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Preliminary observation of chemokine expression in patients with Stanford type A aortic dissection
Cytokine 127 (2020) 154920
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/cytokine
Preliminary observation of chemokine expression in patients with Stanford type A aortic dissection
T
Fudong Fan, Qing Zhou, Jun Pan, Qiang Wang, Hailong Cao, Yunxing Xue, Zhenjun Xu, ⁎ Dongjin Wang Department of Cardiothoracic Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Aortic dissection Chemokines Pathogenesis
Stanford type A Aortic dissection (TAAD) is a deadly cardiovascular disease but the relationship between inflammatory cytokines and disease pathogenesis is still unclear. Observation of the changes of different chemokines may help to explore the etiology of TAAD much further. Clinical data was collected from TAAD patients (TAAD group) and healthy controls (HC group) in our institute between October 2013 and December 2014. Blood sample was harvested from each subject of two groups. The expression levels of eighty chemokines were examined by protein array technology. Then we tested the expressions of macrophage inflammatory protein 1β (MIP-1β), epithelial neutrophil activating peptide 78 (ENA-78), interleukin 16 (IL-16), interferon inducible protein 10 (IP-10), and FMS-like tyrosine kinase 3 (Flt-3) ligand by using luminex technology. Osteopontin (OPN) and monocyte chemotaxis protein (MCP) levels were analyzed by ELISA kits. The mean age of TAAD group is 49.9 ± 11.2 and 48.7 ± 9.9 in HC group, respectively. 76.0% of TAAD patients and 72.0% of healthy controls were male. MIP-1β and ENA-78 expression in TAAD group were significantly lower than that in HC group, while significant increasing IL-16 level was found. Plasma levels of OPN in TAAD group increased remarkably compared with HC group, but MCP-1 and MCP-2 expression significantly decreased. No correlation was shown between serum CRP levels and plasma level of these cytokines by using Spearman analysis. ROC analysis showed that OPN could be indicators for TAAD diagnosis with sensitivity of 0.92 and specificity of 0.99. Our results provide a reasonable way to focus on the chemokines in understanding the pathogenesis of human TAAD.
1. Introduction Stanford type A Aortic dissection (TAAD) is one of dangerous cardiovascular diseases. Although surgery is considered to be the most effective method to save lives, the outcomes is still worse with high rates of morbidity and mortality. In fact, since victims before hospital admission might not be included in the database, the number of TAAD patients could be more than that we thought before [1]. Further etiological exploration is much necessary so as to reduce the incidence of TAAD and improve the clinical outcome. Several commonly diseases have been known to be risk factors of aortic dissection such as systemic hypertension, atherosclerosis, genetic and autoimmune disorders [2]. Abnormal inflammatory response and signal pathway widely exist in the pathogenesis of these diseases [3–5]. Logically speaking, there could be potential relationships between TAAD and inflammatory regulation. Focusing on cytokines expression may help to unveil the concrete formation of TAAD. In the past decade,
⁎
it had been found that a few proteins were involved in the onset and progression of aortic dissection as inflammatory mediators [6,7]. However, most of these studies were concentrated on C-reactive protein (CRP), metalloproteinase family and transforming growth factor-beta signal pathway [6–8]. The expression changes of chemotactic proteins in TAAD patients which might contribute to the formation of dissected vessels were still unclear. Therefore, in this study, we tried to sort out chemokines with abnormal expression by protein microarray and Luminex technique which maybe help to expand key proteins pool concerning the pathogenesis of aortic dissection. 2. Materials and methods 2.1. Patient selection Clinical data was collected from TAAD patients (TAAD group) and healthy controls (HC group) in the Affiliated Drum Tower Hospital of
Corresponding author. E-mail address: [email protected] (D. Wang).
https://doi.org/10.1016/j.cyto.2019.154920 Received 5 January 2019; Received in revised form 26 October 2019; Accepted 4 November 2019 1043-4666/ © 2019 Elsevier Ltd. All rights reserved.
Cytokine 127 (2020) 154920
F. Fan, et al.
Nanjing University Medical School between October 2013 and December 2014. All the patients without coronary artery disease and degenerative valvular heart disease were included. We ruled out the subjects with connective tissue diseases, autoimmune diseases or organ malperfusion and dysfunction. Healthy controls were all enrolled from medical examination center in the same hospital. All the subjects were excluded hypertension, diabetes, autoimmune disease, metabolic disease, coronary artery disease and valvular heart disease. This study was approved by the Ethics committee of the Affiliated Drum Tower Hospital of Nanjing University Medical School, and all subjects gave research authorization.
Table 1 The information on the clinical conditions of both TAAD and control groups.
2.2. Blood samples acquisition
TAAD, type A aortic dissection; HC, healthy control; BMI, body mass index; 2DM, type 2 diabetes
Two milliliters of venous blood were harvested from each subject in TAAD group when patients were admitted to our institution. And for HC group, we acquired same volume of blood sample from each person with fasting in the morning. Every blood sample was heparinized and centrifuged to extract plasma separately stored in −80 ℃.
Variable
TAAD group (n = 50)
HC group (=25)
Age (year) Gender (male/female) BMI (kg/m2) Hypertension (n, %) Hypertriglyceridemia (n, %) Hypercholesterolemia (n, %) Smoking history (n, %) Alcoholism history (n, %) 2-DM (n, %)
49.9 ± 11.2 38/12 24.6 ± 3.6 32, 64% 11, 22% 13, 26% 21, 42% 20, 40% 7, 14%
48.7 ± 9.9 18/7 22.8 ± 3.9 / / / / / /
76.0% of TAAD patients and 72.0% of healthy controls were male. Body mass index (BMI) were 24.6 ± 3.6 and 22.8 ± 3.9 in TAAD and HC group, respectively. There was no difference in age, gender and BMI between the two groups. In all the enrolled TAAD patients, 64% (32/ 50) were combined with hypertension, 22% (11/50) were combined with hypertriglyceridemia and 26% (13/50) were accompanied with hypercholesterolemia. The information on the clinical conditions of both TAAD and control groups have been shown in Table 1.
