameters Normotensive Heart failure P-value Controls (n = 32) Patients (n = 30) Age (years) 53.84 ± 7.14 71.27 ± 13.57 <0.001 Male, n (%) 19 (59.4) 16 (53.3) 0.634 LVEF (%) 65.00 ± 1.88 44.47 ± 12.68 <0.001 Septum (mm) 9.66 ± 0.94 10.67 ± 1.95 0.009 LVEDD (mm) 45.63 ± 1.91 60.10 ± 11.50 <0.001 LV posterior wall (mm) 9.31 ± 0.78 9.97 ± 1.19 0.310 BNP (pg/mL) 26.76 ± 15.37 1151.83 ± 1669.10 <0.001 Systolic blood pressure (mmHg) 121.19 ± 13.08 135.63 ± 20.94 0.014 Diastolic blood pressure (mmHg) 76.16 ± 8.97 78.40 ± 15.05 0.632 Heart rate (b.p.m.) 67.66 ± 7.32 87.27 ± 19.41 <0.001 Total cholesterol (mmol/L) 5.65 ± 1.53 3.85 ± 0.99 0.214 LDL cholesterol (mmol/L) 3.02 ± 1.04 2.14 ± 0.61 0.133 HDL cholesterol (mmol/L) 1.56 ± 0.34 1.07 ± 0.38 <0.001 Triglycerides (mmol/L) 1.44 ± 0.76 1.31 ± 0.89 0.375 Creatinemia (µmol/L) 62.45 ± 15.00 102.02 ± 52.66 <0.001 Fasting blood glucose (mmol/L) 5.47 ± 1.05 6.43 ± 2.40 0.001 White blood cell count (109/L) 5.85 ± 1.64 7.15 ± 2.11 0.009 BNP, brain natriuretic peptide; HDL, high-density lipoprotein; LDL, low-density lipoprotein; LVEDD, left ventricular end diastolic diameter; LVEF, left ventricular ejective fraction. Table 1 Baseline characteristics of normotensive control subjects and heart failure patients Parameters Normotensive Heart failure P-value Controls (n = 32) Patients (n = 30) Age (years) 53.84 ± 7.14 71.27 ± 13.57 <0.001 Male, n (%) 19 (59.4) 16 (53.3) 0.634 LVEF (%) 65.00 ± 1.88 44.47 ± 12.68 <0.001 Septum (mm) 9.66 ± 0.94 10.67 ± 1.95 0.009 LVEDD (mm) 45.63 ± 1.91 60.10 ± 11.50 <0.001 LV posterior wall (mm) 9.31 ± 0.78 9.97 ± 1.19 0.310 BNP (pg/mL) 26.76 ± 15.37 1151.83 ± 1669.10 <0.001 Systolic blood pressure (mmHg) 121.19 ± 13.08 135.63 ± 20.94 0.014 Diastolic blood pressure (mmHg) 76.16 ± 8.97 78.40 ± 15.05 0.632 Heart rate (b.p.m.) 67.66 ± 7.32 87.27 ± 19.41 <0.001 Total cholesterol (mmol/L) 5.65 ± 1.53 3.85 ± 0.99 0.214 LDL cholesterol (mmol/L) 3.02 ± 1.04 2.14 ± 0.61 0.133 HDL cholesterol (mmol/L) 1.56 ± 0.34 1.07 ± 0.38 <0.001 Triglycerides (mmol/L) 1.44 ± 0.76 1.31 ± 0.89 0.375 Creatinemia (µmol/L) 62.45 ± 15.00 102.02 ± 52.66 <0.001 Fasting blood glucose (mmol/L) 5.47 ± 1.05 6.43 ± 2.40 0.001 White blood cell count (109/L) 5.85 ± 1.64 7.15 ± 2.11 0.009 Parameters Normotensive Heart failure P-value Controls (n = 32) Patients (n = 30) Age (years) 53.84 ± 7.14 71.27 ± 13.57 <0.001 Male, n (%) 19 (59.4) 16 (53.3) 0.634 LVEF (%) 65.00 ± 1.88 44.47 ± 12.68 <0.001 Septum (mm) 9.66 ± 0.94 10.67 ± 1.95 0.009 LVEDD (mm) 45.63 ± 1.91 60.10 ± 11.50 <0.001 LV posterior wall (mm) 9.31 ± 0.78 9.97 ± 1.19 0.310 BNP (pg/mL) 26.76 ± 15.37 1151.83 ± 1669.10 <0.001 Systolic blood pressure (mmHg) 121.19 ± 13.08 135.63 ± 20.94 0.014 Diastolic blood pressure (mmHg) 76.16 ± 8.97 78.40 ± 15.05 0.632 Heart rate (b.p.m.) 67.66 ± 7.32 87.27 ± 19.41 <0.001 Total cholesterol (mmol/L) 5.65 ± 1.53 3.85 ± 0.99 0.214 LDL cholesterol (mmol/L) 3.02 ± 1.04 2.14 ± 0.61 0.133 HDL cholesterol (mmol/L) 1.56 ± 0.34 1.07 ± 0.38 <0.001 Triglycerides (mmol/L) 1.44 ± 0.76 1.31 ± 0.89 0.375 Creatinemia (µmol/L) 62.45 ± 15.00 102.02 ± 52.66 <0.001 Fasting blood glucose (mmol/L) 5.47 ± 1.05 6.43 ± 2.40 0.001 White blood cell count (109/L) 5.85 ± 1.64 7.15 ± 2.11 0.009 BNP, brain natriuretic peptide; HDL, high-density lipoprotein; LDL, low-density lipoprotein; LVEDD, left ventricular end diastolic diameter; LVEF, left ventricular ejective fraction. Statistics All data are expressed as mean ± standard deviation (SD). The statistical analysis was performed by SPSS 16.0 (Supplementary material online). Results Angiotensin II increases CXCL1 and CXCR2 expression and CXCR2-positive myelomonocyte infiltration in the heart To investigate the role of CXCL1 and CXCR2 in cardiac remodelling, we first performed microarray analysis to examine the chemokine gene expression in Ang II-infused hearts on Day 1. Among the 12 chemokine genes, CXCL1 was the most markedly upregulated (12.5-fold) in Ang II-infused hearts compared with control hearts (Figure 1A). The increased expression of CXCL1 was validated by quantitative PCR analysis and immunohistochemical staining (Figure 1B and C) in the same hearts. Moreover, its receptor CXCR2 expression at the mRNA and protein levels was also significantly increased in Ang II-infused hearts at different time points (Figure 1D and E). Flow cytometry showed that CXCR2 was predominantly expressed in monocytes/macrophages (77.7%) and neutrophils (70.6%) but not in T cells in the hearts (Figure 1F). Interestingly, Ang II infusion caused a time-dependent increase of CD45+CXCR2+ cells, including CD11b+CXCR2+ monocytes, CD11b+F4/80+CXCR2+ macrophages, and CD11b+Gr-1+CXCR2+ neutrophils in the hearts (Figure 1G). Collectively, these results indicate that Ang II infusion upregulates CXCL1 that may attract CXCR2+ immune cells into the heart. Figure 1 View largeDownload slide Upregualtion of CXCL1 and CXCR2 and myeloid-derived CXCR2-positive cells in angiotensin II-infused mice. (A) The cluster of chemokine mRNA expressions in the hearts after 1 day of angiotensin II infusion. (B) Quantitative real-time polymerase chain reaction analysis of the mRNA expression of chemokines in the same samples (n = 6). (C) Immunohistochemical staining of CXCL1 in heart tissues after 1 day of saline or angiotensin II infusion (n = 3). (D) Quantitative real-time polymerase chain reaction analysis of CXCR2 mRNA level in the hearts after 1, 3, 7, and 14 days of angiotensin II infusion (n = 6). (E) Immunoblotting analysis of CXCR2 protein in hearts (top) and quantification of protein bands (bottom, n = 4). (F) The percentage of CXCR2+ neutrophils, macrophages and T cells in the heart tissues after angiotensin II infusion (n = 6). (G) Flow cytometry analysis of CD45+CXCR2+ cells, CD11b+CXCR2+ monocytes, CD11b+F4/80+CXCR2+ macrophages, CD11b+Gr-1+CXCR2+ neutrophils in the hearts after angiotensin II infusion (left). The percentage of each type cells (right, n = 6). Data are presented as mean ± standard deviation, and n represents number of samples or animals. Figure 1 View largeDownload slide Upregualtion of CXCL1 and CXCR2 and myeloid-derived CXCR2-positive cells in angiotensin II-infused mice. (A) The cluster of chemokine mRNA expressions in the hearts after 1 day of angiotensin II infusion. (B) Quantitative real-time polymerase chain reaction analysis of the mRNA expression of chemokines in the same samples (n = 6). (C) Immunohistochemical staining of CXCL1 in heart tissues after 1 day of saline or angiotensin II infusion (n = 3). (D) Quantitative real-time polymerase chain reaction analysis of CXCR2 mRNA level in the hearts after 1, 3, 7, and 14 days of angiotensin II infusion (n = 6). (E) Immunoblotting analysis of CXCR2 protein in hearts (top) and quantification of protein bands (bottom, n = 4). (F) The percentage of CXCR2+ neutrophils, macrophages and T cells in the heart tissues after angiotensin II infusion (n = 6). (G) Flow cytometry analysis of CD45+CXCR2+ cells, CD11b+CXCR2+ monocytes, CD11b+F4/80+CXCR2+ macrophages, CD11b+Gr-1+CXCR2+ neutrophils in the hearts after angiotensin II infusion (left). The percentage of each type cells (right, n = 6). Data are presented as mean ± standard deviation, and n represents number of samples or animals. Administration of CXCL1 neutralizing antibody prevents angiotensin II-induced cardiac hypertrophy, fibrosis, and inflammation To determine whether the increased expression of CXCL1 could cause adverse cardiac remodelling, WT mice were treated with CXCL1 neutralizing antibody and Ang II infusion (1000 ng/kg/min) for 2 weeks. Administration of CXCL1 antibody significantly decreased Ang II-induced elevation of blood pressure as compared with Immunoglobulin G (IgG)-treated mice (Supplementary material online, Figure S1A). Echocardiography revealed that CXCL1 antibody markedly reversed Ang II-induced cardiac contractile dysfunction as reflected by left ventricular (LV) EF% and fractional shortening (FS%) compared with IgG-treated mice (Figure 2A, Supplementary material online, Table S2). Moreover, Ang II infusion-induced cardiac hypertrophy as indicated by an increase in the heart size, heart weight to tibial length (HW/TL) ratio, the cross-sectional area of myocytes as well as the expression of hypertrophic markers atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) were markedly attenuated in CXCL1 antibody-treated animals (Figure 2B–D). Similarly, there was a significant increase in peripheral and interstitial fibrosis, α-smooth muscle actin (α-SMA)-positive myofibroblasts, and the expression of α-SMA, collagen I and collagen III in CXCL1 antibody-treated animals compared with IgG-treated mice (Figure 2E–G). Interestingly, Ang II-stimulated infiltration of inflammatory cells, especially Mac-2-positive macrophages and CXCR2+ cells and the expression of IL-1β, IL-6, or IL-13 were markedly lower in CXCL1 antibody-treated mice (Figure 2H and I). There was no significant difference in these parameters between two groups after saline infusion (Figure 2A–I). These results suggest that CXCL1 contributes to Ang II-induced cardiac dysfunction and hypertrophic remodelling. Figure 2 View largeDownload slide CXCL1 neutralizing antibody alleviates angiotensin II-induced cardiac hypertrophy, fibrosis and inflammation. Wild-type mice were treated with IgG control or anti-CXCL1 antibody 14 days after saline or angiotensin II infusion. (A) M-mode echocardiography of left ventricular chamber (upper), and measurement of ejection fraction (EF%) and fractional shortening (FS%) (lower, n = 6). (B) Haematoxylin and eosin staining of heart sections (left). The heart weight to tibial length (HW/TL) ratio (right, n = 6). (C) TRITC-labelled wheat germ agglutinin staining of heart sections (left) and quantification of myocyte cross-sectional area (200 cells counted per heart, right, n = 6). (D) Quantitative real-time polymerase chain reaction analysis of atrial natriuretic factor and brain natriuretic peptide mRNA levels in the hearts (n = 6). (E) Masson’s trichrome staining of myocardial fibrosis (left). Quantification of fibrotic area (right, n = 6). Scale bar 50 μm. (F) Immunohistochemical staining of myofibroblasts with α-smooth muscle actin (left) and quantification (right, n = 6). (G) Quantitative real-time polymerase chain reaction analysis of α-smooth muscle actin, collagen I, and collagen III mRNA expression levels in the heart tissues (n = 6). (H) Haematoxylin and eosin and immunohistochemical staining of Mac-2 and CXCR2 in the hearts (left) and the percentage of Mac-2- and CXCR2-positive areas (right, n = 6). (I) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of IL-1β, IL-6, and IL-13 in the hearts (n = 6). Data are presented as mean ± standard deviation, and n represents number of animals. IgG, Immunoglobulin G; TRITC, tetramethylrhodamine. Figure 2 View largeDownload slide CXCL1 neutralizing antibody alleviates angiotensin II-induced cardiac hypertrophy, fibrosis and inflammation. Wild-type mice were treated with IgG control or anti-CXCL1 antibody 14 days after saline or angiotensin II infusion. (A) M-mode echocardiography of left ventricular chamber (upper), and measurement of ejection fraction (EF%) and fractional