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Correlation between serum Dickkopf-1 (DKK1) levels and coronary artery stenosis

Open AccessPublished:October 19, 2022DOI:https://doi.org/10.1016/j.numecd.2022.10.007

      Highlights

      • Correlation between serum Dickkopf-1 (DKK1) & coronary artery stenosis was studied.
      • DKK1 levels of middle- and high-Gensini score groups were significantly higher.
      • DKK1 levels is correlated with the severity of coronary stenosis.
      • Correlation between DKK1 and the inflammatory factors, RBP and PAI-1, was positive.
      • DKK1 level is an independent risk factor for coronary heart disease.

      Abstract

      Background and aims

      To study the correlation between the level of serum Dickkopf-1 (DKK1) and the degree of coronary artery stenosis in patients with coronary atherosclerotic heart disease.

      Methods and results

      In 2018, general data and biochemical indexes of 311 patients who underwent coronary angiography were recorded. Before procedure, arterial blood was drawn and the concentrations of DKK1, retinol binding protein 4 (RBP4), plasminogen activator inhibitor (PAI-1) were measured. Based on coronary angiography results, subjects were divided into a coronary heart disease (CHD) group; and a non-coronary heart disease (non-CHD)group. The CHD group was divided into three subgroups: the low Gensini score; the middle Gensini score; and the high Gensini score subgroups. Compared with those of the non-CHD group, DKK1, RBP4 and PAI-1 of the CHD group were significantly higher, while the OC was lower.
      DKK1,RBP4 and PAI-1 levels of the middle and high Gensini subgroups were significantly higher, compared with that of the low Gensini subgroup. Differences between osteocalcin (OC), beta-isomerized C-terminal telopeptidase (β-CTX), and 25(OH)2D3 of the three subgroups were not significant.
      Correlation between DKK1 and the inflammatory factors, RBP4 and PAI-1, was positive. Correlation between DKK1 and β - CTX, 25(OH)2D3 and OC was not significant. DKK1 was a risk factor for CHD. The degree of coronary artery stenosis was related to DKK1 concentration.

      Conclusions

      Serum DKK1 levels in coronary heart disease patients were significantly higher, and positively correlated with the degree of coronary artery stenosis. DKK1 level is an independent risk factor for coronary heart disease.

      Keywords

      Abbreviations:

      Dickkopf-1 ((DKK1)), Coronary heart disease ((CHD)), Non-coronary heart disease ((non-CHD)), Wingless/Beta Catenin ((Wnt/β-Catenin)), Vascular smooth muscle cells ((VSMCs)), Human umbilical vein endothelial cells ((HUVECs)), Body mass index ((BMI)), β-isomerized C-terminal telopeptides ((β-CTX)), Osteocalcin ((OC)), retinol binding protein 4 ((RBP4)), Plasminogen activator inhibitor ((PAI-1)), Enzyme-linked immunosorbent assay ((ELISA)), Analysis of variance ((ANOVA)), Triglycerides ((TG)), uric acid ((UA)), High density lipoprotein cholesterol ((HDL-C)), Low density lipoprotein cholesterol ((LDL-C)), Fasting blood glucose ((FBG)), Lipoprotein receptor related protein ((LRP)), Glycogen synthase kinase-3β ((GSK3)), Oscillatory shear stress ((OSS)), cAMP response element binding protein ((CREB)), Asymmetric dimethylarginine ((ADMA)), Carotid intima-media thickness ((cIMT)), Endothelial progenitor cells ((EPC))

