Clinical role of serum histone deacetylase 4 measurement in acute ischemic stroke: Relation to disease risk, severity, and prognosis

Abstract Objective Histone deacetylase 4 (HDAC4) is engaged in the pathophysiology of acute ischemic stroke (AIS) through modulating atherosclerosis, inflammation and neurocyte death. This study aimed to investigate the clinical role of HDAC4 in AIS. Methods Serum samples were collected from 176 AIS patients and 80 controls for HDAC4 detection by enzyme‐linked immunosorbent assay (ELISA). In AIS patients, disease severity was assessed by National Institute of Health Stroke Scale (NIHSS) score and their recurrence‐free survival (RFS) and overall survival (OS) were calculated, inflammatory cytokines and adhesion molecules were detected by ELISA. Results HDAC4 was declined in AIS patients vs. controls (p < 0.001), it also had certain ability of distinguishing AIS patients from controls with an area under curve of 0.748 (95% confidence interval: 0.689–0.806). Among AIS patients, HDAC4 was negatively linked with NIHSS score (p < 0.001) but no other clinical features (all p > 0.05). Moreover, HDAC4 was negatively related to interleukin (IL)‐17 (p = 0.010) and tumor necrosis factor alpha (p = 0.001), while it was not correlated with IL‐1β (p = 0.081) or IL‐6 (p = 0.074). Furthermore, HDAC4 was negatively associated with intercellular cell adhesion molecule‐1 (p < 0.001) and vascular cell adhesion molecule‐1 (p = 0.003). During a median follow‐up of 19.0 months, 17 (9.7%) patients had recurrence and 10 (5.7%) patients died. Additionally, high HDAC4 was linked with prolonged RFS (p = 0.044) but not OS (p = 0.079). Conclusion HDAC4 possesses the potential to monitor disease risk, inflammation and estimate recurrence of AIS, while further study with larger scale is needed to verify our findings.


| INTRODUC TI ON
Acute ischemic stroke (AIS) is viewed as one of main causes of neurologic death worldwide. 1,2 In China, stroke is the third highest cause of death with mortality of approximately 1.5 million in 2018, 3 which AIS may result in permanent disability, both functionally and cognitively; meanwhile, the morbidity of AIS is continuous increasing, which causes a huge economical and humanistic burden. 4,5 Over the decades, great advances have made in the strategies for AIS management such as neuroprotective treatment, intravenous thrombolysis, and anticoagulation treatment, while AIS patients still suffer from elevating recurrence and increasing mortality. [6][7][8] In view of that there still exists several challenges in the management of AIS, the identification of reliable biomarker to monitor disease risk, severity, and prognosis is crucial.
Histone deacetylase 4 (HDAC4), a crucial number of epigenetic modifier enzymes, is closely involved in various cellular processes such as apoptosis, senescence, and differentiation. 9 Of note, accumulating researches have reported that HDAC4 is tightly involved in the pathophysiology of AIS. [10][11][12][13][14] For instance, HDAC4 is engaged in progression of atherosclerosis via regulating endothelial cell apoptosis 10 ; moreover, it has been proposed that HDAC4 is able to regulate vascular inflammation 11 ; furthermore, HDAC4 also inhibits neuron cell death in oxygen-glucose deprivation (OGD)-treated mice neurons, indicating its potential of neuroprotection 12 ; in addition, HDAC4 is able to inhibit adhesion molecule (VCAM-1), which is involved in the all stage of atherosclerosis, an important risk factor in pathology of AIS. 15,16 Based on the above-mentioned data, it could be deduced that HDAC4 might play an important clinical role in AIS, while related information is scarce.
Thus, the current study explored the association of HDAC4 with disease risk, disease severity, inflammation, adhesion molecules, and prognosis in AIS patients.

