DOI: https://doi.org/10.21203/rs.3.rs-1473329/v1
Background: Kawasaki disease (KD) has replaced rheumatic fever as the main cause of acquired heart disease in Japanese, American, and Chinese children. Polymorphisms in angiotensin-converting enzyme (ACE) may be associated with susceptibility to KD, but the association of ACE2 with vascular endothelial injury in KD and the possibility for prognosis of vascular injury in KD by evaluating changes serum ACE2 has not yet been assessed. Thus, this study aimed to investigate angiotensin-converting enzyme 2 (ACE2) levels in patients with KD to further investigate the relationship between ACE2 and vascular injury in KD.
Methods: Blood samples were collected from 49 children with KD before intravenous immunoglobulin treatment and 28 healthy children as a control group. Clinical data were collected from the patients and serum ACE2 levels of all participants were measured using enzyme-linked immunosorbent assay.
Results: Serum ACE2 levels were significantly higher in the KD group than in the control group. Serum ACE2 levels were negatively correlated with platelet count (Plt) levels in patients with KD.
Conclusions: Serum ACE2 levels may be related to the severity of coronary artery damage in children with KD.
KD is a systemic vasculitic disease in children under 5 years of age. Clinically known as mucocutaneous lymph node syndrome, which is typically characterized by persistent fever (for more than 5 days), diffuse inflammation of the mucosa (such as dry red rhagadia of the lips and bayberry tongue), non-suppurative conjunctivitis, large non-suppurative cervical lymph nodes (> 1.5 cm in diameter), polymorphic rash, and angioneurotic edema of the extremities[1, 2]. Approximately 25% of untreated children with KD experience coronary artery lesions (CAL), and KD has replaced rheumatic fever as the main cause of acquired heart disease in children in Japan, the United States, and China. Studies have demonstrated that damage to the vascular endothelium occurs in KD[3, 4].
ACE2, a congenic compound of angiotensin-converting enzyme, is expressed on all endothelial cells and also ubiquitously expressed in cardiomyocytes, cardiac fibroblasts, and coronary endothelial cells[5]. Studies have shown that local overexpression of ACE2 improves endothelial function and significantly inhibits the development of early atherosclerosis[6]. In addition, it has been shown that overexpression of ACE2 in Endothelial progenitor cells (EPC) can improve their function by inhibiting apoptosis and oxidative stress[7]. ACE, another M2 family protein belonging to the same metalloprotease family as ACE2, has shown reduced expression in children with KD and significantly increased activity after the application of intravenous immunoglobulin (IVIG) therapy[8]. Polymorphisms in ACE gene may be associated with susceptibility to KD[9]. Therefore, we speculated that ACE2 may also be associated with vascular endothelial injury in KD, and the prognosis of vascular injury in KD can be evaluated by detecting changes ACE2 in serum.
However, the relationship between ACE2 and KD has not yet been studied. Therefore, we investigated the relationship between serum ACE2 levels and coronary artery injury in patients with KD.
Patients were included in the study according KD diagnosis based on diagnostic criteria from the American Heart Association’s 2017 KD guidelines [10]. A total of 49 children (30 boys and 19 girls, mean age: 32 months) with acute febrile KD at the Maternal and Child Health Hospital of Hubei Province were selected, and 28 children of similar age with normal physical examinations (15 boys and 13 girls, mean age: 25 months) were selected as the control group. Children with immune diseases, metabolic diseases, inflammatory diseases, hematological diseases, severe liver and kidney diseases, and other heart diseases were excluded from this study. Blood samples were collected from all patients before and after intravenous immunoglobulin (IVIG) therapy, and CAL was determined by the Z value and lesion surface area from echocardiographic parameter results measured one day before IVIG treatment. Patients with Z values < 2 were included in the KD- no cardiac artery lesions (nCAL) group, those with Z values > 2 were included in the KD- cardiac artery lesions group, and those with Z values > 2.5 were included in the KD with coronary artery aneurysm (KD-CAAs) group [11, 12]. Informed consent was obtained from the guardians of each participant, and the study was approved by the Medical Ethics Committee of the Maternal and Child Health Hospital of Hubei Province.