      1. Introduction

      Atherosclerosis is a chronic inflammatory condition. It is the main pathologic basis of cardiovascular diseases. Plaque rupture and thrombosis may induce acute clinical events such as acute coronary syndrome. Wingless/beta catenin (Wnt/β-Catenin) is involved in the progression of vascular lesions, associated with endothelial dysfunction, macrophage activation, proliferation, and vascular smooth muscle cell migration [
      • Boucher P.
      • Matz R.L.
      • Terrand J.
      Atherosclerosis: gone with the Wnt?.
      ,
      • Liu Y.
      • Neogi A.
      • Mani A.
      The role of Wnt signalling in development of coronary artery disease and its risk factors.
      ,
      • Malgor R.
      • Bhatt P.M.
      • Connolly B.A.
      • Jacoby D.L.
      • Feldmann K.J.
      • Silver M.J.
      • et al.
      Wnt5a, TLR2 and TLR4 are elevated in advanced human atherosclerotic lesions.
      ]. Dickkopf-1 (DKK1), which is an inhibitor of the Wnt/β-Catenin pathway, blocks the Wnt/β-catenin pathway by forming a complex with LRP5/6 [
      • Mani A.
      • Radhakrishnan J.
      • Wang H.
      • Mani A.
      • Mani M.A.
      • Nelson-Williams C.
      • et al.
      LRP6 mutation in a family with early coronary disease and metabolic risk factors.
      ]. In vivo and in vitro studies have confirmed that serum DKK1 play an important role in the development of coronary atherosclerosis and acute ischemic stroke by regulating Wnt/β-Catenin [
      • Liu Y.
      • Neogi A.
      • Mani A.
      The role of Wnt signalling in development of coronary artery disease and its risk factors.
      ,
      • Zhu Z.
      • Guo D.
      • Zhong C.
      • Wang A.
      • Xie X.
      • Xu T.
      • et al.
      Serum DKK-1 (Dickkopf-1) is a potential biomarker in the prediction of clinical outcomes among patients with acute ischemic stroke.
      ,
      • Ueland T.
      • Otterdal K.
      • Lekva T.
      • Halvorsen B.
      • Gabrielsen A.
      • Sandberg W.J.
      • et al.
      Dickkopf-1 enhances inflammatory interaction between platelets and endothelial cells and shows increased expression in atherosclerosis.
      ].
      DKK1 facilitates inflammation, platelet activation, and endothelial dysfunction, and plays an important role during the early stages of atherosclerosis [
      • Di M.
      • Wang L.
      • Li M.
      • et al.
      Dickkopf1 destabilizes atherosclerotic plaques and promotes plaque formation by inducing apoptosis of endothelial cells through activation of ER stress.
      ,
      • Zheng T.F.
      • Liu X.L.
      • Li X.
      • Wang Q.Q.
      • Zhao Y.C.
      • Li X.
      • et al.
      Dickkopf-1 promotes vascular smooth muscle cell proliferation and migration through upregulating UHRF1 during cyclic stretch application.
      ,
      • Motovska Z.
      • Vichova T.
      • Doktorova M.
      • Labos M.
      • Maly M.
      • Widimsky P.
      Serum Dickkopf-1 signaling and calcium deposition in aortic valve are significantly related to the presence of concomitant coronary atherosclerosis in patients with symptomatic calcified aortic stenosis.
      ]. It is secreted by activated platelets in an inflammatory microenvironment of atherosclerotic plaque, inhibits the Wnt/β-catenin pathway in endothelial cells, and contributes to the atherosclerotic process. Di M et al. found overexpression of DKK1 resulted in enlarged and destabilized atherosclerotic lesions and increased apoptosis in aortic as well as carotid plaque. The relative content of vascular smooth muscle cells (VSMCs) and collagen fibers were lowered and plaque size and vulnerability were increased in a group overexpressing DKK1, while the content of VSMCs and collagen fibers were higher in a DKK1-silenced group. The mRNA and protein levels of DKK1 were increased in a time-dependent manner during the reversal of ox-LDL-induced apoptosis by DKK1 in Human umbilical vein endothelial cells (HUVECs), indicating that DKK1 induces apoptosis in HUVECs by activating endoplasmic reticulum stress via the JNK pathway and canonical Wnt signaling [
      • Di M.
      • Wang L.
      • Li M.
      • et al.
      Dickkopf1 destabilizes atherosclerotic plaques and promotes plaque formation by inducing apoptosis of endothelial cells through activation of ER stress.
      ].
      One study detected DKK-1 levels were significantly higher, compared to those of the controls. Elevated DKK-1 levels were significantly associated with premature coronary artery disease during the stable phase, which occurs after one year of the disease [
      • Foulquier S.
      • Daskalopoulos E.P.
      • Lluri G.
      • Hermans K.C.M.
      • Deb A.
      • Blankesteijn W.M.
      WNT signaling in cardiac and vascular disease.
      ]. A prospective, population-based Bruneck Study confirmed that the elevation of baseline DKKI levels was independently associated with the incidence of cardiovascular events after a median follow up period of 15.6 years [
      • Klingenschmid G.
      • Tschiderer L.
      • Himmler G.
      • Rungger G.
      • Brugger S.
      • Santer P.
      • et al.
      Associations of serum dickkopf-1 and sclerostin with cardiovascular events: results from the prospective Bruneck study.
      ]. Further, almost all clinical studies that have been conducted so far suggest that serum levels of DKKI are associated with stable cardiovascular disease.
      Coronary artery stenosis is a marker of the severity of coronary heart disease during the stable phase. Therefore, the current study investigated the correlation between serum DKK1 and the degree of coronary artery stenosis in patients with coronary atherosclerotic heart disease. It will provide a new clue for prevention and treatment of coronary atery diseases.

      2. Methods

      2.1 Subjects

      A total of 311 patients who underwent coronary angiography in the Department of Cardiology at The Affiliated Hospital of ChengDe Medicine University from March to August 2018 were enrolled. These patients included 185 males and 126 females, aged 28–81 years (average age, 59.24 years). Coronary angiography was performed on all the patients and their Gensini score was calculated using the modified scoring schema (Table 1). Based on their coronary angiography results, patients were divided into a non-coronary heart disease group (non-CHD group; n = 105) and a coronary heart disease group (CHD group; n = 206). The CHD group was further divided into three subgroups based on the severity of coronary artery stenosis according to their Gensini score. (i) a low Gensini score group, S1 (2–21 points; n = 70), (ii) a middle Gensini score group, S2 (22–36 points; n = 69), and (iii) a high Gensini score group, S3 (≥37 points; n = 67).
      Table 1Gensini score rule.
      Degree of coronary artery stenosisScoreCoronary lesion siteScore
      ≤25%1LM5
      26%–50%2Proximal LAD or LCX2.5
      51%–75%4Middle LAD1.5
      76%–90%8Distal LAD1
      91%–99%16Middle or distal LAD1
      100%32RCA1
      Subbranch0.5
      LM: left main coronary artery; LAD: left anterior descending; LCX: left circumflex coronary; RCA: right coronary artery.
      Diagnostic criteria for coronary heart disease were based on the guidelines of the American Heart Association as follows: at least one stenosis ≥50% of left main trunk, anterior descending artery, circumflex artery, right coronary artery and its main branches amounts to a diagnosis of coronary heart disease. All patients underwent coronary angiography through the radial or femoral artery, and two specialists jointly assessed the extent of vascular disease.
      Inclusion criteria: (1) more than 18 years old; (2)male and female; (3)underwent coronary angiography in the Department of Cardiology at The Affiliated Hospital of ChengDe Medicine University from March in 2018 for the diagnosis of CHD.
      Exclusion criteria of the study included: (1) Severe infection, trauma, anemia (hemoglobin<120 g/L for adult male and<110 g/L for adult female) [
      • Zhang Zhinan
      • Hao Yushu
      • Zhao Yongqiang
      • Wang Jianxiang
      The HEMATOLOGY(in Chinese).
      ]; (2) Recent (within six months) use of anti-osteoporosis drugs, thyroid hormones, thiazolidinedione, thiazide or cortisol, or other drugs that affect bone metabolism. (3)chronic kidney disease (CKD) Stages III,IV and V,Transaminase level increase for more than three times of the upper normal range (4)Pregnant females;