| Participants
This study serially enrolled 176 first-episode AIS patients between June 2017 and June 2021. The patients with the following criteria were included in the study: (i) confirmed as AIS according to the American Stroke Association Guideline 17 ; (ii) more than 18 years old; (iii) without intracranial hemorrhage based on the images of computed tomography (CT) scan or magnetic resonance angiography (MRA); (iv) volunteered to participate in the study and be followed up regularly.
The patients who met the following conditions were excluded from the study: (i) unwilling to provide peripheral blood (PB) samples for study use; (ii) presented with severe infection; (iii) had a prior history of immune system disease (to eliminate the impact on the assessment of inflammatory cytokines), cancer or malignancy; (iv) pregnancy or nursing mother. In addition, this study also recruited 80 subjects from high-risk population as controls, who had at least two high-risk factors of stroke, including history of smoke, hypertension, hyperlipidemia, hyperuricemia, diabetes mellitus (DM), and chronic kidney disease (CKD). In order to match the demographics of controls with the included AIS patients, the recruitment of controls was started from June 2020 to June 2021. Referring to the age range and gender distribution in the AIS cohort, the age of controls was required to limited in the range of 50-80 years old, and the gender ratio of controls was limited as 3:2 (male vs. female). The controls were ineligible if they met the exclusion criteria for AIS patients or had a history of stroke. The study was admitted by the Ethics Committee. All participants or their statutory guardians signed the informed consents.

| Determination of HDAC4, inflammatory cytokines and adhesion molecules
Within half an hour of after collection, PB samples were centrifuged using centrifuge at 2500 g revolutions per minute for 10 min to isolate serum samples, and were stored at −80℃ for further detec- RFS was defined as the time interval from admission to disease recurrence or patient's death, and OS was defined as the time interval from admission to patient's death.

| Statistical analysis
Statistical analysis and graph construction were, respectively, completed using SPSS V.24.0 (IBM Corp., Armonk, New York, USA) and GraphPad Prism V.6.01 (GraphPad Software Inc., San Diego, CA, USA). Difference of HDAC4 expression between AIS patients and controls were compared using the Mann-Whitney U-test, and receiver operating characteristic (ROC) curve was applied to estimate the ability of HDAC4 expression in identifying different subjects.
Correlation of two variables was assessed using the Mann-Whitney U-test or Spearman's rank correlation test. RFS and OS were described using Kaplan-Meier curves and determined by log-rank test.
p-value < 0.05 was considered statistically significant.

| Clinical features in AIS patients and controls
Among 80 controls, the mean age was 63.

| The association of HDAC4 with prognosis among AIS patients
During a median follow-up of 19.0 months, disease recurred in 17 (9.7%) patients, and 10 (5.7%) patients died. Furthermore, AIS patients were classified as patients with HDAC4 high and patients with HDAC4 low based on the median level of HDAC4 in AIS patients (26.1 pg/ml).
In addition, 1-year and 3-year OS rates were 98.6% and 93.9%, respectively, in patients with HDAC4 high, and they were 97.5% and 82.4%, accordingly in patients with HDAC4 low; moreover, HDAC4 high expression was not correlated with OS (p = 0.079) ( Figure 5B).

| DISCUSS ION
It has been presented that HDAC4 is declined in ODG-treated PC12 cells and ischemic stroke model mice. 18  Until now, recurrence is viewed as a huge challenge for AIS, which causes prolonged hospitalization and elevated mortality. 25 Therefore, the exploration of potential biomarker to predict recurrence is urgent among AIS patients. Surprisingly, in the current study, we found that elevated HDAC4 was correlated with higher RFS but not OS. The possible explanations might be that: (1) elevated HDAC4 could inhibit the disease severity and progression of AIS through suppressing inflammation and restraining the development of atherosclerosis (above-mentioned), which led to better RFS; (2) the events of mortality was relatively low in the current study, thus it might be difficult to observe statistical significance in the correlation between HDAC4 and OS.
There were several limitations in the current study: (1) we did not have a tailed protocol for current study to conduct a regular and stable follow-up, and the follow-up in our study was based on routine schedule and the intention of patients, which might result in high number of patients who lost follow-up; hence, the patients who lost follow-up were treated as censored data (2) the role of HDAC4 in regulatory mechanism of AIS could be explored; (3) the enrolled patients were first-episode AIS patients, hence the clinical role of HDAC4 in recurrent AIS could be further explored; (4) HDAC4 participated in stroke-induced angiogenesis, which was a protective process after stroke 14 ; thus, the correlation of HDAC4 with markers of angiogenesis, such as VEGF, HIF-1 alpha could be explored in the further study; (5) a huge number of patients lost follow-up in the current study; (6) lacking of transcriptive level of HDAC4 detection in the present study.
To be conclusive, HDAC4 possesses the potential to monitor disease risk, inflammation and estimate recurrence of AIS, while further study with larger scale is needed to verify our findings.

CO N FLI C T O F I NTE R E S T
The authors declare they have no conflict of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.