The expression of myeloperoxidase in thrombi is associated with reduced heme oxygenase‐1 induction and worse left ventricular remodeling in patients with acute ST‐elevation myocardial infarction

Abstract Background Myeloperoxidase (MPO) secreted by neutrophils is the enzyme that kills bacteria and other pathogens. Acute myocardial infarction (AMI) is usually caused by thrombosis in response to vulnerable plaque rupture. Circulating MPO was found to be associated with increased mortality in AMI patients. However, the relationship between MPO in thrombi and the prognosis of AMI patients remains unknown. Hypothesis MPO expression in thrombi is associated with the prognosis of patients who underwent primary percutaneous coronary intervention (PCI) after AMI. Methods This study included 41 consecutive patients with acute ST‐elevation myocardial infarction, who successfully underwent primary PCI, during which we collected thrombi remaining in the culprit artery using aspiration catheters. These thrombus samples were fixed, and immunohistochemical staining against MPO and heme oxygenase‐1 (HO‐1) was conducted. Enrolled patients were divided into two groups based on the induction of thrombotic MPO, which was quantified using Image J software. Methods We observed that increased MPO was associated with lower left ventricular ejection fraction (LVEF) and worse LV remodeling in AMI patients. Instead, patients with decreased thrombotic MPO induction had a considerable improvement in LVEF 1 month after discharge (54.4 ± 2.0% vs. 61.1 ± 2.3%, p < 0.01). In the MPO group, a reduction in the thrombotic HO‐1 level contributed to the development of adverse LV remodeling. Logistic regression showed that MPO was a considerable risk factor for adverse LV remodeling (adjusted OR 3.70, p < 0.05). Conclusion MPO expression in thrombi is associated with reduced LVEF and deteriorated LV remodeling in AMI patients, which may be due to HO‐1 suppression in thrombi.


| INTRODUCTION
Acute ST-elevation myocardial infarction (STEMI) remains one of the leading causes of cardiovascular death worldwide. Although the trends of cardiac mortality in developed countries have dropped in recent years, AMI is still considered the second killer in adults in China. To date, we already know that the pathological nature of AMI is caused by thrombi that block the coronary arteries. Subsequent ischaemia and hypoxia in the heart lead to symptomatic chest pain and cardiac malfunction if revascularisation is not applied. A previous study revealed that a high thrombus burden in culprit arteries leads to more complications and worse outcomes. 1 Patients with high thrombus burden are likely to have more major cardiac events and in-stent restenosis after primary stenting. 2 Myeloperoxidase (MPO) secreted by inflammatory cells, such as neutrophils, is an enzyme that kills bacteria and other pathogens in the body. It is produced in the granules of neutrophils and is a hemebinding protein, which acts as a subfamily of peroxidases and produces hypochlorous acid (HOCl) from hydrogen peroxide (H 2 O 2 ) and chloride anion (Cl − ) during the neutrophil respiratory burst. 3 HOCl is cytotoxic and kills bacteria and other pathogens when infections occur. Rashid et al. demonstrated that MPO is a potential molecular imaging and therapeutic target for the detection of high-risk atherosclerotic plaque in mice. 4 A study also reported that circulating MPO is associated with an increase in mortality in patients with AMI, 5 possibly, because the HOCl produced by MPO could promote endothelial dysfunction, apoptosis, and vascular thrombosis. 6 In addition, MPO locally contributes to thrombogenic circumstances by enhancing endothelial cells, which release tissue factors, acting as the initial factor of the coagulation cascade. However, when thrombus forms, neutrophils are locked up with activated platelets, red blood cells, and fibrin. Thus, the function of MPO in thrombus, when AMI occurs, remains unclear. Herein, we hypothesize that the expression of MPO in thrombi is associated with prognosis in patients who underwent primary percutaneous coronary intervention (PCI) after AMI.

| Follow-up after primary PCI
All enrolled patients took prescribed standard medications (including dual antiplatelet, angiotensin-converting-enzyme inhibitors, and beta-blockers) after discharge. We performed follow-up echocardiography