      2.2 Clinical variables and laboratory procedure

      General anthropometric data of the patients were collected. The average of two blood pressure readings, each taken after sitting for 5 min in a resting position, was recorded. The height and weight were measured, and the body mass index (BMI) was calculated according to the formula BMI (kg/m2) = body weight (kg)/height 2 (m2).
      Samples of blood drawn from the radial or femoral artery in the morning after overnight fasting were let stand at room temperature for 2 h and then centrifuged at 3000 rpm for 10 min. The upper portion of the serum supernatant was aspirated and stored at −80 °C until measured.
      Plasma concentrations of β-isomerized C-terminal telopeptides (β-CTX), osteocalcin (OC), and 25-(OH)-D were detected using an electrochemiluminescence method (Roche cobase e601 electrochemiluminescence Immunoanalyzer, Roche Diagnostic GmbH, Mannheim,Germany; Elecsys β-CrossLaps/serum, Elecsys Vitamin D total, Elecsys N-MID Osteocalcin kits ,Roche Diagnostic GmbH, Mannheim,Germany). Plasma levels of DKK-1, retinol binding protein 4 (RBP4) and plasminogen activator inhibitor (PAI-1) in the patients and controls were determined via a commercially available, enzyme-linked immunosorbent assay (ELISA); (Thermo Scientific Multiskan FC,Thermo Fisher, Shanghai,China; ELISA Kit for Dickkopf Related Protein 1,Plasminogen Activator Inhibitor 1 and Retinol Binding Protein 4,Cloud Clone Corp, Shanghai,China) according to the manufacturer's instructions. The differences between intra- and inter-assays of DKK1 were less than 10% and 12%, respectively. Informed consent was obtained from all the participants. Ethics approval was granted.

      2.3 Statistical analysis

      Data for continuous variables are expressed as mean ± standard deviation. Count data are presented as numbers of cases and/or percentages (n/%) or skewed distribution [M(Q25, Q75)]. Continuous variables were tested for normality using the Kolmogorov-Smirnorv test. The Chi-square test, as well as Pearson or Spearman correlation analyses, were utilizied for two group comparisons. Comparisons between the measurement data of groups with skewed distributions were conducted using the rank sum test. Kruskal–Wallis one way analysis of variance (ANOVA) were utilized for multi-group comparisons. Binary Logistic regression was used to detect the correlation between serum DKK1 level and CHD. Multiple ordered logistic regression were used to explore the correlation between serum DKK1 level and the degree of coronary artery stenosis.
      All analyses were conducted using the Statistical Package for Social Sciences (IBM SPSSS tatistics for Windows, version 26.0, Armonk, NY). Statistical significance was set at a p < 0.05.

