Systemic inflammatory changes after pulmonary vein radiofrequency ablation do not alter stem cell mobilization

Abstract

Aims

Aim of this study was to investigate the number of circulating progenitor cells, systemic inflammatory mediators, and myocardial necrosis in patients with paroxysmal atrial fibrillation (AF) undergoing pulmonary vein (PV) isolation by radiofrequency (RF) ablation. Radiofrequency ablation generates a localized myocardial necrosis that might result in a release of inflammatory mediators enhancing progenitor cell mobilization and improving tissue repair.

Methods and results

Blood samples were collected in patients with paroxysmal AF before and after PV isolation. Interleukin (IL)-6, IL-1β, TNF-α, IL-8, IL-10, and IL-12, and stromal derived factor (SDF)-1 were measured by immunoassay. CD34+CD133+, CD117+, and endothelial progenitor cells (EPCs) were analysed by flow cytometry and culture assay. After ablation procedure, a rise in creatine kinase and troponin T levels indicated myocardial necrosis. Leukocyte counts and C-reactive protein and IL-6 levels increased significantly. Myocardial necrosis and inflammatory response correlated with an increase in IL-6 ( P = 0.007). In contrast, SDF-1 levels decreased after RF ablation ( P = 0.004). Yet, no significant changes were observed in IL-1β, TNF-α, IL-8, IL 10, and IL-12 plasma levels or in the number of circulating CD34+CD133+ and CD117+ progenitor cells, whereas EPCs decreased by trend.

Conclusion

Although PV isolation by RF ablation in patients with paroxysmal AF induces a systemic inflammatory response associated with myocardial necrosis, no alterations in circulating progenitor cells were observed. Thus, isolated myocardial necrosis may not be sufficient to account for progenitor cell mobilization.

Inflammation, Progenitor cells, Ablation, Tachyarrhythmias, Cytokines

Introduction

Tissue damage such as myocardial infarction, ¹ severe burns, or bypass surgery ² has been shown to increase the number of circulating progenitor cells. Mobilized from the bone marrow, progenitor cells are recruited to the ischaemic area through specific chemokine and integrin interactions ³ and contribute to neovsacularization and tissue regeneration. ⁴ Since spontaneous progenitor cell mobilization might contribute to a more favourable myocardial remodelling after acute myocardial infarction, the analysis of stem cell mobilizing factors is of particular clinical importance. In addition to myocardial ischaemia, tissue necrosis may contribute to stem cell mobilization by release of growth factors and cytokines. Increased progenitor cell release from the bone marrow is part of the host defence during inflammation as a result of injury-mediated release of stress signals. This release is induced by a wide range of molecules: cytokines such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, SCF, and chemokines such as IL-8, Mip-1α, Gro-β, and stromal derived factor (SDF)-1. ³ Recently, circulating IL-8 was identified as an independent predictor of progenitor cell mobilization in patients with acute myocardial infarction. ¹ IL-8 upregulation occurs in experimental models of myocardial infarction in inflammatory cells of the infarct border zone and in microvascular endothelial cells, ⁵ yet the contribution of vascular injury, myocardial ischaemia, and tissue necrosis to systemic inflammatory changes and subsequent progenitor cell mobilization remains unclear.

In patients with persistent atrial fibrillation (AF), an increase in the number of circulating CD34+ progenitor cells was found, which temporarily decreased after electrical cardioversion. ⁶ Even more pronounced myocardial necrosis followed by infiltration of inflammatory cells was observed after pulmonary vein (PV) isolation by radiofrequency (RF) ablation. ⁷ Aim of this study was to analyse systemic inflammatory changes and circulating progenitor cells after PV isolation in patients with paroxysmal AF.

Methods

Patient selection

The study group comprised 14 patients with paroxysmal AF undergoing segmental electrical isolation of the PVs by RF ablation. The study protocol was approved by institutional Ethics Committee and written informed consent was obtained from all subjects.