shortening (FS%) (lower, n = 6). (B) Haematoxylin and eosin staining of heart sections (left). The heart weight to tibial length (HW/TL) ratio (right, n = 6). (C) TRITC-labelled wheat germ agglutinin staining of heart sections (left) and quantification of myocyte cross-sectional area (200 cells counted per heart, right, n = 6). (D) Quantitative real-time polymerase chain reaction analysis of atrial natriuretic factor and brain natriuretic peptide mRNA levels in the hearts (n = 6). (E) Masson’s trichrome staining of myocardial fibrosis (left). Quantification of fibrotic area (right, n = 6). Scale bar 50 μm. (F) Immunohistochemical staining of myofibroblasts with α-smooth muscle actin (left) and quantification (right, n = 6). (G) Quantitative real-time polymerase chain reaction analysis of α-smooth muscle actin, collagen I, and collagen III mRNA expression levels in the heart tissues (n = 6). (H) Haematoxylin and eosin and immunohistochemical staining of Mac-2 and CXCR2 in the hearts (left) and the percentage of Mac-2- and CXCR2-positive areas (right, n = 6). (I) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of IL-1β, IL-6, and IL-13 in the hearts (n = 6). Data are presented as mean ± standard deviation, and n represents number of animals. IgG, Immunoglobulin G; TRITC, tetramethylrhodamine. Ablation of CXCR2 attenuates angiotensin II-induced cardiac hypertrophy and fibrosis We next tested the role of CXCR2 in regulating cardiac remodelling in WT and CXCR2 KO mice. After 2 weeks of Ang II infusion, systolic blood pressure elevation (Supplementary material online, Figure S1B) and cardiac contractile dysfunction (EF% and FS%) in WT mice were markedly improved in CXCR2 KO mice (Figure 3A, Supplementary material online, Table S3). Moreover, CXCR2 KO significantly reduced Ang II-induced cardiac hypertrophy (increased heart size, HW/TL ratio, myocyte area, and the expression of ANF and BNP) compared with WT mice (Figure 3B–D). Accordingly, LV fibrosis, α-SMA-positive myofibroblasts and the expression of α-SMA, collagen I, and collagen III were less visible in CXCR2 KO hearts (Figure 3E–G). We next determined which signalling pathways are involved in myocyte hypertrophy and fibrosis and found that Ang II-induced increase in the protein levels of p-AKT, p-ERK1/2, p-STAT3, calcineurin A (CaNA), transform growth factor β1 (TGF-β1), or p-Smad2/3 were all remarkably downregulated in CXCR2 KO hearts compared with WT controls (Figure 3H and I). There was no statistically significant difference in these pathological features between two groups at baseline (Figure 3A–I). Figure 3 View largeDownload slide Deficiency of CXCR2 attenuates angiotensin II-induced cardiac hypertrophy and fibrosis. Wild-type (WT) and CXCR2 knockout (CXCR2 KO) mice were infused with saline or angiotensin II for 14 days. (A) M-mode echocardiography of left ventricular chamber (upper), and measurement of ejection fraction (EF%) and fractional shortening (FS%) (lower, n = 6). (B) Representative heart size (left), and heart weight to tibial length (HW/TL) ratio (right, n = 6). (C) TRITC-labelled wheat germ agglutinin staining of heart sections (left), and quantification of myocyte cross-sectional area (200 cells counted per heart, right, n = 6). (D) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of atrial natriuretic factor and brain natriuretic peptide in the heart (n = 6). (E) Massons’ trichrome staining of heart tissues (left), and quantification of fibrotic area (right, n = 6). (F) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of α-smooth muscle actin, collagen I, and collagen III in the heart (n = 6). (G) Immunohistochemical staining of myofibroblasts with α-smooth muscle actin (left) and quantification (right, n = 6). (H, I) Immunoblotting analysis of AKT, ERK1/2, STAT3, calcineurin A (CaNA), transform growth factor β1, and Smad2/3 protein levels in the hearts (left) and quantification (right, n = 4). GAPDH as an internal control. Data are presented as mean ± standard deviation, and n represents number of animals. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TRITC, tetramethylrhodamine. Figure 3 View largeDownload slide Deficiency of CXCR2 attenuates angiotensin II-induced cardiac hypertrophy and fibrosis. Wild-type (WT) and CXCR2 knockout (CXCR2 KO) mice were infused with saline or angiotensin II for 14 days. (A) M-mode echocardiography of left ventricular chamber (upper), and measurement of ejection fraction (EF%) and fractional shortening (FS%) (lower, n = 6). (B) Representative heart size (left), and heart weight to tibial length (HW/TL) ratio (right, n = 6). (C) TRITC-labelled wheat germ agglutinin staining of heart sections (left), and quantification of myocyte cross-sectional area (200 cells counted per heart, right, n = 6). (D) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of atrial natriuretic factor and brain natriuretic peptide in the heart (n = 6). (E) Massons’ trichrome staining of heart tissues (left), and quantification of fibrotic area (right, n = 6). (F) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of α-smooth muscle actin, collagen I, and collagen III in the heart (n = 6). (G) Immunohistochemical staining of myofibroblasts with α-smooth muscle actin (left) and quantification (right, n = 6). (H, I) Immunoblotting analysis of AKT, ERK1/2, STAT3, calcineurin A (CaNA), transform growth factor β1, and Smad2/3 protein levels in the hearts (left) and quantification (right, n = 4). GAPDH as an internal control. Data are presented as mean ± standard deviation, and n represents number of animals. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TRITC, tetramethylrhodamine. Deficiency of CXCR2 blocks angiotensin II-induced cardiac CXCR2+ cell infiltration and inflammatory response To investigate the mechanism by which CXCR2 deletion improves cardiac remodelling, we examined pro-proinflammatory cells that had been recruited by CXCR2 in the heart. Flow cytometry showed that Ang II infusion-induced increase of accumulation of CD45+ myelomonocytes, especially CD11b+F4/80+ macrophages and CD11b+Gr-1+ neutrophils of WT mice was markedly reduced in CXCR2 KO hearts (Figure 4A). Interestingly, CD3+ T cells were similar between WT and CXCR2 KO mice after saline or Ang II infusion (Figure 4A). Haematoxylin and eosin (H&E) staining and quantitative real-time polymerase chain reaction (qPCR) analysis further confirmed that deletion of CXCR2 in mice remarkably abrogated Ang II-induced recruitment of pro-inflammatory cells and the expression of IL-1β, IL-6, tumour necrosis factor-α (TNF-α), and monocyte chemoattractantprotein-1 (MCP-1) compared with WT controls (Figure 4B and C). In addition, we detected NF-κB activation, a key regulator for pro-inflammatory cytokine expression and myocardial injury. Angiotensin II-induced P65 activation was markedly lower in CXCR2 KO mice than in WT hearts (Figure 4D). Thus, these data indicate that CXCR2 mediates accumulation of pro-inflammatory cells that likely contribute to adverse cardiac remodelling and dysfunction. Figure 4 View largeDownload slide CXCR2 deficiency reduces angiotensin II induced inflammatory cells infiltrating into the heart. Wild-type (WT) and CXCR2 knockout (CXCR2 KO) mice were infused with saline or angiotensin II for 14 days. (A) Flow cytometry analysis of CD45+ cells, CD11b+F4/80+ macrophages, CD11b+Gr-1+ neutrophils, and CD3+ T cells in hearts (left). The percentage of gated cells in the total cells (right, n = 6). (B) Haematoxylin and eosin staining of heart sections. (C) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of IL-1β, IL-6, tumour necrosis factor-α, and monocyte chemoattractantprotein-1 in the hearts (n = 6). (D) Immunoblotting blot analysis of p-P65 and P65 protein levels (left) and quantification (right, n = 4). GAPDH as an internal control. Data are presented as mean ± standard deviation, and n represents number of animals. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Figure 4 View largeDownload slide CXCR2 deficiency reduces angiotensin II induced inflammatory cells infiltrating into the heart. Wild-type (WT) and CXCR2 knockout (CXCR2 KO) mice were infused with saline or angiotensin II for 14 days. (A) Flow cytometry analysis of CD45+ cells, CD11b+F4/80+ macrophages, CD11b+Gr-1+ neutrophils, and CD3+ T cells in hearts (left). The percentage of gated cells in the total cells (right, n = 6). (B) Haematoxylin and eosin staining of heart sections. (C) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of IL-1β, IL-6, tumour necrosis factor-α, and monocyte chemoattractantprotein-1 in the hearts (n = 6). (D) Immunoblotting blot analysis of p-P65 and P65 protein levels (left) and quantification (right, n = 4). GAPDH as an internal control. Data are presented as mean ± standard deviation, and n represents number of animals. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Therapeutic administration of CXCR2 inhibitor SB265610 blunts angiotensin II-induced cardiac hypertrophy, fibrosis, and inflammation To further test whether inhibition of CXCR2 improves Ang II-induced adverse cardiac remodelling, WT mice were treated with a CXCR2-specific antagonist SB265610 (2 mg/kg, once a day) and Ang II for 2 weeks. Wild-type mice displayed characteristics of hypertension (Supplementary material online, Figure S1C), cardiac dysfunction (increased EF% and FS%), and hypertrophy (increase in heart size, HW/TL ratio, myocyte area, and the expression of ANF and BNP), which were significantly attenuated in SB265610-treated mice (Figure 5A–D, Supplementary material online, Table S4). Furthermore, less myocardial fibrosis, α-SMA-positive myofibroblasts, and the expression of α-SMA, collagen I and collagen III were obtained in SB265610-treated hearts compared with vehicle-treated controls (Figure 5E–G). There was no significant difference in these parameters between two groups after saline infusion (Supplementary material online, Figure S1C, Figure 5A–G). Figure 5 View largeDownload slide Administration of CXCR2 inhibitor SB265610 improves angiotensin II-induced cardiac remodelling. Wild-type (WT) mice were co-treated with vehicle (DMSO) control or CXCR2 inhibitor SB265610 and angiotensin II for 14 days. (A) M-mode echocardiography of left ventricular chamber (upper), and measurement of ejection fraction (EF%) and fractional shortening (FS%) (lower, n = 6). (B) Representative heart size (left) and heart weight to tibial length (HW/TL) ratio (right, n = 6). (C) TRITC-labelled wheat germ agglutinin staining of heart sections (left) and quantification of myocyte cross-sectional area (200 cells counted per heart, right, n = 6). (D) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of atrial natriuretic factor and brain natriuretic peptide in the hearts (n = 4). (E) Masson trichrome staining of heart tissues (left) and quantification of fibrotic area (right, n = 6). (F) Immunohistochemical staining of myofibroblasts with α-smooth muscle actin (left) and quantification (right, n = 6). (G) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of α-smooth muscle actin, collagen I, and collagen III in the heart (n = 4). Data are presented as mean ± standard deviation, and n represents number of