Predictive value of plasma ephrinB2 levels for amputation risk following endovascular revascularization in peripheral artery disease

Introduction

Peripheral arterial disease (PAD) of the lower extremities is a chronic disease primarily characterized by atherosclerosis, eventually leading to acute or chronic limb ischemia. More than 200 million people around the world suffer PAD, and the number is keep growing (Lawall et al., 2016). As the aging population continues to grow dramatically, PAD has been already emerged as a critical socioeconomic concern in China. The most common symptoms of the lower limb PAD is intermittent claudication, which is painful and harm of the walking ability severely. Moreover, symptoms like resting pain and ulcers on the foot/leg may start to manifest, potentially resulting in amputation of the affected limb (Reinecke et al., 2015). Furthermore, the development of PAD is often combined with carotid artery stenosis and coronary artery stenosis, exhibiting very higher risk of ischemic events and death compared to other cardiovascular diseases (Saenz-Pipaon et al., 2021).

There are large of scale studies demonstrated that the development of PAD is associated with atherosclerosis-related inflammation caused by pro-inflammatory cytokines and inflammatory signaling pathways (Hansson & Hermansson, 2011). Neutrophils have traditionally been regarded as bystanders or biomarkers of cardiovascular diseases (Silvestre-Roig et al., 2020), associated with innate immunity, while lymphocytes are typically involved in adaptive immune responses. Recent studies suggest that changes in the neutrophil-to-lymphocyte ratio (NLR) to some extent reflect the severity of inflammation in the body and are correlated with the prognosis of vascular reconstructive surgery (Machado et al., 2018).

The erythropoietin-producing human hepatocellular (Eph) receptors tyrosine kinases and their membrane-bound ligands, the ephrins, play a role not only in the communication signaling pathways between pericytes and endothelial cells but also in the inflammatory responses. EphrinB2, a member of the Eph receptor family, is thought to be involved in the regulation of inflammation in atherosclerosis (Funk & Orr, 2013; Lehoux & Jones, 2016). A previous study found that ephrinB2 expression was significantly increased in human atherosclerosis through gene expression analysis of atherosclerotic plaques in human carotid endarterectomy tissue (Sakamoto et al., 2008). Meanwhile, it was found that ephrinB2 expression elevates in the atherosclerotic endothelium of mouse aorta compared to non-atherosclerotic areas (Braun et al., 2011). In addition, in diabetes, an increase in the ephrin-B2 signaling pathway in peripapillary cells mediates retinal vascular inflammation (Coucha et al., 2020). However, the role of ephrinB2 related signaling in lower extremity lower extremity PAD remains unclear.

In our study, we investigated the expression levels of ephrinB2 in the plasma of lower extremity PAD patients. Additionally, we explored its ability to predict amputation in those post endovascular revascularization patients combined with clinical follow-up data.

Materials and Methods

Results

A total of 133 cases of lower extremity PAD patients were eligible for inclusion and the clinical characteristics of lower extremity PAD patients included in the study are summarized in Table 1. The mean age was 66.5 ± 12.1 years; 81.2% were either current or former smokers, and comorbidities such as hypertension (63.9%), diabetes (36.8%), and coronary artery disease (32.3%) were recorded. It was noted that there were no significant differences in drug usage across patients of different Rutherford categories.

Elevation of plasma ephrinB2 level in lower extremity PAD patients

To evaluate the plasma ephrinB2 expression in lower extremity PAD patients, we conducted Western blot analysis to examine the samples collected. We observed a significant upregulation of ephrinB2 expression in lower extremity PAD patients compared to healthy people as control (p < 0.001) (Figs. 1A, 1B). Further analysis reveals a significant difference in the expression levels of ephrinB2 in plasma between Rutherford category 3 and 4 (p = 0.023), while no significant difference is observed between categories 4–6 (Fig. 1C).

High levels of plasma ephrinB2 indicate a higher risk of postoperative amputation events

During the 30-month follow-up, 11 individuals had amputations and 14 individuals died. Among the deceased, six died from cardiac pathologies, five from COVID-19, two from cancer, and one from sepsis.

