The relationship between endothelial progenitor cells and pulmonary hypertension in children with congenital heart disease CURRENT STATUS:

Background: To assess the relationship between endothelial progenitor cells (EPCs) in peripheral blood and pulmonary hypertension (PH) in children with congenital heart disease (CHD) and explore the possibility of applying EPCs to treatment of PH in children with CHD. Methods: In this study, a total of 173 cases with CHD (from 0 to 6 years old) were collected. According to the right heart catheterization of mean pulmonary arterial pressure (mPAP) results, these cases were divided into PH groups (including high PH group, n=32, and the middle PH group, n=30) and non-PH group (n=111). We took their peripheral blood for flow cytometry, and counted the number of EPCs(CD133+/KDR+ cells). The number of EPCs /μL of peripheral blood was calculated using the following formula: EPCs /μL =WBC /L × lymphocytes % × EPCs % × 10-6. Results: The median EPCs of the non-PH group, middle PH group and high PH group are 1.86/μL, 1.30 / μL and 0.98/μL, respectively. The mPAP decreases steadily as the level of EPCs increases (P<0.05). After adjustment of gender, age and BMI, the EPCs was significantly associated with a decreased risk of high PH (OR=0.37, 95%CI: 0.16-0.87, P<0.05). However, EPCs was not significantly associated with middle PH (P>0.05). Conclusion: The findings revealed that the EPCs and high PH in CHD patients correlate significantly and EPCs may become an effective treatment for PH in CHD patients.EPCs may be a protective factor of high PH for children with CHD.

Pulmonary hypertension (PH) is a common clinical disease which can exist independently. It is mainly the manifestation of many diseases when they progress to a certain stage, namely secundum PH [1].
In clinic, left-to-right shunt congenital heart disease (CHD) will increase the right cardiac blood flow, thus damaging the pulmonary artery and causing PH [2]. The statistics show that about 4%-10% of CHD patients develop PH [3][4][5][6][7]. The global prevalence of PH is about 1%, and 80% of the patients live in developing countries. The prevalence of CHD-related PH is about 25 people per million in the general population. The deaths of patients with both PH and CHD are at least twice as many as those without PH [1]. PH is very common in children, mainly due to CHD [2,7,8].
In 1997, Asahara et al. [9] first isolated a kind of cells from human peripheral blood that could be cultured into endothelial cells in vitro. Stimulated by various factors, these cells entered the peripheral blood from the bone marrow to participate in the formation of new blood vessels and repair the damaged endothelial cells. These cells were called endothelial progenitor cells (EPCs). EPCs are bone-marrow-derived cells with the ability to differentiate into endothelial cells, repair and regenerate damaged pulmonary blood vessels. Previous experimental studies have found that EPCs can be applied to the treatment of lung injury [10][11][12]. There have been many studies demonstrating that the number of EPCs is related to PH, but the results are inconsistent [13][14][15][16]. To our knowledge, there are few studies on the relationship between EPCs and PH in children with CHD. The purpose of this study was to investigate the relationship between the number of EPCs and PH in children with CHD and to explore the possibility of applying EPCs to treatment of PH in children with CHD.

Measurements
According to the "2010 ESC Guidelines for the Management of Grown-up Congenital Heart Disease", the diagnosis of CHD was based on echocardiography [17]. Mean pulmonary arterial pressure (mPAP) was measured by right heart catheterization. According to "the 6th World Symposium on Pulmonary Hypertension" [18], patients were divided into PH groups (including high PH group and the middle PH group) and the non-PH group (n = 111). Non-PH was defined as mPAP<20mmHg (111 patients); the middle PH was defined as 20mmHg ≤mPAP≤25mmHg (30 patients) and the high PH was defined as mPAP>25mmHg (32 patients). were applied for each sample as a negative control. After incubation, the lysing solution (MasterTech, USA) was used to eliminate erythrocytes cells. Horizontal centrifuge (Shanghai anting China), 1200 RPM was employed to centrifuge the samples 3 times. Finally, the number of CD133+/KDR+ cells was counted. The number of monocytes in human peripheral blood is about 0.30-0.80×10 9 /L; the proportion of EPCs is about 0.01 %; the number of EPCs is estimated to be 0.30-1.00 /μL [19,20].
Thus, the number of EPCs < 1.00 /μL was used as the control. The number of EPCs /μL of peripheral blood was calculated by using the following formula: EPCs/μL = WBC/L × lymphocytes% × EPCs% × 10 -6 [9] . Body mass index (BMI) was calculated as the subject's weight (kg) divided by the height squared (meters).

