HMGB1 as a biomarker for myeloproliferative neoplasm complicated with atherosclerosis

Abstract

Objective

The study analyzed the relationship between serum levels of HMGB1 and biochemical indices related to atherosclerosis, clarifying the diagnostic value of HMGB1 in cases of myeloproliferative neoplasm (MPN) accompanied by atherosclerosis (AS).

Methods

The bone marrow samples and serum were obtained from patients with MPN. qPCR was used to detect the level of HMGN1 mRNA in bone marrow mononuclear cells, while ELISA was used to measure the level of HMGB1 in serum. Additionally, relevant biochemical indices of the patients were collected and the correlation between these indices and HMGB1 levels was assessed.

Results

Compared with the control group, MPN patients exhibited significantly elevated levels of HMGB1 in both bone marrow mononuclear cells and serum. The Pearson correlation analysis revealed a negative correlation between serum HMGB1 levels and both HDL-C and ApoA1. The ROC curve analysis demonstrated that serum HMGB1 had an AUC of 0.929 (P < 0.001) for predicting the complication of AS in MPN patients, with a sensitivity of 100.00% and a specificity of 78.05%. Furthermore, logistic regression analysis showed that serum HMGB1 levels were statistically significant in determining the presence of AS in MPN patients (P < 0.05).

Conclusion

Elevated levels of HMGB1 in the bone marrow and serum of MPN patients demonstrate a correlation with biochemical indices related to AS. This finding may serve as a valuable reference for predicting AS complications in MPN patients.

Introduction

Myeloproliferative neoplasm (MPN) represents a class of clonal, chronic proliferative diseases that arise from hematopoietic stem cells in the bone marrow. These diseases are characterized primarily by the unlimited clonal proliferation of one or more series of granulocytes, erythrocytes, and megakaryocytes in the bone marrow. The peripheral blood shows a significant increase in the levels of leukocytes, erythrocytes, or platelets, accompanied by enlargement of the liver, spleen, or lymph nodes. Additionally, there is a risk of progression to acute leukemia [1]. A key pathogenic mechanism underlying MPN involves mutations in driver genes, including JAK2, MPL, and CALR, which trigger the activation of the JAK-STAT pathway. This, in turn, initiates the inflammation pathway, resulting in chronic inflammation [2].

HMGB1, also known as high mobility group protein 1, is a chromatin-associated protein that derives its name from its remarkable ability to migrate rapidly in polyacrylamide gel electrophoresis with no signs of aggregation. Within the cell nucleus, HMGB1 binds to chromatin and can shuttle from the nucleus to the cytoplasm under various stress conditions. Subsequently, it can be released into the extracellular environment, where it plays a pivotal role in mediating inflammatory and immune responses [3]. In the nucleus, HMGB1 is a DNA companion that maintains chromosome structure and function [4]. In the cytoplasm, HMGB1 promotes autophagy by interacting with the Beclin1 protein [5]. Studies have demonstrated that the expression of HMGB1 is related to sepsis, malignant tumors, as well as autoimmune diseases, and its level may reflect the severity of inflammation and tissue injury [6,7,8].

As an essential cytokine in inflammation and immunity, HMGB1 has been proven to act as a core mediator in the pathogenesis of atherosclerosis (AS), with its release triggering significant inflammatory responses driven by endothelial cells and infiltrating immune cells [9]. In addition, HMGB1 can also mediate plaque formation by stimulating macrophage migration and is a key factor in the development and instability of atherosclerotic plaques [10]. However, it remains unclear whether HMGB1 can serve as a risk warning factor for MPN patients with concurrent AS.

Therefore, this study focuses on MPN patients as the study subjects, aiming to investigate the expression levels of HMGB1 and its correlation with biochemical indices linked to AS. By doing so, the study seeks to elucidate the role and diagnostic value of HMGB1 in MPN complicated with AS.