We investigated the predictive roles of ephrinB2 and its combination with NLR in this study. The ROC analysis revealed that plasma ephrinB2 levels and NLR have potential predictive value for amputation events within 30 months after lower extremity PAD endovascular revascularization, with AUCs of 0.659 (p = 0.047; 95% confidence interval (CI) [0.502~0.861]) and 0.730 (p = 0.012; 95% CI [0.559~0.900]), respectively. When the test results for ephrinB2 and NLR were considered jointly, the ROC analysis revealed an AUC of 0.811 (p < 0.001; 95% CI [0.675–0.918]), indicating higher predictive capability (Fig. 2A, Table 2). However, when ephrinB2 is applied individually, it did not demonstrate a predictive role in postoperative 30-month mortality events (p = 0.153, 95% CI [0.383–0.714]) (Fig. 2B, Table 2). To further study the relationship between plasma ephrin levels and amputation events within 30 months after revascularization, as shown in Fig. 2C, we conducted a logistic analysis. The results indicated a significant correlation between ephrinB2 expression and the 30-month post-amputation rate. Furthermore, even after adjusting for age, gender, and other clinical and laboratory parameters, the association remained statistically significant.

Table 2:

ROC analysis of predictive indicators for amputation and mortality within 30 months after vascular reconstruction.

Variables	AUC	95% Confidence interval	P value	Cutoff	Sensitivity	Specificity
Amputation
EphrinB2	0.659	[0.502–0.861]	0.047	2.13	63.6	68.9
NLR	0.730	[0.559–0.900]	0.012	7.3	54.5	91.0
EphrinB2+NLR	0.811	[0.675–0.918]	<0.001		81.8	89.6
Mortality
EphrinB2	0.549	[0.383–0.714]	0.153	1.29	78.6	37.8
NLR	0.584	[0.305–0.410]	0.105	2.57	78.6	47.9
EphrinB2+NLR	0.661	[0.515–0.806]	0.049		71.4	68.9

DOI: 10.7717/peerj.17531/table-2

Note:

NLR, neutrophil lymphocyte ratio; Significant P-values were presented in bold.

Depending on the optimal cut-off value according to ROC for the 30 months amputation obtained from Youden’s index (2.13 for plasma ephrinB2 level), the outcomes were further analyzed after dividing the patients into paired groups: low-ephrinB2/high-ephrinB2. As shown in Table 3, in the high-ephrinB2 group, there was a higher incidence of amputation events within 30 months after endovascular revascularization, while there was no difference in mortality events.

Table 3:

Univariate analysis of plasma ephrinB2 level and all adverse event occurrences during the study period for all patients.

EphrinB2 = 2.13	Amputation	Mortality
LOW-ephrinB2 vs. High-ephrinB2	4/88 (4.54%) vs. 7/45 (15.56%)	8/88 (9.09%) vs. 6/45 (13.33%)
	p = 0.0438	p = 0.5522
	OR: 3.868;	OR: 1.538;
	95% CI [1.115–12.25]	95% CI [0.480–5.030]

DOI: 10.7717/peerj.17531/table-3

The Kaplan-Meier plot for the 30 months amputation and mortality based on the optimal cut-off value of plasma ephrinB2 level for all patients is shown in Fig. 3. The results indicated that the high ephrinB2 group had a higher amputation risk (p = 0.048).

Discussion

In this study, we investigated the relationship between plasma ephrinB2 levels and lower extremity PAD patients. Our findings revealed that patients with lower extremity PAD had significantly higher plasma ephrinB2 levels compared to the healthy control group. Additionally, correlation analysis demonstrated a positive relationship between plasma ephrinB2 levels and some hematological markers including white blood cell counts, neutrophil counts, as well as NLR. Logistic regression analysis suggested that higher preoperative plasma ephrinB2 levels might be indicative of an increased probability of amputation within 30 months after endovascular revascularization. Moreover, ROC curve analysis indicated that the combined use of plasma ephrinB2 levels and NLR showed higher predictive accuracy (AUC = 0.811) for amputation events within this timeframe, compared to NLR alone (AUC = 0.730). Also, the ephrinB2-NLR combination is higher in specificity and sensitivity to predict amputation risk compare to ephrinB2 or NLR alone.