Statistical analysis
The data were expressed as the mean ± standard deviation for normally distributed continuous variables and compared by one-way analyses of variance. Median values (inter-quartile range) were used and compared by Mann-Whitney U test when the parameters exhibited skewed deviations. The number and proportions in each subgroup of categorical variables were compared by the Mann-Whitney U test. The multiple comparisons in each subgroup were made by Bonferroni test or Student-Newman-Keuls test. A multinomial logit analysis was performed to explore relationships between the number of EPCs and PH severity after adjustment of age, gender, and BMI. All P-values less than 0.05 were considered to be statistically significant. All of the analyses were performed using SAS 9.4 (SAS Institute Inc., Cary, NC, USA).

Results
In the present study, a total of 173 CHD patients met all analysis criteria, including 111 (64.16%) non-PH patients, 30(17.34%) middle PH patients and 32(18.50%) high PH patients. The general characteristics of the non-PH group, middle PH group and high PH group are shown in Table 1. The median age of the non-PH patients, middle PH patients and high PH patients are 2 years, 2 years, and 0 years, respectively. The median age was lower in the high PH group than in the middle PH and non-PH groups(Student-Newman-Keuls test, all P<0.05). BMI, WBC, and lymphocyte are similar in the three subgroups (all P>0.05). The proportions of EPCs>1.00/μL of the non-PH group, middle PH group and high PH group are 70.27, 70.00 and 50.00, respectively, and the proportions of EPCs is significantly reduced among the high PH patients (P = 0.05). With the non-PH group as a reference, the associations between EPCs and mPAP levels were estimated in multinomial logistic models (Table   2). EPCs was significantly associated with a decreased risk of high PH (OR = 0.42, 95%CI: 0.19-0.95, P<0.05). We next adjusted age and gender and examined the associations between EPCs and mPAP levels, and the results showed that EPCs was significantly associated with a decreased risk of high PH (OR = 0.38, 95%CI: 0.17-0.87, P<0.05). After adjustment of gender, age and BMI, the EPCs was also significantly associated with a decreased risk of high PH (OR = 0.37, 95%CI: 0.16-0.87, P<0.05).
However, EPCs was not significantly associated with middle PH (P>0.05).