2.3. Protein array analysis We numbered all plasma samples of each group. Four plasma samples from TAAD group and four from HC group were randomly selected by computer. The expression levels of eighty chemokines were examined by protein array technology according to manual instructions (human cytokine array kit/AAH-CYT-G5-4, RayBiotech, Inc, Norcross, GA). And the data was analyzed by the supporting software.
3.2. Chemokine expression by protein array technology For the detection of different chemokine expressions, we used a protein assay and analyzed by fold change > 1.5 and < 0.66, as well as P < 0.05, then we found 34 factors that were significant differently expressed between TAAD patients and controls (Fig. 1).
2.4. Chemokines expression examination Chemokines expression in all plasma samples including macrophage inflammatory protein 1β (MIP-1β), epithelial neutrophil activating peptide 78 (ENA-78), interleukin 16 (IL-16), interferon inducible protein 10 (IP-10) and FMS-like tyrosine kinase 3 (Flt-3) ligand were analyzed by luminex technology following the kit protocol (Human Cytokine/Chemokine Magnetic Bead Panel kit/HCYTOMAG-60K/62K, Millipore Sigma, Burlington, MA). We tested the expression of osteopontin (OPN) and monocyte chemotaxis proteins (MCPs) in plasma by using the specific enzyme linked immunosorbent assay kit (ELISA, eBioscience, Thermo Fisher Scientific, Waltham, MA) according to the operative instructions.
3.3. MIP-1β, ENA-78, IL-16, IP-10 and Flt-3 ligand levels Plasma levels of MIP-1β and ENA-78 in TAAD group were significantly lower than that in HC group (MIP-1β: 40.8 ± 20.1 pg/ml vs. 68.3 ± 53.2 pg/ml, P < 0.01, Fig. 2A; ENA-78: 135.4 ± 127.9 pg/ml vs. 259.4 ± 197.1 pg/ml, P < 0.001, Fig. 2B), while obviously increased IL-16 level was found in TAAD group (137.8 ± 251.9 pg/ml vs. 17.2 ± 15.3 pg/ml, P < 0.05, Fig. 2C). Serum concentrations of IP-10 and Flt-3 ligand between TAAD group and HC group had no difference (IP-10: 487.4 ± 646.1 pg/ml vs. 319.8 ± 218.7 pg/ml, Fig. 2D; Flt-3 ligand: 11.4 ± 42.1 pg/ml vs. 36.7 ± 87.8 pg/ml, Fig. 2E, both P > 0.05). There was no correlation between these chemokines and serum CRP levels by Spearman analysis (data not shown).
2.5. Statistical analysis Categorical data are presented as frequencies and percentages and continuous data as the mean ± standard deviation. We used a t-test to make statistical analysis of continuous data for normal distributions and the Mann-Whitney U test for non-normal distributions. The MannWhitney test was used for analysis of categorical data. Spearman analysis was used to determine the correlations between cytokines and CRP levels. Statistical analyses were performed with SPSS version 16.0 and GraphPad Prism version 4.3 software packages. P values less than 0.05 were considered significant.
3.4. OPN expression It was shown that plasma levels of OPN in TAAD group increased significantly compared with HC group (234.9 ± 20.5 pg/ml vs. 25.2 ± 4.6 pg/ml, P < 0.001, Fig. 3). Spearman correlation analysis showed that there was no correlation between plasma OPN concentration and serum CRP level (data not shown).
3. Results 3.1. Enrolled subjects
3.5. MCPs expression
Fifty TAAD patients were eligible for this study. The time from symptoms onset to hospitalization is 5–79 h (average 13.4 ± 12.9 h). This is also the time lag between symptoms onset and blood sampling. Twenty-five healthy controls (HC group) were also included. All TAAD patients underwent surgical treatment and the time from hospitalization to operation is 2–72 h (average 7.9 ± 6.7 h). The mean age of TAAD group is 49.9 ± 11.2 and 48.7 ± 9.9 in HC group, respectively.
Plasma levels of MCP-1 and MCP-2 remarkably decreased in TAAD group compared to HC group (MCP-1: 318.9 ± 35.0 pg/ml vs. 420.3 ± 50.6 pg/ml, P < 0.05, Fig. 4A; MCP-2: 20.6 ± 8.0 pg/ml vs. 34.0 ± 18.3 pg/ml, P < 0.001, Fig. 4B), while MCP-4 had no change (45.1 ± 4.5 pg/ml vs. 51.5 ± 14.8 pg/ml, P > 0.05, Fig. 4C). No correlation between serum CRP levels and plasma MCPs concentrations by using the Spearman correlation analysis (data not shown). 2
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Fig. 1. Different expressed proteins between health controls (samples 1–4) and TAAD patients (samples 5–8).
evaluated whether the level of single-protein expression would allow us to discriminate patients with TAAD from healthy controls. As shown in Table 2, the area under the curve (AUC) for the expression level of OPN and IL-16 was 0.9898 and 0.7714 separately. The value of one minus AUC (1-AUC) for the expression level of MIP-1β, MCP-1, ENA-78 and
3.6. Diagnostic value of chemokines for TAAD patients The absence of association between these chemokines and CRP levels supported that the changed expression of chemokines might be constitutional for TAAD patients. Using ROC analysis, we then 3
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Fig. 2. MIP-1β, ENA-78, IL-16, IP-10 and Flt-3 ligand expressions and the relation to CRP level. A, TAAD patients showed significant lower levels of plasma MIP-1β compared to HC group. B, TAAD patients showed significant lower levels of plasma ENA-78 compared to HC group. C, plasma IL-16 significantly increased in TAAD patients. D, plasma IP-10 had no change between TAAD patients and HC. E, plasma Flt-3L showed no difference between TAAD and HC groups. *, P < 0.5; **, P < 0.01; ***, P < 0.001. Table 2 The area under the ROC curve of different chemokine expressions. Chemokine
AUC/1-AUC
95% CI
P value
OPN IL-16 MIP-1β MCP-1 ENA-78 MCP-2
0.9898 0.7714 0.6404 0.8020 0.8068 0.8364
0.9759–1.0000 0.6643–0.8784 0.4957–0.7851 0.7023–0.9016 0.6821–0.9315 0.7192–0.9536
0.0071 0.0446 0.0732 0.0514 0.0678 0.0581
OPN, Osteopontin; IL-16, interleukin 16; MIP-1β, macrophage inflammatory protein 1β; MCP-1, monocyte chemotaxis protein 1; ENA-78, epithelial neutrophil activating peptide 78; MCP-2, monocyte chemotaxis protein 2; ROC, receiver operating characteristic; AUC, area under curve.