Materials and methods

Sample collection

This was a retrospective observational study. 52 patients diagnosed with MPN from 2023 to 2024 were included, with 30 IDA patients as the control group. The age and sex of patients across the different groups were similar. The diagnosis of MPNs is based on the WHO criteria. All patients exhibited mutations in JAK2, MPL, or CALR genes and demonstrated an increase in one or more blood cell lineages in their peripheral blood. Notably, no patient had received prior treatment before sample collection. All participants were recruited from the Second Affiliated Hospital of Qiqihar Medical College and had not received any treatment before recruitment. The bone marrow and peripheral blood samples were collected from all participants, from which bone marrow mononuclear cells were extracted and serum was preserved. Patients’ basic information and biochemical indices were also documented. Ethical approval was obtained from the ethics committee of the Second Affiliated Hospital of Qiqihar Medical College, and all patients provided written informed consent.

Extraction of bone marrow mononuclear cells

Four milliliters of bone marrow blood were combined with lymphocyte separation fluid (biosharp, #BL590, China) in a 2:1 ratio, followed by centrifugation at 2500 rpm for 15 min. The resulting white membrane layer was then transferred to an EP tube and centrifuged again at 12,000 rpm for 2 min. After discarding the supernatant, the precipitate was collected, representing the bone marrow mononuclear cells.

QPCR

Total RNA was extracted from bone marrow mononuclear cells using TRIzol reagent (TIANGEN, #DP424, China), and its concentration was determined with a NanoDrop 2000. Reverse transcription was performed using FastKing gDNA Dispelling RT SuperMix (TIANGEN, #KR118, China) following the manufacturer’s instructions, and qPCR was performed using SuperReal PreMix Plus (SYBR Green) (TIANGEN, #FP205-02, China). The qPCR cDNA was normalized using the 2-ΔΔCT method. The primer sequences used were: HMGB1, F: 5′-ATCCCAATGCACCCAAGAGG-3′, R: 5′-CAATGGACAGGCCAGGATGT-3′; GAPDH, F: 5′-GTCTCCTCTGACTTCAACAGCG-3′, R: 5′-ACCACCCTGTTGCTGTAGCCAA-3′.

ELISA

Serum samples were collected from MPN and IDA patients. The levels of HMGB1 (CUSABIO, #CSB-E08223 h, China) were measured by ELISA kits, following the instructions.

Statistical analysis

All statistical analyses were performed using SPSS 26.0 (USA). Continuous variables with a normal distribution were expressed as mean ± SD or median with interquartile range, while categorical variables were expressed as percentages. Normal distribution was assessed using the Shapiro–Wilk test. The qualitative variables were analyzed using the chi-square test. The diagnostic value of HMGB1 was evaluated using the receiver operating characteristic (ROC) curve, with the cut-off value determined using the Youden index (specificity + sensitivity−1). Logistic regression analysis was performed and statistical significance was defined as P < 0.05.

Results

HMGB1 expression is upregulated in MPN

QPCR analysis revealed significantly elevated mRNA expression levels of HMGB1 in the bone marrow of MPN patients compared to the control group (Fig. 1A). Given that HMGB1 is a secreted protein, we also measured its levels in the peripheral blood serum of the participants. ELISA results demonstrated that HMGB1 levels in the serum of MPN patients were significantly higher than those in the control group and AS patients (Fig. 1B), thus confirming the overexpression of HMGB1 in MPN.

Fig. 1

HMGB1 Expression is Upregulated in MPN. A The qPCR assay revealed the expression of HMGB1 mRNA expression in bone marrow mononuclear cells, normalized to GAPDH. ^***P < 0.0001 vs. Control group (n = 30 controls and 52 MPN patients). B ELISA measurements demonstrated serum levels of HMGB1 in MPN and AS patients. ^***P < 0.0001 vs. Control group (n = 30 controls, 52 MPN patients, and 30 AS patients)

HMGB1 is correlated with AS in MPN

To investigate the role of HMGB1 in MPN complicated with AS, we collected relevant biochemical indices from the patients and conducted a correlation analysis with serum HMGB1 levels. The results showed a negative correlation between HMGB1 and HDL-C and ApoA1 (Fig. 2A, B). HDL-C is renowned for its potent anti-atherosclerotic properties, with ApoA1 serving as its primary structural and functional protein. This indicates that the high expression of HMGB1 in MPN patients is correlated with the initiation and progression of AS.

