Research Article Open Access
Serum Wnt5a Level is Associated with In-Stent Restenosis in PCI Patients
Weidong Ma1, Shan Jia1, Chunyan Zhang1, Zhihui Yao1, Yan Zhang1, Congxia Wang1*
11Department of Cardiovascular Medicine, The Second Affiliated hospital of Xi’an Jiao tong University
*Corresponding author: Congxia Wang, MD, Professor, The second affiliated hospital of Xi’an Jiao tong University, 157, Xi 5 Road, Xi’an, Shaanxi,People’s Republic of China, Tel: +86 13892838729; Fax: No FAX number; E mail: @
Received: March 06,2019; Accepted: March 15, 2019; Published: March 19 , 2019
Citation: Wang C, Weidong Ma, Jia S, Chunyan Z, et al. (2019) Serum Wnt5a Level is Associated with In-Stent Restenosis in PCI Patients. J Clin Trial Cardiol 6(1): 1-5.DOI: 10.15226/2374-6882/6/1/00157
Abstract
Objective: To explore the association between serum Wnt5a level and ISR, and to assess the possibility of Wnt5a to be a predictor of ISR.

Methods: 184 bare metal stent (BMS) implanted patients were enrolled in this case-control study. According to coronary angiography results, all patients were divided into 2 groups: in-stent restenosis (ISR) group and non-ISR group. Serum Wnt5a levels were determined using enzyme-linked immune sorbent assay (ELISA).

Results: Serum Wnt5a levels were higher in ISR group than those in non-ISR group, and were associated with the Gensini score. ISR rate was the highest in the upper tertile of Wnt5a. Multivariate logistic regression indicated that Wnt5a was an independent risk factor of ISR. For diagnosing ISR, the area under ROC curve was 0.817. The cutoff value of Wnt5a diagnosing ISR was 25.83ng/L, with the specificity of 60.68% and sensitivity of 89.55%.

Conclusion: Serum Wnt5a levels is associated with ISR in PCI patients with BMS implantation. Wnt5a is an independent risk factor of ISR, and may act as a biomarker for diagnosing ISR in BMS implanted patients.

Keywords: WNT5A; In-Stent Restenosis; Percutaneous coronary intervention; Bare metal stent.
Introduction
Over the past decades, percutaneous coronary intervention (PCI) with stent implantation is an important treatment for symptomatic coronary artery disease (CAD). However, in-stent restenosis (ISR), which is the most common complication of PCI, plagues cardiologists and patients. Although drug-eluting stents (DES) have been widely used in reducing the incidence of ISR, 5%~10% DES implanted patients are still in the risk of ISR which needs for further revascularization [1]. Therefore, finding the risk factor of ISR is of great significance for predicting and preventing ISR.

ISR occurs in an average of 12 months in DES implanted patients and 6 months in bare-metal stents (BMS) implanted patients post-PCI [2]. The occurrence of ISR is a complex patho physiological process, and its mechanism is still not quite clear. Vascular injury post-PCI is an important initiating factor, which subsequently induced inflammatory response and vascular reparative process [3]. The vascular response to PCI is associated with cellular functions in many cell types, including endothelial cells and vascular smooth muscle cells (VSMCs) [4].

The Wnt signal transduction cascade plays an important role in many physiological and patho physiological processes. It’s a main regulator of development of mammals, and affect cell functions in many cell types [5]. Wnt proteins can activate two different pathways. The canonical Wnt signaling dependents on β-catenin, while non-canonical Wnt signaling is β-cateninindependent. Wnt5a, which can activate non-canonical Wnt signaling, is a member of Wnt family, and it has recently attracted attention of researchers. It is reported that Wnt5a may be implicated in the regulation of cell proliferation and migration in endothelial cells and VSMCs, which is critical for the occurrence of ISR [6, 7]. However, the clinical significance of Wnt5a in ISR is still not clear.