Electrophysiological study

After written informed consent, patients underwent electrophysiological study under conscious sedation with midazolam and fentanyl or propofol during continuous monitoring of blood pressure and O ₂ saturation. Vascular access was obtained through both femoral veins. An octapolar electrode catheter (XPT™, C.R. Bard, Covington, GA, USA) was positioned in coronary sinus (CS). Following transseptal puncture, two steerable catheters were positioned in the left atrium (LA) under the guidance of transseptal sheaths (Preface Multipurpose, Biosense Webster, Diamond Bar, CA, USA or SL, St Jude Medical, Eschborn, Germany) that were continuously perfused with heparinized saline: an irrigated tip ablation catheter (Celsius Thermocool, Biosense Webster) and a circular mapping catheter equipped with 10 or 14 1 mm electrodes (Lasso, Biosense Webster or Orbiter PV, C.R. Bard) were used. Anticoagulation was initiated with a bolus of 5000 IU heparin and adjusted to maintain the activated clotting time between 280 and 310 s. Pulmonary vein isolation was performed as described previously. ⁸ For segmental PV mapping and isolation, a distally circular shaped circumferential catheter and an irrigated 3.5 mm tip ablation catheter (Thermocool Navistar™, Biosense Webster) were positioned in the LA. Ablation was guided by circumferential mapping and anatomical three-dimensional reconstruction of LA and PV using the CARTO (Biosense Webster) or NavX system (St Jude Medical). Irrigated RF energy with a flow rate of 20–30 mL/min was delivered for ≤2 min for lesions aiming at isolating PV. The maximum temperature was set to 43°C, and a maximum power of 30 W was applied. The endpoint of PV ablation was electrical-isolation-signified by the abolition or dissociation of PV potentials. Routinely, all PVs were targeted for electrical isolation. Venous blood samples were obtained before and 18 h after PV ablation.

Immunoassays

Concentrations of IL-1β, TNF-α, IL-6, IL-8, IL-10, IL-12, and stromal derived factor-1 (SDF-1) were determined by immunoassays (CBA Human Inflammation Kit, BD Biosciences, San Diego, USA; SDF-1 immunoassay, Quantikine, R&D Systems, Minneapolis, MN, USA). Detection limits were 7.2 pg/mL for IL-1β, 3.7 pg/mL for TNF-α, 2.5 pg/mL for IL-6, 3.6 pg/mL for IL-8, 3.3 pg/mL for IL-10, 1.9 pg/mL for IL-12, and 18 pg/mL for SDF-1. Intra-assay variability for the lower assay range were <10%.

Flow cytometry

To analyse circulating progenitor cells, mononuclear cells enriched from citrate phosphate dextrose acid-anticoagulated blood samples by Ficoll (Ficoll-Paque PLUS, GE Healthcare, Freiburg, Germany) density gradient centrifugation were stained according to a modified protocol of the European Working Group on Clinical Cell Analysis. ¹ Vital CD133+CD34+CD45−7AAD- progenitor cells were determined by staining with FITC-conjugated anti-CD34, PE-conjugated anti-CD133, APC-conjugated anti-CD45 and 7AAD (BD Biosciences), and mesenchymal progenitor cells were determined by staining with PerCP-conjugated anti-CD117 (BD Biosciences). Surface expression of the SDF-1 receptor CXCR4 on CD34+ cells was analysed using FITC-conjugated anti-CXCR4 (R&D Systems), PE-conjugated anti-CD133, PerCP-conjugated anti-CD45, and APC-conjugated anti-CD34. Fluorescence isotype-matched antibodies were used as controls. Flow cytometric analysis was performed using a FACS Calibur (Becton Dickinson, Mountain View, CA, USA). Fluorescence intensity of at least 200 000 cells was recorded and analysed using CellQuest (BD, Heidelberg, Germany) software.

Culture assay of endothelial progenitor cells

Mononuclear cells were cultured on fibronectin-coated cover slides in EGM-2 medium (Cambrex Clonetics, Baltimore, MD, USA) using 5 × 10 ⁶ cells per 24-well plate as described before. ⁹ After 7 days of culture, endothelial progenitor cells (EPCs) were analysed by determining cell counts in 5 randomly selected 10-fold magnified fields from duplicate wells.