animals. DMSO, dimethylsulfoxide; TRITC, tetramethylrhodamine. Figure 5 View largeDownload slide Administration of CXCR2 inhibitor SB265610 improves angiotensin II-induced cardiac remodelling. Wild-type (WT) mice were co-treated with vehicle (DMSO) control or CXCR2 inhibitor SB265610 and angiotensin II for 14 days. (A) M-mode echocardiography of left ventricular chamber (upper), and measurement of ejection fraction (EF%) and fractional shortening (FS%) (lower, n = 6). (B) Representative heart size (left) and heart weight to tibial length (HW/TL) ratio (right, n = 6). (C) TRITC-labelled wheat germ agglutinin staining of heart sections (left) and quantification of myocyte cross-sectional area (200 cells counted per heart, right, n = 6). (D) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of atrial natriuretic factor and brain natriuretic peptide in the hearts (n = 4). (E) Masson trichrome staining of heart tissues (left) and quantification of fibrotic area (right, n = 6). (F) Immunohistochemical staining of myofibroblasts with α-smooth muscle actin (left) and quantification (right, n = 6). (G) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of α-smooth muscle actin, collagen I, and collagen III in the heart (n = 4). Data are presented as mean ± standard deviation, and n represents number of animals. DMSO, dimethylsulfoxide; TRITC, tetramethylrhodamine. Consistent with the findings from CXCR2 KO mice, flow cytometry confirmed that SB265610 treatment also significantly reduced myocardial infiltration of CD45+ myelomonocytes, CD11b+F4/80+ macrophages and CD11b+Gr-1+ neutrophils compared with vehicle-treated controls after Ang II infusion (Supplementary material online, Figure S2A). Moreover, Ang II-induced myocardial recruitment of pro-inflammatory cells and the expression of IL-1β, IL-6, TNF-α, and MCP-1 were markedly lower in SB265610-treated mice than in vehicle-treated animals (Supplementary material online, Figure S2B and C). Bone marrow-derived CXCR2-deficient cells attenuates angiotensin II-induced cardiac hypertrophy, fibrosis, and inflammation To directly test whether CXCR2+ myeloid cells affect cardiac remodelling, we created various chimeric mice by BM transplant and infused them with Ang II for the ensuing 2 weeks. WT mice reconstituted with CXCR2 KO BM exhibited a significant decrease in systolic blood pressure (Supplementary material online, Figure S3), improvement of contractile function (EF% and FS%) (Figure 6A, Supplementary material online, Table S5), hypertrophy (HW/TL ratio and myocyte area) (Figure 6B and C) and fibrosis (collagen area and α-SMA-positive myofibroblasts) (Figure 6E and F), infiltration of pro-inflammatory cells (Figure 6H) and the mRNA expression of ANF, BNP, α-SMA, collagen I and collagen III, IL-6, IL-1β, TNF-α and MCP-1 (Figure 6D, G, I) as compared to WT mice reconstituted with WT BM. Similar effects were confirmed in CXCR2 KO mice reconstituted with CXCR2 KO BM (Figure 6A–I). Conversely, reconstitution of CXCR2 KO mice with WT BM fully restored Ang II-induced pathological changes (Figure 6A–I). These findings suggest that BM-derived CXCR2 cells predominantly contribute to the development of hypertrophic remodelling in this model. Figure 6 View largeDownload slide Bone marrow-derived CXCR2-deficient cells prevent angiotensin II-induced cardiac remodelling. Wild-type (WT) or CXCR2 knockout (KO) mice were given bone marrow from CXCR2 knockout or wild-type mice, respectively, and then infused with angiotensin II for 2 weeks. (A) M-mode echocardiography of left ventricular chamber (upper) and measurement of ejection fraction (EF%) and fractional shortening (FS%) (lower, n = 6). (B) Haematoxylin and eosin staining of heart sections (left) and heart weight to tibial length (HW/TL) ratio (right, n = 6). (C) TRITC-labelled wheat germ agglutinin staining of heart sections (left) and quantification of myocyte cross-sectional area (200 cells counted per heart, right, n = 6). (D) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of atrial natriuretic factor and brain natriuretic peptide in the hearts (n = 6). (E) Massons’ trichrome staining of heart tissues (left) and quantification of fibrotic area (right, n = 6). (F) Immunohistochemical staining of myofibroblasts with α-smooth muscle actin (left) and quantification (right, n = 6). (G) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of α-smooth muscle actin, collagen I, and collagen III in the hearts (n = 6). (H) Haematoxylin and eosin staining of heart sections. (I) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of IL-1β, IL-6, tumour necrosis factor-α, and monocyte chemoattractantprotein-1 in the hearts (n = 4). Data are presented as mean ± standard deviation, and n represents number of animals. TRITC, tetramethylrhodamine. Figure 6 View largeDownload slide Bone marrow-derived CXCR2-deficient cells prevent angiotensin II-induced cardiac remodelling. Wild-type (WT) or CXCR2 knockout (KO) mice were given bone marrow from CXCR2 knockout or wild-type mice, respectively, and then infused with angiotensin II for 2 weeks. (A) M-mode echocardiography of left ventricular chamber (upper) and measurement of ejection fraction (EF%) and fractional shortening (FS%) (lower, n = 6). (B) Haematoxylin and eosin staining of heart sections (left) and heart weight to tibial length (HW/TL) ratio (right, n = 6). (C) TRITC-labelled wheat germ agglutinin staining of heart sections (left) and quantification of myocyte cross-sectional area (200 cells counted per heart, right, n = 6). (D) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of atrial natriuretic factor and brain natriuretic peptide in the hearts (n = 6). (E) Massons’ trichrome staining of heart tissues (left) and quantification of fibrotic area (right, n = 6). (F) Immunohistochemical staining of myofibroblasts with α-smooth muscle actin (left) and quantification (right, n = 6). (G) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of α-smooth muscle actin, collagen I, and collagen III in the hearts (n = 6). (H) Haematoxylin and eosin staining of heart sections. (I) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of IL-1β, IL-6, tumour necrosis factor-α, and monocyte chemoattractantprotein-1 in the hearts (n = 4). Data are presented as mean ± standard deviation, and n represents number of animals. TRITC, tetramethylrhodamine. CXCR2-deficient macrophages inhibit cardiomyocyte hypertrophy and myofibroblast differentiation in vitro We next determined whether CXCR2 influences macrophage migration and activation in vitro. A transwell migration assay showed that CXCL1 treatment significantly enhanced migration of WT macrophages (Φ), but it had no significant effect on CXCR2 KO macrophages (Supplementary material online, Figure S4A). Quantitative real-time polymerase chain reaction and enzyme linked immunosorbent assay (ELISA) analysis confirmed that Ang II-induced up-regulation of IL-1β, IL-6, TGF-β1, ICAM-1, VCAM-1, or MCP-1 at both the mRNA and protein levels in WT macrophages was markedly attenuated in CXCR2 KO macrophages compared with WT cells (Supplementary material online, Figure S4B and C). Moreover, activation of P65 was also significantly inhibited in CXCR2 KO macrophages compared with WT cells after Ang II infusion (Supplementary material online, Figure S4D). To test whether CXCR2+ macrophages directly influenced cardiomyocyte (CM) hypertrophy and myofibroblast differentiation in vitro, WT or CXCR2 KO macrophages were co-cultured with WT neonatal rat cardiac myocytes (CMs) or fibroblasts (CFs), respectively. After 24 h of Ang II treatment, co-culture of CMs with CXCR2 KO macrophages had a significant reduction in CM size, the expression of ANF, BNP and β-MHC, and the protein levels of p-AKT, p-ERK1/2, p-STAT3 and CaNA compared with co-culture of CMs with WT macrophages (Supplementary material online, Figure S4E–G). Meanwhile, co-culture of CFs with CXCR2 KO macrophages markedly attenuated Ang II-induced increase in the mRNA levels of α-SMA, collagen I, collagen III or TGF-β1 compared with co-culture of CFs with WT macrophages (Supplementary material online, Figure S4H). Similarly, the protein levels of α-SMA, collagen I, collagen III, TGF-β1, or p-Smad2/3 were remarkably lower in co-culture of CFs with CXCR2 KO macrophages after Ang II treatment (Supplementary material online, Figure S4I). Serum CXCL1 level and CXCR2+ inflammatory cells are increased in heart failure patients with hypertension To test whether CXCR2+ monocytes and CXCL1 are important in human hypertensive HF, we analysed serum CXCR2+ immune cells, CXCL1 level, and other cardiovascular risk factors in HF patients (n = 30) and normal individuals (n = 32). Heart failure patients were older and had higher total cholesterol, LDL, triglyceride, and lower HDL concentrations compared with normal controls (Table 1). Flow cytometry analysis revealed that the numbers of circulating CD45+ myeloid cells, including CD182(CXCR2)+ cells, CD14+ monocytes, CD14+CD182+ monocytes, CD14+CD64+CD80+ monocytes, CD14+CD64+CD80+CD182+ monocytes, CD14+CD11b+CD209+ monocytes, CD14+CD11b+CD209+CD182+ monocytes, and CXCL1 level were significantly higher in HF patients than in normal individuals (Figure 7A–I). After adjusting for gender, age, total cholesterol, LDL, HDL, and triglycerides, multivariable logistic regression models indicated that there was a statistically significant association between the number of CXCR2+ cells, including CD45+ cells [odds ratio (OR) 1.162], CD14+CD182+ monocytes (OR 1.556), CD64+CD80+CD182+ monocytes (OR 1.030), CD11b+CD209+CD182+ monocytes (OR 1.106), CXCL1 level (OR 1.386), and HF (Table 2). Table 2 Results of the multivariable logistic regression models of CXCR2+ cells on heart failure Model Odds ratio (95% CI) P-value CD45+ CD182+cells 1.162 (1.001–1.349) 0.048 CD45+ CD14+CD182+ monocytes 1.556 (1.061–2.282) 0.024 CD45+ CD64+CD80+CD182+ monocytes 1.030 (1.005–1.055) 0.018 CD45+ CD11b+CD209+CD182+ monocytes 1.106 (1.020–1.200) 0.015 Serum CXCL-1 protein levels (pg/mL) 1.386 (1.003–1.915) 0.048 Model Odds ratio (95% CI) P-value CD45+ CD182+cells 1.162 (1.001–1.349) 0.048 CD45+ CD14+CD182+ monocytes 1.556 (1.061–2.282) 0.024 CD45+ CD64+CD80+CD182+ monocytes 1.030 (1.005–1.055) 0.018 CD45+ CD11b+CD209+CD182+ monocytes 1.106 (1.020–1.200) 0.015 Serum CXCL-1 protein levels (pg/mL) 1.386 (1.003–1.915) 0.048 All models adjusted for age, systolic blood pressure, HDL cholesterol, creatinemia, fasting blood glucose, and white blood cell count. CI, confidence interval; HDL, high-density lipoprotein. Table 2 Results of the multivariable logistic regression models of CXCR2+ cells on heart failure Model Odds ratio (95% CI) P-value CD45+ CD182+cells 1.162 (1.001–1.349) 0.048 CD45+ CD14+CD182+ monocytes 1.556 (1.061–2.282) 0.024 CD45+ CD64+CD80+CD182+ monocytes 1.030 (1.005–1.055) 0.018 CD45+ CD11b+CD209+CD182+ monocytes 1.106 (1.020–1.200) 0.015 Serum CXCL-1 protein levels (pg/mL) 1.386 (1.003–1.915) 0.048 Model Odds ratio (95% CI) P-value CD45+ CD182+cells 1.162 (1.001–1.349) 0.048 CD45+ CD14+CD182+ monocytes 1.556 (1.061–2.282) 0.024 CD45+ CD64+CD80+CD182+ monocytes 1.030 (1.005–1.055) 0.018 CD45+ CD11b+CD209+CD182+ monocytes 1.106 (1.020–1.200) 