Fig. 3. OPN expressions in TAAD patients. Plasma OPN significantly increased in TAAD patients compared to HC group. ***, P < 0.001.
MCP-2 was 0.6404, 0.8020, 0.8068 and 0.8364 respectively because the values of AUC for these chemokines expressions were all below 0.5. However, there were no significant differences in the AUC values for the expression levels of MIP-1β, MCP-1, ENA-78 and MCP-2. According to a rough guide for assessing the utility of a biomarker based on its ROC curve, OPN could be a good indicator for TAAD diagnosis, with a sensitivity of 0.92 and specificity of 0.99 (Fig. 5). 4. Discussion Further exploration of the TAAD pathogenesis is vital to reduce the mortality and improve clinical outcomes. Previous studies about TAAD showed that overexpression of MMPs in the media of aortic vessel wall lead to proteins degradation and dysfunction of vascular smooth muscle cells (VSMCs) [8]. The excessive activation of transforming growth factor-β (TGF-β) signaling pathway could result in the abnormal structure of aortic vessel tissue which may promote the initiation and progression of TAAD [1,9]. However, few articles were concentrating on the plasma chemokine expressions. In this study, the changes of several chemokine expression levels were found, suggesting the
Fig. 5. Area under the receiver operating characteristic curves (AUC) for detection of TAAD by reference to different chemokine expressions.
Fig. 4. MCPs expressions in TAAD patients. A, plasma MCP-1 markedly increased in TAAD patients compared to healthy subjects. B, plasma MCP-2 significantly decreased in TAAD patients compared to HC group. C, MCP-4 showed no difference between the two groups. **, P < 0.01; ***, P < 0.001. 4
Cytokine 127 (2020) 154920
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5. Conclusion
relationship between these proteins and TAAD formation. Unlike previous studies to detect some specific cytokines in patients’ plasma, we firstly used protein array analysis to screen changed chemokines among 80 proteins. The primary screening showed that there were significant differently expressed in 34 cytokines between TAAD patients and healthy controls (Fig. 1). Then we determined some chemokines by luminex technology (MIP-1β, ENA-78, IL-16, IP-10, and Flt3L) as well as ELISA (MCP-1, MCP-2, MCP-4 and OPN). At this step we furthermore confirmed that MIP-1β, ENA-78, MCP-1 and MCP-2 were significantly decreased, while OPN and IL-16 were remarkably increased in TAAD group. MIP-1β is one of proteins crucial for immune responses towards infection and inflammation that as well participates in the regulation of VSMCs function [10]. Some articles reported that the expression of MIP-1β increased in the people with hypertension and atherosclerotic arteries, which is associated with the higher cardiovascular events [3,11]. ENA-78 is one of the chemokines capable of activating neutrophil and preventing cells apoptosis [13]. In patients with idiopathic pulmonary fibrosis (IPF), ENA-78 was able to advance angiogenesis. When ENA-78 was depleted from IPF tissue specimens, angiogenic activity was significant attenuated [12]. ENA-78 was also involved in endothelial inflammation contributing to acute coronary syndromes (ACS). Utilization of atorvastatin could reduce ENA-78 expression in endothelial cells and improve prognosis of ACS patients [13]. Five members of the family of MCPs have been identified so far. MCP-1, MCP-2, MCP-3, MCP-4 and MCP-5 constitute a subfamily within the CC chemokines. In the primary screening and consequent verification, decreased MCP-1 and MCP-2 were found in TAAD patients. It seemed to be a paradox with what we found in the current study since several cardiovascular diseases such as hypertension and atherosclerotic were all risk factors of TAAD. We speculated that the lower plasma level of these proteins might be associated with higher expression in the aortic tissues. Presumably the aggregation of chemokines in vessel wall could reduce the amount in plasma. However, little publication disclosed the relationship between these cytokines and TAAD pathogenesis which need to be explored. OPN is not merely an essential protein to osteogenesis but also a mediator with diverse functions. OPN was closely associated with hypertension-induced vascular remodeling [14]. The increased OPN was found in patients with coronary and aortic atherosclerosis contributing to accelerate plaque progression [15,16]. It has been reported that as a synthetic mark, OPN was relevant to the functional phenotypic switching of aortic medial VSMCs. The expression of OPN in the vessel tissue of patients with aortic dissection was remarkably elevated [17]. Therefore, OPN could be a vital cytokine related to the functional regulation of VSMCs. IL-16 is a typical inflammatory cytokine, which could promote the production of IL-6, TNF-α and other cytokines. The significant increased OPN and IL-16 may predict the formation of TAAD. To verify this conclusion, we performed ROC analysis and finally found that there were significant statistical differences in OPN and IL16 based on the AUC. However, according to the ROC curve, OPN could be used as a biomarker for the diagnosis of TAAD with a sensitivity of 0.92 and specificity of 0.99. There were several limits in our article. Firstly, here is just an observational study about TAAD patients and healthy controls. So there must be inevitable bias in the enrollment of subjects. Secondly, because the period between symptoms onset and hospitalization in each TAAD patient was much different, inevitably, the time lag of blood samples was large, which might have some effects on the final results. Lastly, we could not acquire the aortic wall tissues from healthy people. The tissues from patients undergoing coronary arteries bypass grafting were not really normal and cannot be used as control. That was why we just analyzed the expression of chemokines in blood. The analysis of the tissues acquired from donors for heart transplantation and TAAD patients could promote further exploration of TAAD pathogenesis.
