Heme is involved in the systemic inflammatory response following radiofrequency ablation of hepatic hemangiomas

Objective Radiofrequency ablation (RFA) is an effective and minimally invasive treatment for managing hepatic hemangiomas. Systemic inflammatory response syndrome (SIRS) often occurs with hemoglobinuria, and its underlying pathophysiological mechanism is unknown. Heme can trigger inflammation by inducing the generation of reactive oxygen species (ROS) and the production of inflammatory mediators. We therefore investigated whether circulating heme is involved in SIRS following RFA of hepatic hemangiomas. Methods We enrolled 65 patients with hepatic hemangioma who underwent RFA. Serum concentrations of free heme, ROS, and tumor necrosis factor α (TNF-α) were measured after RFA. Univariate analysis and a multivariate binary logistic regression model were used to evaluate the contribution of 17 risk factors for SIRS after RFA. Results Fifty-nine (59/65, 90.8%) patients developed hemoglobinuria, among which 25 (25/59, 42.4%) experienced SIRS shortly after RFA. In the SIRS group, the serum concentrations of heme, ROS, and TNF-α were immediately elevated after RFA compared with baseline and slowly regained their normal levels 3 days after RFA. Moreover, the concentrations of circulating heme significantly correlated with those of ROS (r = 0.805, P < 0.001) and TNF-α (r = 0.797, P < 0.001). Multivariate analysis showed that the volume of hemangioma [odds ratio (OR) = 1.293, P = 0.031], time of ablation (OR = 1.194, P = 0.029) as well as the concentrations of heme (OR = 1.430, P = 0.017), ROS (OR = 1.251, P = 0.031), and TNF-α (OR = 1.309, P = 0.032) were significantly associated with SIRS. Conclusion Circulating heme was associated with the induction of ROS and the production of TNF-α, which may contribute to the induction of SIRS following RFA of hepatic hemangiomas.


Abstract
Background Radiofrequency ablation (RFA) is an effective and minimally invasive treatment for managing hepatic hemangiomas. Systemic inflammatory response syndrome (SIRS) often occurs with hemoglobinuria, and its underlying pathophysiological mechanism is unknown. Heme can trigger inflammation by inducing the generation of reactive oxygen species (ROS) and the production of inflammatory mediators. We therefore investigated whether circulating heme is involved in SIRS following RFA of hepatic hemangiomas.
Methods We enrolled 65 patients with hepatic hemangioma who underwent RFA. Serum concentrations of free heme, ROS, and tumor necrosis factor α (TNF-α) were measured after RFA. Conclusions Circulating heme was associated with the induction of ROS and the production of TNF-α, which may contribute to the induction of SIRS following RFA of hepatic hemangiomas.

Background
Hepatic hemangiomas are the most common benign tumors of the liver, with an incidence ranging from 0.4% to 20% among the general population [1,2]. Fortunately, most lesions are small, asymptomatic, and do not require surgical intervention [2]. However, giant hemangiomas (diameters ≥5 cm) pose a higher risk of rupture, which presents with uncontrollable abdominal pain. The size of such lesions may increase during follow-up, potentially requiring radical intervention [3,4].
Radiofrequency ablation (RFA) is increasingly used for managing hepatic hemangiomas because of its unique advantages compared with other therapies. The advantages include minimal invasiveness, low cost, low incidence of complications, short duration of hospitalization, and increased patient compliance [5][6][7]. However, the nearly unavoidable hemolysis after RFA, attributable to the generous blood supply of hepatic hemangiomas, is a major disadvantage. Moreover, the incidence of systemic inflammatory response syndrome (SIRS) is high among patients who experience hemoglobinuria after RFA and may be accompanied by acute respiratory distress syndrome and severe myocardial dysfunction [7,8]. Unfortunately, the underlying pathophysiological mechanism is unknown. Intravascular hemolysis, or the destruction of red blood cells (RBCs) in the circulation, can occur in numerous diseases, including the acquired hemolytic anemias, sickle cell disease, and thalassemia, as well as during transfusion reactions, pre-eclampsia, and infections. During hemolysis, heme derived from hemoglobin (Hb) accumulates because of the inability of detoxification systems to sufficiently scavenge it [9]. Evidence indicates that Hb-derived heme may serve as an endogenous danger signal, triggering inflammation whenever red blood cells (RBCs) are destroyed, and Hb is released into extracellular compartments [10,11].
Free heme mediates diverse pathological effects, including increased production of reactive oxygen species (ROS) and inflammatory mediators such as TNF-as well as upregulation of the production of endothelial cell adhesion molecules [12][13][14][15][16]. These factors can lead to inflammation in sterile and infectious conditions, contributing to the pathogenesis of hemolytic diseases, subarachnoid hemorrhage, malaria, and sepsis [12,13]. However, we are unaware of studies that address whether heme contributes to the incidence of SIRS after patients undergo RFA to treat hepatic hemangioma.
The goal of the present study is to answer this question.