      3. Results

      3.1 Characteristics of the CHD and non-CHD groups

      We enrolled 206 CHD patients and 105 non-CHD individuals (controls). Characteristics of the two groups are presented (Table 2). The CHD group was older, with higher triglycerides (TG) and uric acid (UA) levels, as well as lower high density lipoprotein cholesterol (HDL-C) levels, than the control group. There were no significant differences between sex composition, diabetes prevalence, hypertension prevalence, SBP, DBP, and BMI of the CHD group and non-CHD groups.
      Table 2Comparison of Baseline data in CHD and non-CHD group.
      non-CHD (n = 105)CHD (n = 206)P
      Age (y)57.64 ± 10.0860.06 ± 8.680.028
      Gender (male/Female)50/55135/710.102
      Diabetes (%)14 (13%)60 (29%)0.134
      Hypertension (%)48 (46%)140 (68%)0.243
      SBP (mmHg)135.89 ± 19.52139.88 ± 19.370.087
      DBP (mmHg)79.77 ± 13.2080.52 ± 12.230.62
      BMI (kg/m2)25.06 ± 3.2325.25 ± 3.430.632
      TG (mmol/l)1.38 (1.02–2.0)1.70 (1.12–2.74)0.003
      TC (mmol/l)3.92 (3.48–4.8)3.96 (3.29–4.7)0.365
      FBG (mmol/l)6.34 (5.39–7.7)6.86 (5.37–9.12)0.072
      LDL-C (mmol/l)2.11 (1.74–2.59)2.05 (1.55–2.61)0.326
      HDL-C (mmol/l)1.18 (1.01–1.41)1.10 (0.95–1.29)0.004
      LPa (mmol/l)89.3 (18.2,141.6)106.65 (48.4,332.0)0.105
      APO-A (mmol/l)1.33 (1.06,1.57)1.18 (1.05,1.39)0.165
      APO-B (mmol/l)0.84 (0.66,1.01)0.60 (0.51,0.78)<0.05
      UA (μmol/l)296 (228–350)313 (262–380)0.033
      Ca (mmol/l)2.3 (2.23–2.36)2.29 (2.23–2.25)0.346
      ALP (U/l)73 (59–89)74 (61–86)0.96
      DKK1 (ng/ml)1.14 (0.46–2.03)5.66 (4.05–7.46)<0.05
      RBP4 (ng/ml)2.18 (1.35–2.75)11.81 (7.13–17.96)<0.05
      PAI1 (ng/ml)198.65 (113.10–316.31)364.85 (259.21–458.73)<0.05
      β-CTX (ng/ml)0.24 (0.16–0.42)0.23 (0.16–0.36)0.394
      OC (ng/ml)15.03 (10.35–19.28)12.53 (9.98–17.56)0.041
      25- (OH)-D (nmol/l)52.09 (41.57–62.75)51.18 (42.89–61.80)0.808
      Smoker number (%)33 (31.4%)111 (53.9%)<0.05
      Family history of premature cardiovascular and cerebrovascular disease (%)19 (18.1%)41 (19.9%)0.413
      History of cerebrovascular disease (%)7 (6.67%)42 (20.4%)0.01
      History of depression (%)2 (1.92%)0 (0)0.113
      subjects treated with antiplatelet agents99 (94.0%)206 (100%)0.01
      subjects treated with beta-blockers40 (38.1%)114 (55.3%)0.03
      subjects treated with RAS modulators21 (20%)84 (40.7%)<0.05
      subjects treated with statins98 (93.1%)206 (100%)<0.05
      Data are presented as mean (SD) or number (%) or mediam. SBP, Systolic blood pressure; DBP, Diastolic blood pressure; BMI, Body mass index; TG, Triglycerides; TC, Total cholesterol; FBG, Fasting blood glucose; LDL-C, Low-density lipoprotein cholesterol; UA, Uric acid; ALP, Alkaline phosphatase; DKK1, dickkopf-1; RBP4, Retinol-binding protein 4; PAI1, plasminogen activator inhibitor 1.
      Serum DKK1 concentration of the CHD group was significantly higher than that of the non-CHD group [5.66 (4.05–7.46) vs 1.14 (0.46–2.03) P < 0.05]; [Fig. 1]. Compared with the non-CHD group, RBP4 [11.81 (7.13–17.96) vs 2.18 (1.35–2.75), P < 0.05] and PAI-1 [364.85 (259.21–458.73) vs 198.65 (113.10–316.31), P < 0.05] were significantly higher in the CHD group, whereas OC [15.03 (10.35–19.28) vs 12.53 (9.98–17.56), P = 0.041] was lower in the CHD group. There were no significant differences between β-CTX and 25-(OH)-D of the two groups.
      Figure 1
      Figure 1Comparison of DKK1 concentrations between CHD and non-CHD groups. The central line represents the median of the distribution, the boxes span from 25th to 75th percentiles. Outliers (open circles) are defined as a score between 1.5 and 3 box lengths away from the upper edge of the box. Extreme scores (asterisks) are defined as a score that is greater than 3 box lengths away from the upper edge of the box. The concentration of DKK1 in CHD group were higher than that in non-CHD group, P < 0.05. DKKI, Dickkopf-1; CHD, Coronary heart disease.