Our study found that plasma MIP-1β, ENA-78, MCP-1 and MCP-2 were significantly decreased but OPN and IL-16 were remarkably increased in TAAD patients, suggesting chemokines disorder in the pathogenesis of TAAD. OPN could be used as a biomarker for the diagnosis of TAAD. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments None. Sources of funding This study was supported by the Medical Science and Technology Development Foundation, Nanjing Department of Health, China (No. YKK13071), and the National Natural Science Foundation of China (No. 81800408). References [1] C.A. Nienaber, R.E. Clough, N. Sakalihasan, T. Suzuki, R. Gibbs, F. Mussa, et al., Aortic dissection, Nat. Rev. Dis. Primers 2 (2016) 16053, https://doi.org/10.1038/nrdp. 2016.53. [2] R.H. Mehta, T. Suzuki, P.G. Hagan, E. Bossone, D. Gilon, A. Llovet, et al., Predicting death in patients with acute type A aortic dissection, Circulation 105 (2002) 200–206, https:// doi.org/10.1161/01.cir.0000043546.26296.d2. [3] S. Cagnin, M. Biscuola, C. Patuzzo, E. Trabetti, A. Pasquali, P. Laveder, et al., Reconstruction and functional analysis of altered molecular pathways in human atherosclerotic arteries, BMC Genom. 10 (2009) 13, https://doi.org/10.1186/1471-2164-10-13. [4] A. Yadav, V. Saini, S. Arora, MCP-1: chemoattractant with a role beyond immunity: a review, Clin. Chim. Acta 411 (2010) 1570–1579, https://doi.org/10.1016/j.cca.2010.07. 006. [5] E.R. Gimba, T.M. Tilli, Human osteopontin splicing isoforms: known roles, potential clinical applications and activated signaling pathways, Cancer Lett. 331 (2013) 11–17, https://doi.org/10.1016/j.canlet.2012.12.003. [6] F. del Porto, M. Proietta, L. Tritapepe, F. Miraldi, A. Koverech, P. Cardelli, et al., Inflammation and immune response in acute aortic dissection, Ann. Med. 42 (2010) 622–629, https://doi.org/10.3109/07853890.2010.518156. [7] F. Luo, X.L. Zhou, J.J. Li, R.T. Hui, Inflammatory response is associated with aortic dissection, Ageing Res. Rev. 8 (2009) 31–35, https://doi.org/10.1016/j.arr.2008.08.001. [8] N. Cifani, M. Proietta, L. Tritapepe, C. Di Gioia, L. Ferri, M. Taurino, et al., Stanford-A acute aortic dissection, inflammation, and metalloproteinases: a review, Ann. Med. 47 (2015) 441–446, https://doi.org/10.3109/07853890.2015.1073346. [9] J.D. Humphrey, Possible mechanical roles of glycosaminoglycans in thoracic aortic dissection and associations with dysregulated transforming growth factor-β, J. Vasc. Res. 50 (2013) 1–10, https://doi.org/10.1159/000342436. [10] R. Cheung, M. Malik, V. Ravyn, B. Tomkowicz, A. Ptasznik, R.G. Collman, An arrestindependent multi-kinase signaling complex mediates MIP-1beta/CCL4 signaling and chemotaxis of primary human macrophages, J. Leukoc. Biol. 86 (2009) 833–845, https://doi. org/10.1189/jlb.0908551. [11] Y. Tatara, M. Ohishi, K. Yamamoto, A. Shiota, N. Hayashi, Y. Iwamoto, et al., Macrophage inflammatory protein-1beta induced cell adhesion with increased intracellular reactive oxygen species, J. Mol. Cell. Cardiol. 47 (2009) 104–111, https://doi.org/10.1016/j. yjmcc.2009.03.012. [12] M.P. Keane, J.A. Belperio, M.D. Burdick, J.P. Lynch, M.C. Fishbein, R.M. Strieter, ENA-78 is an important angiogenic factor in idiopathic pulmonary fibrosis, Am. J. Respir. Crit. Care Med. 164 (2001) 2239–2242, https://doi.org/10.1164/ajrccm.164.12.2104106. [13] I. Zineh, A.L. Beitelshees, G.J. Welder, W. Hou, N. Chegini, J. Wu, et al., Epithelial neutrophil-activating peptide (ENA-78), acute coronary syndrome prognosis, and modulatory effect of statins, PLoS One 3 (2008) e3117, https://doi.org/10.1371/journal. pone.0003117. [14] L.E. de Castro Brás, Osteopontin: a major player on hypertension-induced vascular remodeling, J. Mol. Cell Cardiol. 85 (2015) 151–152, https://doi.org/10.1016/j.yjmcc. 2015.05.020. [15] A. Mazzone, M.S. Parri, D. Giannessi, M. Ravani, M. Vaghetti, P. Altieri, et al., Osteopontin plasma levels and accelerated atherosclerosis in patients with CAD undergoing PCI: a prospective clinical study, Coron. Artery. Dis. 22 (2011) 179–187, https:// doi.org/10.1097/mca.0b013e3283441d0b. [16] Y. Momiyama, R. Ohmori, Z.A. Fayad, T. Kihara, N. Tanaka, R. Kato, et al., Associations between plasma osteopontin levels and the severities of coronary and aortic atherosclerosis, Atherosclerosis 210 (2010) 668–670, https://doi.org/10.1016/j.atherosclerosis. 2009.12.024. [17] L. Wang, J. Zhang, W. Fu, D. Guo, J. Jiang, Y. Wang, Association of smooth muscle cell phenotypes with extracellular matrix disorders in thoracic aortic dissection, J. Vasc. Surg. 56 (2012) 1698–1709, https://doi.org/10.1016/j.jvs.2012.05.084.