Materials And Methods
Patients and blood sample collection From January 2016 to December 2018, 65 patients with hepatic hemangiomas underwent RFA at our institution. The inclusion criteria for RFA are described in our previous study [17]. These patients did not experience significant heart, lung, liver, kidney abnormities, or other serious concomitant diseases before undergoing RFA.
RFA was performed using internally cooled cluster electrodes, Cool-tip ACTC 2025 (for laparoscopic procedures) or ACTC 1525 (for CT-guided percutaneous procedures) electrodes, and an RF generator (Covidien Healthcare, Dublin, Ireland).
Blood cell count, C-reactive protein (CRP), urine analysis, and routine biochemical tests were used to evaluate liver and renal function before RFA and 1 h and 1-3 days after RFA. Chemical analysis of Hb and flow cytometry were used to detect RBCs. Hemoglobinuria was diagnosed if the Hb and RBC tests were positive and negative, respectively [18]. Wine-colored hemoglobinuria can be visually observed, and deep-yellow hemoglobinuria can only be detected using routine urine analysis. Blood samples were collected in heparinized tubes before RFA and 1 h and 1-3 days after RFA. After sampling, the serum was prepared using centrifugation, divided into aliquots, and stored at -70 ℃ for testing serum concentrations of heme, ROS, and TNF-α.
All patients granted written informed consent before treatment. The Research and Ethics Committee of Beijing Chao-yang Hospital, Capital Medical University approved this study, which was conducted in accordance with the standards of the Declaration of Helsinki.

Ablated volume of a hemangioma
The ablated volume of a hemangioma, which was considered equivalent to the volume of the hemangioma before RFA, was determined according to the results of contrast-enhanced computed tomography (CT) or magnetic resonance imaging (MRI) before RFA to correlate the ablated volume with SIRS. Lesion volumes were calculated as follows: volume = X × Y × Z × π/6, where X, Y, and Z are the maximum diameters (supine vertical, sagittal, and coronal planes) of the tumor measured using CT or MRI [20].

Histology of Excised Hemangiomas
Hemangioma tissues were laparoscopically resected after RFA [21]. The tissues in the region of and "adjacent ablated hepatic hemangioma," located < 1.0 cm from the ablation tissues were collected.

Measurements of heme, ROS and TNF-α
We passed serum samples through a Microcon YM-3 column (Millipore, Solarbio, Beijing, China) (60 min at 14 ℃, 21000g) to remove proteins >3 kDa. We quantified free heme in these protein-depleted sera using a chromogenic assay in accordance with the manufacturer's instructions (

Statistical analysis
The values of continuous variables are presented as the mean ± standard deviation (SD) or median values with interquartile ranges, and those of categorical variables are presented as percentages.
Categorical data were compared using the Chi-square test or Fisher's exact test, and continuous data were compared using the Student t test or the Wilcoxon rank-sum test. Pearson's correlation coefficient was determined to assess the significance of correlations between variables. Univariate and multivariate logistic regression analyses were conducted with the diagnosis of SIRS as a dependent variable. Only parameters that were significantly associated with SIRS identified by univariate logistic analysis were included in the multivariate logistic regression model, and P < 0.05 indicates a significant difference. Statistical analysis was performed using SPSS version 17.0 for Windows (SPSS, Chicago, IL, USA). All descriptive graphs were generated using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, USA).

Results
Patients' characteristics RFA was successful for all patients. Of 65 patients, 59 (90.8%) developed hemoglobinuria shortly after RFA. Subsequently, the color of urine, or that determined using routine urine analysis, recovered gradually after reaching baseline values and hydration treatment in 1-2 days. Major complications caused by acute kidney injury occurred. Of 59 patients with hemoglobinuria, 25 (42%) patients experienced SIRS. Fluid replacement, dieresis, and glucocorticoids were administered, and 24 (96.0%) patients recovered within 3-4 days. One patient developed severe myocardial dysfunction. Chest Xray showed the butterfly sign of alveolar edema with cardiomegaly. Therapy was immediately initiated, including high-flow oxygen inhalation delivered through a face mask, intravenous (IV) diuretics, IV methylprednisolone, and short-term antibiotic treatment, after which clinical symptoms gradually disappeared, and heart and lung function slowly improved. This patient was discharged at 9 days after RFA. Long-term sequelae were not observed in patients during the 6-month follow-up.
All patients were classified according to the diagnostic criteria stated above into the SIRS group or Non-SIRS group. The patients' clinical parameters are summarized in Table 1.

Hemolysis in adjacent ablated hepatic hemangiomas
In hepatic hemangioma tissue, blood vessels were characterized by spongy hyperplasia, endothelial cells were arranged along the vascular wall, and the RBCs in the lumen were normal (HE, 200×) ( Figure 1A). The "adjacent ablated hepatic hemangioma" tissue did not exhibit coagulative necrosis, the endothelial cells were disordered along the wall of the blood vessels, and partial burning and deformation of blood cells in the were accompanied by their destruction in the lumen (HE, 200×) ( Figure 1B).