Discussion
PH is a pathophysiological syndrome caused by known or unknown factors, characterized by pulmonary vascular contraction, remodeling and in situ thrombosis. Generally, endothelial dysfunction, oxidative stress, inflammation, and angiogenesis disorders are considered as the main causes. The progressive increase of pulmonary vascular resistance restricts blood flow and causes an abnormal increase of pulmonary artery pressure, eventually leading to hemodynamic changes in pulmonary circulation, impaired right heart function, and even death [2,21,22]. The previous PH is defined as mPAP≥25mmHg measured by right heart catheterization when the subject is at rest in a lying position [23]. According to the study, the normal mPAP value is 14±3.30mmHg when people are resting, and the mPAP should not exceed 20mmHg under normal circumstances even considering age, gender, race, and other related factors. In our study, the mPAP is 14.82±2.04mmHg in non-PH groups, similar to the above study. Recent studies have found that patients with mPAP ranging 20mmHg<mPAP≤25mmHg have a significantly increased risk of disease progression. Therefore, 20mmHg<mPAP≤25mmHg can be considered as the early stage of pulmonary vascular disease [18,24].
Some studies have suggested that peripheral blood EPCs may be derived from bone marrow EPCs, and blood sampling is a relatively non-invasive method compared with bone marrow puncture [25,26]. Therefore, studies on EPCs have shifted from bone marrow EPCs to peripheral blood EPCs in recent years. It can be seen that EPCs in peripheral blood are biomarkers of various pathophysiological states. Because flow cytometry is relatively sensitive to the identification of biomarkers, it has high universality and usability. During the past decade, this technology has developed rapidly and become the mainstream method for separation, classification, and analysis of EPCs [27][28][29]. EPCs are counted by flow cytometry using different markers or combinations of them.
There is still a considerable controversy over the phenotype of EPCs. Currently, the common expression of CD34, CD133, and VEGFR2 is most widely used for the identification of endothelial progenitor cells. The human expression of VEGFR2 is also known as KDR [16,19,24]. CD133 is expressed in early endothelial progenitor cells but absent in mature endothelial cells, which is the surface marker of endothelial progenitor cells [30,31]. KDR is an important marker of endothelial tissue [32,33]. However, CD34 expressed in endothelial cells at any stage cannot be used as a specific marker of endothelial progenitor cells [12,27,33]. Therefore, flow cytometry was used in this study to define CD133+/KDR+ cells as endothelial progenitor cells. It has been proved that CD133+/KDR+ cells can differentiate endothelial cells in vitro and in vivo, contributing to the reendothelialization of the left heart, and promoting endothelial regeneration at the site of ischemia and vascular injury [12]. However, since there is no unified definition and classification of endothelial progenitor cell surface markers at present, some researchers have used combinations of other surface markers to identify different EPCs subgroups [19,20,34].
In the present study, both unadjusted and adjusted mPAP decline steadily with the increase in the level of EPCs. In the high PH group (>25mmHg) the risk of PH is significantly higher than that in the non-PH group (P<0.05), regardless of adjusting gender, age, and BMI or not. However, a significant difference in risk of PH between EPCs and the middle PH group (20-25mmHg) is not found (P>0.05).
PH severity is negatively correlated with the number of EPCs, suggesting that the reduction of EPCs increases the risk of PH among CHD patients. At present, there are few studies on the relationship between EPCs and PH in children with CHD. In the study by Zhu et al., [1,14] the number of EPCs is reduced in idiopathic PH, which is consistent with our study. Liu et al. [15] also found that EPCs in PH combined with the chronic obstructive pulmonary disease was decreasing. However, Schiavon et al. [13] concluded that elevated EPCs in patients with end-stage PH may be associated with a long course of illness, leading to a compensatory proliferation of EPCs. Other surface markers have been used to identify other EPCs subsets. Some scholars argue that reduced EPCs levels may lead to endothelial dysfunction in CHD patients, triggering PH. Due to the continuous damage of pulmonary artery endothelial cells caused by PH, EPCs are mobilized to repair them, and then EPCs are gradually exhausted. The higher the mPAP is, the more EPCs are consumed, while EPCs cells are reduced, thereby affecting the repair of pulmonary artery endothelial cells and causing a vicious cycle [16,35].
Sen et al. [12] believe EPCs to be an important marker of cardiovascular diseases.
Through this study, in patients with CHD, due to the increase of blood flow or accelerated flow rate in the pulmonary artery, endothelial cells are damaged or subject to dysfunction and EPCs are largely used to repair endothelial cells, leading to a decrease in the number. EPCs can be assumed to be a protective factor of PH and associated with pulmonary artery pressure. According to the present study, no difference in the middle PH group is probably because that the condition of these patients is relatively mild, and the peripheral blood EPCs are enough to repair the endothelial injury; so the EPCs are not excessively consumed. Recent studies have found that EPCs play an important role in maintaining vascular homeostasis, reversing pulmonary vascular remodeling and promoting angiogenesis; their function is to participate in the differentiation of vascular smooth muscle cells and potential cardiomyocytes by releasing cytokines, growth factors, and chemokines [34,36,37]. In addition, various cytokines and VEGF may inhibit the mobilization of bone marrow EPCs and indirectly reduce peripheral EPCs [38,39]. By releasing angiogenic factors, anti-apoptotic factors and antiinflammatory factors, some cells can be differentiated or have paracrine to play the therapeutic role of progenitor cells [10,16]. Thus, EPCs have been used in treating PH in animal studies and achieved good results [40][41][42]. Lavoie et al. [43] applied EPCs in the experimental treatment of idiopathic PH patients and found that the pulmonary artery pressure decreased to varying degrees, but could not be completely reduced to a normal level. Hence, the treatment should still be combined with pulmonary artery antihypertensive drugs. It has provided a good prospect for the radical treatment of PH.
According to our study, EPCs may be a protective factor of PH in children with CHD. Therefore, for patients with congenital heart disease complicated with pulmonary hypertension, if the pulmonary artery pressure cannot be reduced to a normal level after surgery, EPCs may be an effective treatment.
Strengths and limitation: There are few studies about the relationship between EPCs and PH in children with CHD. A rigorous experimental design was carried out strictly. However, there are many types of CHD in the study, which may affect the final results. We will continue to conduct research on a specific CHD to obtain more scientific results. Due to the low incidence of PH in children with CHD, the sample size of the experiment was small which may affect the results. In the further experiment, we will collect more samples to confirm our conclusion.

Conclusion
The current study revealed the relationship between EPCs and PH in children with CHD. The findings suggest that a reduction in children with CHD may be associated with an increase in mPAP. There was a significant correlation between EPCs and high PH in CHD patients and EPCs may become an effective treatment for PH in CHD patients.

Ethics approval and consent to participate
The study was conducted in accordance with the Declaration of Helsinki and approved by the local ethics committee of Qingdao Women and Children's Hospital (No. QFELL-KY-2019-034). All the parents' patient has given their Parental consent for this study.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
The National Natural Science Foundation of China (No. 81770316) and Shandong Taishan Scholarship (2017) provided funding for the project. The funding bodies had no role in the development of the study design, analysis, interpretation, or in the writing of the manuscript or the decision to submit it for publication.

Authors' contributions
HXS had primary responsibility for patient screening, enrollment, outcome assessment, and drafted the manuscript. GJL performed the data analysis and drafted the manuscript. ZHD reviewed and revised the paper. ZB contributed to patient screening and enrollment. ZHJ contributed to the outcome assessment. GL summarized clinical data. SLP was responsible for study conception and design. All authors read and approved the final manuscript.