5
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/cytokine
Preliminary observation of chemokine expression in patients with Stanford type A aortic dissection
T
Fudong Fan, Qing Zhou, Jun Pan, Qiang Wang, Hailong Cao, Yunxing Xue, Zhenjun Xu, ⁎ Dongjin Wang Department of Cardiothoracic Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Aortic dissection Chemokines Pathogenesis
Stanford type A Aortic dissection (TAAD) is a deadly cardiovascular disease but the relationship between inflammatory cytokines and disease pathogenesis is still unclear. Observation of the changes of different chemokines may help to explore the etiology of TAAD much further. Clinical data was collected from TAAD patients (TAAD group) and healthy controls (HC group) in our institute between October 2013 and December 2014. Blood sample was harvested from each subject of two groups. The expression levels of eighty chemokines were examined by protein array technology. Then we tested the expressions of macrophage inflammatory protein 1β (MIP-1β), epithelial neutrophil activating peptide 78 (ENA-78), interleukin 16 (IL-16), interferon inducible protein 10 (IP-10), and FMS-like tyrosine kinase 3 (Flt-3) ligand by using luminex technology. Osteopontin (OPN) and monocyte chemotaxis protein (MCP) levels were analyzed by ELISA kits. The mean age of TAAD group is 49.9 ± 11.2 and 48.7 ± 9.9 in HC group, respectively. 76.0% of TAAD patients and 72.0% of healthy controls were male. MIP-1β and ENA-78 expression in TAAD group were significantly lower than that in HC group, while significant increasing IL-16 level was found. Plasma levels of OPN in TAAD group increased remarkably compared with HC group, but MCP-1 and MCP-2 expression significantly decreased. No correlation was shown between serum CRP levels and plasma level of these cytokines by using Spearman analysis. ROC analysis showed that OPN could be indicators for TAAD diagnosis with sensitivity of 0.92 and specificity of 0.99. Our results provide a reasonable way to focus on the chemokines in understanding the pathogenesis of human TAAD.
1. Introduction Stanford type A Aortic dissection (TAAD) is one of dangerous cardiovascular diseases. Although surgery is considered to be the most effective method to save lives, the outcomes is still worse with high rates of morbidity and mortality. In fact, since victims before hospital admission might not be included in the database, the number of TAAD patients could be more than that we thought before [1]. Further etiological exploration is much necessary so as to reduce the incidence of TAAD and improve the clinical outcome. Several commonly diseases have been known to be risk factors of aortic dissection such as systemic hypertension, atherosclerosis, genetic and autoimmune disorders [2]. Abnormal inflammatory response and signal pathway widely exist in the pathogenesis of these diseases [3–5]. Logically speaking, there could be potential relationships between TAAD and inflammatory regulation. Focusing on cytokines expression may help to unveil the concrete formation of TAAD. In the past decade,
⁎
it had been found that a few proteins were involved in the onset and progression of aortic dissection as inflammatory mediators [6,7]. However, most of these studies were concentrated on C-reactive protein (CRP), metalloproteinase family and transforming growth factor-beta signal pathway [6–8]. The expression changes of chemotactic proteins in TAAD patients which might contribute to the formation of dissected vessels were still unclear. Therefore, in this study, we tried to sort out chemokines with abnormal expression by protein microarray and Luminex technique which maybe help to expand key proteins pool concerning the pathogenesis of aortic dissection. 2. Materials and methods 2.1. Patient selection Clinical data was collected from TAAD patients (TAAD group) and healthy controls (HC group) in the Affiliated Drum Tower Hospital of
Corresponding author. E-mail address: [email protected] (D. Wang).
https://doi.org/10.1016/j.cyto.2019.154920 Received 5 January 2019; Received in revised form 26 October 2019; Accepted 4 November 2019 1043-4666/ © 2019 Elsevier Ltd. All rights reserved.
Cytokine 127 (2020) 154920
F. Fan, et al.
Nanjing University Medical School between October 2013 and December 2014. All the patients without coronary artery disease and degenerative valvular heart disease were included. We ruled out the subjects with connective tissue diseases, autoimmune diseases or organ malperfusion and dysfunction. Healthy controls were all enrolled from medical examination center in the same hospital. All the subjects were excluded hypertension, diabetes, autoimmune disease, metabolic disease, coronary artery disease and valvular heart disease. This study was approved by the Ethics committee of the Affiliated Drum Tower Hospital of Nanjing University Medical School, and all subjects gave research authorization.
Table 1 The information on the clinical conditions of both TAAD and control groups.
2.2. Blood samples acquisition
TAAD, type A aortic dissection; HC, healthy control; BMI, body mass index; 2DM, type 2 diabetes
Two milliliters of venous blood were harvested from each subject in TAAD group when patients were admitted to our institution. And for HC group, we acquired same volume of blood sample from each person with fasting in the morning. Every blood sample was heparinized and centrifuged to extract plasma separately stored in −80 ℃.
Variable
TAAD group (n = 50)
HC group (=25)
Age (year) Gender (male/female) BMI (kg/m2) Hypertension (n, %) Hypertriglyceridemia (n, %) Hypercholesterolemia (n, %) Smoking history (n, %) Alcoholism history (n, %) 2-DM (n, %)
49.9 ± 11.2 38/12 24.6 ± 3.6 32, 64% 11, 22% 13, 26% 21, 42% 20, 40% 7, 14%
48.7 ± 9.9 18/7 22.8 ± 3.9 / / / / / /
76.0% of TAAD patients and 72.0% of healthy controls were male. Body mass index (BMI) were 24.6 ± 3.6 and 22.8 ± 3.9 in TAAD and HC group, respectively. There was no difference in age, gender and BMI between the two groups. In all the enrolled TAAD patients, 64% (32/ 50) were combined with hypertension, 22% (11/50) were combined with hypertriglyceridemia and 26% (13/50) were accompanied with hypercholesterolemia. The information on the clinical conditions of both TAAD and control groups have been shown in Table 1.
