Elevated Circulating Fibrocytes Is a Marker of Left Atrial Fibrosis and Recurrence of Persistent Atrial Fibrillation

Figures

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  Figure 1. Overview of the workflow for the present study. AF indicates atrial fibrillation; MMPs, matrix metalloproteinases.

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  Figure 2. The proportion of circulating fibrocytes linked to the degree of left atrium (LA) low‐voltage area and recurrence rate after ablation in patients with persistent disease. All CD45+ leukocytes were gated for analysis. Representative images of flow cytometry displaying circulating fibrocytes, determined by the population of cells expressing CD45, CD34, and collagen I relative to the whole population of leukocytes (CD45+) in the fresh blood from sinus rhythm (SR) controls (B), patients with paroxysmal AF (PaAF; C), or patients with persistent atrial fibrillation (PeAF; D), respectively. Isotype negative control is shown in (A). Patients with persistent atrial fibrillation (AF) displayed a higher percentage of circulating fibrocytes compared with patients with paroxysmal AF or SR controls (E). Representative images of 3‐dimensional CARTO showing the electroanatomic map in the LA (F). The red color indicates low‐voltage tissues, while the purple color indicates healthy tissues (F). Univariate linear regression analyzed the correlation between circulating fibrocyte percentage and LA fibrosis (G). The percentage of low‐voltage area was calculated by the area of red color relative to the…

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  Figure 3. Circulating fibrocyte differentiation in the cell culture. Representative images of differentiated fibrocytes cultured from sinus rhythm (SR) controls (A and B), patients with paroxysmal atrial fibrillation (PaAF; C and D), or patients with persistent atrial fibrillation (PeAF; E and F) at a magnification of ×20 and ×40. Spindle‐shaped cells were myofibroblasts that were differentiated from circulating fibrocytes.

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  Figure 4. Quantitation and purification of differentiated fibrocytes following cell culture. Differentiated fibrocytes were determined by the population of cells expressing CD45 and collagen I relative to the whole population of leukocytes (CD45+) in the cultured cells from sinus rhythm (SR) controls, patients with paroxysmal atrial fibrillation (PaAF), or patients with persistent atrial fibrillation (PeAF). Representative forward and side scatter properties of adherent cells after 12 days of culture by flow cytometry was used to separate the 3 populations (A: R1, R2, and R3). Isotype controls are presented in the left panels (B, D, and F). Double positive cells were identified as differentiated fibrocytes (R1, C), CD45‐positive cells were leukocytes (R2, E), and double negative cells were accepted as cell fragments (R3, G). Quantitation of differentiated fibrocytes in the group, patients with paroxysmal atrial fibrillation (PaAF), and patients with persistent atrial fibrillation (PeAF) are presented in (H). Data are presented as mean±SD. *P<0.05 vs SR controls, † P<0.05 vs PaAF.

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  Figure 5. Differentiated fibrocytes from patients with atrial fibrillation (AF) were in a hyperactivated status. The mRNA levels of α‐smooth muscle actin (α‐SMA) (A), collagen I (B), collagen III (C), and fibronectin (D) of sorted differentiated fibrocytes from sinus rhythm (SR) controls, patients with paroxysmal atrial fibrillation (PaAF), and patients with persistent atrial fibrillation (PeAF) were measured by real‐time polymerase chain reaction analysis. Representative immunofluorescence showed protein levels of α‐SMA in differentiated fibrocytes from SR controls (E), patients with PaAF (F) or PeAF (G). Quantitation of total α‐SMA fluorescent intensity is presented in (H). Migrated cells in transwell assays were stained by 4′,6‐diamidino‐2‐phenylindole (DAPI; I through K). Differentiated fibrocytes from patients with PeAF (K) presented an enhanced migratory ability at 36 hours compared with patients with PaAF (J) and SR controls (I). Quantitation of migrated cell number is presented in (L). Data are presented as mean±SD (n=4–6 per group). *P<0.05 vs SR controls, † P<0.05 vs PaAF.

