Angiopoietin‐2 and hemocompatibility‐related adverse events during percutaneous left ventricular assist device supports

Despite considerable improvement in survival in heart failure patients receiving left ventricular assist devices (LVADs), hemocompatibilityrelated adverse events (HRAEs) including bleeding and thromboembolic evens remain as unsolved issues. Inappropriate activation of the inflammatory and angiogenesis cascade including angiopoietin-2 (Ang-2) seems to have a considerable association with the development of arteriovenous malformation and gastrointestinal bleeding during LVAD supports. Ang-2 is considerably associated with the plasma B-type natriuretic peptide (BNP) levels in the heart failure cohort. Furthermore, Ang-2 is inappropriately elevated relative to BNP levels in some LVAD patients. However, its prognostic impact on HRAEs remains uninvestigated. In this study, we investigated the prognostic impact of Ang-2 level relative to BNP (Ang-2/BNP) obtained immediately after percutaneous LVAD implantation on future occurrences of HRAE.


| INTRODUCTION
Despite considerable improvement in survival in heart failure patients receiving left ventricular assist devices (LVADs), hemocompatibilityrelated adverse events (HRAEs) including bleeding and thromboembolic evens remain as unsolved issues. 1,2 Inappropriate activation of the inflammatory and angiogenesis cascade including angiopoietin-2 (Ang-2) seems to have a considerable association with the development of arteriovenous malformation and gastrointestinal bleeding during LVAD supports. 3 Ang-2 is considerably associated with the plasma B-type natriuretic peptide (BNP) levels in the heart failure cohort. Furthermore, Ang-2 is inappropriately elevated relative to BNP levels in some LVAD patients. 4 However, its prognostic impact on HRAEs remains uninvestigated. In this study, we investigated the prognostic impact of Ang-2 level relative to BNP (Ang-2/BNP) obtained immediately after percutaneous LVAD implantation on future occurrences of HRAE.

| Patient selection
In this prospective study, consecutive patients who received percutaneous LVAD between August 2018 and February 2019 were included. Indication of percutaneous LVAD therapy is determined by the attending cardiologists. In brief, candidates had cardiogenic shock refractory to guideline-directed medical therapy. All participants gave informed consents, and the institutional ethical review board approved this study beforehand. We affirm that this manuscript is an honest, accurate, and transparent account of the study being reported, that no important aspects of the study have been omitted, and that any discrepancies from the study as planned.
The original data are available when required and considered to be appropriate.

| Variables collection
In addition to the baseline characteristics data, Ang-2/BNP was measured from patients' plasma within 3 days following LVAD implantation, using Human Angiopoietin-2 Quantikine ELISA Kit.

| Outcomes
A primary endpoint was set as any occurrence of HRAEs during LVAD supports. HRAEs consist of gastrointestinal bleeding, symptomatic stroke with image findings, and device thrombosis medically or surgically managed, according to the INTERMACS definition. 2 Death, device explantation, or 30-day follow-up was censored.

| Statistical analyses
Statistical analyses were performed using SPSS Statistics 22 (SPSS Inc, Armonk, Illinois). Continuous variables were expressed as median and interquartile. Teruhiko Imamura and Makiko Nakamura contributed equally to this work.
The impact of Ang-2/BNP on HRAE was investigated as a primary concern. Receiver operating characteristics analysis was performed to calculate a cutoff of Ang-2/BNP for the occurrence of HRAE. Kaplan-Meier analyses and log-rank tests were performed to compare freedom from HRAE between high Ang-2/BNP and low Ang-2/BNP groups. Cox proportional hazard ratio regression analyses were performed to investigate the impact of high Ang-2/BNP on the occurrence of HRAE by adjusting for age, which is another well-known risk factor of HRAE.

| Ang-2 and BNP
The inflammatory system has a considerable association with advanced heart failure: Ang-2 and BNP have a strong correlation in the heart failure cohort. 5 Our team recently demonstrated that Ang-2 was inappropriately elevated relative to the BNP level during LVAD supports compared to the heart failure cohort, 4 probably due to the stimulation of Ang-2 activity via hematological instability. 6 This is a rationale of why we used Ang-2/BNP as a variable instead of Ang-2 alone. The detailed mechanism of the variety of Ang-2/BNP levels among each individual remains unknown.

| Ang-2/BNP and HRAE
The association between Ang-2 and gastrointestinal bleeding during LVAD supports is receiving great concern. Inflammatory and angiogenesis cascade including Ang-2 might stimulate a formation of arteriovenous malformation and increase the risk of gastrointestinal bleeding. 3 In this study, we demonstrated that the inappropriately elevated Ang-2 level predicted future bleedings. In other words, Ang-2 is inappropriately activated immediately following LVAD implantation in such high-risk patients.
Elevated Ang-2/BNP level was associated with also the occurrence of stroke with high specificity. The detailed mechanism requires further investigations, but our team previously hypothesized the association among right ventricular failure, chronic inflammation, and stroke during LVAD supports. 7 As a major marker of

| Limitations and future concerns
We should state that the study is a proof of concept, and the finding should be validated in larger scale studies. We adjusted for age alone, given its considerable impact on HRAE and small sample size, 2 and we cannot deny any other confounders. We showed an association between Ang-2/BNP and HRAEs, but the causality remains unknown with a lack of detailed data explaining it. We observed just for 30 days, given that the devices were percutaneous ones, and the applicability of our findings to other durable LVADs remains uncertain.
Nevertheless, our findings would give us a clue to risk-stratify patients for the future occurrence of HRAE during LVAD supports.
Furthermore, any therapeutic intervention to improve the elevated Ang-2, including omega-3 fatty acid or any other more specific agents, 8 might reduce the risk of HRAE.

| CONCLUSION
Elevated Ang-2 level soon after percutaneous LVAD implantation was associated with future HRAEs. The clinical implication to intervene in Ang-2 would be a future concern.
FUNDING Teruhiko Imamura receives grant support from JSPS KAKENHI: JP20K17143, which had no involvements in conducting the study and preparing the draft.

AUTHOR CONTRIBUTIONS
Conceptualization: Teruhiko Imamura Teruhiko Imamura has full access to all of the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis.

TRANSPARENCY STATEMENT
Teruhiko Imamura affirms that this manuscript is an honest, accurate, and transparent account of the study being reported, that no important aspects of the study have been omitted, and that any discrepancies from the study as planned have been explained.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.