In this study, we aim to explore the association between serum Wnt5a level and ISR, and to assess the possibility of Wnt5a to be a predictor of ISR in patients with BMS implantation.
Materials and Methods
Study Population
We enrolled 184 patients who had undergone BMS implantation in cardiovascular department of the second affiliated hospital of Xi’an Jiao tong University from June 2016 to June 2017. All patients underwent subsequent Coronary Angiography (CAG) 12 months after PCI, and were divided into 2 groups according to the CAG results: ISR group (n=67) and non-ISR group (n=117). Baseline parameters were collected at admission, including sex, age, Body Mass Index (BMI), Systolic Blood Pressure (SBP), Diastolic Blood Pressure (DBP) and risk factors of CAD. Current smokers were defined as patients who smoked ≥10 cigarettes per day. Patients whose blood pressure over 140/90 mmHg, or who had been diagnosed with hypertension, were considered current hypertension. The definition of hyperlipidemia was described previously [8]. ISR was defined as diameter stenosis >50% at the stent segment or 5mm segment adjacent to the stent [1]. The exclusion criteria including: (1) dysfunction of liver or kidney; (2) cancer; (3) autoimmune disease; (4) severe infection; (5) diabetes; (6) heart failure; (7) valvular heart disease; (8) cardiomyopathy. The patients provided written informed consent after explanation of the study, and this study was performed under medical ethics.
CAG and Gensini score
All patients had undergone subsequent CAG. CAG and Gensini score assessment was performed as previous described [9].
Blood sample and laboratory test
Blood samples were obtained the day after admission (after 8 hours fasting). The standard methods was performed to determine serum levels of Blood Urea Nitrogen (BUN), Creatinine (Cr), Fasting Blood Glucose (FBG), Total Cholesterol (TC), Triglyceride (TG), High Density Lipoprotein (HDL) and Low Density Lipoprotein (LDL). Serum Wnt5a (Cusabio Technology LLC, Wuhan, China) and IL-6 (Protein tech Group, Inc, America) levels were determined by enzyme-linked immunosorbent assay (ELISA).
Statistical analysis
Statistical analyses were performed by using SPSS 18.0 software. Continuous data was expressed as mean±SD, and Student’s t-test was applied to compare means between groups. One-way ANOVA with the LSD post hoc multiple comparisons were applied to compare means among 3 or more groups. Categorical data was expressed as numbers and percentages, and Chi-square test was used to seek difference between groups. Spearman correlation analysis was used to explore the relationship between Wnt5a and Gensini scores. Univariate and multivariate logistic regression analysis was used to explore the relationship between Wnt5a and ISR. The potential of Wnt5a diagnosing ISR was assessed by ROC curve analysis. P< 0.05 was considered statistically different.
Results
There are 67 patients in ISR group and 117 patients in non-ISR group. The clinical characteristics are listed in Table 1. No significant differences were observed between 2 groups regarding gender, sex, BMI, SBP, DBP and risk factors of CAD. CAG results showed that there are no significant differences regarding culprit vessel between 2 groups. Gensini score is significantly higher in ISR group (66.75±12.28) than that in non-ISR group (33.11±10.96). As for laboratory test, FBG, BUN, Cr, TC, TG, HDL, LDL are all at similar levels in 2 groups. Serum Wnt5a and IL-6 levels are significantly higher in ISR group than those in non- ISR group. Spearman correlation analysis showed that Wnt5a is associated with Gensisn score (r=0.580, P< 0.001, Figure 1).
Table 1: Baseline characteristics of patients

 

ISR(n=67)

Non-ISR(n=117)

P

Sex (male %)

47 (70.15%)

73 (62.39%)

0.288

Age

62.28±10.76

60.50±8.77

0.223

BMI

21.69±1.85

21.51±2.13

0.566

SBP

132.79±19.68

131.20±15.60

0.546

DBP

76.93±11.57

75.65±10.06

0.435

Gensini score

66.75±12.28

33.11±10.96

<0.001

Risk factors of CAD

Smoking

44 (65.67%)

66 (56.41%)

0.218

Hyperlipidemia

38 (56.72%)

53 (45.30%)

0.136

Hypertension

38 (56.72%)

52 (44.44%)

0.109

Culprit vessel

LM

5 (7.6%)

7 (5.98%)

0.696

LAD

55 (82.09%)

89 (76.07%)

0.341

LCX

35 (52.24%)

47 (40.17%)

0.113

RCA

49 (73.13%)

86 (73.50%)

0.956

Laboratory test

FBG(mmol/L)

5.22±0.63

5.12±0.59

0.294

BUN(mmol/L)

5.04±1.23

4.81±1.27

0.233

Cr(μmol/L)