Changes of heme, ROS and TNF-α in patients with hemangioma
In the SIRS group, increased concentrations of serum heme (7.72 ± 0.61 μM) were detected 1 h after ablation compared with preoperative concentrations (0.49 ± 0.31 μM) and slowly decreased from the day after RFA. The peak value occurred 1 h after RFA. In the non-SIRS group, heme concentrations after RFA were slightly higher compared with those before RFA, but the difference was not significant ( Figure.  were slightly higher (20.92 ± 3.42 μM) after RFA, although the difference was not significant ( Figure   2B).

Discussion
Traditionally, surgical resection and surgical enucleation are the most frequently used treatments of choice for hepatic hemangiomas. However, it is important to consider that many of the trade-offs encountered during the treatment of cancer are not applicable to hepatic hemangiomas because of their benign natural history. Thus, a highly effective but morbid treatment would represent a poor choice for most patients. RFA is safe, well tolerated, and effective for treating most patients with hepatic hemangiomas [4][5][6]. However, the main disadvantage of RFA is the nearly unavoidable hemolysis attributable to the generous blood supply of a hepatic hemangioma [17]. For example, depending on the severity of hemolysis, hemoglobinuria, hemolytic jaundice, anemia, or renal damage can occur [7,8]. Further, adverse events associated with SIRS often occur in patients with hemoglobinuria after they undergo RFA to treat hepatic hemangioma, particularly for hemangiomas ≥10 cm. Whether hemolysis and SIRS are pathologically associated is unknown. Here we discovered an important contribution of heme to RFA-induced SIRS.
Heme is a ubiquitous molecular complex of iron and the tetrapyrrole protoporphyrin IX [12,13]. When bound to hemoproteins, heme plays an essential role in numerous biological processes in aerobic organisms, such as from reversible binding of oxygen to electron transport molecules of the respiratory chain [12]. After hemolysis, extracellular Hb is readily oxidized by ferrous Hb to yield ferric Hb (methemoglobin), which readily releases free heme. Evidence supports the conclusion that inflammatory mechanisms driven by heme may play a fundamental role in the pathophysiology of hemolytic diseases [16,22]. For example, heme is directly cytotoxic and can activate specific receptors and signaling pathways to promote the generation of ROS and induce inflammation and programed cell death [9,16,23]. Studies using models of infectious and noninfectious diseases show that heme can induce monocytes and macrophages to secrete TNF-α through TLR4-mediated signaling, which induces inflammation [14,15,24]. These results highlight the great potential importance of studies of the molecular mechanisms of heme-induced inflammation and cell death aimed on identifying new therapeutic targets.
In the present study, pathology verified that RFA of hepatic hemangiomas led to massive destruction of RBCs and intravascular hemolysis. Specifically, 59 patients developed hemoglobinuria, among which 25 experienced SIRS. We detected significant increases in the concentrations of circulating heme, ROS, and TNF-α in the SIRS group 1 h after ablation, which decreased to pre-RAF concentrations after 3 days. Further, the concentrations of circulating heme were significantly associated with those of ROS and TNF-α. Multivariate analysis identified baseline patients' characteristics 1 h after undergoing RFA that were associated with the risk of SIRS such as sex, age, concomitant illness, volume of a hemangioma, distribution of the lesion, RFA, time of RFA, hemoglobinuria, and the concentrations of ALT, AST, total bilirubin, BUN, creatinine, CRP, heme, ROS, and TNF-α. Multivariate analysis showed that the volume of a hemangioma, time of RFA, heme, ROS, and TNF-α were independent risk factors of SIRS. We thus raise the possibility that the release of heme from damaged RBCs caused by RFA induces SIRS, which represents a novel mechanism of RFArelated SIRS.
However, there are limitations to the present study. First, the sample size was relatively small, which decreased statistical power. Second, cell culture and animals model of hepatic hemangioma are required to mimic clinical RFA. Third, our results are from a single center, which may have introduced selection bias that may explain our results.

Conclusions
We found that damage to RBCs caused by RFA released extracellular heme to the peripheral circulation, which induced the production of ROS and inflammatory cytokines that may contribute to SIRS after RFA of hepatic hemangiomas. Recognition of the inflammatory burden of hemolytic processes during this treatment will likely serve as a foundation for developing new approaches for use in combination with established therapies aimed at treating hemolytic diseases, with the goal of

Consent for publication
Not applicable

Availability of data and materials
The data used and analyzed during the current study are included in this published article, and are also available from the corresponding author on reasonable request. Technical appendix, statistical code, and dataset are also available from the corresponding author on reasonable request. data angalysis; JG revised the manuscript. All authors read and approved the final manuscript.