| RESULTS
We successfully conducted MPO and HO-1 immunochemical staining and found that all subjects were positive in MPO and HO-1 staining ( Figure S1). The enrolled patients were divided into two groups  2193.0 ± 582.1) and higher incidence of single-vessel diseases (52.6% vs. 36.4%) than their counterparts. Finally, in-hospital echo test was performed in both groups. The results showed no differences in LVEF, LVDED, LVSED, LVDEV, or LVSED between the two groups (Table 1).
Two deaths were recorded (one patient died from cardiogenic shock before discharge; the other died from major cardiovascular events at 2 months after discharge).
During follow-up, we performed 100% echo test at 1 month and 85% echo test at 3 months in all enrolled patients ( remained not significantly different between the two groups. Moreover, to reveal LV remodeling after MI in these patients, we performed a statistical analysis of these cardiac parameters in each group ( Figure 1(A)). Interestingly, in the MPO-group, we found a significant increase in LVEF 1 month after discharge (61.1 ± 2.3 vs. 54.4 ± 2.0, p < 0.01). This improvement in LVEF was caused by a slight increase in LVDEV and a decrease in LVESD. Nevertheless, in the MPO+ group, LVEF remained unchanged 1 month after the procedure and decreased at 3 months of follow-up (Figure 1(B)), which may be due to considerable increases in LVEDV and LVESD, indicating LV enlargement and dysfunction.
Immunostaining revealed a higher level of HO-1 in thrombi collected from the MPO-group than from the MPO+ group (Figure 2(A)).
Furthermore, we measured the levels of circulating total bilirubin, which is a product of heme degradation catalyzed by HO-1. However, we did not find any differences in terms of the levels of circulating  (Figure 2(B)).

In-hospital Follow-up
In  LV remodeling is commonly observed in patients with AMI. 14 Parameters that evaluate LV remodeling by echo are powerful predictors of prognosis for these patients. In these parameters, LVESV was reported as the major determinant of survival after recovery from myocardial infarction. 15 LV adverse remodeling, normally determined as an increase in LVEDV, is the main cause of heart failure after an AMI with ST-segment elevation. Reindl et al. revealed that following primary PCI, an increase of 10% of LVEDV appeared to be associated with the occurrence of major adverse cardiovascular events, including all-cause death, re-infarction, stroke, and new-onset congestive heart failure. 14 Inflammation is a key component of tissue healing. Clinicians measured inflammatory markers, for example, interleukin (IL)-1β, before PCI, 2 days after PCI, and 7 days after PCI and found that IL-1β levels predicted LVESV index and LVEDV index tested by cardiovascular magnetic resonance at 1 year. 16 In our cohort, due to the small sample size, we did not observe a significant incidence in major adverse cardiovascular events between the two groups during the follow-up period. Nevertheless, in the MPO+ group, patients had increased LVEDV and decreased LVEF, which was consistent with previous findings. This evidence suggests that even if primary PCI was performed in both groups, the inflammatory levels indicated by thrombotic MPO played an important role in affecting the development of adverse LV remodeling. As a result, further understanding of the mechanistic insights of MPO in AMI appears to be urgent.
HO-1 is one of the main protein enzymes that degrade heme in the body. 17 A previous pathological study revealed increased HO-1 levels in atherosclerotic plaques. In a clinical study, Novo et al.
showed that increased HO-1 levels led to reduced severity of coronary artery diseases in patients with STEMI. 18 As such, HO-1 is considered as a protective enzyme against oxidative stress that causes cardiovascular diseases. 19 Additionally, aged and ovariectomised ani-

| STUDY LIMITATIONS
In this study, the number of enrolled patients was very limited. This is because, according to the European society of Cardiology Guidelines for the management of AMI, thrombotic aspiration is only recommended for patients with high thrombus burden. In our hospital, we did not conduct thrombectomy in every patient who underwent primary PCI, and we therefore strongly considered this work as a pilot study. However, before starting this study, we had carefully performed statistical power calculations using average values and found that the current number of enrolled patients had a 100% statistical power. Further clinical investigation with a larger sample size must provide solid evidence in the future. In addition, follow-up was completed at 3 months; regardless of our finding that thrombotic MPO played an important role in the development of adverse LV remodeling to some extent, we failed to observe the long-term effect of thrombotic MPO on cardiovascular death or major cardiac events. A longer follow-up is currently on-going, and future data could help us further clarify the true functions of MPO in thrombus.

| CONCLUSIONS
Our findings strongly support that the inflammation indicated by MPO may suppress HO-1 levels and worsen the recovery of cardiac functions in patients with AMI. We conclude that the expression of MPO in thrombi is associated with reduced HO-1 induction and worse LV remodeling in patients with acute STEMI.

ACKNOWLEDGMENT
We would like to thank Dr Jinwei Ni, Dr Xiaoxiang Yan and Dr Zhenbin Zhu for sample collection, thank Professor Weifeng Shen for suggestions on manuscript modification and thank National Natural Science Foundation of China for funding.

CONFLICT OF INTEREST
None.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.