Blood samples from patients with KD were collected before and after IVIG treatment, while blood samples from the healthy control group were collected during routine health visits. The collected venous blood samples were centrifuged at 1500 rpm for 5 min, and the supernatant was collected, labeled, aliquoted, and stored at -80°C until testing.
ACE2 serum concentrations were measured in all participants using enzyme-linked immunosorbent assay kits (R&D, USA) according to the manufacturer's instructions. All samples were analyzed in duplicate. Clinical parameters, including white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hb), Plt, neutrophil percentage (N), lymphocyte percentage (L), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), aspartate aminotransferase (AST), alanine aminotransferase (ALT), procalcitonin (PCT) and creatine kinase-MB (CK-MB), were also collected.
According to the Kolmogorov-Smirnov test, the data from this study conformed to a normal Gaussian distribution. All data are shown as mean ± Standard error of mean (SEM) or number and percentage (n, %). Differences between the groups were assessed using an unpaired 2-tailed t-test. All statistical analyses were performed using SPSS software (version 21.0; SPSS, Inc., Chicago, IL, USA). Statistical significance was set at p < 0.05.
There was no significant difference in age or sex between the KD with control groups (unpublished data). In addition, 49 patients with KD were further divided into two groups according to the Z values into CAL groups: KD with CAL (KD-CAL, n = 22) and KD without CAL (KD-nCAL, n = 27), and CAA groups: KD with CAA (KD-CAA, n = 16) and KD without CAA (KD-nCAL, n = 33).
Serum ACE2 levels were measured between 0.03 and 67.44 pg/ml. As shown in Fig. 1.a, serum ACE2 concentration was significantly higher in the KD group (6.56 ± 1.52 pg/ml) than in the control group (1.21 ± 0.28 pg/ml) (p = 0.001). Figure 1b shows that ACE2 levels were higher in the KD-CAL group (9.65 ± 2.91 pg/ ml) compared with those of the KD-nCAL group (4.05 ± 1.28 pg/ml) (p = 0.089), but the difference was not statistically significant. Figure 1c shows that ACE2 levels were higher in the KD-CAA group (9.58 ± 3.43pg/ml) compared with those of the KD-NCAA group (5.10 ± 1.51 pg/ml) (p = 0.171), but the difference was not statistically significant. Figure 1d shows that ACE2 content after IVIG (2.48 ± 2.46 pg/ ml) was lower than prior to IVIG (3.79 ± 3.92 pg/ml), but the difference was not statistically significant (p = 0.294).
There was no significant correlation between ACE2 serum levels and white blood cells, red blood cells, hemoglobin, platelets, CRP, ALT, AST, L%, N%, LDH, or CK-MB (p > 0.05); however, serum ACE2 levels were negatively correlated with Plt levels in patients with KD (p < 0.05). (Table 1)As shown in the table below, the levels of CK-MB in the KD-CAL group were significantly higher than those in the nCAL group; however, the other clinical data were not statistically different between the two groups.(Table 2)