0.015 Serum CXCL-1 protein levels (pg/mL) 1.386 (1.003–1.915) 0.048 All models adjusted for age, systolic blood pressure, HDL cholesterol, creatinemia, fasting blood glucose, and white blood cell count. CI, confidence interval; HDL, high-density lipoprotein. Figure 7 View largeDownload slide Serum CXCL1 levels and blood CXCR2+ cells are increased in patients with heart failure. (A–H) Flow cytometric analysis of circulating inflammatory cells, including CD45+ CD182 (CXCR2)+ cells, CD45+CD14+ monocytes, CD45+CD14+CD182+ monocytes, CD45+CD14+CD64+CD80+ monocytes, CD45+CD14+CD64+CD80+CD182+ monocytes, CD45+CD14+CD11b+CD209+ monocytes, and CD45+CD14+CD11b+CD209+CD182+ monocytes in normotensive people (n = 32) and heart failure patients (n = 30). (I) ELISA assay of serum CXCL1 levels in normotensive people (n = 32) and heart failure patients (n = 30). Data are presented as mean ± standard deviation and n represents number of persons. Figure 7 View largeDownload slide Serum CXCL1 levels and blood CXCR2+ cells are increased in patients with heart failure. (A–H) Flow cytometric analysis of circulating inflammatory cells, including CD45+ CD182 (CXCR2)+ cells, CD45+CD14+ monocytes, CD45+CD14+CD182+ monocytes, CD45+CD14+CD64+CD80+ monocytes, CD45+CD14+CD64+CD80+CD182+ monocytes, CD45+CD14+CD11b+CD209+ monocytes, and CD45+CD14+CD11b+CD209+CD182+ monocytes in normotensive people (n = 32) and heart failure patients (n = 30). (I) ELISA assay of serum CXCL1 levels in normotensive people (n = 32) and heart failure patients (n = 30). Data are presented as mean ± standard deviation and n represents number of persons. Discussion Here, we demonstrated for the first time that CXCL1–CXCR2 signalling induces myocardial recruitment of monocytes, which is the key step for initiating Ang II-induced cardiac remodelling. Angiotensin II infusion significantly upregulated CXCL1 expression, which promoted the infiltration of BM-derived CXCR2+ monocytes into the heart tissues and produced multiple pro-inflammatory cytokines, thereby causing cardiac remodelling and dysfunction. Conversely, therapeutic blocking of CXCL1 and CXCR2 or ablation of CXCR2 markedly attenuated these effects. Thus, our novel evidence supports a strong causative role of CXCL1–CXCR2 axis in the pathogenesis of cardiac remodelling. A working model is illustrated in Figure 8. It is well known that pro-inflammatory cells play pivotal roles in the development of cardiovascular diseases.14 Circulating monocytes are the precursors of Ly6Chi inflammatory monocytes in mice, which are the equivalent to CD14+ monocytes in humans.15 Previous studies defined M1/M2 macrophages by labelling CD64 and CD80 for M1 or CD11b and CD209 for M2 in polarized human peripheral blood monocytes in vitro and other tissues but not in the context of CD14+ cells.16,17 Here, we analysed several subpopulations of CD14+ monocytes in human samples, and found that these cells were significantly increased in HF patients (Figure 7). Recently, our data and that of other researchers have demonstrated that CD11b+Gr-1+ or CD11b+Ly6Chi monocytes and CD11b+F4/80+ macrophages are critical early mediators of Ang II-induced vascular dysfunction, arterial hypertension and cardiac fibrosis in mice.12,13,18–20 Chemokines are reported to be critically involved in leucocyte trafficking to the inflammatory sites, and their principal targets are haematopoietic cells.5 Interestingly, these chemokines are markedly upregulated upon heart injury to induce a chemotactic response in vivo.5 CXCL1 has been reported to exert a critical role in different cardiovascular diseases by regulating the recruitment of neutrophils, T lymphocytes and monocytes.11,21 In this study, we extended previous findings and found that CXCL1 was also significantly upregulated in Ang II-infused hearts (Figure 1) and HF patients (Figure 8), which recruited CXCR2+ monocytes into the heart tissues leading to cardiac remodelling, but this effect was prevented by CXCL1 neutralizing antibody (Figure 2). Further studies are required to determine how CXCL1 is upregulated by Ang II in the heart. Figure 8 View largeDownload slide A working model for CXCL1 to recruit CXCR2+ monocytes into the heart, which initiate and aggravate angiotensin II-induced inflammation, cardiac hypertrophy, and fibrosis leading to cardiac remodelling. Inhibition of CXCL1 by neutralizing antibody (nAb) or CXCR2 by inhibitor SB265610 prevents these effects. Figure 8 View largeDownload slide A working model for CXCL1 to recruit CXCR2+ monocytes into the heart, which initiate and aggravate angiotensin II-induced inflammation, cardiac hypertrophy, and fibrosis leading to cardiac remodelling. Inhibition of CXCL1 by neutralizing antibody (nAb) or CXCR2 by inhibitor SB265610 prevents these effects. CXCL1 exerts multiple biological functions via binding to its CXCR2 receptor, which also serves as a receptor for other CXCL subfamily members.5 CXCR2 is reported to be express on the surface of both immune and non-immune cells, including leucocytes, T lymphocytes, CMs, and CFs, and it regulates the functions of these cells. Interestingly, CXCR2 activation plays critical roles in the recruitment of leucocytes and neutrophils, which are involved in the pathogenesis of myocardial infarction and atherosclerosis.8,9 Our recent data indicated that CXCR2 mainly mediates infiltration of monocytes into the injured arteries leading to hypertension.11 Here, we further demonstrated that genetic and pharmacological inhibition of CXCR2 effectively reduced Ang II-induced infiltration of monocytes/macrophages and cardiac remodelling through inhibition of multiple signalling pathways in vivo and in vitro (Figures 3–6, Supplementary material online, Figure S4). Overall, our results suggest that CXCR2+ monocytes/macrophages directly contribute to Ang II-induced cardiac remodelling and dysfunction. This study identified for the first time that CXCL1–CXCR2 axis induced recruitment of monocytes/macrophages into the heart thereby leading to cardiac remodelling and dysfunction in response to Ang II. Circulating CXCR2+ monocytes and CXCL1 level were significantly higher in HF patients. Therapeutic targeting of CXCL1 or CXCR2 signalling prevented adverse cardiac remodelling and dysfunction, thus representing an attractive new strategy for treating hypertensive HF. Supplementary material Supplementary material is available at European Heart Journal online. Funding National Natural Science Foundation of China (81630009 and 81330003 to H.-H.L; 81500303 to L.W.), Dalian high level Talents Innovation and Entrepreneurship Projects (2015R019), and the Chang Jiang Scholar Program of China (T2011160 to H.-H.L.). Conflict of interest: none declared. 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Google Scholar CrossRef Search ADS PubMed Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png European Heart Journal Oxford University Press

CXCL1–CXCR2 axis mediates angiotensin II-induced cardiac hypertrophy and remodelling through regulation of monocyte infiltration

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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.
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Abstract

Abstract Aims Chemokine-mediated monocyte infiltration into the damaged heart represents an initial step in inflammation during cardiac remodelling. Our recent study demonstrates a central role for chemokine receptor CXCR2 in monocyte recruitment and hypertension; however, the role of chemokine CXCL1 and its receptor CXCR2 in angiotensin II (Ang II)-induced cardiac remodelling remain unknown. Methods and results Angiotensin II (1000 ng kg−1 min−1) was administrated to wild-type (WT) mice treated with CXCL1 neutralizing antibody or CXCR2 inhibitor SB265610, knockout (CXCR2 KO) or bone marrow (BM) reconstituted chimeric mice for 14 days. Microarray revealed that CXCL1 was the most highly upregulated chemokine in the WT heart at Day 1 after Ang II infusion. The CXCR2 expression and the CXCR2+ immune cells were time-dependently increased in Ang II-infused hearts. Moreover, administration of CXCL1 neutralizing antibody markedly prevented Ang II-induced hypertension, cardiac dysfunction, hypertrophy, fibrosis, and macrophage accumulation compared with Immunoglobulin G (IgG) control. Furthermore, Ang II-induced cardiac remodelling and inflammatory response were also significantly attenuated in CXCR2 KO mice and in WT mice treated with SB265610 or transplanted with CXCR2-deficienct BM cells. Co-culture experiments in vitro further confirmed that CXCR2 deficiency inhibited macrophage migration and activation, and attenuated Ang II-induced cardiomyocyte hypertrophy and fibroblast differentiation through multiple signalling pathways. Notably, circulating CXCL1 level and CXCR2+ monocytes were higher in patients with heart failure compared with normotensive individuals. Conclusions Angiotensin II-induced infiltration of monocytes in the heart is largely mediated by CXCL1–CXCR2 signalling which initiates and aggravates cardiac remodelling. Inhibition of CXCL1 and/or CXCR2 may represent new therapeutic targets for treating hypertensive heart diseases. Chemokine CXCL1 , Receptor CXCR2 , Monocyte infiltration , Angiotensin II , Inflammation , Cardiac remodelling Translational perspective Our present study reveals that CXCL1–CXCR2 axis is essential for monocyte infiltration into the heart and cardiac remodelling induced by angiotensin II. Blocking of CXCL1 and CXCR2 activation or ablation of CXCR2 effectively prevents adverse cardiac remodelling. Thus, pharmacological targeting of CXCL1–CXCR2 signalling may represent a novel therapeutic strategy for treating hypertensive heart diseases. Introduction Adverse cardiac remodelling is now recognized as a determinant of the clinical course of heart failure (HF), which is still one of the leading causes of death worldwide.1 This process is mainly influenced by haemodynamic load and neuro-hormonal activation.2 Among the neuro-humoral factors, angiotensin (Ang) II, a key component of the renin–angiotensin system (RAS), plays an important role in the pathogenesis of cardiac remodelling in a variety of diseases.3 Interestingly, pharmacological or genetic interventions that inhibit RAS activity have been shown to reduce cardiac inflammation and improve HF.3 There is growing evidence to support the critical role of inflammatory cells especially monocytes/macrophages in the pathophysiology of HF in both animal and human research.4 Therefore, it is important to identify the mechanisms that attract these cells into the heart after hypertensive stress to prevent cardiac dysfunction. Chemokines are a family of inflammatory cytokines that have ability to induce the directional migration and activation of leucocytes. They are classified into four distinct classes, based on a shared cysteine motif: CC (CCL1–28), CXC (CXCL1–17), CX3C (CX3C1), and C (XCL1-2). Chemokines exert their biological functions via cell–surface receptors, which are designated as CCRs, CXCRs, CX3CRs, and XCRs. Increasing evidence suggests that these chemokines play a fundamental role in recruiting neutrophils, monocytes, and lymphocytes to the injured