Other methods

Full blood count, C-reactive protein as well as serum creatin kinase (CK), and troponin T (TnT) were determined in the clinical chemistry laboratory.

Statistical analysis

Differences between two matched samples were tested by Friedmann’s test followed by Wilcoxon’s matched-pairs signed-ranks test. Differences between two groups were tested by two-sided t -test. A P -value <0.05 was regarded as significant.

Results

Patients and procedural characteristics

In the present study, 14 patients with paroxysmal AF undergoing PV isolation AF were included. The mean age of the study population was 58 ± 9 years. None of the patients was diagnosed with coronary artery disease. All patients had normal left ventricular function with a mean shortening fraction of 37.3 ± 6.1%. Diameters of the LA were not enlarged with an average of 40.1 ± 4.1 mm. Mean number of AF episodes per week was 2.9 ± 2.9 with a mean duration of 8.3 ± 7.8 h. The study population is further described in Table  1 .

Table 1

Baseline characteristics of study patients ( n = 14)

Age, years	58 ± 9
Sex, M/F	9/5
CAD, n	0
MI, n	0
Number of AF episodes per week, n	2.9 ± 2.9
Mean duration of AF episodes, h	8.3 ± 7.8
LA diameter, mm	40.1 ± 4.1
Left ventricular shortening fraction, %	37.3 ± 6.1
Hypertension, n	5
Diabetes, n	0
Hypercholesteraemia, n	6
Smoking, n	1
Beta-blocker, n	14
ACE-inhibitors, n	4
Amiodaron, n	0
Statins, n	4
Calcium antagonists, n	0
Phenprocoumon, n	14

AF, atrial fibrillation; CAD, coronary artery disease; MI, history of myocardial infarction.

For PV isolation, procedure time was 276 ± 67 min, number of RF applications 75 ± 30, and duration of RF ablation 3010 ± 1604 s. Applied contrast media was 152 ± 64 mL and fluoroscopic time was 53 ± 20 min. Effective isolation of the PVs was achieved in all 14 patients and all patients were in sinus rhythm after the procedure. No major complications such as pericardial tamponade, major bleeding, death, or cerebral event occurred.

Myocardial necrosis and systemic inflammation

In patients with paroxysmal AF and PV isolation, a significant increase in CK and TnT levels ( Figure  1 ) occurred. Similarly, leukocyte count, C-reactive protein levels, and IL-6 plasma concentrations were elevated ( Figure  2 ), whereas plasma concentrations of IL-1β, TNF-α, IL-8, IL-12, and IL-10 remained unchanged throughout the procedure ( Table 2 ). The increase in IL-6 was correlated with the increase in TnT ( R = 0.82, P = 0.0005). In contrast, SDF-1 levels decreased after PV ablation and the decrease in SDF-1 was negatively correlated with the increase in TnT plasma levels ( R = −0.87, P < 0.0001; Figure 3 ).

Figure 1

Pulmonary vein ablation induces myocardial necrosis with a significant increase of myocardial markers creatine kinase (CK) and troponin T (TnT). After pulmonary vein ablation, myocardial markers creatine kinase ( A ) and troponin T ( B ) are significantly elevated compared with pre-procedural values.

Figure 2

Pulmonary vein ablation induced interleukin-6-associated systemic inflammation. Leukocytes ( A ), C-reactive protein ( B ), and interleukin-6 (IL-6) ( C ) are significantly increased after pulmonary vein ablation.

Figure 3

Myocardial necrosis by pulmonary vein ablation induced significant decrease in stromal derived factor-1 (SDF-1) levels correlating with post-procedural troponin T (TnT) levels. Stromal derived factor-1 levels significantly decreased after pulmonary vein ablation ( A ). Changes in stromal derived factor-1 plasma concentrations were significantly correlated with changes in troponin T ( B ).