Fig. 2

HMGB1 is Correlated with AS in MPN. A, B The relationship between HMGB1 and the expression levels of HDL-C or ApoA1 in 30 controls and 52 MPN patients

Diagnostic value of HMGB1 in MPN complicated with AS

The diagnostic value of serum HMGB1 levels for MPN complicated with AS was assessed using ROC curve analysis. The data showed that HMGB1 can effectively evaluate the risk of AS development in MPN patients, with a cut-off value of 105.92 (sensitivity: 100.00%, specificity: 78.05%) and an area under the curve (AUC) of 0.929 (Fig. 3). Furthermore, logistic regression analysis was conducted with sex, age, and serum HMGB1 levels as independent variables, and the occurrence of AS as the dependent variable. The results revealed that sex and age were not statistically significant, while serum HMGB1 levels were statistically significant (P < 0.05). Specifically, for each unit increase in HMGB1, the risk of developing AS increased by 1.033 times (Table 1).

Fig. 3

The Diagnostic Value of HMGB1 in MPN Complicated AS. Receiver operator characteristic (ROC) curve illustrating the expression level of HMGB1 in the Control group/the MPN group

Table 1 Logistic regression analysis of patients’ sex, age and HMGB1

Discussion

Myeloproliferative neoplasm (MPN) is clonal hematopoietic stem cells characterized by the proliferation of one or more cell lineages. Genetically, the mutations in the JAK2 V617 F, MPL, and CALR driver genes can activate the JAK-STAT pathway, subsequently activating the inflammatory pathway. Activating the inflammatory pathway can also affect the JAK2 V617 F mutation, fostering disease progression [11]. Wang et al. [12] demonstrated that the JAK2 V617 F mutation in hematopoietic cells accelerates the development of atherosclerosis in hyperlipidemic mice. Plaque composition analysis indicated that the JAK2 mutation increased neutrophil infiltration in early atherosclerotic lesions and enhanced neutrophil rolling and adhesion capabilities [12]. This suggests that MPN patients are more prone to developing AS than those without the disorder.

HMGB1 functions in the cell membrane, cytoplasm, and nucleus, but its principal activities occur in the extracellular space [13]. Research has established that extracellular HMGB1 primarily originates from two sources. First, it is secreted by monocytes/macrophages and pituitary cells under inflammatory stimulus, mediating local and systemic inflammatory responses [14]. Second, it is released into the extracellular space during cell death or injury [15]. Substantial evidence indicates that HMGB1 is overexpressed in plaque tissue, and the inflammation it mediates promotes plaque development and exacerbates inflammation within the plaques, marking it as a novel predictor of atherosclerosis [16,17,18].

MPN progression stems from various factors, including genetic alterations and chronic inflammation. Complications such as systemic inflammation, cardiovascular diseases, and organ fibrosis can heighten the risk of MPN progression. In MPN, HMGB1 can activate inflammatory signaling pathways, driving pathological processes including smooth muscle cell proliferation, lipid accumulation, and fibrous matrix expansion [16]. Furthermore, HMGB1 can facilitate cell proliferation and migration, thereby contributing to the formation and progression of atherosclerotic plaques [16,17,18]. In MPN patients, abnormal expression and release of HMGB1 may intensify this pathological process. Therefore, inhibiting the release and activity of HMGB1 could potentially mitigate the inflammatory response and the development of atherosclerosis in MPN patients.

We quantified HMGB1 mRNA levels in bone marrow using qPCR and HMGB1 levels in serum using ELISA. The results revealed a significant increase in HMGB1 expression in MPN patients compared to the control group and AS group. Additionally, serum HMGB1 levels were negatively correlated with HDL-C and ApoA1. The epidemiological data indicate that decreased HDL-C levels contribute to the development of AS [19,20,21]. ApoA1, a key component of HDL-C, also exhibits anti-atherosclerotic abilities [22, 23]. The reduced levels of both HDL-C and ApoA1 in MPN patients further imply the potential for MPN to be complicated with AS, which is likely related to the upregulation of HMGB1.

In conclusion, our study highlights HMGB1 as a promising biomarker with significant diagnostic value for MPN complicated with AS, enabling better identification of high-risk individuals. However, it is essential to acknowledge the study’s limitations, primarily its relatively small sample size. Therefore, further large-scale prospective studies are necessary to validate HMGB1’s potential as a diagnostic and evaluative biomarker for MPN complicated with AS.