heart.5 Interestingly, several chemokines [MIP-1a, MIP-1β, RANTES, CXCL1 and interleukin (IL)-8, and CXCL5] and their receptors (CCR1, CCR2, CCR5, CCR7, CXCR1, CXCR2, and CX3CR) have been found in animal models and patients with hypertrophy and HF, and some of them are closely related to the disease severity.6,7 Among them, CXC chemokine CXCL1 and its receptor CXCR2 play a critical role in promoting the recruitment of neutrophils and monocytes/macrophages into the injured heart and arterial wall, thereby inducing myocardial infarction, ischaemia/reperfusion injury, atherosclerosis, and hypertension.8–10 Recent studies have demonstrated that monocytes have been implicated in the pathogenesis of Ang II-induced hypertension and cardiac remodelling.4,11–13 Therefore, it is important to explore whether the CXCL1–CXCR2 axis induces recruitment of monocytes into the heart and has a critical role in Ang II-induced cardiac remodelling. In this study, we showed that bone marrow (BM)-derived CXCR2+ monocytes are involved in Ang II-induced adverse cardiac remodelling and dysfunction. The levels of CXCL1 and CXCR2 were markedly upregulated in Ang II-induced hypertrophic heart and blood of HF patients. The inhibition of CXCL1 and CXCR2 or deletion of CXCR2 significantly reduced Ang II-induced infiltration of monocytes into the heart thereby improving cardiac remodelling and dysfunction. Therefore, these results provide novel evidence supporting that inhibition of CXCL1–CXCR2 signalling exerts a cardio-protective role and representing a new therapeutic target for hypertensive cardiac remodelling. Methods Animals Wild-type (WT) mice (C57BL/6J, male) and CXCR2 knockout mice [CXCR2 KO, B6.129S2(C)-Cxcr2tm1Mwm/J] at 8–12 weeks of age were used to establish the cardiac remodelling model by subcutaneous infusion of Ang II at a dose of 1000 ng/kg/min as described previously.13 The anti-CXCL1 antibody (100 μg/mice/day) and CXCR2 inhibitor SB265610 (2 mg/kg/day) were administrated intraperitoneally to WT mice beginning 1 day before Ang II infusion and continued during Ang II treatment (Supplementary material online). Flow cytometry The inflammatory cells in the heart tissues were analysed by flow cytometry (Supplementary material online). Histological study Heart samples, fixed in 4% paraformaldehyde and embedded in paraffin, were used for the quantification of cardiac hypertrophy, fibrosis, and inflammatory cells (Supplementary material online). Bone marrow chimeric mice Chimeric mice were used to examine the contribution of BM-derived CXCR2-positive cells to cardiac hypertrophic remodelling (Supplementary material online). Human study populations We explored the patients with HF with reduced ejection fraction (EF%) (n = 32) and age- and sex-matched control subjects (n = 30) in a monocentric clinical cohort between August 2017 and October 2017. Heart failure patients were diagnosed according to the 2016 ESC Guidelines.1 The baseline characteristics of control subjects and patients are indicated in Table 1 (Supplementary material online). Table 1 Baseline characteristics of normotensive control subjects and heart failure patients Parameters Normotensive Heart failure P-value Controls (n = 32) Patients (n = 30) Age (years) 53.84 ± 7.14 71.27 ± 13.57 <0.001 Male, n (%) 19 (59.4) 16 (53.3) 0.634 LVEF (%) 65.00 ± 1.88 44.47 ± 12.68 <0.001 Septum (mm) 9.66 ± 0.94 10.67 ± 1.95 0.009 LVEDD (mm) 45.63 ± 1.91 60.10 ± 11.50 <0.001 LV posterior wall (mm) 9.31 ± 0.78 9.97 ± 1.19 0.310 BNP (pg/mL) 26.76 ± 15.37 1151.83 ± 1669.10 <0.001 Systolic blood pressure (mmHg) 121.19 ± 13.08 135.63 ± 20.94 0.014 Diastolic blood pressure (mmHg) 76.16 ± 8.97 78.40 ± 15.05 0.632 Heart rate (b.p.m.) 67.66 ± 7.32 87.27 ± 19.41 <0.001 Total cholesterol (mmol/L) 5.65 ± 1.53 3.85 ± 0.99 0.214 LDL cholesterol (mmol/L) 3.02 ± 1.04 2.14 ± 0.61 0.133 HDL cholesterol (mmol/L) 1.56 ± 0.34 1.07 ± 0.38 <0.001 Triglycerides (mmol/L) 1.44 ± 0.76 1.31 ± 0.89 0.375 Creatinemia (µmol/L) 62.45 ± 15.00 102.02 ± 52.66 <0.001 Fasting blood glucose (mmol/L) 5.47 ± 1.05 6.43 ± 2.40 0.001 White blood cell count (109/L) 5.85 ± 1.64 7.15 ± 2.11 0.009 Parameters Normotensive Heart failure P-value Controls (n = 32) Patients (n = 30) Age (years) 53.84 ± 7.14 71.27 ± 13.57 <0.001 Male, n (%) 19 (59.4) 16 (53.3) 0.634 LVEF (%) 65.00 ± 1.88 44.47 ± 12.68 <0.001 Septum (mm) 9.66 ± 0.94 10.67 ± 1.95 0.009 LVEDD (mm) 45.63 ± 1.91 60.10 ± 11.50 <0.001 LV posterior wall (mm) 9.31 ± 0.78 9.97 ± 1.19 0.310 BNP (pg/mL) 26.76 ± 15.37 1151.83 ± 1669.10 <0.001 Systolic blood pressure (mmHg) 121.19 ± 13.08 135.63 ± 20.94 0.014 Diastolic blood pressure (mmHg) 76.16 ± 8.97 78.40 ± 15.05 0.632 Heart rate (b.p.m.) 67.66 ± 7.32 87.27 ± 19.41 <0.001 Total cholesterol (mmol/L) 5.65 ± 1.53 3.85 ± 0.99 0.214 LDL cholesterol (mmol/L) 3.02 ± 1.04 2.14 ± 0.61 0.133 HDL cholesterol (mmol/L) 1.56 ± 0.34 1.07 ± 0.38 <0.001 Triglycerides (mmol/L) 1.44 ± 0.76 1.31 ± 0.89 0.375 Creatinemia (µmol/L) 62.45 ± 15.00 102.02 ± 52.66 <0.001 Fasting blood glucose (mmol/L) 5.47 ± 1.05 6.43 ± 2.40 0.001 White blood cell count (109/L) 5.85 ± 1.64 7.15 ± 2.11 0.009 BNP, brain natriuretic peptide; HDL, high-density lipoprotein; LDL, low-density lipoprotein; LVEDD, left ventricular end diastolic diameter; LVEF, left ventricular ejective fraction. Table 1 Baseline characteristics of normotensive control subjects and heart failure patients Parameters Normotensive Heart failure P-value Controls (n = 32) Patients (n = 30) Age (years) 53.84 ± 7.14 71.27 ± 13.57 <0.001 Male, n (%) 19 (59.4) 16 (53.3) 0.634 LVEF (%) 65.00 ± 1.88 44.47 ± 12.68 <0.001 Septum (mm) 9.66 ± 0.94 10.67 ± 1.95 0.009 LVEDD (mm) 45.63 ± 1.91 60.10 ± 11.50 <0.001 LV posterior wall (mm) 9.31 ± 0.78 9.97 ± 1.19 0.310 BNP (pg/mL) 26.76 ± 15.37 1151.83 ± 1669.10 <0.001 Systolic blood pressure (mmHg) 121.19 ± 13.08 135.63 ± 20.94 0.014 Diastolic blood pressure (mmHg) 76.16 ± 8.97 78.40 ± 15.05 0.632 Heart rate (b.p.m.) 67.66 ± 7.32 87.27 ± 19.41 <0.001 Total cholesterol (mmol/L) 5.65 ± 1.53 3.85 ± 0.99 0.214 LDL cholesterol (mmol/L) 3.02 ± 1.04 2.14 ± 0.61 0.133 HDL cholesterol (mmol/L) 1.56 ± 0.34 1.07 ± 0.38 <0.001 Triglycerides (mmol/L) 1.44 ± 0.76 1.31 ± 0.89 0.375 Creatinemia (µmol/L) 62.45 ± 15.00 102.02 ± 52.66 <0.001 Fasting blood glucose (mmol/L) 5.47 ± 1.05 6.43 ± 2.40 0.001 White blood cell count (109/L) 5.85 ± 1.64 7.15 ± 2.11 0.009 Parameters Normotensive Heart failure P-value Controls (n = 32) Patients (n = 30) Age (years) 53.84 ± 7.14 71.27 ± 13.57 <0.001 Male, n (%) 19 (59.4) 16 (53.3) 0.634 LVEF (%) 65.00 ± 1.88 44.47 ± 12.68 <0.001 Septum (mm) 9.66 ± 0.94 10.67 ± 1.95 0.009 LVEDD (mm) 45.63 ± 1.91 60.10 ± 11.50 <0.001 LV posterior wall (mm) 9.31 ± 0.78 9.97 ± 1.19 0.310 BNP (pg/mL) 26.76 ± 15.37 1151.83 ± 1669.10 <0.001 Systolic blood pressure (mmHg) 121.19 ± 13.08 135.63 ± 20.94 0.014 Diastolic blood pressure (mmHg) 76.16 ± 8.97 78.40 ± 15.05 0.632 Heart rate (b.p.m.) 67.66 ± 7.32 87.27 ± 19.41 <0.001 Total cholesterol (mmol/L) 5.65 ± 1.53 3.85 ± 0.99 0.214 LDL cholesterol (mmol/L) 3.02 ± 1.04 2.14 ± 0.61 0.133 HDL cholesterol (mmol/L) 1.56 ± 0.34 1.07 ± 0.38 <0.001 Triglycerides (mmol/L) 1.44 ± 0.76 1.31 ± 0.89 0.375 Creatinemia (µmol/L) 62.45 ± 15.00 102.02 ± 52.66 <0.001 Fasting blood glucose (mmol/L) 5.47 ± 1.05 6.43 ± 2.40 0.001 White blood cell count (109/L) 5.85 ± 1.64 7.15 ± 2.11 0.009 BNP, brain natriuretic peptide; HDL, high-density lipoprotein; LDL, low-density lipoprotein; LVEDD, left ventricular end diastolic diameter; LVEF, left ventricular ejective fraction. Statistics All data are expressed as mean ± standard deviation (SD). The statistical analysis was performed by SPSS 16.0 (Supplementary material online). Results Angiotensin II increases CXCL1 and CXCR2 expression and CXCR2-positive myelomonocyte infiltration in the heart To investigate the role of CXCL1 and CXCR2 in cardiac remodelling, we first performed microarray analysis to examine the chemokine gene expression in Ang II-infused hearts on Day 1. Among the 12 chemokine genes, CXCL1 was the most markedly upregulated (12.5-fold) in Ang II-infused hearts compared with control hearts (Figure 1A). The increased expression of CXCL1 was validated by quantitative PCR analysis and immunohistochemical staining (Figure 1B and C) in the same hearts. Moreover, its receptor CXCR2 expression at the mRNA and protein levels was also significantly increased in Ang II-infused hearts at different time points (Figure 1D and E). Flow cytometry showed that CXCR2 was predominantly expressed in monocytes/macrophages (77.7%) and neutrophils (70.6%) but not in T cells in the hearts (Figure 1F). Interestingly, Ang II infusion caused a time-dependent increase of CD45+CXCR2+ cells, including CD11b+CXCR2+ monocytes, CD11b+F4/80+CXCR2+ macrophages, and CD11b+Gr-1+CXCR2+ neutrophils in the hearts (Figure 1G). Collectively, these results indicate that Ang II infusion upregulates CXCL1 that may attract CXCR2+ immune cells into the heart. Figure 1 View largeDownload slide Upregualtion of CXCL1 and CXCR2 and myeloid-derived CXCR2-positive cells in angiotensin II-infused mice. (A) The cluster of chemokine mRNA expressions in the hearts after 1 day of angiotensin II infusion. (B) Quantitative real-time polymerase chain reaction analysis of the mRNA expression of chemokines in the same samples (n = 6). (C) Immunohistochemical staining of CXCL1 in heart tissues after 1 day of saline or angiotensin II infusion (n = 3). (D) Quantitative real-time polymerase chain reaction analysis of CXCR2 mRNA level in the hearts after 1, 3, 7, and 14 days of angiotensin II infusion (n = 6). (E) Immunoblotting analysis of CXCR2 protein in hearts (top) and quantification of protein bands (bottom, n = 4). (F) The percentage of CXCR2+ neutrophils, macrophages and T cells in the heart tissues after angiotensin II infusion (n = 6). (G) Flow cytometry analysis of CD45+CXCR2+ cells, CD11b+CXCR2+ monocytes, CD11b+F4/80+CXCR2+ macrophages, CD11b+Gr-1+CXCR2+ neutrophils in the hearts after angiotensin II infusion (left). The percentage of each type cells (right, n = 6). Data are presented as mean ± standard deviation, and n represents number of samples or animals. Figure 1 View largeDownload slide Upregualtion of CXCL1 and CXCR2 and myeloid-derived CXCR2-positive cells in angiotensin II-infused mice. (A) The cluster of chemokine mRNA expressions in the hearts after 1 day of angiotensin II infusion. (B) Quantitative real-time polymerase chain reaction analysis of the mRNA expression of chemokines in the same samples (n = 6). (C) Immunohistochemical staining of CXCL1 in heart tissues after 1 day of saline or angiotensin II infusion (n = 3). (D) Quantitative real-time polymerase chain reaction analysis of CXCR2 mRNA level in the hearts after 1, 3, 7, and 14 days of angiotensin II infusion (n = 6). (E) Immunoblotting analysis of CXCR2 protein in hearts (top) and quantification of protein bands (bottom, n = 4). (F) The percentage of CXCR2+ neutrophils, macrophages and T cells in the heart tissues after angiotensin II infusion (n = 6). (G) Flow cytometry analysis of CD45+CXCR2+ cells, CD11b+CXCR2+ monocytes, CD11b+F4/80+CXCR2+ macrophages, CD11b+Gr-1+CXCR2+ neutrophils in the hearts after