      3.2 Elevated serum DKK-1 levels were increased with the degree of coronary artery stenosis

      The characteristics of the three subgroups were compared based on the severity of coronary artery stenosis [Table 3]. Compared with the low Gensini score group, the DKK1 level in the middle Gensini score group and the high-Gensini score group were significantly increased [Fig. 2].
      Table 3Comparison of Baseline data of three subgroups in CHD group.
      S1 (n = 70)S2 (n = 69)S3 (n = 67)P
      Age (y)59 (54∼64)62 (53∼65)61 (55∼68)0.323
      Gender (male/Female)47/2347/2241/260.658
      Diabetes (%)15 (21%)25 (36%)20 (30%)0.158
      Hypertension (%)47 (67%)46 (66%)47 (70%)0.895
      SBP (mmHg)137 (124∼150)142 (131∼157)137 (123∼155)0.170
      DBP (mmHg)81 (71∼88)84 (73∼93)79 (71∼87)0.182
      BMI(kg/m2)24.2 (23.1∼27.1)24.8 (22.9∼27.2)25.4 (23.5∼28.3)0.393
      TG (mmol/l)1.62 (1.09∼2.76)1.80 (0.99∼2.91)1.75 (1.14∼2.56)0.884
      TC (mmol/l)3.93 (3.34∼4.67)3.98 (3.20∼4.48)3.94 (3.22∼4.82)0.833
      FBG (mmol/l)6.72 (5.42∼9.03)6.57 (5.37∼8.86)7.22 (5.36∼9.50)0.783
      HDL-C (mmol/l)1.11 (0.96∼1.25)1.13 (0.96∼1.31)1.06 (0.93∼1.29)0.333
      LDL-C (mmol/l)1.91 (1.66∼2.53)2.06 (1.45∼2.51)2.06 (1.59∼2.72)0.601
      UA (μmol/l)308.8 (263.6∼391.8)315.4 (255.8∼360.5)310.0 (266.9∼387.0)0.632
      Ca (mmol/l)2.3 (2.24∼2.35)2.27 (2.23∼2.34)2.29 (2.23∼2.35)0.59
      ALP(U/l)70 (59∼85)74 (61∼92)73 (59∼89)0.66
      β-CTX (ng/ml)0.19 (0.12∼0.28)0.26 (0.16∼0.38)0.26 (0.17∼0.37)a0.288
      OC (ng/ml)12.46 (9.35∼17.10)12.09 (10.12∼17.26)13.10 (10.48∼18.33)0.027
      25-(OH)-D (nmol/l)53.85 (46.84∼66.10)49.70 (41.82∼58.22)48.49 (39.80∼60.86)a0.604
      DKK1 (ng/ml)4.61 (3.35∼6.38)6.13 (3.90∼7.57)6.45 (5.09∼7.76)<0.05
      RBP4 (ng/ml)7.51 (4.90∼14.62)15.38 (7.97∼21.41)13.27 (9.14∼16.74)<0.05
      PAI1 (ng/ml)256.63 (198.48∼326.9)398.65 (303.32∼465.26)456.06 (367.68∼515.90)<0.05
      Data are presented as mean (SD) or number (%) or median. SBP, Systolic blood pressure; DBP, Diastolic blood pressure; BMI, Body mass index; TG, Triglycerides; TC, Total cholesterol; FBG, Fasting blood glucose; LDL-C, Low-density lipoprotein cholesterol; UA, Uric acid; ALP, Alkaline phosphatase; β-CTX, β collagen specific sequences; 25-(OH)-D,25-hydroxyl vitamin D; OC, Osteocalcin; DKK1, dickkopf-1; RBP4, Retinol-binding protein 4; PAI1, plasminogen activator inhibitor 1.
      Figure 2
      Figure 2Comparison of the median concentrations of DKK1 in the three subgroups of the CHD group. 1, low Gensini score group; 2, middle Gensini score group, 3, high Gensini score. The central line represents distribution median, the boxes span from 25th to 75th percentiles. Outliers (open circles) are defined as a score that is between 1.5 and 3 box lengths away from the upper edge of the box. Extreme scores (asterisks) are defined as a score that is greater than 3 box lengths away from the upper edge of the box. A, B and C represent the comparison of the median concentrations of DKK1 in three subgroups of CHD group, respectively. In the high Gensini score group, the median concentrations of DKK1 were higher; P < 0.05.
      The high Gensini score group had higher OC. Correlation analysis between circulating levels of DKK1 and β-CTX, OC and 25-(OH)-D did not reveal any relevant correlations [Table 4]. No significant differences were detected among total cholesterol (TC), TG, low density lipoprotein cholesterol (LDL-C), fasting blood glucose (FBG), and UA of the three groups.

      3.3 Serum DKK1 concentration is correlated with serum RBP4 and PAI-1

      Serum RBP4 and PAI-1 levels in the CHD group were elevated. Compared with those of the low Gensini score group, the RBP4 and PAI-1 levels were significantly higher in the middle and high Gensini score groups[Table 3]. Serum DKK1 concentration was inversely correlated with serum PAI-1 (Rs = 0.083; P = 0.005) and RBP4 (Rs = 0.494; P < 0.001) concentrations [Table 4].
      Table 4Correlations between DKK1 and RBP4,PAI-1 and bone turnover markers.
      DKK1
      RsP
      RBP40.494<0.001
      PAI-10.0830.005
      β-CTX−0.0590.304
      OC−0.0460.417
      25-(OH)-D−0.0180.757

      3.4 DKK1 level was associated with degree of coronary artery stenosis

      When adjusted for age, gender, BMI, FBG and biochemical indexes, such as TG, TC, LDL-C, HDL-C, diabetes mellitus and hypertension history, logistic regression analysis showed that DKK1 was a risk factor for coronary heart disease (OR = 2.309; 95% CI 1.866–2.858; P < 0.001); [Table 5].
      Table 5The results of logistic regression analysis of factors for CHD and the multiple ordered logistic regression of risk factors for degree of coronary artery stenosis.
      Coronary heart diseaseCoronary artery stenosis
      ORP95% CIORP95%CI
      β-CTX0.8780.9250.059–12.9820.660.220.005–0.664
      25-(OH)-D1.0090.8220.936–1.0860.9950.6430.976–1.015
      OC0.9890.350.966–1.0121.1140.0011.046–1.186
      DKK12.309<0.0011.866–2.8581.1630.0121.035–1.309
      Adjustments were made for age, gender, BMI, FBG, TG, TC, LDL-C, HDL-C, history of diabetes mellitus and hypertension. The severity of coronary artery stenosis was evaluated according to their Gensini score. Gensini score was calculated using the modified scoring schema (Table 1).
      Multiple ordered logistic regression analysis, which was performed with age, gender, BMI, FBG, TG, TC, LDL-C, HDL-C, β-CTX, OC, 25- (OH)-D, and DKK1, diabetes and hypertension as independent variables, indicated that the levels of DKK1 (OR:1.163 [1.035, 1.309]; P = 0.012), and OC (OR:1.114 [1.046, 1.186]; P = 0.001) were associated with the degree of coronary artery stenosis. Further, the higher the level of DKK1, the more serious the coronary artery stenosis, and the coefficient was 0.151. When the level of DKK1 was increased by 1 ng/ml, the degree of increase in coronary artery stenosis was 1.163 [ Table 5].