Table 2

Cytokines and progenitor cells

	Pre-procedural	Post-procedural	P -value
Leukocytes (/µL)	4.48 ± 1.81	7.33 ± 2.38	0.007
C-reactive protein (mg/L)	2.40 ± 2.90	20.10 ± 9.19	0.0001
CK (U/L)	117 ± 49.8	192 ± 105	0.007
TnT (ng/mL)	0.21 ± 0.80	1.14 ± 0.87	0.003
IL-1β	ND	ND
TNF-α	ND	ND
IL-6 (pg/mL)	1.06 ± 2.48	12.4 ± 15.3	0.007
IL-8 (pg/mL)	12.6 ± 19.2	8.96 ± 3.26	0.50
IL-12 (pg/mL)	0.81 ± 2.70	1.01 ± 3.29	0.50
IL-10 (pg/mL)	1.22 ± 2.33	1.74 ± 2.31	0.47
SDF-1 (pg/mL)	2155 ± 427	1687 ± 348	0.004
Cells (/µL)
CD133+CD34+	0.76 ± 0.54	0.64 ± 0.51	0.38
CD117+	0.11 ± 0.23	0.07 ± 0.86	0.85
CXCR4+ (mean fluorescence)	29 ± 66	14 ± 40	0.45
EPC, n	30 ± 24	17 ± 15	0.09

AF, atrial fibrillation; CK, creatine kinase; TnT, Troponin T; IL, interleukin; SDF, stromal derived factor; EPC, endothelial progenitor cell; ND, not detectable.

Circulating CD34+CD133+, CD117+, and endothelial progenitor cells

After PV ablation in the patients with paroxysmal AF, no changes in the number of circulating CD34+CD133+, CD34+CXCR4+, and CD117+ progenitor cells were observed. However, there was a trend towards decrease in the number of EPCs after ablation ( Table 2 ).

Discussion

Major findings of our study are as follows ¹ : myocardial necrosis after PV isolation by RF ablation in patients with paroxysmal AF was associated with an increase in circulating IL-6 and a decrease in SDF-1, whereas cytokines concentrations of IL-1β, TNF-α, IL-8, IL-10, and IL-12 were not affected. ² This pro-inflammatory response did not alter the number of circulating CD34+CD133+, CD34+CXCR4+, and CD117+ progenitor cells.

Myocardial necrosis by radiofrequency ablation therapy stimulates IL-6 release and subsequent systemic inflammatory changes

Radiofrequency catheter ablation has been shown to be effective in several cardiac arrhythmias. In early studies, the extent of myocardial injury was considered small, due to the small lesion size and the relatively limited number of RF lesions applied. ^{10 , 11} As more complex tachycardias were treated and procedures rendered more extensive with longer application times creating larger lesions, the myocardial damage increased. Serial blood samples taken from patients undergoing RF ablation for different arrhythmias showed significant increases in TnT and CK up to 48 h after the procedure. ^12–14 The elevation in markers of myocardial damage was correlated with the size of the ablation lesions measured by the amount of applied energy and the number of RF applications. ¹³ Accordingly, we found a significant increase of TnT and CK levels in all patients undergoing PV isolation, indicating extensive tissue damage. Myocardial necrosis provoked predominantly an IL-6 release with subsequent rise in C-reactive protein and the number of circulating leukocytes. Other cytokines such as IL1β, IL-8, IL-10, IL-12, and TNF-α that are known to relate to pro-inflammatory responses in sepsis ¹⁵ and acute coronary syndromes ^{1 , 16} remained unchanged after RF ablation. Specific tissue injury, for example necrosis, ischaemia, or endotoxin, may, thus, stimulate unique patterns of cytokine release that provoke distinct repair mechanisms. Earlier studies found C-reactive protein levels to be higher in patients with AF and showed that prevalence and frequency of AF is linked to systemic inflammatory stages. ^17–19 Recent studies, analysing non-antiarrhythmic drugs for the prevention of AF, found anti-inflammatory drugs to be attractive candidates. ²⁰ Against this background, it is tempting to speculate that the frequently observed early recurrence of AF after ablation therapy may be triggered by a transient inflammation, and anti-inflammatory drugs might be one possible therapeutic strategy.