angiotensin II infusion (left). The percentage of each type cells (right, n = 6). Data are presented as mean ± standard deviation, and n represents number of samples or animals. Administration of CXCL1 neutralizing antibody prevents angiotensin II-induced cardiac hypertrophy, fibrosis, and inflammation To determine whether the increased expression of CXCL1 could cause adverse cardiac remodelling, WT mice were treated with CXCL1 neutralizing antibody and Ang II infusion (1000 ng/kg/min) for 2 weeks. Administration of CXCL1 antibody significantly decreased Ang II-induced elevation of blood pressure as compared with Immunoglobulin G (IgG)-treated mice (Supplementary material online, Figure S1A). Echocardiography revealed that CXCL1 antibody markedly reversed Ang II-induced cardiac contractile dysfunction as reflected by left ventricular (LV) EF% and fractional shortening (FS%) compared with IgG-treated mice (Figure 2A, Supplementary material online, Table S2). Moreover, Ang II infusion-induced cardiac hypertrophy as indicated by an increase in the heart size, heart weight to tibial length (HW/TL) ratio, the cross-sectional area of myocytes as well as the expression of hypertrophic markers atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) were markedly attenuated in CXCL1 antibody-treated animals (Figure 2B–D). Similarly, there was a significant increase in peripheral and interstitial fibrosis, α-smooth muscle actin (α-SMA)-positive myofibroblasts, and the expression of α-SMA, collagen I and collagen III in CXCL1 antibody-treated animals compared with IgG-treated mice (Figure 2E–G). Interestingly, Ang II-stimulated infiltration of inflammatory cells, especially Mac-2-positive macrophages and CXCR2+ cells and the expression of IL-1β, IL-6, or IL-13 were markedly lower in CXCL1 antibody-treated mice (Figure 2H and I). There was no significant difference in these parameters between two groups after saline infusion (Figure 2A–I). These results suggest that CXCL1 contributes to Ang II-induced cardiac dysfunction and hypertrophic remodelling. Figure 2 View largeDownload slide CXCL1 neutralizing antibody alleviates angiotensin II-induced cardiac hypertrophy, fibrosis and inflammation. Wild-type mice were treated with IgG control or anti-CXCL1 antibody 14 days after saline or angiotensin II infusion. (A) M-mode echocardiography of left ventricular chamber (upper), and measurement of ejection fraction (EF%) and fractional shortening (FS%) (lower, n = 6). (B) Haematoxylin and eosin staining of heart sections (left). The heart weight to tibial length (HW/TL) ratio (right, n = 6). (C) TRITC-labelled wheat germ agglutinin staining of heart sections (left) and quantification of myocyte cross-sectional area (200 cells counted per heart, right, n = 6). (D) Quantitative real-time polymerase chain reaction analysis of atrial natriuretic factor and brain natriuretic peptide mRNA levels in the hearts (n = 6). (E) Masson’s trichrome staining of myocardial fibrosis (left). Quantification of fibrotic area (right, n = 6). Scale bar 50 μm. (F) Immunohistochemical staining of myofibroblasts with α-smooth muscle actin (left) and quantification (right, n = 6). (G) Quantitative real-time polymerase chain reaction analysis of α-smooth muscle actin, collagen I, and collagen III mRNA expression levels in the heart tissues (n = 6). (H) Haematoxylin and eosin and immunohistochemical staining of Mac-2 and CXCR2 in the hearts (left) and the percentage of Mac-2- and CXCR2-positive areas (right, n = 6). (I) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of IL-1β, IL-6, and IL-13 in the hearts (n = 6). Data are presented as mean ± standard deviation, and n represents number of animals. IgG, Immunoglobulin G; TRITC, tetramethylrhodamine. Figure 2 View largeDownload slide CXCL1 neutralizing antibody alleviates angiotensin II-induced cardiac hypertrophy, fibrosis and inflammation. Wild-type mice were treated with IgG control or anti-CXCL1 antibody 14 days after saline or angiotensin II infusion. (A) M-mode echocardiography of left ventricular chamber (upper), and measurement of ejection fraction (EF%) and fractional shortening (FS%) (lower, n = 6). (B) Haematoxylin and eosin staining of heart sections (left). The heart weight to tibial length (HW/TL) ratio (right, n = 6). (C) TRITC-labelled wheat germ agglutinin staining of heart sections (left) and quantification of myocyte cross-sectional area (200 cells counted per heart, right, n = 6). (D) Quantitative real-time polymerase chain reaction analysis of atrial natriuretic factor and brain natriuretic peptide mRNA levels in the hearts (n = 6). (E) Masson’s trichrome staining of myocardial fibrosis (left). Quantification of fibrotic area (right, n = 6). Scale bar 50 μm. (F) Immunohistochemical staining of myofibroblasts with α-smooth muscle actin (left) and quantification (right, n = 6). (G) Quantitative real-time polymerase chain reaction analysis of α-smooth muscle actin, collagen I, and collagen III mRNA expression levels in the heart tissues (n = 6). (H) Haematoxylin and eosin and immunohistochemical staining of Mac-2 and CXCR2 in the hearts (left) and the percentage of Mac-2- and CXCR2-positive areas (right, n = 6). (I) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of IL-1β, IL-6, and IL-13 in the hearts (n = 6). Data are presented as mean ± standard deviation, and n represents number of animals. IgG, Immunoglobulin G; TRITC, tetramethylrhodamine. Ablation of CXCR2 attenuates angiotensin II-induced cardiac hypertrophy and fibrosis We next tested the role of CXCR2 in regulating cardiac remodelling in WT and CXCR2 KO mice. After 2 weeks of Ang II infusion, systolic blood pressure elevation (Supplementary material online, Figure S1B) and cardiac contractile dysfunction (EF% and FS%) in WT mice were markedly improved in CXCR2 KO mice (Figure 3A, Supplementary material online, Table S3). Moreover, CXCR2 KO significantly reduced Ang II-induced cardiac hypertrophy (increased heart size, HW/TL ratio, myocyte area, and the expression of ANF and BNP) compared with WT mice (Figure 3B–D). Accordingly, LV fibrosis, α-SMA-positive myofibroblasts and the expression of α-SMA, collagen I, and collagen III were less visible in CXCR2 KO hearts (Figure 3E–G). We next determined which signalling pathways are involved in myocyte hypertrophy and fibrosis and found that Ang II-induced increase in the protein levels of p-AKT, p-ERK1/2, p-STAT3, calcineurin A (CaNA), transform growth factor β1 (TGF-β1), or p-Smad2/3 were all remarkably downregulated in CXCR2 KO hearts compared with WT controls (Figure 3H and I). There was no statistically significant difference in these pathological features between two groups at baseline (Figure 3A–I). Figure 3 View largeDownload slide Deficiency of CXCR2 attenuates angiotensin II-induced cardiac hypertrophy and fibrosis. Wild-type (WT) and CXCR2 knockout (CXCR2 KO) mice were infused with saline or angiotensin II for 14 days. (A) M-mode echocardiography of left ventricular chamber (upper), and measurement of ejection fraction (EF%) and fractional shortening (FS%) (lower, n = 6). (B) Representative heart size (left), and heart weight to tibial length (HW/TL) ratio (right, n = 6). (C) TRITC-labelled wheat germ agglutinin staining of heart sections (left), and quantification of myocyte cross-sectional area (200 cells counted per heart, right, n = 6). (D) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of atrial natriuretic factor and brain natriuretic peptide in the heart (n = 6). (E) Massons’ trichrome staining of heart tissues (left), and quantification of fibrotic area (right, n = 6). (F) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of α-smooth muscle actin, collagen I, and collagen III in the heart (n = 6). (G) Immunohistochemical staining of myofibroblasts with α-smooth muscle actin (left) and quantification (right, n = 6). (H, I) Immunoblotting analysis of AKT, ERK1/2, STAT3, calcineurin A (CaNA), transform growth factor β1, and Smad2/3 protein levels in the hearts (left) and quantification (right, n = 4). GAPDH as an internal control. Data are presented as mean ± standard deviation, and n represents number of animals. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TRITC, tetramethylrhodamine. Figure 3 View largeDownload slide Deficiency of CXCR2 attenuates angiotensin II-induced cardiac hypertrophy and fibrosis. Wild-type (WT) and CXCR2 knockout (CXCR2 KO) mice were infused with saline or angiotensin II for 14 days. (A) M-mode echocardiography of left ventricular chamber (upper), and measurement of ejection fraction (EF%) and fractional shortening (FS%) (lower, n = 6). (B) Representative heart size (left), and heart weight to tibial length (HW/TL) ratio (right, n = 6). (C) TRITC-labelled wheat germ agglutinin staining of heart sections (left), and quantification of myocyte cross-sectional area (200 cells counted per heart, right, n = 6). (D) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of atrial natriuretic factor and brain natriuretic peptide in the heart (n = 6). (E) Massons’ trichrome staining of heart tissues (left), and quantification of fibrotic area (right, n = 6). (F) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of α-smooth muscle actin, collagen I, and collagen III in the heart (n = 6). (G) Immunohistochemical staining of myofibroblasts with α-smooth muscle actin (left) and quantification (right, n = 6). (H, I) Immunoblotting analysis of AKT, ERK1/2, STAT3, calcineurin A (CaNA), transform growth factor β1, and Smad2/3 protein levels in the hearts (left) and quantification (right, n = 4). GAPDH as an internal control. Data are presented as mean ± standard deviation, and n represents number of animals. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TRITC, tetramethylrhodamine. Deficiency of CXCR2 blocks angiotensin II-induced cardiac CXCR2+ cell infiltration and inflammatory response To investigate the mechanism by which CXCR2 deletion improves cardiac remodelling, we examined pro-proinflammatory cells that had been recruited by CXCR2 in the heart. Flow cytometry showed that Ang II infusion-induced increase of accumulation of CD45+ myelomonocytes, especially CD11b+F4/80+ macrophages and CD11b+Gr-1+ neutrophils of WT mice was markedly reduced in CXCR2 KO hearts (Figure 4A). Interestingly, CD3+ T cells were similar between WT and CXCR2 KO mice after saline or Ang II infusion (Figure 4A). Haematoxylin and eosin (H&E) staining and quantitative real-time polymerase chain reaction (qPCR) analysis further confirmed that deletion of CXCR2 in mice remarkably abrogated Ang II-induced recruitment of pro-inflammatory cells and the expression of IL-1β, IL-6, tumour necrosis factor-α (TNF-α), and monocyte chemoattractantprotein-1 (MCP-1) compared with WT controls (Figure 4B and C). In addition, we detected NF-κB activation, a key regulator for pro-inflammatory cytokine expression and myocardial injury. Angiotensin II-induced P65 activation was markedly lower in CXCR2 KO mice than in WT hearts (Figure 4D). Thus, these data indicate that CXCR2 mediates accumulation of pro-inflammatory cells that likely contribute to adverse cardiac remodelling and dysfunction. Figure 4 View largeDownload slide CXCR2 deficiency reduces angiotensin II induced inflammatory cells infiltrating into the heart. Wild-type (WT) and CXCR2 knockout (CXCR2 KO) mice were infused with saline or angiotensin II for 14 days. (A) Flow cytometry analysis of CD45+ cells, CD11b+F4/80+ macrophages, CD11b+Gr-1+ neutrophils, and CD3+ T cells in hearts (left). The percentage of gated cells in the total cells (right, n = 6). (B) Haematoxylin and eosin staining of heart sections. (C) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of IL-1β, IL-6, tumour necrosis factor-α, and monocyte chemoattractantprotein-1 in the hearts (n = 6). (D) Immunoblotting blot analysis of p-P65 and P65 protein levels (left) and quantification (right, n = 4). GAPDH as an internal control. Data are presented as mean ± standard deviation, and n represents number of animals. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Figure 4 View largeDownload slide CXCR2 deficiency reduces angiotensin II induced inflammatory cells infiltrating into the heart. Wild-type (WT) and CXCR2 knockout (CXCR2 KO) mice were infused with saline or angiotensin II for 14 days. (A) Flow cytometry analysis of CD45+ cells, CD11b+F4/80+ macrophages, CD11b+Gr-1+ neutrophils, and CD3+ T cells in hearts (left). The percentage of gated cells in the total cells (right, n = 6). (B) Haematoxylin and eosin staining of heart sections. (C) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of IL-1β, IL-6, tumour necrosis factor-α, and monocyte chemoattractantprotein-1 in the hearts (n = 6). (D) Immunoblotting blot analysis of p-P65 and P65 protein levels (left) and quantification (right, n = 4). GAPDH as an internal control. Data are presented as mean ± standard deviation, and n represents number of animals. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Therapeutic administration of CXCR2 inhibitor SB265610 blunts angiotensin II-induced cardiac hypertrophy, fibrosis, and inflammation To further test whether inhibition of CXCR2 improves Ang II-induced adverse cardiac remodelling, WT mice were treated with a CXCR2-specific antagonist SB265610 (2 mg/kg, once a day) and Ang II for 2 weeks. Wild-type mice displayed characteristics of hypertension (Supplementary material online, Figure S1C), cardiac dysfunction (increased EF% and FS%), and hypertrophy (increase in heart size, HW/TL ratio, myocyte area, and the expression of ANF and BNP), which were significantly attenuated in SB265610-treated mice (Figure 5A–D, Supplementary material online, Table S4). Furthermore, less myocardial fibrosis, α-SMA-positive myofibroblasts, and the expression of α-SMA, collagen I and collagen III were obtained in SB265610-treated hearts compared with vehicle-treated controls (Figure 5E–G). There was no significant difference in these parameters between two groups after saline infusion (Supplementary material online, Figure S1C, Figure 5A–G). Figure 5 View largeDownload slide Administration of CXCR2 inhibitor SB265610 improves angiotensin II-induced cardiac remodelling. Wild-type (WT) mice were co-treated with vehicle (DMSO) control or CXCR2 inhibitor SB265610 and angiotensin II for 14 days. (A) M-mode echocardiography of left ventricular chamber (upper), and measurement of ejection fraction (EF%) and fractional shortening (FS%) (lower, n = 6). (B) Representative heart size (left) and heart weight to tibial length (HW/TL) ratio (right, n = 6). (C) TRITC-labelled wheat germ agglutinin staining of heart sections (left) and quantification of myocyte cross-sectional area (200 cells counted per heart, right, n = 6). (D) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of atrial natriuretic factor and brain natriuretic peptide in the hearts (n = 4). (E) Masson trichrome staining of heart tissues (left) and quantification of fibrotic area (right, n = 6). (F) Immunohistochemical staining of myofibroblasts with α-smooth muscle actin (left) and quantification (right, n = 6). (G) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of α-smooth muscle actin, collagen I, and collagen III in the heart (n = 4). Data are presented as mean ± standard deviation, and n represents number of animals. DMSO, dimethylsulfoxide; TRITC, tetramethylrhodamine. Figure 5 View largeDownload slide Administration of CXCR2 inhibitor SB265610 improves angiotensin II-induced cardiac remodelling. Wild-type (WT) mice were co-treated with vehicle (DMSO) control or CXCR2 inhibitor SB265610 and angiotensin II for 14 days. (A) M-mode echocardiography of left ventricular chamber (upper), and measurement of ejection fraction (EF%) and fractional shortening (FS%) (lower, n = 6). (B) Representative heart size (left) and heart weight to tibial length (HW/TL) ratio (right, n = 6). (C) TRITC-labelled wheat germ agglutinin staining of heart sections (left) and quantification of myocyte cross-sectional area (200 cells counted per heart, right, n = 6). (D) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of atrial natriuretic factor and brain natriuretic peptide in the hearts (n = 4). (E) Masson trichrome staining of heart tissues (left) and quantification of fibrotic area (right, n = 6). (F) Immunohistochemical staining of myofibroblasts with α-smooth muscle actin (left) and quantification (right, n = 6). (G) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of α-smooth muscle actin, collagen I, and collagen III in the heart (n = 4). Data are presented as mean ± standard deviation, and n represents number of animals. DMSO, dimethylsulfoxide; TRITC, tetramethylrhodamine. Consistent with the findings from CXCR2 KO mice, flow cytometry confirmed that SB265610 treatment also significantly reduced myocardial infiltration of CD45+ myelomonocytes, CD11b+F4/80+ macrophages and CD11b+Gr-1+ neutrophils compared with vehicle-treated controls after Ang II infusion (Supplementary material online, Figure S2A). Moreover, Ang II-induced myocardial recruitment of pro-inflammatory cells and the expression of IL-1β, IL-6, TNF-α, and MCP-1 were markedly lower in SB265610-treated mice than in vehicle-treated animals (Supplementary material online, Figure S2B and C). Bone marrow-derived CXCR2-deficient cells attenuates angiotensin II-induced cardiac hypertrophy, fibrosis, and inflammation To directly test whether CXCR2+ myeloid cells affect cardiac remodelling, we created various chimeric mice by BM transplant and infused them with Ang II for the ensuing 2 weeks. WT mice reconstituted with CXCR2 KO BM exhibited a significant decrease in systolic blood pressure (Supplementary material online, Figure S3), improvement of contractile function (EF% and FS%) (Figure 6A, Supplementary material online, Table S5), hypertrophy (HW/TL ratio and myocyte area) (Figure 6B and C) and fibrosis (collagen area and α-SMA-positive myofibroblasts) (Figure 6E and F), infiltration of pro-inflammatory cells (Figure 6H) and the mRNA expression of ANF, BNP, α-SMA, collagen I and collagen III, IL-6, IL-1β, TNF-α and MCP-1 (Figure 6D, G, I) as compared to WT mice reconstituted with WT BM. Similar effects were confirmed in CXCR2 KO mice reconstituted with CXCR2 KO BM (Figure 6A–I). Conversely, reconstitution of CXCR2 KO mice with WT BM fully restored Ang II-induced pathological changes (Figure 6A–I). These findings suggest that BM-derived CXCR2 cells predominantly contribute to the development of hypertrophic remodelling in this model. Figure 6 View largeDownload slide Bone marrow-derived CXCR2-deficient cells prevent angiotensin II-induced cardiac remodelling. Wild-type (WT) or CXCR2 knockout (KO) mice were given bone marrow from CXCR2 knockout or wild-type mice, respectively, and then infused with angiotensin II for 2 weeks. (A) M-mode echocardiography of left ventricular chamber (upper) and measurement of ejection fraction (EF%) and fractional shortening (FS%) (lower, n = 6). (B) Haematoxylin and eosin staining of heart sections (left) and heart weight to tibial length (HW/TL) ratio (right, n = 6). (C) TRITC-labelled wheat germ agglutinin staining of heart sections (left) and quantification of myocyte cross-sectional area (200 cells counted per heart, right, n = 6). (D) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of atrial natriuretic factor and brain natriuretic peptide in the hearts (n = 6). (E) Massons’ trichrome staining of heart tissues (left) and quantification of fibrotic area (right, n = 6). (F) Immunohistochemical staining of myofibroblasts with α-smooth muscle actin (left) and quantification (right, n = 6). (G) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of α-smooth muscle actin, collagen I, and collagen III in the hearts (n = 6). (H) Haematoxylin and eosin staining of heart sections. (I) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of IL-1β, IL-6, tumour necrosis factor-α, and monocyte chemoattractantprotein-1 in the hearts (n = 4). Data are presented as mean ± standard deviation, and n represents number of animals. TRITC, tetramethylrhodamine. Figure 6 View largeDownload slide Bone marrow-derived CXCR2-deficient cells prevent angiotensin II-induced cardiac remodelling. Wild-type (WT) or CXCR2 knockout (KO) mice were given bone marrow from CXCR2 knockout or wild-type mice, respectively, and then infused with angiotensin II for 2 weeks. (A) M-mode echocardiography of left ventricular chamber (upper) and measurement of ejection fraction (EF%) and fractional shortening (FS%) (lower, n = 6). (B) Haematoxylin and eosin staining of heart sections (left) and heart weight to tibial length (HW/TL) ratio (right, n = 6). (C) TRITC-labelled wheat germ agglutinin staining of heart sections (left) and quantification of myocyte cross-sectional area (200 cells counted per heart, right, n = 6). (D) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of atrial natriuretic factor and brain natriuretic peptide in the hearts (n = 6). (E) Massons’ trichrome staining of heart tissues (left) and quantification of fibrotic area (right, n = 6). (F) Immunohistochemical staining of myofibroblasts with α-smooth muscle actin (left) and quantification (right, n = 6). (G) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of α-smooth muscle actin, collagen I, and collagen III in the hearts (n = 6). (H) Haematoxylin and eosin staining of heart sections. (I) Quantitative real-time polymerase chain reaction analysis of the mRNA levels of IL-1β, IL-6, tumour necrosis factor-α, and monocyte chemoattractantprotein-1 in the hearts (n = 4). Data are presented as mean ± standard deviation, and n represents number of animals. TRITC, tetramethylrhodamine. CXCR2-deficient macrophages inhibit cardiomyocyte hypertrophy and myofibroblast differentiation in vitro We next determined whether CXCR2 influences macrophage migration and activation in vitro. A transwell migration assay showed that CXCL1 treatment significantly enhanced migration of WT macrophages (Φ), but it had no significant effect on CXCR2 KO macrophages (Supplementary material online, Figure S4A). Quantitative real-time polymerase chain reaction and enzyme linked immunosorbent assay (ELISA) analysis confirmed that Ang II-induced up-regulation of IL-1β, IL-6, TGF-β1, ICAM-1, VCAM-1, or MCP-1 at both the mRNA and protein levels in WT macrophages was markedly attenuated in CXCR2 