      4. Discussion

      The results of our study indicated that serum DKK1 concentration in the CHD group was significantly higher than that in the non-CHD group. Binary logistic regression indicated that increased serum DKK1 levels were associated with coronary heart disease. We further investigated the association between DKK1 and coronary artery stenosis. Our results demonstrated that the DKK1 concentration in the high Gensini score group was significantly higher than that in the low Gensini score group, which indicated that the correlation between DKK1 concentration and coronary artery stenosis was a positive one. DKK1 concentration increased with the severity of coronary stenosis.
      DKK1 is highly expressed in macrophages and endothelial cells present in atherosclerotic lesions. In vivo and in vitro studies have investigated the role played by DKK-1 in the inflammatory interaction between platelets and endothelial cells as a major modulator of WNT signaling, in atherogenesis and plaque destabilization [
      • Ueland T.
      • Otterdal K.
      • Lekva T.
      • Halvorsen B.
      • Gabrielsen A.
      • Sandberg W.J.
      • et al.
      Dickkopf-1 enhances inflammatory interaction between platelets and endothelial cells and shows increased expression in atherosclerosis.
      ]. WNTs are secreted glycoproteins which are present in virtually all animals. WNT signaling includes the “canonical” WNT/β-catenin signaling pathway and the “non-canonical” planar cell polarity and WNT/Ca2+ signaling pathways. The WNT/β-catenin signaling pathway plays an important role in many biological processes. When WNT binds with Frizzled receptors and low-density lipoprotein receptor related protein (LRP) 5/6 coreceptors, the gene Disheveled (Dsh/Dvl, in Drosophila and vertebrates, respectively) is activated. Activation of Dvl inhibits glycogen synthase kinase-3β (GSK3) activity and disrupts the adenomatous polyposis coil/Axin/GSK3 complex, resulting in β-catenin accumulating in the cytoplasm, and subsequently translocating to the nucleus. In the nucleus, β-catenin binds to LEF/TCF thereby stimulating the expression of WNT target genes [
      • Foulquier S.
      • Daskalopoulos E.P.
      • Lluri G.
      • Hermans K.C.M.
      • Deb A.
      • Blankesteijn W.M.
      WNT signaling in cardiac and vascular disease.
      ]. The phosphorylation of LRP5/6 is a key step in the initiation of the WNT/β-catenin signaling. DKK1, which is a soluble glycoprotein, inhibits LRP5/6 and plays a key role in the regulation of the WNT signaling pathway. DKK1 directly inhibits WNT protein activity by competing with it to bind to the LRP receptor, or by indirectly binding to the LRP receptor via the kremen receptor, containing a kringle domain to form trimer, thereby reducing intracellular signal transmission by the WNT protein, which blocks the canonical WNT/β-catenin cytokine transmission pathway as well as the c-jun amino terminal kinase and WNT/plane cell polar transmission pathways [
      • Mao B.
      • Wu W.
      • Davidson G.
      • Li M.
      • Mechler B.M.
      • Delius H.
      • et al.
      Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling.
      ,
      • Mao B.
      • Wu W.
      • Li Y.
      • Hoppe D.
      • Stannek P.
      • Glinka A.
      • et al.
      LDL-receptor-related protein 6 is a receptor for Dickkopf proteins.
      ].
      Atherosclerosis is a common arteriosclerotic vascular disease. It forms the pathological basis of coronary heart disease, cerebrovascular disease and other ischemic cardiovascular and cerebrovascular diseases [
      • Viola J.
      • Soehnlein O.
      Atherosclerosis-A matter of unresolved inflammation.
      ]. It is an important factor associated with morbidity and mortality. Serum DKK1 played a role in the process of atherosclerosis [
      • He X.W.
      • Wang E.
      • Bao Y.Y.
      • Wang F.
      • Zhu M.
      • Hu X.F.
      • et al.
      High serum levels of sclerostin and Dickkopf-1 are associated with acute ischaemic stroke.
      ,
      • Gaudio A.
      • Privitera F.
      • Pulvirenti I.
      • Canzonieri E.
      • Rapisarda R.
      • Fiore C.E.
      The relationship between inhibitors of the Wnt signalling pathway (sclerostin and Dickkopf-1) and carotid intima-media thickness in postmenopausal women with type 2 diabetes mellitus.
      ]. Meanwhile, a large number of studies have shown that serum DKK-1 level is closely associated with atherosclerotic diseases, such as early myocardial infarction [
      • Goliasch G.
      • Wiesbauer F.
      • Kastl S.P.
      • Katsaros K.M.
      • Blessberger H.
      • Maurer G.
      • et al.
      Premature myocardial infarction is associated with low serum levels of Wnt-1.
      ,
      • Register T.C.
      • Hruska K.A.
      • Divers J.
      • Bowden D.W.
      • Palmer N.D.
      • et al.
      Plasma Dickkopf (DKK1) concentrations negatively associate with atherosclerotic calcified plaque in african americans with type 2 diabetes.
      ] and ischemic cerebrovascular disease [
      • Seifert-Held T.
      • Pekar T.
      • Gattringer T.
      • Simmet N.E.
      • Scharnagl H.
      • Fazekas F.
      • et al.
      Circulating Dickkopf-1 in acute ischemic stroke and clinically stable cerebrovascular disease.
      ].
      Endothelial dysfunction involving VSMCs and arterial calcification are the main factors associated with the mechanism underlying atherosclerosis. Many basic studies have confirmed the effect of DKK1 on endothelial cells, VSMCs and vascular calcification [
      • Zheng T.F.
      • Liu X.L.
      • Li X.
      • Wang Q.Q.
      • Zhao Y.C.
      • Li X.
      • et al.
      Dickkopf-1 promotes vascular smooth muscle cell proliferation and migration through upregulating UHRF1 during cyclic stretch application.