Circulating progenitor cells remain unchanged during the systemic inflammatory reaction after radiofrequency ablation therapy

Progenitor cell mobilization from the bone marrow is induced by the release of mobilizing factors followed by a process in which adhesion interactions of progenitor cells to bone marrow stromal cells are disrupted by proteolytic enzymes. ^21–25 Different mechanisms have been found to initiate the mobilization cascade, such as vascular trauma due to burns or coronary artery bypass grafting, ischaemia, and myocardial infarction. ^{1 , 2 , 26–28}

During acute myocardial infarction, vascular trauma, thrombosis, ischaemia, and myocardial necrosis provoke the release of IL-8, VEGF, and G-CSF that enhance mobilization of progenitor cells from the bone marrow. One specific aim of this study was to investigate the influence of isolated myocardial necrosis induced by RF ablation therapy as a possible mobilizing factor on circulating progenitor cells. The number of circulating mesenchymal and haematopoietic cells remained unchanged after the procedure.

Interactions of the chemokine SDF-1 with its receptor CXCR4 on progenitor cells have been implicated as a principal axis regulating migration, and mobilization of haematopoietic progenitor cells during steady-state homeostasis and injury. ³ We, therefore, sought to measure SDF-1 plasma levels and CXCR4 expression on circulating progenitor cells. CXCR4 expression was not altered after RF ablation. This was unexpected, since stimulation with interleukin-6 (IL-6) upregulates CXCR4 expression by CD34 ⁺ cells, leading to their increased SDF-1-mediated migration and homing. ²⁹ Moreover, IL-6 has been identified to contribute to maintenance and expansion of haematopoietic progenitor cells in vitro . ³⁰ These changes might not be achieved with the transient increase in IL-6 observed in the patients after PV isolation.

Compared with pre-procedural values, SDF-1 levels decreased significantly after PV isolation. This decline in SDF-1 levels negatively correlated with corresponding changes in TnT plasma levels and may be directly related to myocardial necrosis through PV isolation. The underlying mechanisms remain to be unravelled. Stromal derived factor which is expressed by stroma cells from different tissues is upregulated in response to tissue hypoxia and damage signal attracting circulating haematopoietic progenitor cells through activation of specific integrin molecules. A role for SDF-1 in progenitor cell recruitment from the bone marrow to peripheral blood has been proposed, since G-CSF-mediated mobilization causes an imbalance between the expression of SDF-1 and CXCR4, SDF-1 gene transfer enhances the number of circulating progenitor cells, and overexpression of SDF-1 in ischaemic tissues has been found to enhance progenitor cell recruitment from the blood. ³ Accordingly, we found a trend towards decrease in circulating EPCs after PV isolation.

Study limitations

One limitation of the study is that in addition to myocardial necrosis, other peri-procedural factors such as the invasive procedure itself, the administered volume, contrast dye, and flouroscopy time may have contributed to IL-6 increase and SDF-1 decrease. Furthermore, effects of ablation induced myocardial necrosis and accompanying inflammation on progenitor cell mobilization only be extrapolated with caution to the myocardial necrosis of the ventricle occurring in myocardial infarction, as ischaemia and vascular trauma are absent or minimal during ablation.

Conclusion and clinical implications

Since other investigations showed an association between the prevalence and frequency of AF and systemic inflammatory changes, it is tempting to speculate that the frequently observed early recurrence of AF after ablation therapy may be affected by a transient inflammation, and anti-inflammatory drugs might be one possible therapeutic strategy.

Funding

The study was supported by grants from the Deutsche Herzstiftung, Frankfurt am Main, Germany and the Wilhelm Sander-Stiftung, Munich, Germany.

Acknowledgements

We thank Mrs B. Campbell, A. Stobbe, and C. Bauer for invaluable technical assistance.

Conflict of interest : none declared.