KO macrophages compared with WT cells (Supplementary material online, Figure S4B and C). Moreover, activation of P65 was also significantly inhibited in CXCR2 KO macrophages compared with WT cells after Ang II infusion (Supplementary material online, Figure S4D). To test whether CXCR2+ macrophages directly influenced cardiomyocyte (CM) hypertrophy and myofibroblast differentiation in vitro, WT or CXCR2 KO macrophages were co-cultured with WT neonatal rat cardiac myocytes (CMs) or fibroblasts (CFs), respectively. After 24 h of Ang II treatment, co-culture of CMs with CXCR2 KO macrophages had a significant reduction in CM size, the expression of ANF, BNP and β-MHC, and the protein levels of p-AKT, p-ERK1/2, p-STAT3 and CaNA compared with co-culture of CMs with WT macrophages (Supplementary material online, Figure S4E–G). Meanwhile, co-culture of CFs with CXCR2 KO macrophages markedly attenuated Ang II-induced increase in the mRNA levels of α-SMA, collagen I, collagen III or TGF-β1 compared with co-culture of CFs with WT macrophages (Supplementary material online, Figure S4H). Similarly, the protein levels of α-SMA, collagen I, collagen III, TGF-β1, or p-Smad2/3 were remarkably lower in co-culture of CFs with CXCR2 KO macrophages after Ang II treatment (Supplementary material online, Figure S4I). Serum CXCL1 level and CXCR2+ inflammatory cells are increased in heart failure patients with hypertension To test whether CXCR2+ monocytes and CXCL1 are important in human hypertensive HF, we analysed serum CXCR2+ immune cells, CXCL1 level, and other cardiovascular risk factors in HF patients (n = 30) and normal individuals (n = 32). Heart failure patients were older and had higher total cholesterol, LDL, triglyceride, and lower HDL concentrations compared with normal controls (Table 1). Flow cytometry analysis revealed that the numbers of circulating CD45+ myeloid cells, including CD182(CXCR2)+ cells, CD14+ monocytes, CD14+CD182+ monocytes, CD14+CD64+CD80+ monocytes, CD14+CD64+CD80+CD182+ monocytes, CD14+CD11b+CD209+ monocytes, CD14+CD11b+CD209+CD182+ monocytes, and CXCL1 level were significantly higher in HF patients than in normal individuals (Figure 7A–I). After adjusting for gender, age, total cholesterol, LDL, HDL, and triglycerides, multivariable logistic regression models indicated that there was a statistically significant association between the number of CXCR2+ cells, including CD45+ cells [odds ratio (OR) 1.162], CD14+CD182+ monocytes (OR 1.556), CD64+CD80+CD182+ monocytes (OR 1.030), CD11b+CD209+CD182+ monocytes (OR 1.106), CXCL1 level (OR 1.386), and HF (Table 2). Table 2 Results of the multivariable logistic regression models of CXCR2+ cells on heart failure Model Odds ratio (95% CI) P-value CD45+ CD182+cells 1.162 (1.001–1.349) 0.048 CD45+ CD14+CD182+ monocytes 1.556 (1.061–2.282) 0.024 CD45+ CD64+CD80+CD182+ monocytes 1.030 (1.005–1.055) 0.018 CD45+ CD11b+CD209+CD182+ monocytes 1.106 (1.020–1.200) 0.015 Serum CXCL-1 protein levels (pg/mL) 1.386 (1.003–1.915) 0.048 Model Odds ratio (95% CI) P-value CD45+ CD182+cells 1.162 (1.001–1.349) 0.048 CD45+ CD14+CD182+ monocytes 1.556 (1.061–2.282) 0.024 CD45+ CD64+CD80+CD182+ monocytes 1.030 (1.005–1.055) 0.018 CD45+ CD11b+CD209+CD182+ monocytes 1.106 (1.020–1.200) 0.015 Serum CXCL-1 protein levels (pg/mL) 1.386 (1.003–1.915) 0.048 All models adjusted for age, systolic blood pressure, HDL cholesterol, creatinemia, fasting blood glucose, and white blood cell count. CI, confidence interval; HDL, high-density lipoprotein. Table 2 Results of the multivariable logistic regression models of CXCR2+ cells on heart failure Model Odds ratio (95% CI) P-value CD45+ CD182+cells 1.162 (1.001–1.349) 0.048 CD45+ CD14+CD182+ monocytes 1.556 (1.061–2.282) 0.024 CD45+ CD64+CD80+CD182+ monocytes 1.030 (1.005–1.055) 0.018 CD45+ CD11b+CD209+CD182+ monocytes 1.106 (1.020–1.200) 0.015 Serum CXCL-1 protein levels (pg/mL) 1.386 (1.003–1.915) 0.048 Model Odds ratio (95% CI) P-value CD45+ CD182+cells 1.162 (1.001–1.349) 0.048 CD45+ CD14+CD182+ monocytes 1.556 (1.061–2.282) 0.024 CD45+ CD64+CD80+CD182+ monocytes 1.030 (1.005–1.055) 0.018 CD45+ CD11b+CD209+CD182+ monocytes 1.106 (1.020–1.200) 0.015 Serum CXCL-1 protein levels (pg/mL) 1.386 (1.003–1.915) 0.048 All models adjusted for age, systolic blood pressure, HDL cholesterol, creatinemia, fasting blood glucose, and white blood cell count. CI, confidence interval; HDL, high-density lipoprotein. Figure 7 View largeDownload slide Serum CXCL1 levels and blood CXCR2+ cells are increased in patients with heart failure. (A–H) Flow cytometric analysis of circulating inflammatory cells, including CD45+ CD182 (CXCR2)+ cells, CD45+CD14+ monocytes, CD45+CD14+CD182+ monocytes, CD45+CD14+CD64+CD80+ monocytes, CD45+CD14+CD64+CD80+CD182+ monocytes, CD45+CD14+CD11b+CD209+ monocytes, and CD45+CD14+CD11b+CD209+CD182+ monocytes in normotensive people (n = 32) and heart failure patients (n = 30). (I) ELISA assay of serum CXCL1 levels in normotensive people (n = 32) and heart failure patients (n = 30). Data are presented as mean ± standard deviation and n represents number of persons. Figure 7 View largeDownload slide Serum CXCL1 levels and blood CXCR2+ cells are increased in patients with heart failure. (A–H) Flow cytometric analysis of circulating inflammatory cells, including CD45+ CD182 (CXCR2)+ cells, CD45+CD14+ monocytes, CD45+CD14+CD182+ monocytes, CD45+CD14+CD64+CD80+ monocytes, CD45+CD14+CD64+CD80+CD182+ monocytes, CD45+CD14+CD11b+CD209+ monocytes, and CD45+CD14+CD11b+CD209+CD182+ monocytes in normotensive people (n = 32) and heart failure patients (n = 30). (I) ELISA assay of serum CXCL1 levels in normotensive people (n = 32) and heart failure patients (n = 30). Data are presented as mean ± standard deviation and n represents number of persons. Discussion Here, we demonstrated for the first time that CXCL1–CXCR2 signalling induces myocardial recruitment of monocytes, which is the key step for initiating Ang II-induced cardiac remodelling. Angiotensin II infusion significantly upregulated CXCL1 expression, which promoted the infiltration of BM-derived CXCR2+ monocytes into the heart tissues and produced multiple pro-inflammatory cytokines, thereby causing cardiac remodelling and dysfunction. Conversely, therapeutic blocking of CXCL1 and CXCR2 or ablation of CXCR2 markedly attenuated these effects. Thus, our novel evidence supports a strong causative role of CXCL1–CXCR2 axis in the pathogenesis of cardiac remodelling. A working model is illustrated in Figure 8. It is well known that pro-inflammatory cells play pivotal roles in the development of cardiovascular diseases.14 Circulating monocytes are the precursors of Ly6Chi inflammatory monocytes in mice, which are the equivalent to CD14+ monocytes in humans.15 Previous studies defined M1/M2 macrophages by labelling CD64 and CD80 for M1 or CD11b and CD209 for M2 in polarized human peripheral blood monocytes in vitro and other tissues but not in the context of CD14+ cells.16,17 Here, we analysed several subpopulations of CD14+ monocytes in human samples, and found that these cells were significantly increased in HF patients (Figure 7). Recently, our data and that of other researchers have demonstrated that CD11b+Gr-1+ or CD11b+Ly6Chi monocytes and CD11b+F4/80+ macrophages are critical early mediators of Ang II-induced vascular dysfunction, arterial hypertension and cardiac fibrosis in mice.12,13,18–20 Chemokines are reported to be critically involved in leucocyte trafficking to the inflammatory sites, and their principal targets are haematopoietic cells.5 Interestingly, these chemokines are markedly upregulated upon heart injury to induce a chemotactic response in vivo.5 CXCL1 has been reported to exert a critical role in different cardiovascular diseases by regulating the recruitment of neutrophils, T lymphocytes and monocytes.11,21 In this study, we extended previous findings and found that CXCL1 was also significantly upregulated in Ang II-infused hearts (Figure 1) and HF patients (Figure 8), which recruited CXCR2+ monocytes into the heart tissues leading to cardiac remodelling, but this effect was prevented by CXCL1 neutralizing antibody (Figure 2). Further studies are required to determine how CXCL1 is upregulated by Ang II in the heart. Figure 8 View largeDownload slide A working model for CXCL1 to recruit CXCR2+ monocytes into the heart, which initiate and aggravate angiotensin II-induced inflammation, cardiac hypertrophy, and fibrosis leading to cardiac remodelling. Inhibition of CXCL1 by neutralizing antibody (nAb) or CXCR2 by inhibitor SB265610 prevents these effects. Figure 8 View largeDownload slide A working model for CXCL1 to recruit CXCR2+ monocytes into the heart, which initiate and aggravate angiotensin II-induced inflammation, cardiac hypertrophy, and fibrosis leading to cardiac remodelling. Inhibition of CXCL1 by neutralizing antibody (nAb) or CXCR2 by inhibitor SB265610 prevents these effects. CXCL1 exerts multiple biological functions via binding to its CXCR2 receptor, which also serves as a receptor for other CXCL subfamily members.5 CXCR2 is reported to be express on the surface of both immune and non-immune cells, including leucocytes, T lymphocytes, CMs, and CFs, and it regulates the functions of these cells. Interestingly, CXCR2 activation plays critical roles in the recruitment of leucocytes and neutrophils, which are involved in the pathogenesis of myocardial infarction and atherosclerosis.8,9 Our recent data indicated that CXCR2 mainly mediates infiltration of monocytes into the injured arteries leading to hypertension.11 Here, we further demonstrated that genetic and pharmacological inhibition of CXCR2 effectively reduced Ang II-induced infiltration of monocytes/macrophages and cardiac remodelling through inhibition of multiple signalling pathways in vivo and in vitro (Figures 3–6, Supplementary material online, Figure S4). Overall, our results suggest that CXCR2+ monocytes/macrophages directly contribute to Ang II-induced cardiac remodelling and dysfunction. This study identified for the first time that CXCL1–CXCR2 axis induced recruitment of monocytes/macrophages into the heart thereby leading to cardiac remodelling and dysfunction in response to Ang II. Circulating CXCR2+ monocytes and CXCL1 level were significantly higher in HF patients. Therapeutic targeting of CXCL1 or CXCR2 signalling prevented adverse cardiac remodelling and dysfunction, thus representing an attractive new strategy for treating hypertensive HF. Supplementary material Supplementary material is available at European Heart Journal online. Funding National Natural Science Foundation of China (81630009 and 81330003 to H.-H.L; 81500303 to L.W.), Dalian high level Talents Innovation and Entrepreneurship Projects (2015R019), and the Chang Jiang Scholar Program of China (T2011160 to H.-H.L.). Conflict of interest: none declared. 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Google Scholar CrossRef Search ADS PubMed Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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European Heart JournalOxford University Press

Published: Mar 5, 2018

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