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 . 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 [27–29]. 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 re-endothelialization of the left heart, and promoting endothelial regeneration at the site of ischemia and vascular injury . 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. also found that EPCs in PH combined with the chronic obstructive pulmonary disease was decreasing. However, Schiavon et al. 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.  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 anti-inflammatory 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 [40–42]. Lavoie et al.  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.