|
ACE2 |
||
|---|---|---|
|
r |
p |
|
|
WBC (103/uL) RBC (103/uL) Hb (g/L) Plt (103/uL) CRP (mg/dL) ESR (mm/h) ALT (U/L) AST (U/L) L% N% Ck-Mb (U/L) PCT (ng/ml) LDH (U/L) |
-0.252 -0.064 -0.036 -0.307 -0.109 0.103 -0.068 -0.049 0.213 -0.191 -0.100 -0.06 -0.002 |
0.081 0.693 0.805 0.032* 0.466 0.492 0.642 0.739 0.176 0.237 0.502 0.715 0.991 |
| Abbreviations: WBC, white blood cell; RBC, red blood cell; Hb, hemoglobin; Plt, platelet count, CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; ALT, alanine transferase; AST, aspartate aminotransferase; L%, lymphocyte percentage; N%, neutrophil percentage; Ck-Mb, creatine kinase-MB; PCT, procalcitonin; LDH, lactate dehydrogenase | ||
|
KD-CAL |
KD-nCAL |
P |
|
|---|---|---|---|
|
WBC (103/ul) RBC (103/ul) Hb (g/L) CRP (mg/dL) N% L% Plt (103/ul) ALT (U/L) AST (U/L) LDH (U/L) CK-MB (U/L) PCT (ng/ml) ESR (mm/h) |
15.16 ± 4.71 4.00 ± 0.44 105.50 ± 12.14 54.20 ± 36.90 68.23 ± 18.61 24.56 ± 15.39 399.05 ± 161.40 55.16 ± 53.43 39.58 ± 25.64 84.82 ± 66.50 0.92 ± 0.95 5.15 ± 8.10 70.86 ± 23.89 |
14.25 ± 6.28 3.99 ± 0.36 108.93 ± 8.95 73.33 ± 66.92 64.91 ± 19.14 27.67 ± 15.95 370.81 ± 107.92 53.66 ± 84.28 42.26 ± 41.65 57.59 ± 64.15 0.20 ± 0.22 1.39 ± 2.08 72.76 ± 29.62 |
0.578 0.928 0.262 0.247 0.583 0.525 0.468 0.943 0.793 0.281 0.027* 0.052 0.812 |
| Abbreviations: WBC, white blood cell; RBC, red blood cell; Hb, hemoglobin; CRP, C-reactive protein; L%, lymphocyte percentage; N%, neutrophil percentage; Plt, platelet count; ALT, alanine transferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase; Ck-Mb, creatine kinase-MB; PCT, procalcitonin; ESR, erythrocyte sedimentation rate | |||
ACE2 has been the first congenic compound of an angiotensin-converting enzyme discovered in recent years[13]. It is an important member of the renin-angiotensin system, which plays an important pathological role in cardiovascular and cerebrovascular diseases[14, 15][16]. Mainly expressed in the cardiovascular system, it can convert Angiotensin II to Angiotensin (1–7)[17, 18]; reduce macrophage infiltration; reduce monocyte MCP-1, IL-6, TNF-α, nuclear factor-kappaB (NF-κB), VCAM-1 and ROS levels; inhibit apoptosis, and increase NO release. It protects the endothelial cells and prevents the development of atherosclerotic plaques in vivo[19] and can regulate vascular function by regulating the release of nitric oxide and oxidative stress[20, 21]. Several studies have demonstrated the role of ACE2 in vascular injury. However, the potential role of ACE2 in KD has not yet been reported; therefore, the main purpose of this study was to detect the expression of ACE2 in children with KD and investigate the relationship between serum ACE2 levels and coronary artery injury in patients with KD. Our results showed that serum ACE2 levels were significantly higher in patients with KD than in the controls. Serum ACE2 levels in patients with KD were also positively correlated with CK-MB.