      ,
      • Motovska Z.
      • Vichova T.
      • Doktorova M.
      • Labos M.
      • Maly M.
      • Widimsky P.
      Serum Dickkopf-1 signaling and calcium deposition in aortic valve are significantly related to the presence of concomitant coronary atherosclerosis in patients with symptomatic calcified aortic stenosis.
      ,
      • Kim K.I.
      • Park K.U.
      • Chun E.J.
      • Choi S.I.
      • Youn T.J.
      • Cho G.Y.
      • et al.
      A novel biomarker of coronary atherosclerosis: serum DKK1 concentration correlates with coronary artery calcification and atherosclerotic plaques.
      ]. Disturbed oscillatory flow increases DKK1 expression of HUVECs by generating oscillatory shear stress (OSS). Knockdown or silencing DKK1 via lentiviral genes attenuates OSS-induced increase in monocyte adhesion and endothelial tight junction impairment, thereby attenuating atherogenesis in ApoE−/− mice [
      • Li M.
      • Liu X.
      • Zhang Y.
      • Di M.
      • Wang H.
      • Wang L.
      • et al.
      Upregulation of Dickkopf1 by oscillatory shear stress accelerates atherogenesis.
      ]. DKK-1 is associated with soluble CD40L, a marker reflecting platelet-mediated inflammation and asymmetric dimethylarginine (ADMA), a marker reflecting endothelial dysfunction, and urinary 11-dehydro-thromboxane B2, which is a marker of platelet activation in vivo [
      • Lattanzio S.
      • Santilli F.
      • Liani R.
      • Vazzana N.
      • Ueland T.
      • Di Fulvio P.
      • et al.
      Circulating dickkopf-1 in diabetes mellitus: association with platelet activation and effects of improved metabolic control and low-dose aspirin.
      ]. All of the studies shown that DKK1 facilitates inflammation, platelet activation, and endothelial dysfunction, and plays an important role in the development of atherosclerosis.
      RBP4 and PAI-1 play an important role in the occurrence and development of coronary heart disease. RBP4 expression is an important marker of inflammatory response [
      • Munkhtulga L.
      • Nakayama K.
      • Utsumi N.
      • Yanagisawa Y.
      • Gotoh T.
      • Omi T.
      • et al.
      Identification of a regulatory SNP in the retinol binding protein 4 gene associated with type 2 diabetes in Mongolia.
      ,
      • Farjo K.M.
      • Farjo R.A.
      • Halsey S.
      • Moiseyev G.
      • Maet J.X.
      Retinol-binding protein 4 induces inflammation in human endothelial cells by an NADPH oxidase and nuclear factor kappa B-dependent and retinol-independent mechanism.
      ], which is associated with early endothelial dysfunction. PAI-1 antigen, a single chain glycoprotein produced by endothelial cells and hepatocytes, is an effective inhibitor of plasminogen activation and fibrinolysis. PAI-1 expression was increased in atherosclerotic lesions [
      • Chandler W.L.
      Stratton JR Laboratory evaluation of fibrinolysis in patients with a history of myocardial infarction Am.
      ,
      • Schneiderman J.
      • Sawdey M.S.
      • Keeton M.R.
      • Bordin G.M.
      • Bernstein E.F.
      Dilley RB, et al.Increased typel plasminogen activator inhibitor gene expression in atherosclerotic human.
      ]. PAI-1 inhibits the clearance of fibrin mediated by plasminogen,the overexpression and release of PAI-1 promotes the deposition of fibrin on the arterial wall. Studies of pathological processes have indicated the presence of a large number of fibrin related peptides in coronary atherosclerosis related lesions, which promote the growth of plaques by stimulating the adhesion, migration, and proliferation of macrophages and smooth muscle cells as well as plaque growth, by combining with low-density lipoproteins. Therefore, decreased local fibrinolysis reduces the degradation and metastasis of fibrin. Chronic deposition of fibrin for long periods of time causes repetitive damage to blood vessels, facilitates the invasion of the fibrin matrix by fibroblasts, leads to collagen deposition and, together with a large number of accumulated matrix proteins, causes the fibrosis of tissues and rigidity of tube walls, thereby promoting the development of atherosclerosis [
      • Rerolle I.P.
      • Hertig A.
      • Nguyen G.
      • Rondeau E.P.
      Plasrninogen activator inhibitor 1 is a potential target in renal fibrogenesis.Kidney.
      ].
      Liu et al., found that compared with those of healthy people without coronary heart disease, the serum RBP4 concentrations of patients with coronary heart disease were significantly increased [
      • Liu Y.
      • Wang D.
      • Chen H.
      • Min X.
      Circulating retinol binding protein 4 is associated with coronary lesion severity of patients with coronary artery disease.
      ]. Our study showed similar results. Our study we also found that the concentrations of RBP4 and PAI-1 in the high Gensini score group were significantly higher than those in the low Gensini score group. One previous study confirmed that DKK1 exerted a beneficial effect by preventing atherosclerosis via inhibition of HMG-CoA reductase and non-steroidal isoprenoid intermediates. DKK-1 mediates the regulation of 21% of statin-modulated proteins, including PAI-1 [
      • Pontremoli M.
      • Brioschi M.
      • Baetta R.
      • Ghilardi S.
      • Banfi C.
      Identification of DKK-1 as a novel mediator of statin effects in human endothelial cells.
      ]. We found that serum DKKI concentration was associated with serum PAI-1 and RBP4 concentrations.
      Atherosclerosis and osteoporosis are coexisting clinical conditions affected by aging. They share the same pathophysiological mechanism [
      • Garcia-Martin A.
      • Reyes-Garcia R.