73.77±16.43

70.80±14.56

0.206

TC(mmol/L)

4.14±0.97

4.18±1.09

0.818

TG(mmol/L)

1.78±1.17

1.65±1.02

0.411

HDL(mmol/L)

1.04±0.27

1.10±0.28

0.139

LDL(mmol/L)

2.61±0.87

2.57±0.80

0.758

Wnt5a(ng/L)

32.48±7.75

24.63±4.72

<0.001

IL-6(pg/ml)

48.51±3.44

45.60±2.77

<0.001

BMI: Body Mass Index; SBP: Systolic Blood Pressure; DBP: Diastolic Blood Pressure; LM: Left Main Coronary Artery; LAD: Left Anterior Descending Branch; LCX: Left Circumflex Branch; RCA: Right Coronary Artery FBG: Fasting Blood Glucose; BUN: Blood Urea Nitrogen; Cr: Creatinine; TC: Total Cholesterol; TG:Triglyceride; HDL: High Density Lipoprotein; LDL: Low Density Lipoprotein
Figure 1: Correlation between Wnt5a and Gensini scores
Spearman correlation analysis showed that serum Wnt5a levels were associated with Gensini scores (r=0.580, P< 0.001).
To explore whether Wnt5a is associated with ISR, we divided all patients into 3 groups according to the textiles of serum Wnt5a levels: tertile 1 (Wnt5a level≤24.39 ng/L, n=61); tertile 2 (24.39< Wnt5a level≤28.75 ng/L, n=62); tertile 3 (Wnt5a level >28.75 ng/L, n=61). We assessed the ISR rates among 3 groups. As shown in Figure 2, the ISR rate in tertile 3 (38/61) is significantly higher than that in tertile 2 (25/62). The ISR rate in tertile 2 is significantly higher than that in tertile 1 (4/61)
Figure 2: ISR rate in different tertile of serum Wnt5a levels
ISR rate was the highest in tertile 3 (38/61) than those in tertile 2 (25/62) and tertile 3 (4/61).
To further explore whether Wnt5a is a risk factor of ISR, we performed logistic regression analysis. As shown in Table 2, Wnt5a is associated with the presence of ISR (OR=1.287; 95% CI: 1.179- 1.404, P< 0.001) as indicated by Univariate logistic regression. After adjusting for sex, age, BMI, risk factors of CAD, serum lipid parameters and IL-6, Wnt5a is independently associated with the presence of ISR (OR=1.256; 95% CI: 1.129-1.397, P< 0.001).
Table 2: Univariate and multivariate logistic regression of Wnt5a and ISR risk

 

OR (95% CI)

P

Univariate

1.287(1.179,1.404)

<0.001

Adjusted model1a

1.287(1.177,1.408)

<0.001

Adjusted model 2b

1.284(1.174,1.405)

<0.001

Adjusted model 3c

1.256(1.129,1.397)

<0.001

OR : odds ratio
Model1a: Adjusted for sex, age and BMI
Model2b: Adjusted for model 1 and risk factors of CAD
Model3c: Adjusted for model 2 and TC, TG, LDL, HDL and IL-6
Then we performed ROC analysis to assess the possibility of Wnt5a predicting ISR. The area under ROC curve was 0.817 (95% CI: 0.757-0.876, P< 0.001). The cutoff value of Wnt5a predicting ISR was 25.83 ng/L, with the specificity of 60.68% and the sensitivity of 89.55% (Figure 3).
Figure 3: SROC curve for Wnt5a predicting ISR
Discussion
ISR is a major complication of PCI, which adversely affects patients’ life quality and prognosis. Exploring the risk factors of ISR is of great clinical significance for predicting and preventing the occurrence of ISR. In the current study, we found that serum Wnt5a level is associated with Gensini score in PCI patients with BMS implantation, which indicated that Wnt5a may play a role in the development of coronary artery stenosis. It is reported that Wnt5a was involved in various physiological processes such as proliferation, migration, inflammation and lipid metabolism, each of which is essential for the development of atherosclerosis [10]. Moreover, we report for the first time that serum Wnt5a is an independent risk factor of ISR, and there is possibility of Wnt5a to be a predictor of ISR in BMS implanted patients.