ACE2 is mainly present on the vascular endothelium, and in children with KD, it is also shed from the vascular endothelium into the serum, which in turn causes an increase in ACE2 in the serum. This finding is similar to that of ACE2 in coronary atherosclerosis and hypertensive heart disease [5, 22] [23] can induce endothelial cell apoptosis [19]and activate NF-κB through myeloid differentiation gene response 88- dependent and -independent pathways by upregulating the expression of Toll-like receptor 4 (TLR4) on the surface of dendritic cells, initiating inflammatory cytokine transcription, mediating inflammatory mediator secretion, apoptosis, and producing oxygen-free radicals production [24]. The mRNA expression of TLR4 and the levels of its related factors in children in the acute phase of KD are significantly higher than those in normal children [25]. In this study, the content of ACE2 in the serum of children with KD was significantly increased, which indicated that the up-regulation of serum ACE2 may be the mechanism of vascular injury and inflammation in the acute phase KD. In addition, the level of ACE2 in KD-CAL and KD-CAA was higher than that in the control group; however, this difference was not statistically significant, which may have been related to the small number of samples or the expression of ACE2 on a variety of cells. One recent study [26] has demonstrated that the expression of ACE2 is mainly in the endothelial tissues of arteries, arterioles, heart, and kidney, but is also found on the vascular smooth muscle of tubular epithelial cells, intrarenal arteries, and coronary vessels. This indicates that ACE2 may be insensitive and specific for the formation of CAL in KD patients; however, ACE2 may lead to the formation of CAL, and our study found that the level of ACE2 in KD-CAA was also increased. Its increasing trend was more obvious than that in CAL group, which may be because the vascular injury and aneurysm formation in KD is caused by inflammatory factors and oxidative stress. ACE2 has been reported to have antioxidant effects in most vascular injuries [20, 27]; hence, we speculate that the negative results of this study may have been related to the small sample size.
In this study, correlation analysis showed a negative correlation between serum ACE2 and platelet levels in patients with KD. In general, the platelet count of children with KD gradually increased from the first week of onset, peaked in the 2nd–3rd week of onset, and then gradually decreased. Platelet activation level was enhanced and closely related to cardiovascular injury and mortality. The increase in platelet activation level may be involved in coronary artery injury in children with KD, and 1% – 2% of children with KD had decreased platelets after onset, suggesting that those with thrombocytopenia were prone to CAA[28]. In children with KD, ACE2 on the vascular endothelium sheds into the serum and loses the effect of antagonizing platelets, further leading to platelet aggregation on the vascular endothelium. This in turn induces the formation of thrombosis on the vascular endothelium, which leads to vasodilatation and vascular endothelial injury, indicating that ACE2 may be related to vascular endothelial injury and the formation of hemangioma.
In addition, our study found that the CK-MB levels in the KD-CAL group were significantly higher than those in the KD-NCAL group. CK-MB is a marker for clinical judgment of myocarditis and myocardial injury; its increase is associated with the formation of coronary artery aneurysms [29, 30]. However, the level of ACE2 was also significantly increased in the KD-CAL group, suggesting that ACE2 may lead to the formation of CAL, leading to vascular endothelial injury. However, our study did not find a specific link between ACE2 and CK-MB in the KD-CAL group, which needs to be confirmed in further studies.
In conclusion, this study provides the first evidence that serum ACE2 levels are significantly increased and are correlated with platelet count in patients with KD. These results suggest that high ACE2 levels may play a major role in the inflammatory response and vascular injury in the acute phase of KD. However, whether and how ACE2 can cause CAL, and the association of ACE2-related genes with vascular endothelial injury needs to be further studied.
Acknowledgements
We thank the patients and their family for their contribution. All authors read and approved the final manuscript including the authorship list.
Funding
This research was funded by the Hubei Provincial Natural Science Foundation (Grant Number:2021CFB558 ) and Hubei Province health and family planning scientific research project(Grant Number:WJ2018H0142). Hubei Provincial Natural Science Foundation and Hubei Province health and family planning scientific research project were not directly involved in study design, collection, analysis, and interpretation of the data, writing of the manuscript, or the decision to submit the article for publication.
Competing interests
The authors have no relevant financial or non-financial interests to disclose.
Author contributions
Yi Gan, Junhua Shu and Xiaoqin Zhou contributed to the study conception and design. Material preparation, data collection and analysis were performed by Yi Gan, Yawei Feng, Heng Li, Guirong Wang and Maidina Anni. Danna Tu supervised the whole process of experiment design, data analysis, article writing, and submission. The first draft of the manuscript was written by Yawei Feng and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Ethics approval
This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Medical Ethics Committee of the Maternal and Child Health Hospital of Hubei Province (2021/XM010).
Consent to participate
Informed consent was obtained from all individual participants included in the study.
Consent to publish
The authors affirm that human research participants provided informed consent for publication of the data.