2.3. Protein array analysis We numbered all plasma samples of each group. Four plasma samples from TAAD group and four from HC group were randomly selected by computer. The expression levels of eighty chemokines were examined by protein array technology according to manual instructions (human cytokine array kit/AAH-CYT-G5-4, RayBiotech, Inc, Norcross, GA). And the data was analyzed by the supporting software.
3.2. Chemokine expression by protein array technology For the detection of different chemokine expressions, we used a protein assay and analyzed by fold change > 1.5 and < 0.66, as well as P < 0.05, then we found 34 factors that were significant differently expressed between TAAD patients and controls (Fig. 1).
2.4. Chemokines expression examination Chemokines expression in all plasma samples including macrophage inflammatory protein 1β (MIP-1β), epithelial neutrophil activating peptide 78 (ENA-78), interleukin 16 (IL-16), interferon inducible protein 10 (IP-10) and FMS-like tyrosine kinase 3 (Flt-3) ligand were analyzed by luminex technology following the kit protocol (Human Cytokine/Chemokine Magnetic Bead Panel kit/HCYTOMAG-60K/62K, Millipore Sigma, Burlington, MA). We tested the expression of osteopontin (OPN) and monocyte chemotaxis proteins (MCPs) in plasma by using the specific enzyme linked immunosorbent assay kit (ELISA, eBioscience, Thermo Fisher Scientific, Waltham, MA) according to the operative instructions.
3.3. MIP-1β, ENA-78, IL-16, IP-10 and Flt-3 ligand levels Plasma levels of MIP-1β and ENA-78 in TAAD group were significantly lower than that in HC group (MIP-1β: 40.8 ± 20.1 pg/ml vs. 68.3 ± 53.2 pg/ml, P < 0.01, Fig. 2A; ENA-78: 135.4 ± 127.9 pg/ml vs. 259.4 ± 197.1 pg/ml, P < 0.001, Fig. 2B), while obviously increased IL-16 level was found in TAAD group (137.8 ± 251.9 pg/ml vs. 17.2 ± 15.3 pg/ml, P < 0.05, Fig. 2C). Serum concentrations of IP-10 and Flt-3 ligand between TAAD group and HC group had no difference (IP-10: 487.4 ± 646.1 pg/ml vs. 319.8 ± 218.7 pg/ml, Fig. 2D; Flt-3 ligand: 11.4 ± 42.1 pg/ml vs. 36.7 ± 87.8 pg/ml, Fig. 2E, both P > 0.05). There was no correlation between these chemokines and serum CRP levels by Spearman analysis (data not shown).
2.5. Statistical analysis Categorical data are presented as frequencies and percentages and continuous data as the mean ± standard deviation. We used a t-test to make statistical analysis of continuous data for normal distributions and the Mann-Whitney U test for non-normal distributions. The MannWhitney test was used for analysis of categorical data. Spearman analysis was used to determine the correlations between cytokines and CRP levels. Statistical analyses were performed with SPSS version 16.0 and GraphPad Prism version 4.3 software packages. P values less than 0.05 were considered significant.
3.4. OPN expression It was shown that plasma levels of OPN in TAAD group increased significantly compared with HC group (234.9 ± 20.5 pg/ml vs. 25.2 ± 4.6 pg/ml, P < 0.001, Fig. 3). Spearman correlation analysis showed that there was no correlation between plasma OPN concentration and serum CRP level (data not shown).
3. Results 3.1. Enrolled subjects
3.5. MCPs expression
Fifty TAAD patients were eligible for this study. The time from symptoms onset to hospitalization is 5–79 h (average 13.4 ± 12.9 h). This is also the time lag between symptoms onset and blood sampling. Twenty-five healthy controls (HC group) were also included. All TAAD patients underwent surgical treatment and the time from hospitalization to operation is 2–72 h (average 7.9 ± 6.7 h). The mean age of TAAD group is 49.9 ± 11.2 and 48.7 ± 9.9 in HC group, respectively.
Plasma levels of MCP-1 and MCP-2 remarkably decreased in TAAD group compared to HC group (MCP-1: 318.9 ± 35.0 pg/ml vs. 420.3 ± 50.6 pg/ml, P < 0.05, Fig. 4A; MCP-2: 20.6 ± 8.0 pg/ml vs. 34.0 ± 18.3 pg/ml, P < 0.001, Fig. 4B), while MCP-4 had no change (45.1 ± 4.5 pg/ml vs. 51.5 ± 14.8 pg/ml, P > 0.05, Fig. 4C). No correlation between serum CRP levels and plasma MCPs concentrations by using the Spearman correlation analysis (data not shown). 2
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Fig. 1. Different expressed proteins between health controls (samples 1–4) and TAAD patients (samples 5–8).
evaluated whether the level of single-protein expression would allow us to discriminate patients with TAAD from healthy controls. As shown in Table 2, the area under the curve (AUC) for the expression level of OPN and IL-16 was 0.9898 and 0.7714 separately. The value of one minus AUC (1-AUC) for the expression level of MIP-1β, MCP-1, ENA-78 and
3.6. Diagnostic value of chemokines for TAAD patients The absence of association between these chemokines and CRP levels supported that the changed expression of chemokines might be constitutional for TAAD patients. Using ROC analysis, we then 3
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Fig. 2. MIP-1β, ENA-78, IL-16, IP-10 and Flt-3 ligand expressions and the relation to CRP level. A, TAAD patients showed significant lower levels of plasma MIP-1β compared to HC group. B, TAAD patients showed significant lower levels of plasma ENA-78 compared to HC group. C, plasma IL-16 significantly increased in TAAD patients. D, plasma IP-10 had no change between TAAD patients and HC. E, plasma Flt-3L showed no difference between TAAD and HC groups. *, P < 0.5; **, P < 0.01; ***, P < 0.001. Table 2 The area under the ROC curve of different chemokine expressions. Chemokine
AUC/1-AUC
95% CI
P value
OPN IL-16 MIP-1β MCP-1 ENA-78 MCP-2
0.9898 0.7714 0.6404 0.8020 0.8068 0.8364
0.9759–1.0000 0.6643–0.8784 0.4957–0.7851 0.7023–0.9016 0.6821–0.9315 0.7192–0.9536
0.0071 0.0446 0.0732 0.0514 0.0678 0.0581
OPN, Osteopontin; IL-16, interleukin 16; MIP-1β, macrophage inflammatory protein 1β; MCP-1, monocyte chemotaxis protein 1; ENA-78, epithelial neutrophil activating peptide 78; MCP-2, monocyte chemotaxis protein 2; ROC, receiver operating characteristic; AUC, area under curve.