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  Figure 6. Differentially expressed genes in differentiated fibrocytes among 3 groups. The mRNA levels of 20 candidate genes in the differentiated fibrocytes (myofibroblasts) were examined using real‐time polymerase chain reaction analysis. Relative to the sinus rhythm (SR) groups, levels of CXC chemokine receptor type 4 (CXCR4), chemokine (C‐C motif) ligand 2 (CCL2), matrix metalloproteinase (MMP) 2, transforming growth factor β1 (TGF‐β1), interleukin (IL) 8, platelet‐derived growth factor receptor B (PDGFRB), CX3C chemokine receptor 1 (CX3CR1), transforming growth factor β receptor 1 (TGF‐βR1), and MMP‐8 in SR controls, patients with paroxysmal atrial fibrillation (PaAF), and patients with persistent atrial fibrillation (PeAF) are shown in (A through I). Data are presented as mean±SD (n=4–6 per group). *P<0.05 vs SR controls, † P<0.05 vs PaAF.

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  Figure 7. Circulating fibrocytes showed variations of differentiation in response to different sources of plasma. Leukocytes from patients with sinus rhythm (SR) or persistent atrial fibrillation were cultured in DMEM supplemented with plasma from SR controls or patients with persistent atrial fibrillation (AF). Plasma from patients with AF enhanced differentiational ability of SR fibrocytes compared with culture in its own plasma, while AF fibrocytes showed a reduced capacity to differentiate in response to SR plasma. Data are presented as mean±SD (n=4–6 per group). *P<0.05 vs AF plasma.

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  Figure 8. Changes in plasma cytokines and chemokines. Plasma levels of transforming growth factor β1 (TGF‐β1), stromal cell–derived factor 1 (SDF‐1), interleukin (IL)8, matrix metalloproteinase (MMP) 2, MMP‐8, and serum amyloid P (SAP) were measured by ELISA kit and are presented by dot plots (A through F). Data are presented as mean±SD. *P<0.05 vs sinus rhythm (SR) controls, † P<0.05 vs paroxysmal atrial fibrillation (PaAF). PeAF indicates persistent atrial fibrillation.

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  Figure 9. Transforming growth factor β1 (TGF‐β1) promoted circulating fibrocytes to differentiate into myofibroblasts. Representative images of differentiated fibrocytes from sinus rhythm (SR) controls cultured with or without TGF‐β1 (A and B). Spindle‐shaped cells are recognized as myofibroblasts that differentiated from circulating fibrocytes. The portion of myofibroblasts (CD45+/collagen I+ cells) was examined by flow cytometry (C and D), and quantitation is shown in (E). Representative images of differentiated fibrocytes from SR controls under the condition of TGF‐β1 treatment, with or without transforming growth factor β receptor 1 (TGF‐βR1) knockdown are shown in (F and G). The portion of CD45+/collagen I+ cells infected by short hairpin control (shControl) or short hairpin TGF‐βRa (shTGF‐βR1) was analyzed by flow cytometry and is presented in (H and I). Quantitation of differentiated fibrocytes is shown in (J). Data are presented as mean±SD (n=3–5 per group). *P<0.05 vs vehicle or shControl.

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  Figure 10. Stromal cell–derived factor 1 (SDF‐1) promoted migration of fibrocytes. Circulating fibrocytes from sinus rhythm (SR) controls were loaded into the upper chamber of transwells for 36 hours after addition of SDF‐1 or vehicle to the lower chamber of transwell. Representative images of migrated fibrocytes were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) and are shown in (A and B). Quantitation of migrated cells is shown in (C). Fibrocytes with short hairpin control (shControl) or short hairpin CXC chemokine receptor type 4 (shCXCR4) were assessed to the transwell assay under the stimulation of SDF‐1 for 36 hours. Representative images of migrated fibrocytes stained with DAPI are shown in (D and E). Quantitation of migrated cells is presented in (F). Data are presented as mean±SD (n=3–5 per group). *P<0.05 vs vehicle or shControl.