      • Garcia-Fontana B.
      • Morales-Santana S.
      • Coto-Montes A.
      • Munoz-Garach M.
      • et al.
      Relationship of Dickkopf1 (DKK1) with cardiovascular disease and bone metabolism in caucasian type 2 diabetes mellitus (correction).
      ,
      • Paschou S.A.
      • Anagnostis P.
      • Vryonidou A.
      • Goulis D.G.
      Diabetes and atherosclerosis: old players in a new field, osteoporosis.
      ]. Mineralization of extracellular matrix was observed in both atherosclerosis and osteoporosis. Cellular events that occur during bone metabolism are closely associated with atheromatous plaque [
      • Schweighofer N.
      • Aigelsreiter A.
      • Trummer O.
      • Graf-Rechberger M.
      • Hacker N.
      • Kniepeiss D.
      • et al.
      Direct comparison of regulators of calcification between bone and vessels in humans.
      ]. Thus, the hypothesis of “bone-cardiovascular axis” was developed [
      • Verheyen N.
      • Grübler M.R.
      • Catena C.
      • Fahrleitner-Pammer A.
      • van Ballegooijen A.J.
      The bone-cardiovascular Axis: mechanisms and clinical relevance.
      ]. OC is a marker of osteogenesis which is excreted by osteoblasts. Reportedly, the numbers of endothelial progenitor cells (EPC) co-expressing the osteoblastic marker osteocalcin [OC (+) EPC] were increased in patients with early and late coronary atherosclerosis. OC (+) EPCs were retained in coronary circulation [
      • Gössl M.
      • Mödder U.I.
      • Gulati R.
      • Rihal C.S.
      • Prasad A.
      • Loeffler D.
      • et al.
      Coronary endothelial dysfunction in humans is associated with coronary retention of osteogenic endothelial progenitor cells.
      ], and the number of circulating endothelial progenitor cells were correlated with the degree of calcification [
      • Collin J.
      • Gössl M.
      • Matsuo Y.
      • Cilluffo R.R.
      • Flammer A.J.
      • Loeffler D.
      • et al.
      Osteogenic monocytes within the coronary circulation and their association with plaque vulnerability in patients with early atherosclerosis.
      ]. Our study showed that OC levels in the CHD group were decreased compared with those of the non-CHD group. Multiple ordered logistic regression showed that OC was associated with the degree of coronary artery stenosis.
      β-CTX is a bone turnover marker, expressing osteoblast activity. It is also a biomarker of collagen turnover. Type I collagen, which is the predominant fibrillar collagen of the myocaridium, plays an important role in heart remodeling. Degradation of myocardial collagen results in ventricular dilation and ventricular stiffness reduction. Higher β-CTX levels are associated with a significantly higher risk of cardiovascular death/heart failure in patients with non-ST elevated acute coronary syndrome, over a median follow-up time of 12 months. β-CTX in the top quartile is associated with cardiovascular death and new or worsening heart failure [
      • Zelniker T.A.
      • Jarolim P.
      • Scirica B.M.
      • Braunwald E.
      • Park J.G.
      • Das S.
      • et al.
      Biomarker of collagen turnover (C-terminal telopeptide) and prognosis in patients with non-ST -elevation acute coronary syndromes.
      ]. In our study, no correlation was found between the β-CTX levels with CHD and between the β-CTX levels with the severity of coronary artery stenosis. The results demonstrate,as a part of type I collagen, β-CTX maybe more correlated with myocardial injury than simple vascular disease.
      There were some limitations in our study. First,our study was case-controlled, it was be set by certain limitations. Prospective follow-up observation is needed to further clarify the causal relationship between DKK1 and the degree of coronary artery stenosis. The mechanism underlying the effect exerted by DKK1 on atherosclerosis needs further investigation pertaining to the involvement of molecular biological and genetic factors. Second,the patients both with and without ACS were enrolled in the study,for there were only 12 CHD patients without ACS, so we did not do the subgroup analysis according to the subtypes of CHD to further investigate the difference in the relationship of DKK1,ACS and stable angina.

      5. Conclusions

      The severity of coronary stenosis increased with DKK1 concentration. The DKK1 levels of the middle and high Gensini score groups were significantly increased, compared with that of the low Gensini score group. The concentrations of RBP4 and PAI-1 in the CHD group were significantly higher than those in the non-CHD group. Serum DKKI concentration was associated with serum PAI-1 and RBP4 and PAI-1 concentrations. The levels of OC in the CHD group were decreased, compared with those of the non-CHD group. OC was associated with the degree of coronary artery stenosis. The level of serum DKK1 was increased in patients with coronary heart disease, which was positively correlated with the degree of coronary artery stenosis.

      Role of the funding source

      The study was supported by Hebei Provincial Department of Science and Technology, Technology Innovation Guidance Project-Science and Technology Work Conference ; Chengde Science and Technology Department, Science and Technology support program (No. 201606A063 ). The funders of the study had no role in the design of the study, data collection, data analysis or data interpretation and the article publication.

      Data statement

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

      Declaration of competing interest

      The authors declare no potential conflicts of interests with respect to research, authorship, and/or publication of this article.

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