Fig. 3. OPN expressions in TAAD patients. Plasma OPN significantly increased in TAAD patients compared to HC group. ***, P < 0.001.
MCP-2 was 0.6404, 0.8020, 0.8068 and 0.8364 respectively because the values of AUC for these chemokines expressions were all below 0.5. However, there were no significant differences in the AUC values for the expression levels of MIP-1β, MCP-1, ENA-78 and MCP-2. According to a rough guide for assessing the utility of a biomarker based on its ROC curve, OPN could be a good indicator for TAAD diagnosis, with a sensitivity of 0.92 and specificity of 0.99 (Fig. 5). 4. Discussion Further exploration of the TAAD pathogenesis is vital to reduce the mortality and improve clinical outcomes. Previous studies about TAAD showed that overexpression of MMPs in the media of aortic vessel wall lead to proteins degradation and dysfunction of vascular smooth muscle cells (VSMCs) [8]. The excessive activation of transforming growth factor-β (TGF-β) signaling pathway could result in the abnormal structure of aortic vessel tissue which may promote the initiation and progression of TAAD [1,9]. However, few articles were concentrating on the plasma chemokine expressions. In this study, the changes of several chemokine expression levels were found, suggesting the
Fig. 5. Area under the receiver operating characteristic curves (AUC) for detection of TAAD by reference to different chemokine expressions.
Fig. 4. MCPs expressions in TAAD patients. A, plasma MCP-1 markedly increased in TAAD patients compared to healthy subjects. B, plasma MCP-2 significantly decreased in TAAD patients compared to HC group. C, MCP-4 showed no difference between the two groups. **, P < 0.01; ***, P < 0.001. 4
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5. Conclusion
relationship between these proteins and TAAD formation. Unlike previous studies to detect some specific cytokines in patients’ plasma, we firstly used protein array analysis to screen changed chemokines among 80 proteins. The primary screening showed that there were significant differently expressed in 34 cytokines between TAAD patients and healthy controls (Fig. 1). Then we determined some chemokines by luminex technology (MIP-1β, ENA-78, IL-16, IP-10, and Flt3L) as well as ELISA (MCP-1, MCP-2, MCP-4 and OPN). At this step we furthermore confirmed that MIP-1β, ENA-78, MCP-1 and MCP-2 were significantly decreased, while OPN and IL-16 were remarkably increased in TAAD group. MIP-1β is one of proteins crucial for immune responses towards infection and inflammation that as well participates in the regulation of VSMCs function [10]. Some articles reported that the expression of MIP-1β increased in the people with hypertension and atherosclerotic arteries, which is associated with the higher cardiovascular events [3,11]. ENA-78 is one of the chemokines capable of activating neutrophil and preventing cells apoptosis [13]. In patients with idiopathic pulmonary fibrosis (IPF), ENA-78 was able to advance angiogenesis. When ENA-78 was depleted from IPF tissue specimens, angiogenic activity was significant attenuated [12]. ENA-78 was also involved in endothelial inflammation contributing to acute coronary syndromes (ACS). Utilization of atorvastatin could reduce ENA-78 expression in endothelial cells and improve prognosis of ACS patients [13]. Five members of the family of MCPs have been identified so far. MCP-1, MCP-2, MCP-3, MCP-4 and MCP-5 constitute a subfamily within the CC chemokines. In the primary screening and consequent verification, decreased MCP-1 and MCP-2 were found in TAAD patients. It seemed to be a paradox with what we found in the current study since several cardiovascular diseases such as hypertension and atherosclerotic were all risk factors of TAAD. We speculated that the lower plasma level of these proteins might be associated with higher expression in the aortic tissues. Presumably the aggregation of chemokines in vessel wall could reduce the amount in plasma. However, little publication disclosed the relationship between these cytokines and TAAD pathogenesis which need to be explored. OPN is not merely an essential protein to osteogenesis but also a mediator with diverse functions. OPN was closely associated with hypertension-induced vascular remodeling [14]. The increased OPN was found in patients with coronary and aortic atherosclerosis contributing to accelerate plaque progression [15,16]. It has been reported that as a synthetic mark, OPN was relevant to the functional phenotypic switching of aortic medial VSMCs. The expression of OPN in the vessel tissue of patients with aortic dissection was remarkably elevated [17]. Therefore, OPN could be a vital cytokine related to the functional regulation of VSMCs. IL-16 is a typical inflammatory cytokine, which could promote the production of IL-6, TNF-α and other cytokines. The significant increased OPN and IL-16 may predict the formation of TAAD. To verify this conclusion, we performed ROC analysis and finally found that there were significant statistical differences in OPN and IL16 based on the AUC. However, according to the ROC curve, OPN could be used as a biomarker for the diagnosis of TAAD with a sensitivity of 0.92 and specificity of 0.99. There were several limits in our article. Firstly, here is just an observational study about TAAD patients and healthy controls. So there must be inevitable bias in the enrollment of subjects. Secondly, because the period between symptoms onset and hospitalization in each TAAD patient was much different, inevitably, the time lag of blood samples was large, which might have some effects on the final results. Lastly, we could not acquire the aortic wall tissues from healthy people. The tissues from patients undergoing coronary arteries bypass grafting were not really normal and cannot be used as control. That was why we just analyzed the expression of chemokines in blood. The analysis of the tissues acquired from donors for heart transplantation and TAAD patients could promote further exploration of TAAD pathogenesis.