The mechanism of ISR is not fully elucidated. Kumatsu et al., investigated 11 stented coronary arteries in 11 patients. Histological findings indicated that inflammation and neointima proliferation is critical for the formation of ISR [11]. After PCI, the presence of stents can induce acute inflammation. Adhesion of acute inflammatory cells, including neutrophils and monocytes, is a character of early vascular response. Over weeks, the chronic inflammatory cells, including macrophages, replace acute inflammatory cells [4, 12]. Wnt5a is an important regulator of inflammation. Chiristman el al. reported that Wnt5a expressed in advanced atherosclerosis plaques, which is characterized by macrophage aggregation [13]. Recombinant Wnt5a could induce inflammatory cytokines production of macrophages via TLR4 dependent pathways, including IL-6 [14]. Our data showed that serum IL-6 levels, an important inflammatory cytokine, is higher in ISR group than those in non-ISR group. Multivariate logistic regression suggested that Wnt5a is independently associated with the presence of ISR, even adjusted for IL-6. This result indicates that Wnt5a may affect ISR by some other mechanism except for inflammation.

Neointima tissue proliferation is an important mechanism of ISR. This pathological process involves functional changes in various vascular wall cells. As the inner layer of arteries, endothelial cells play important roles in ISR process. Vascular endothelium integrity is an important prerequisite for protection of blood vessel [15]. However, PCI operation, including barotraumas and stimulation of stent, could damage vascular endothelium, which could induce the proliferation, migration and apoptosis of endothelial cells [16]. Wnt5a is reported to be a regulator of endothelial cells. Masckauchan el al., found that Wnt5a is expressed in human endothelial cells, and it could induce the proliferation and survival of endothelial cells. Endothelial cells migration was inhibited by reduced Wnt5a expression [17]. Kim et al. found that Wnt5a induced release of inflammatory cytokines, such as IL-6 and IL-8, via Wnt/Ca2+ signaling pathway. This result suggests that Wnt5a may act as an inflammatory mediator in endothelial cells [18].

Endothelial cell layer can inhibits VSMCs proliferation and intimal hyperplasia under normal circumstance, and VSMCs express high levels of contractile proteins. After vascular injury, phenotypic transformation may occur in VSMCs, which is characterized by increased level of proliferation, migration and extracellular matrix synthesis [19]. It is reported that the stable neointima contains 20% VSMCs [20], suggests that VSMCs play an important role in ISR. Pandey et al. found that Wnt5a mRNA was expressed in human VSMCs, as well as its receptors, such as Fzd1, Fzd2 and Fzd5 [21]. This result suggests that Wnt5a may be implicated in ISR via regulating VSMCs functions. Qin el al. treated VSMC with siRNA of Wnt5a, and found that total and free cholesterol content in VSMC exposed to oxLDL increased significantly, whereas recombinant Wnt5a treatment had the opposite effect [22]. This result suggests that Wnt5a could regulate cholesterol accumulation in VSMC. DirenZo el al. found that application of recombinant Wnt5a, Wnt2b, Wnt4 or Wnt9a stimulated VSMC proliferation via β-catenin dependent pathways [7]. However, it seems controversial that recombinant Wnt5a or over expression of Wnt5a cannot increase the proliferation of VSMC in another study [23]. Further study is needed to clarify Wnt5a functions in VSMC and underlying mechanisms.

Some limitations need to be noticed. First, this was a singlecenter study with a relatively small study population. It is necessary to confirm our conclusion in a large sample size study in the future. Second, as a case-control study, we cannot elucidate a causal relationship. Prospective and experimental studies are needed to fully elucidate the relationship between serum Wnt5a levels and ISR and its underlying mechanisms. Third, we did not measure serum Wnt5a levels over time. The dynamic changes in Wnt5a levels may help clarify the role of Wnt5a in the prognosis of ISR.

In conclusion, our data suggests that serum Wnt5a levels are associated with ISR in PCI patients with BMS implantation. Wnt5a is an independent risk factor of ISR, and it may act as a predict biomarker of ISR in BMS implanted patients. Our findings provide new insights into the clinical significance of the Wnt signaling pathway. Further study is needed to elucidate the relationship between Wnt5a and ISR and its underlying mechanisms.
Acknowledgement
The study was funded by The National Natural Science Foundation of China (No. 81273878).
Declarations
No conflict of interest.
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