PAD not only has a negative impact on the health and quality of life of patients but also imposes a significant economic burden on society (Criqui & Aboyans, 2015). Numerous studies have indicated a close association between the occurrence and development of PAD and the systemic inflammatory response (Aday & Matsushita, 2021; Brevetti et al., 2010; Fort-Gallifa et al., 2016). Previous studies have shown that during inflammation process, the expression of ephrinB2 in capillaries is upregulated. Activating ephrinB2 ligands can lead to a decrease in the integrity of endothelial cell junctions and enhance the pro-inflammatory phenotype of the endothelium (Liu et al., 2014; Sweet et al., 2013). Furthermore, under conditions of pre-atherosclerotic blood flow, ephrinB2 is upregulated and functions as a chemoattractant, facilitating white blood cell migration even in the absence of other chemotactic factors (Cao et al., 2018). ADAM10 is a cell surface shedding enzyme that regulates physiological processes, including Notch signaling (Tsai et al., 2014). Despite some studies suggesting that ephrinB2 can be cleaved by the enzyme Adam10 to release active proteins in specific circumstances (Lagares et al., 2017; Mueller et al., 2021), the connection between plasma-free ephrinB2 levels and the activation of inflammatory cells remains unclear. Nevertheless, this connection may play a role in the pathogenesis of peripheral arterial disease. Lower extremity PAD often leads to ischemia and necrosis in the affected endovascular supply area, with endovascular revascularization being a common treatment option. However, despite undergoing revascularization, some patients may still require amputation due to already gangrene tissue or deterioration later. Therefore, predicting amputation after vascularization is important for physicians and patients. Gary et al. (2013) conducted a study involving 2,121 lower extremity PAD patients and found that an elevated NLR is significantly associated with an increased risk of critical limb ischemia (CLI). Shin et al. (2017) conducted a study on 381 patients with acute myocardial infarction (AMI) who underwent percutaneous coronary intervention (PCI), which showed that elevated NLR and CRP levels after PCI in AMI patients are associated with an increased risk of long-term mortality, suggesting that the combined use of both markers enhances the predictive value for long-term prognosis. In our study, we observed a significant increase in NLR with disease severity. Therefore, we chose NLR as an additional variable and used ROC curve analysis to assess its predictive capabilities when used alone and in combination with ephrinB2 for amputation events. We found that the combined application of plasma ephrinB2 levels and NLR demonstrated higher predictive accuracy for amputation events both in sensitivity and specificity, providing new insights into the role of plasma ephrinB2 in lower extremity PAD.

Membrane-bound ephrinB2 and its receptor EphB4 play a crucial role in blood and lymphatic vessel formation under both normal and pathological conditions (Abéngozar et al., 2012; Masumura et al., 2009), and they are indispensable for arterial remodeling. Our results revealed that elevated levels of free ephrinB2 in plasma may indicate the development of lower extremity PAD or worse postoperative outcomes following vascularization. This phenomenon may be attributed to ephrinB2 related molecular signaling transduction in mechanism that led to the cleavage of ephrinB2 by ADAM10 on endothelial cells, resulting in reduced endothelial cell stability and hindered neovascularization. Additionally, free ephrinB2 may also participate in the inflammatory response. Therefore, further in-depth mechanism study is needed to elucidate the role of ephrinB2 in lower extremity PAD.

This study has several limitations. Firstly, the study design is cross-sectional, which only allows for correlations between variables and does not establish causal relationships. Secondly, the study is a single-center investigation with a relatively small sample size, and the participants were exclusively recruited from specific hospitals and underwent rigorous screening. This led to a relatively small and varied number of patients in each Rutherford grade, which may affect the accuracy of our results. Additionally, due to the profound impact of the COVID-19 pandemic on postoperative survival outcomes during the study period, it may not accurately reflect the predictive role of plasma ephrinB2 under normal circumstances. Therefore, further research on the association between plasma ephrinB2 levels and postoperative patient mortality is still needed.

Conclusions

In summary, we revealed elevated levels of plasma ephrinB2 in lower extremity PAD patients and explored its potential association with atherosclerotic inflammation. This study investigated the predictive capability of ephrinB2, both independently and in combination with NLR, for forecasting amputation events occurring within 30 months following lower extremity PAD endovascular revascularization. This study may offer a novel approach for predicting post-PAD amputation events and broaden the scope of research on the significance of ephrinB2 molecule in lower extremity PAD.

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