Our study found that plasma MIP-1β, ENA-78, MCP-1 and MCP-2 were significantly decreased but OPN and IL-16 were remarkably increased in TAAD patients, suggesting chemokines disorder in the pathogenesis of TAAD. OPN could be used as a biomarker for the diagnosis of TAAD. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments None. Sources of funding This study was supported by the Medical Science and Technology Development Foundation, Nanjing Department of Health, China (No. YKK13071), and the National Natural Science Foundation of China (No. 81800408). References [1] C.A. Nienaber, R.E. Clough, N. Sakalihasan, T. Suzuki, R. Gibbs, F. Mussa, et al., Aortic dissection, Nat. Rev. Dis. Primers 2 (2016) 16053, https://doi.org/10.1038/nrdp. 2016.53. [2] R.H. Mehta, T. Suzuki, P.G. Hagan, E. Bossone, D. Gilon, A. Llovet, et al., Predicting death in patients with acute type A aortic dissection, Circulation 105 (2002) 200–206, https:// doi.org/10.1161/01.cir.0000043546.26296.d2. [3] S. Cagnin, M. Biscuola, C. Patuzzo, E. Trabetti, A. Pasquali, P. Laveder, et al., Reconstruction and functional analysis of altered molecular pathways in human atherosclerotic arteries, BMC Genom. 10 (2009) 13, https://doi.org/10.1186/1471-2164-10-13. [4] A. Yadav, V. Saini, S. Arora, MCP-1: chemoattractant with a role beyond immunity: a review, Clin. Chim. Acta 411 (2010) 1570–1579, https://doi.org/10.1016/j.cca.2010.07. 006. [5] E.R. Gimba, T.M. Tilli, Human osteopontin splicing isoforms: known roles, potential clinical applications and activated signaling pathways, Cancer Lett. 331 (2013) 11–17, https://doi.org/10.1016/j.canlet.2012.12.003. [6] F. del Porto, M. Proietta, L. Tritapepe, F. Miraldi, A. Koverech, P. Cardelli, et al., Inflammation and immune response in acute aortic dissection, Ann. Med. 42 (2010) 622–629, https://doi.org/10.3109/07853890.2010.518156. [7] F. Luo, X.L. Zhou, J.J. Li, R.T. Hui, Inflammatory response is associated with aortic dissection, Ageing Res. Rev. 8 (2009) 31–35, https://doi.org/10.1016/j.arr.2008.08.001. [8] N. Cifani, M. Proietta, L. Tritapepe, C. Di Gioia, L. Ferri, M. Taurino, et al., Stanford-A acute aortic dissection, inflammation, and metalloproteinases: a review, Ann. Med. 47 (2015) 441–446, https://doi.org/10.3109/07853890.2015.1073346. [9] J.D. Humphrey, Possible mechanical roles of glycosaminoglycans in thoracic aortic dissection and associations with dysregulated transforming growth factor-β, J. Vasc. Res. 50 (2013) 1–10, https://doi.org/10.1159/000342436. [10] R. Cheung, M. Malik, V. Ravyn, B. Tomkowicz, A. Ptasznik, R.G. Collman, An arrestindependent multi-kinase signaling complex mediates MIP-1beta/CCL4 signaling and chemotaxis of primary human macrophages, J. Leukoc. Biol. 86 (2009) 833–845, https://doi. org/10.1189/jlb.0908551. [11] Y. Tatara, M. Ohishi, K. Yamamoto, A. Shiota, N. Hayashi, Y. Iwamoto, et al., Macrophage inflammatory protein-1beta induced cell adhesion with increased intracellular reactive oxygen species, J. Mol. Cell. Cardiol. 47 (2009) 104–111, https://doi.org/10.1016/j. yjmcc.2009.03.012. [12] M.P. Keane, J.A. Belperio, M.D. Burdick, J.P. Lynch, M.C. Fishbein, R.M. Strieter, ENA-78 is an important angiogenic factor in idiopathic pulmonary fibrosis, Am. J. Respir. Crit. Care Med. 164 (2001) 2239–2242, https://doi.org/10.1164/ajrccm.164.12.2104106. [13] I. Zineh, A.L. Beitelshees, G.J. Welder, W. Hou, N. Chegini, J. Wu, et al., Epithelial neutrophil-activating peptide (ENA-78), acute coronary syndrome prognosis, and modulatory effect of statins, PLoS One 3 (2008) e3117, https://doi.org/10.1371/journal. pone.0003117. [14] L.E. de Castro Brás, Osteopontin: a major player on hypertension-induced vascular remodeling, J. Mol. Cell Cardiol. 85 (2015) 151–152, https://doi.org/10.1016/j.yjmcc. 2015.05.020. [15] A. Mazzone, M.S. Parri, D. Giannessi, M. Ravani, M. Vaghetti, P. Altieri, et al., Osteopontin plasma levels and accelerated atherosclerosis in patients with CAD undergoing PCI: a prospective clinical study, Coron. Artery. Dis. 22 (2011) 179–187, https:// doi.org/10.1097/mca.0b013e3283441d0b. [16] Y. Momiyama, R. Ohmori, Z.A. Fayad, T. Kihara, N. Tanaka, R. Kato, et al., Associations between plasma osteopontin levels and the severities of coronary and aortic atherosclerosis, Atherosclerosis 210 (2010) 668–670, https://doi.org/10.1016/j.atherosclerosis. 2009.12.024. [17] L. Wang, J. Zhang, W. Fu, D. Guo, J. Jiang, Y. Wang, Association of smooth muscle cell phenotypes with extracellular matrix disorders in thoracic aortic dissection, J. Vasc. Surg. 56 (2012) 1698–1709, https://doi.org/10.1016/j.jvs.2012.05.084.
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